WO2013151735A1 - Method to mitigate long-qt arrhythmias - Google Patents

Abstract

Screening for compounds capable of mitigating LQTS includes incubating cardiomyocytes, providing a test population, and contacting a candidate compound. An outward potassium ion current is measured in response to a depolarizing voltage. If the outward potassium current is increased relative to an outward potassium current in a control population, then it is determined that the candidate compound is capable of treating LQTS. In another embodiment, a method includes contacting an agent that prolongs an action potential to a first test population. After contacting the candidate compound, an action potential is measured. If a duration of the action potential is reduced towards a control duration, then the candidate compound is capable of mitigating LQTS. Some embodiments include determining that the action potential in a second test population with the candidate but not the agent is not different from action potential of a control population.

Description

METHOD TO MITIGATE LONG-QT ARRHYTHMIAS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of United States Provisional Application No. 61619983, filed April 4, 2012, of which its entirety is incorporated herein.

BACKGROUND OF THE INVENTION

[0002] Congenital long-QT syndrome is a genetic disorder encompassing a family of mutations that can lead to aberrant ventricular electrical activity. These genetic mutations are called "channelopathies"; they are the responsible genes that encode for protein channels that regulate the flow of sodium, potassium, and calcium ions in and out of the cardiac myocyte. The result of these mutations is an increased risk of ventricular arrhythmia, specifically torsade de pointes that can lead to syncope, aborted cardiac arrest, and sudden cardiac death.

[0003] Acquired long-QT and secondary arrhythmias can result from cardiac ischemia, bradycardia and metabolic abnormalities such as low serum potassium or calcium

concentration Acquired long-QT can also result from treatment with certain medications, including antibiotics, antihistamines, general anesthetics, and, most commonly,

antiarrhythmic medications. (Zipes D P, Am. J. Cardiol. v59, pp6E-31E, 1987). Indeed, many promising drugs for treating a plethora of different diseases have been abandoned in the early stages of drug testing because of adverse side effects related to causing acquired long-QT.

[0004] Cardiac arrhythmias are a common cause of morbidity and mortality, accounting for approximately 11% of all natural deaths (e.g., see Kannel WB, Kannel C, Paffenbarger RS Jr, Cupples LA., "Heart rate and cardiovascular mortality: the Framingham Study" Am Heart J. vl l3, no.6, ppl489-94, Jun 1987, hereinafter Kannel 1987; and Willich SN, Stone PH, Muller JE, Toiler GH, Crowder J, Parker C, Rutherford JD, Turi ZG, Robertson T,

Passamani E, et al., "High-risk subgroups of patients with non-Q wave myocardial infarction based on direction and severity of ST segment deviation," Am Heart J, vl 14, no.5, ppl 110- 9, Nov 1987, hereinafter Willich 1987). In general, presymptomatic diagnosis and treatment of individuals with life-threatening ventricular tachyarrhythmias is poor; and, in some cases, medical management actually increases the risk of arrhythmia and death (Cardiac

Arrhythmia Suppression Trial II Investigators, 1992). These factors make early detection of individuals at risk for cardiac arrhythmias and arrhythmia prevention high priorities.

Although long-QT (LQT) is not a common diagnosis, ventricular arrhythmias are very common; more than 300,000 United States citizens die suddenly every year (Kannel 1987; Willich 1987) and, in many cases, the underlying mechanism may be aberrant cardiac repolarization.

SUMMARY OF THE INVENTION

[0005] Applicants have determined that early after de-polarizations can lead to LQT and can be effectively treated by activating potassium channels, such as outward rectifying potassium two pore channels (abbreviated K2p channels), to induce outward potassium ion currents.

[0006] Techniques are provided for screening for compounds capable of treating long- QT syndrome.

[0007] In a first set of embodiments, a method includes isolating cardiomyocytes and incubating the cardiomyocytes under conditions suitable for measuring ion currents. A test population of one or more cardiomyocytes is provided. A candidate compound is contacted to the test population. An outward potassium ion current from a cardiomyocyte in the test population is measured in response to applying a depolarizing voltage. It is determined whether the outward potassium current is increased in the test population relative to an outward potassium current in a control population of one or more cardiomyocytes in response to applying the depolarizing voltage. If the outward potassium current is increased in the test population relative to the control population, then it is determined that the candidate compound is capable of mitigating long-QT syndrome.

[0008] In a second set of embodiments, a method includes isolating cardiomyocytes and incubating the cardiomyocytes under conditions suitable for measuring action potential. A first test population of cardiomyocytes is provided. A positive ion channel blocker is contacted to the first test population; and, a candidate compound is contacted to the first test population. An action potential in response to applying an excitation voltage is measured in the first test population; and, it is determined whether an early afterdepolarization occurs or is reduced in the action potential of the first test population. If the early afterdepolarization is substantively reduced or absent, then it is determined that the candidate compound is capable of treating long-QT syndrome.

[0009] In some embodiments of the second set of embodiments, the method includes providing a second test population of cardiomyocytes; and, contacting the candidate compound to the second test population. An action potential in response to applying an excitation voltage is measured in the second test population. If the early afterdepolarization is substantively absent and the action potential in the second test population is not substantively different from action potential in a control population of cardiomyocytes, then it is determined that the candidate compound is capable of treating long-QT syndrome.

[0010] In other embodiments, an apparatus or system is configured to perform one or more steps of one or more of the above methods.

[0011] Still other aspects, features, and advantages of the invention are readily apparent from the following detailed description, simply by illustrating a number of particular embodiments and implementations, including the best mode contemplated for carrying out the invention. The invention is also capable of other and different embodiments, and its several details can be modified in various obvious respects, all without departing from the spirit and scope of the invention. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present invention is illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings and in which like reference numerals refer to similar elements and in which:

[0013] FIG. 1A and FIG. IB are block diagrams that illustrate an example experimental setup for measuring an electrocardiogram (ECG) and a corresponding example ECG, according to an embodiment;

[0014] FIG. 1C and FIG. ID are block diagrams that illustrate an example experimental setup for measuring an action potential (AP) and a corresponding example AP trace, according to an embodiment;

[0015] FIG. 2A and FIG. 2B are block diagrams that illustrate an example experimental setup for measuring a patch clamped ion current and a corresponding example ion current trace, according to an embodiment;

[0016] FIG. 3 is a flow chart that illustrates an example method of screening for compounds capable of treating long-QT syndrome, according to one embodiment;

[0017] FIG. 4 is a flow chart that illustrates an example method of screening for compounds capable of treating long-QT syndrome, according to another embodiment;

[0018] FIG. 5A and FIG. 5B are graphs that illustrate example experimental AP traces resulting from use of a compound screened for treating long-QT syndrome in a canine, according to an embodiment;

[0019] FIG. 6 is a graph that illustrates example AP traces that express prolonged afterdepolarization and with and without a compound screened for treating long-QT syndrome in a guinea pig, according to an embodiment;

[0020] FIG. 7 is a graph that illustrates example dose dependent curves showing reduction of elongation of AP duration (in percent) with dose of a compound screened for treating long-QT syndrome in a guinea pig, according to an embodiment;

[0021] FIG. 8A through FIG. 8D are graphs that illustrate example experimental sodium cyanide induced K⁺ currents measured in cardiomyocytes as affected by various K⁺ current blocking factors (blockers), according to an embodiment; [0022] FIG. 8E is a bar graph that illustrates example effectiveness of various blockers to sodium ion currents induced by sodium cyanide, according to an embodiment;

[0023] FIG. 9A through FIG. 9C are graphs that illustrate example computed AP traces resulting from changes in ion channel currents based on a model of AP in canines, according to an embodiment;

[0024] FIG. 10 is a block diagram that illustrates a computer system upon which an embodiment of the invention may be implemented; and

[0025] FIG. 11 illustrates a chip set upon which an embodiment of the invention may be implemented.

DETAILED DESCRIPTION

[0026] A method, composition of matter, article of manufacture and apparatus are described for screening for compounds capable of mitigating long-QT (LQT) syndrome. In the following description, for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known structures and devices are shown in block diagram form in order to avoid unnecessarily obscuring the present invention. Various references are cited below. In each case the cited reference is hereby incorporated in its entirety as if fully set forth herein, except for terminology inconsistent with that used herein.

[0027] Some embodiments of the invention are described below in the context of artificially induced LQT in cardiomyocytes by administration of a rapid potassium ion channel blocker drug, dofetilide, screening the drug ONO (i.e. 4-chloro-2-[[(E)-3-(4- pentylphenyl)prop-2-enoyl]amino]benzoic acid, and mitigation of the induced LQT by administration of ONO. However, the invention is not limited to this context. In other embodiments, other drugs (such as terfenidine, erythromycin, quinidine or others, e.g., see Redfern et al., " Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development," Cardiovascular Res., v58, pp 32-45, 2003) are used to induce LQT or early afterdepolarization (EAD) in a population of myocytes, or myocytes genetically disposed to LQT are used, or myocytes not inclined to LQT are used, other compounds are screened, and other compounds that pass the screen are administered in therapeutically effective amounts or prophylactically effective amounts for genetically induced or acquired long QT and the arrhythmias resulting from long QT. [0028] The following terms as used herein have the corresponding meanings given here. TABLE 1. Definitions of terms used herein

response to the passage of an action potential

nucleic acid a molecule comprising a sequence of one or more repeating chemical units known as "nucleotides" or "bases." There are four bases in deoxyribonucleic acid (DNS): adenine, thymine, cytosine, and guanine, represented by the letters A, T, C and G, respectively. In Ribonucleic acid (RNA) the base uracil (U) replaces the base thymine (T).

OMIM Online Mendelian Inheritance in Man, a database of human genes and genetic disorders developed by staff at Johns Hopkins University and hosted on the internet.

ONO 4-chloro-2-[[(E)-3-(4-pentylphenyl)prop-2-enoyl]amino]benzoic acid

Polarized A membrane that maintains an electrical potential difference membrane (polarization) across the membrane

prophylactically an amount of a therapeutic agent, which, when administered to a effective amount subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of a disease or set of one or more symptoms, or reducing the likelihood of the onset (or reoccurrence) of the disease or set of symptoms. The full prophylactic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations protein a generic term referring to native protein, fragments, peptides, or

analogs of a polypeptide sequence. Synonym used herein includes "polypeptide." Hence, native protein, fragments, and analogs are species of a polypeptide genus

sample includes any biological specimen obtained from a subject

subject An organism that is an object of a method or material, including

mammals, e.g., humans, dogs, cows, horses, kangaroos, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. Synonyms used herein include "patient" and "animal" substantively Not negligible for a particular purpose

therapeutic agent any agent administered with the intent of preventing or treating a

specific disease or diseases or some manifestation of same.

therapeutically an amount of a therapeutic agent, which achieves an intended effective amount therapeutic effect in a subject., e.g., eliminating or reducing the severity of a disease or set of one or more symptoms The full therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations treating taking steps to obtain beneficial or desired results, including clinical results, such as alleviating or ameliorating one or more symptoms of a disease; diminishing the extent of disease; delaying or slowing disease progression; ameliorating and palliating or stabilizing a metric

(statistic) of disease. "Treatment" refers to the steps taken.

1. Overview

[0029] Applicants have determined that LQT measured in mammals is related to early afterdepolarizations (EAD) recorded in vitro, and that EAD are mitigated without adverse effects on normal action potential repolarization by compounds that increase outward rectifying potassium current measured in individual cardiomyocytes.

[0030] Applicants have developed methods to screen compounds for their capability to mitigate LQT.

[0031] In one set of embodiments, the method comprises measuring action potentials of in vitro heart tissue exposed to a candidate compound in two samples, a first tissue sample from a normal heart, and a second tissue sample from the normal heart exposed to a positive ion current blocker, such as dofetilide, which induces EAD. If the candidate compound shortens the action potential duration in the second tissue sample but does not substantively affect the action potential duration in the first tissue sample, then the compound is capable of mitigating long QT syndrome, whether the long QT syndrome is congenital or induced by medication. A capable compound is determined to be viable if the compound also passes safety metrics.

[0032] In another set of embodiments, the method comprises measuring outward rectifying potassium current in an individual cardiomyocyte with the candidate compound. If the candidate compound induces an increased outward rectifying potassium current compared to a control, then the compound is capable of mitigating long QT syndrome, whether the long QT syndrome is congenital or induced by medication. A capable compound is determined to be viable if the compound also passes safety metrics.

[0033] In some of these embodiments, proper dosing for the capable candidate compound is determined by further action potential measurements in heart tissue samples from the normal heart exposed to a positive ion current blocker, such as the potassium ion current blocker dofetilide.

1.1 Apparatus overview

[0034] FIG. 1A and FIG. IB are block diagrams that illustrate an example experimental setup for measuring an electrocardiogram (ECG) and a corresponding example ECG, according to an embodiment. FIG. 1A is a block diagram that illustrates an example experiential setup 100 to measure the electrical activity of the heart 192 over a period of time, as detected by electrodes 112 attached to the outer surface of the skin of a subject 190 and recorded by processing equipment 110 external to the body and connected to the electrodes 112 by corresponding leads 114. In some embodiments, the processing equipment 110 is specialized equipment; and, in some embodiments, the processing equipment 110 is or includes a computer system, as described in more detail below with reference to FIG. 10. A graph of measured electric field as a function of time detected at one or more electrodes 112 through leads 114 is called the electrocardiogram (ECG, also abbreviated as EKG). Although FIG. 1A depicts a subject 190 and the subject's heart 192 for purposes of illustrating deployment of system 100, neither is part of the system 100. In some embodiments, a drug to induce or treat LQT is introduced to the subject at or near the heart. In such embodiments, the experimental setup includes drug infusion equipment 116 configured to administer the drug to the heart 192 of the subject, e.g., intravenously or by catheter 115 disposed in or near the heart 192. In some embodiments, the drug infusion equipment is controlled by the processing equipment 110.

[0035] FIG. IB is a block diagram that illustrates an example normal ECG trace 120 for a single heartbeat, according to an embodiment. Time increases to the right and electric field strength increases vertically. The ECG trace 120 shows several labeled features including an initial increase P 101 in electric field followed in sequence by a dip Q 103, a spike R 105, another dip S 107 and a final increase T 109. Certain intervals are defined by these labeled features, including PR interval 121 from beginning of P 101 to beginning of Q 103, QRS complex from beginning of Q 103 to end of S 107, ST segment from end of S 107 to beginning of T 109, and QT interval from beginning of Q 103 to end of T 109. An RR interval refers to a time interval from the peak of R 105 to the peak of R in the next heartbeat, not shown. A rate corrected QT (abbreviated QTc) is given by the ratio of the QT interval to the square root of the RR interval. LQT occurs when the QT interval, or QTc, is

substantively prolonged relative to normal variations.

[0036] FIG. 1C and FIG. ID are block diagrams that illustrate an example experimental setup for measuring an action potential (AP) and a corresponding example AP trace, according to an embodiment. FIG. 1C is a block diagram that illustrates an example experimental setup 130 for measuring action potential in a sample of heart tissue 194. The heart tissue 194, comprising multiple cardiomyocytes, is isolated and incubated in vitro in an in vitro vessel 195 under conditions suitable for measuring action potential, as described in more detail below in the methods section. The in vitro vessel 195 is at electrical ground.

[0037] A pulse generator 132 is electrically connected to electrode 134 to introduce a driving voltage pulse. The pulse traverses the tissue as a propagating voltage termed the action potentials (AP) that is sustained by ions flowing through ion channels in the membranes of the cardiomyocytes. As described below, the ions utilized by cardiomyocytes to propagate the AP include sodium ions Na⁺, calcium ions Ca⁺ and potassium ions K²⁺, as described in more detail below. Several voltage gated ion channels are involved in the opening and closing to allow the various ions to pass through the membrane to support and shape the action potential. The AP is measured at electrode 138 (that pierces the membranes of one or more cardiomyocites in tissue sample 194) by a voltage detector 136. [0038] The measured AP is recorded by processor 140, such as computer system described below with reference to FIG. 10 or chipset described below with reference to FIG. 11. In some embodiments, the processor 140 also controls the timing and shape of the driving voltage pulse generated by the pulse generator 132. Although FIG. 1C depicts heart tissue 194 and in vitro vessel 195 for purposes of illustrating the deployment of system 130, neither is part of the system 130.

[0039] FIG. ID is a graph 141 that illustrates an example normal measured AP, according

_3 to an embodiment. The horizontal axis 142 is time (e.g., in milliseconds, ms, 1 ms = 10 seconds) increasing to the right. The vertical axis is voltage (e.g., in milliVolts, mV, 1 mV =

_3

10 volts) increasing upward, with the value zero indicated by a horizontal dashed line. At rest, the interior of the membrane is at a non-zero rest polarization (e.g., - 80 mV for cardiomyocytes due to slow potassium leakage through the membrane.). The AP trace 150 is plotted. Upon approach of the pulse, the relative positive voltage causes one or more voltage gated ion channel types to open which allows corresponding ion types to flow and depolarize the membrane for a duration called the AP duration 154 until the rest polarization is restored.

[0040] Myocyte membranes incorporate many well known ion channels, which are membrane proteins that reconfigure under different conditions to either block or assist the up gradient or down gradient passage of a particular ion. There are over 300 types of ion channels in various living cells. Ion channels may be classified by the nature of their gating (the conditions that cause them to open or close), the species of ions passing through those gates (e.g., sodium, calcium and potassium), the number of gates (pores) and localization of proteins.

[0041] Voltage-gated ion channels open and close in response to membrane potential.

Voltage-gated sodium channels include at least 9 members and are largely responsible for action potential creation and propagation. The pore-forming a subunits are very large (up to 4,000 amino acids) and consist of four homologous repeat domains (TIV) each comprising six transmembrane segments (S 1-S6) for a total of 24 transmembrane segments. Voltage- gated calcium channels include 10 members, though these members are known to co- assemble with α₂δ, β, and γ subunits. These channels play an important role in linking muscle excitation with contraction. The a subunits have an overall structural resemblance to those of the sodium channels and are equally large. Voltage-gated potassium channels (Kv) include almost 40 members, which are further divided into 12 subfamilies. These channels are known mainly for their role in re -polarizing the cell membrane following action potentials. For some potassium channels, the a subunits have six transmembrane segments, homologous to a single domain of the sodium channels. Correspondingly, they assemble as tetramers to produce a functioning channel. For K2p channels, the gene encodes two pore domains and the subunits assemble as dimmers.

[0042] A number of specific human gene loci have been identified that are associated with congenital LQT syndrome. The most common causes of congenital LQT syndrome are mutations in the genes KCNQ1 (LQT1), KCNH2 (LQT2), and SCN5A (LQT3). Table 2 is a list of all known human genes associated with LQT syndrome and effects on corresponding ion channels.

Table 2. Human genes associated with LQT syndrome.

[0043] To determine which ion channel types are opening at what times and for how long, ion currents are measured directly in some embodiments. FIG. 2A and FIG. 2B are block diagrams that illustrate an example experimental setup for measuring a patch clamped ion current and a corresponding example ion current trace, according to an embodiment. FIG. 2A is a block diagram that illustrates an example patch clamp experimental setup 260. The patch clamp uses one electrode and a feedback circuit to keep the cell's membrane potential at a level set by an experimenter so that voltage gated ion currents can be determined. The patch clamp 260 operates by negative feedback. The membrane potential amplifier, voltage amplifier 264a, measures membrane voltage at patch electrode 268 that pierces the membrane of a cell (here a myocyte) and sends output to the feedback amplifier 264b. The feedback amplifier 264b subtracts the membrane voltage from the command voltage that is received from the voltage (signal) generator 262. This difference signal is amplified and output is sent into the myocyte via the same patch electrode 268. The current is measured at ammeter 266. The patch electrode 268 include electrolyte in a glass needle to restore the ionic balance inside and outside the membrane. In some embodiments, the voltage generator is controlled by processor 270, such as the computer system of FIG. 10 or chip set of FIG. 11, which also records the current measured at ammeter 266. Although FIG. 2A depicts the cardiomyocyte 296 and in vitro vessel 297, neither is part of the patch clamp experimental setup 260.

[0044] FIG. 2B is a graph 271 that illustrates an example ion current trace 280 measured from a voltage gated ion channel under the influence of a voltage signal 276. Ion current is often normalized by dividing by membrane electrical capacitance. Also plotted is the voltage output by generator 262. The horizontal axis 272 indicates time (e.g., in seconds). The lower vertical axis 274 indicates voltage (e.g., in mV); and, the upper vertical axis 278 indicates

-12

normalized current in picoAmperes (pA, 1 pA = 10 amperes) per picoFarads (pF, 1 pF =

-12

10 farads). In response to non zero voltage 276, an ion channel opens and ions flow, thus producing a non zero current 280, at least for some time.

[0045] Although devices are depicted in FIG. 1A, FIG. 1C and FIG. 2A as integral blocks in a particular arrangement for purposes of illustration, in other embodiments one or more devices, or portions thereof, are arranged in a different manner, or are omitted, or one or more different devices are included.

1.2 Method overview

[0046] FIG. 3 is a flow chart that illustrates an example method 300 of screening for compounds capable of mitigating long-QT syndrome, according to one embodiment.

Although steps are depicted in FIG. 3, and in subsequent flowchart FIG. 4, as integral steps in a particular order for purposes of illustration, in other embodiments, one or more steps, or portions thereof, are performed in a different order, or overlapping in time, in series or in parallel, or are omitted, or one or more additional steps are added, or the method is changed in some combination of ways. [0047] In step 301, as described in more detail in the next subsection, cardiomyocyte tissue is isolated and incubated under conditions suitable for measuring action potential and used to provide two test populations of cardiomyocytes.

[0048] In step 303, an agent that prolongs the action potential, such as potassium channel blocker dofetilide is contacted to the first test population. Dofetilide is marketed under the trade name Tikosyn by Pfizer and has the IUPAC name of N-[4-[2-[2-[4- (methanesulfonamido)phenoxy]ethyl-methyl-amino]ethyl]phenyl]methanesulfonamide. From experiments described below, it has been determined that such contact leads to early afterdepolarizations (EAD) in measured action potentials, which is related to LQT. In some embodiments, step 303 includes measuring the action potential of the first test population to characterize the extent of EAD, e.g., using methods described in detail in the next subsection.

[0049] In step 305, a candidate compound is contacted to the first test population, e.g., after establishing the first population is subject to EAD, using any method known in the art, such as described in the next subsection. In step 307, after contacting the candidate compound, the action potential is measured in the first test population in response to applying an excitation voltage, e.g., using the methods described in the next subsection.

[0050] In step 311, it is determined whether EAD is substantively reduced or eliminated in the first test population compared to a population with EAD, such a known historical EAD response or the response measured during step 303. If no substantive reduction is observed, then in step 313 the compound is determined to be not capable of treating or preventing LQT. For example, in some embodiments, EAD is substantively reduced if EAD is reduced by more than about 95% . In some embodiments, this condition is considered satisfied if the APD is reduced to within normal limits. Reductions of about 50% or more of the difference between the APD in step 311 and that in normal conditions is considered a good criterion. After step 313 the screen ends for the particular candidate compound; and another candidate compound is screened starting with step 301.

[0051] If substantive reduction or elimination is observed, then in step 321 the compound is contacted to the second test population that has not been contacted by the positive ion channel blocker. In step 323, it is determined whether an action potential of the second test population is substantively affected, e.g., changed by more than about 10% In some embodiments, step 323 is based on historical information about the effect of the candidate compound on AP or normal myocytes or cardiomyocytes. In some embodiments, step 323 includes measuring the action potential of the second test population before contacting the candidate compound to characterize the normal AP, e.g., using methods described in detail in the next subsection.

[0052] If it is determined, in step 323, that an action potential of the second test population is substantively affected, then in step 313, as described above, it is determined that the compound is not capable of mitigating LQT. If it is determined, in step 323, that an action potential of the second test population is not substantively affected, then in step 325 it is determined that the compound is capable of mitigating LQT and the screen ends for the candidate compound; and, an different candidate compound is screened starting with step 301.

[0053] In some embodiments, step 325 includes determining a relationship between dose and an amount of reduction of the EAD or an amount of deviations from normal action potential amplitude or duration, or some combination.

[0054] In some embodiments, a screened compound determined to be capable of treating or preventing LQT is administered to a patient in a therapeutically effective dose or a prophylactically effective dose.

[0055] FIG. 4 is a flow chart that illustrates an example method 450 of screening for compounds capable of mitigating long-QT syndrome, according to another embodiment.

[0056] In step 451, as described in more detail in the next subsection, cardiomyocytes are isolated and incubated under conditions suitable for measuring ion currents, such as patch clamp measurements of voltage gated ion currents, and used to provide a test populations of cardiomyocytes.

[0057] In step 455, a candidate compound is contacted to the test population, such as described in the next subsection. In step 457, after contacting the candidate compound, the outward potassium ion current, such as a two-pore outward rectifying potassium (K2p) channel current, is measured in the first test population in response to applying a gating voltage, e.g., using the methods described in the next subsection. For example, in some embodiments, in step 455 the outward potassium (K⁺) ion current is blocked using a K⁺ channel blocker, such as zinc (Zn) or quinidine, as described in more detail below. In some embodiments, the sodium current is blocked by setting the voltage signal at -40 mV or more. In addition, the outward calcium channel is also blocked, e.g. using nisoldipine as described in the next subsection. When the sodium channel and calcium channel are blocked, the outward positive ion current is attributed to potassium ion flow through K2p channels, for reasons given in more detail below.

[0058] In step 461, it is determined whether outward potassium ion current is increased compared to a population of normal cardiomyocytes, such a known historical measurements. In some embodiments, step 461 includes measuring outward potassium ion current in a control population of cardiomyocytes not contacted by the candidate compound. If no substantive increase is observed, then in step 463 the compound is determined to be not capable of treating or preventing LQT. For example, in some embodiments, outward potassium ion current is substantively increased if increased by more than about 10%. [After step 463 the screen ends for the particular candidate compound; and another candidate compound is screened starting with step 451.

[0059] If it is determined, in step 461, that an outward potassium ion current is

substantively increased, then in step 465 it is determined that the compound is capable of mitigating LQT and the screen ends for the candidate compound; and, an different candidate compound is screened starting with step 451.

[0060] In some embodiments, step 465 includes determining a relationship between dose and an amount of potassium current.

[0061] In some embodiments, a screened compound determined to be capable of treating or preventing LQT is administered to a patient in a therapeutically effective dose or a prophylactically effective dose.

1.3 Detailed materials and methods

[0062] Specific materials and methods used in many embodiments are described in this section. Many are described in Maria N. Obreztchikova , Eugene A. Sosunov , Alexei Plotnikov, Evgeny P. Anyukhovsky , Ravil Z. Gainullin , Peter Danilo Jr. , Zi-Ho Yeom, Richard B. Robinson, Michael R. Rosen, "Developmental changes in IKr and IKs contribute to age-related expression of dofetilide effects on repolarization and proarrhythmia,"

Cardiovascular Research, v59, pp339-350, 2003 (hereinafter Obtreztchikova 2003).

[0063] In vivo (also called in situ) experiments were conducted with canines or guinea pigs using similar protocols. For example, following anesthesia with thiopental (also known as sodium thiopental, better known as Sodium Pentothal™, a trademark of Abbott Laboratories, Abbott Park, Illinois), 17 milligrams per kilogram weight of animal intravenous (mg/kg i.v. , the animals were then intubated, and ventilated with isoflurane (i.e. 2-chloro-2- (difluoromethoxy)-l,l,l-trifluoro-ethane) 1.5-3.0% and oxygen. The femoral artery was catheterized to monitor blood pressure and both cephalic veins were catheterized to maintain drug infusion and facilitate collection of blood samples. A heating pad was used to maintain body temperature.

[0064] Seven electrocardiographic leads were recorded using "Dr. Vetter PC-EKG" software (Dr. Vetter, Baden-Baden, Germany). Recordings are made before and after dofetilide infusion and before and after contact with a candidate compound. ECG parameters were measured from at least 10 consecutive complexes at every experimental time point and averaged values were analyzed. Rate correction of the QT interval was done using Bazett's formula (Bazett HC. "An analysis of the time-relations of electrocardiograms". Heart v7, pp 353-370, 1920). QT and QTc dispersion were calculated from seven ECG leads (I-aVFand V10) as difference between maximal (QTmax) and minimal (QTmin) intervals measured in simultaneously recorded leads. Data were analyzed as average dispersion calculated in three consecutive complexes.

[0065] Dofetilide (a gift of Helopharm, Berlin, Germany) was administered as a 0.1 mg/kg i.v. bolus followed by 0.1 milligrams per kilogram of animal weight per minute (mg/kg/min) infusion for 10 min. The infusion was terminated if ventricular arrhythmias developed.

[0066] In young and adult dogs, 1 to 2 milliliters (ml) of blood was drawn before dofetilide administration and at the end of drug infusion. In neonates, blood was drawn only at the end of drug infusion. When an arrhythmia occurred, a blood sample was obtained at that time and the infusion was terminated. Blood samples were heparinized and centrifuged at 10,000 revolutions per minute (rpm) for 30 min and stored at -70 °C. [0067] Plasma drug levels were determined by high-performance liquid chromatography (HPLC). Dofetilide was extracted from plasma with a liquid-liquid extraction column (EXTRELUT™ NT1, Merck, Germany) as follows: 600 ml of plasma sample was applied to the column and equilibrated for 10 min, followed by diethyl ether 1 ml for 10 min. This elution process was repeated three times. Eluted solvent was evaporated under a stream of nitrogen. The dry residue was re-suspended in 200 ml of acetonitrile (J. T. BAKER

CHEMICAL COMPANY CORPORATION™, New Jersey, USA) (gradient A) and 0.1% trifluoroacetic acid (S IGM A- ALDRICH CO. LLC™, St. Louis, Missouri) (gradient B). Finally, a 30-ml aliquot was injected into the HPLC system.

[0068] Dofetilide concentrations were measured with a HP 1100 series system, (Hewlett- Packard, Germany) and a HYPERSIL® BDS-C , 125x2.0 mm I.D., 3 μιη particle size column (AGILENT, USA). The UV detection wavelength was 224+/- 5 nanometers (nm), and flow-rate, 0.25 ml/min. The calibration was linear from 10 to 1250 nanograms per milliliter (ng/ ml).

[0069] For multicellular tissue preparations, e.g., from canine or guinea pigs, animals were anesthetized with sodium pentobarbital, 30 mg/kg i.v. (young and adult) or 40 mg/kg i.p. (neonate). Their hearts were removed through a left lateral thoracotomy and immersed in cold Tyrode' s solution (SIGMA- ALDRICH CO. LLC™, St. Louis, Missouri) equilibrated with 95% 0₂ and 5% C0₂ and containing (in units of millimolar per liter, mmol/ 1): NaCl 131, NaHC0₃ 18, KC1 4, CaCl₂ 2.7, MgCl₂ 0.5, NaH₂P0₄ 1.8 and dextrose 5.5. Epicardial, endocardial, and midmyocardial strips (-10x5x0.5 to 1 mm) were filleted with surgical blades parallel to the surface of the left ventricular free wall. The preparations were placed in a tissue bath and superfused with control Tyrode' s solution (37 °C, pH 7.35+0.05). Solution was pumped at 12 ml/min, changing chamber content three times /min. The bath was connected to ground via a 3 M KCl/Ag/AgCl junction.

[0070] For action potential (AP) measurements, preparations were impaled with 3 molar per liter (mol/ 1) KCl-filled glass capillary microelectrodes having tip resistances equal to 10 to 20 megaohm (ΜΩ equal to 10⁶ ohms.). The electrodes were coupled by an Ag/AgCl junction to an amplifier with high input impedance and capacitance neutralization. Transmembrane action potentials were digitized with an analog-to-digital converter (D-210, DATAQ INSTRUMENTS™, Akron, OH) and stored on a personal computer for subsequent analysis. For stimulation of preparations, standard techniques were used to deliver square- wave pulses 1.0 milliseconds (ms) in duration and 1.5 times threshold through bipolar TEFLON™ (from 3M of Minneapolis MN)-coated silver electrodes. To investigate frequency-dependence of drug effects, preparations were driven at cycle lengths (CLs) from 4000 to 250 ms in sequence. Each frequency was maintained for 3 min before data were collected.

[0071] Experiments began after 3 h of equilibration in control Tyrode's solution. The effects of dofetilide 10 ⁸ to 10 ⁶ mol/1 and of the Ι_& and I_Ks blocker azimilide (a gift of Procter and Gamble) (5x10 ⁶ mol/1 in the presence of dofetilide, 10 ⁶ mol/1) were studied. Measurements of drug effects commenced after 30 min equilibration at each concentration.

[0072] For single myocyte preparations, canine LV subepicardial myocytes were isolated. A collagenase perfusion method reported previously was used (e.g., see Charpentier F, Liu Q-Y, Rosen MR, Robinson RB, "Age-related differences in beta-adrenergic regulation of repolarization in canine epicardial myocytes," Am J Physiol v271, ppHl 174-H1181, 1996, hereinafter Charpentier 1996). Briefly, a wedge of left ventricular free wall was dissected, and the first or second branch of the left circumflex coronary artery was cannulated. After 10 to 15 min of collagenase perfusion for adults and 6 min for young, the epicardial layer (~1 to 2 mm) was removed, placed in a beaker, minced, incubated in fresh collagenase solution and agitated with 95% 0₂ and 5% C0₂ for 5 to 15 min. Incubation was repeated 3 to 5 times, and the supernatant from each digestion was centrifuged. Isolated cells were stored at room temperature in buffer solution.

[0073] For ion current recordings, myocytes were placed in a heated bath and superfused with a modified Tyrode's solution containing (in mmol/ 1): NaCl 140, KC1 5.4, CaCl₂ 1.8, MgCl₂ 1, HEPES 5, glucose 10 (pH 7.4 adjusted with NaOH) at 35 °C. Delayed rectifier potassium current (I_K) was recorded via whole cell patch clamp using a personal computer equipped with pClamp 8 software, DigiData 1200 series interface and Axopatch ID amplifier (Axon Instruments). Borosilicate glass pipettes had tip resistances of 1 to 2.5 ΜΩ. L-type Ca²⁺ current was blocked with 10 mol/ 1 nisoldipine. Na⁺ current was inactivated by holding cells at -40 mV, or using one of the Na⁺ current blockers described below with reference to FIG. 8A through FIG. 8E. The pipette solution contained (in mmol/ 1) KOH 60, KC1 80, aspartate 40, HEPES 5, EGTA 10, MgATP 5, Na creatinine phosphate 5, CaCl₂ 0.65 (pH was adjusted to 7.2 with 1 M NaOH). Currents were recorded during 5 s depolarizing test pulses ranging from -20 to +55 mV in 15 mV increments and upon repolarization to holding potential -40 mV. Pulses were applied at 20 s intervals to ensure deactivation of tail currents. The protocol was run before and after exposure of a cell to dofetilide, 10 ⁶ mol/ 1. Where indicated, chromanol 293B (ira«5'-N-[6-cyano-3,4-dihydro-3-hydroxy-2,2-dimethyl-2H-l- benzopyran-4-yl]-N-methyl-ethanesulfonamide, a gift of AVENTIS PHARMA™,

Germany), 10 ⁵ mol/ 1, was added to dofetilide and the protocol was repeated. I_& was obtained by digital subtraction from controls of currents recorded in the presence of dofetilide. The dofetilide-resistant current was considered I_Ks.

[0074] In adult myocytes, in which I_Ks was observed, current rundown was checked by comparing total I_K tail at 25 to -55 mV from 2 to 8 min after rupture (measurement window). No significant rundown was seen. (slow potassium current) was not observed in most young myocytes. The possibility of this being a rundown artifact was tested by recording from three myocytes using the perforated patch method Charpentier 1996. defined as a chromanol- sensitive outward tail current, was observed in one of the three cells. This incidence is comparable to that which was found using ruptured patch in two of 10 young myocytes.

[0075] The current-voltage (I-V) relationship was determined by first fitting tail currents normalized to cell capacitance, with a double-exponential fit of the form given by Equation 1.

I_t = Ao + A] e -t/τΙ) + A₂ e⁽^²) . (1)

The voltage dependence of Ιχ_τ (rapid potassium current) activation was then determined by fitting these amplitude values with a Boltzmann function given by Equation 2.

/ = /_max/{ l+ exp[(V_1/2 - Vt)/k] } (2) to obtain /max , V_1/2 and k. Statistical comparison was done on the average of the individual fit parameters. Each cell's activation curve was then normalized by that cell's /_max value to generate an average conductance graph.

[0076] Data are expressed as mean + S.E.M. Values for ionic currents were corrected for cell capacitance, which was 20+1 picoFarad (pF, for number of sample, n, =10) for young and 207+6 pF (n=16) for adult myocytes. One-way or two-way analysis of variance for repeated or non-repeated measures was used for data analysis, with Bonferroni's test when the F-value permitted. Significance of incidence of early afterdepolarization was evaluated with Fisher's exact test. Post-hoc analysis was used to estimate dofetilide effects on ECG parameters across age groups using the Bonferroni test where variances were equal and Games-Howell where variances were unequal. P<0.05 was considered significant.

[0077] The work by Obtreztchikova 2003 indicated that the extent to which elongation of AP duration, dispersion and EADs are manifested across the life cycle in the absence and presence of Ιχ_τ blocking drugs (and in the absence or presence of disease) appear to be the ultimate determinants of proarrhythmia expression, such as LQT.

2. Example Embodiments

[0078] In illustrated embodiments, the compound ONO was screened using the methods of FIG. 3 and FIG. 4, described above, and found capable of mitigating LQT, at least in some animals, by both methods. These embodiments also illustrate the relationship of the two methods to each other and to LQT.

[0079] FIG. 5A through FIG. 5B are graphs that illustrate example experimental AP traces resulting from use of a compound screened for mitigating long-QT syndrome in a canine, according to an embodiment. FIG. 5A is a graph 500 that illustrates example action potential (AP) in canine cardiomyocyte tissue, according to an embodiment of some steps of method 300. The horizontal axis 502 indicates elapsed time in milliseconds (ms) from arrival of the excitation voltage. The vertical axis 504 indicates voltage in milliVolts (mV) from zero (indicated by dashed line). As can be seen, the rest state before arrival of the excitation voltage is a polarization voltage of about -80mV. The arrival of the excitation voltage at time t=0 opens one or more ion channels that allow ions to flow that depolarize the membrane potential of the cardiomyocyte tissue. Traces are shown for a normal control tissue represented by AP trace 511, tissue contacted by potassium channel blocker dofetilide (as in step 303 of method 300) represented by AP trace 512, and tissue contacted by both dofetilide and ONO (as in step 305 of method 300) represented by AP trace 513.

[0080] After an action potential duration (APD) of about 200 ms, the normal AP trace 511 re-polarizes to the rest polarization state. However, the dofetilide alone AP trace 512, after beginning to re-polarize near 200 ms, suddenly depolarizes again, giving an example of early afterdepolarization (EAD). Upon application of the screened compound ONO, the AP trace 513 (e.g., as measured during step 307 of method 300) shows near normal repolarization and substantively absent EAD. Thus, in step 311 of method 300 in this embodiment, it is determined that the compound ONO substantively reduces EAD.

[0081] FIG.5B is a graph 520 that illustrates example effect of screened compound on action potential (AP) in canine cardiomyocyte tissue, according to an embodiment of other steps of method 300. The horizontal axis 502, vertical axis 504 and AP trace 511 are as described for FIG. 5A. Traces are shown for a normal control tissue represented by AP trace 511, and normal tissue contacted by ONO alone (as in step 321 of method 300) represented by AP trace 523. Upon application of the screened compound ONO alone, the AP trace 523 (e.g., as measured during step 323 of method 300) shows near normal repolarization and no substantive effect on the normal AP. Thus, in step 323 of method 300 in this embodiment, it is determined that the compound ONO does not substantively affect normal polarization. The compound ONO is determined to be a compound capable of mitigating LQT, at least in some circumstances.

[0082] FIG. 6 is a graph 600 that illustrates example AP traces with afterdepolarization prolonged AP and with and without compound ONO screened for treating long-QT syndrome in a guinea pig, according to an embodiment. Here the prolonged action potential was induced by dofetilide. The horizontal axis 602 indicates elapsed time in milliseconds; and the vertical axis 604 indicates voltage in mV, with zero voltage indicated by a horizontal dashed line. AP trace 611 indicates action potential of a control tissue from the guinea pig with normal action potential depolarizing a rest polarization of -80 mV. After an action potential duration (APD) of about 223 ms, normal AP trace 611 re-polarizes to the rest polarization state. However, upon contact with dofetilide alone at a dose of 1 microMole (μΜ) represented by AP trace 612, after beginning to re -polarize, the trace 612 extends the duration of repolarization by about 77 ms, indicative of a long action potential (in a single myocyte which would be equivalent to long Q-T on the ECG). Upon subsequent contact with the screened compound ONO at a dose of 300 nanoMoles (nM), the AP trace 613 shows faster than normal repolarization at 181 ms. Upon application of the screened compound ONO alone at the same concentration (0.3 μΜ =300 nM), the AP trace 614 shows near normal repolarization after 218 ms and no substantive effect on the normal AP. These traces corroborate the results obtained for ONO in canine tissue and depicted in FIG. 4A through FIG. 4D.

[0083] FIG. 7 is a graph 720 that illustrates example dose dependent curves showing reduction of AP duration (in percent) with dose of a compound screened for treating long-QT syndrome in a guinea pig, according to an embodiment. The logarithmic horizontal axis 722 is concentration of ONO in log Moles base 10. The vertical axis is percentage reduction in AP duration (APD). Above or below each point is the number of measurements averaged to produce the point plotted. Trace 731 indicates the reduction of dofetilide-induced extended- APD at half effective dose of ONO (114 nM for returning the APD from the prolonged level to the control level) is contacted to tissue.. The higher the dose (e.g., concentration) of ONO from 10^"7'5 M to 10^"6 M, the greater the reduction of the extended- APD from 0% to 60%. Trace 732 indicates the reduction of APD duration is negligible when ONO alone is contacted to normal heart tissue across all measured doses.

[0084] FIG. 8A through FIG. 8D are graphs that illustrate example experimental sodium cyanide induced K⁺ ion currents measured in cardiomyocytes as affected by various sodium current blocking factors (blockers), according to an embodiment. These plots demonstrate how K⁺ currents can be blocked so that the effect of a compound on potassium channel ion currents can be isolated. The horizontal axis 602 indicates elapsed time in minutes. The vertical axis 804 indicates normalized current in picoAmperes per picoFarad (pA/pF).

[0085] FIG. 8 A is a graph 810 that illustrates example K⁺ ion current 815 in response to 2 milliMoles (mM) of sodium cyanide (NaCN) applied during time line 811. The K⁺ current increases over time but is substantively turned off when contacted by 5 nanoMoles (nM) of

2_

zinc ion (Zn ) as indicated by time line segments 812. FIG. 8B is a graph 820 that illustrates example K⁺ current 825 in response to 2 miUiMoles (mM) of sodium cyanide

2_

(NaCN) applied during time line 821 and 5 nanoMoles (nM) of zinc ion (Zn ) applied during time line 822. FIG. 8C is a graph 830 that illustrates example K⁺ current 835 in response to 2 miUiMoles (mM) of sodium cyanide (NaCN) applied during time line 831 and 0.5 miUiMoles (mM) of Quinidine applied during time line 832. FIG. 8D is a graph 840 that illustrates example K⁺ current 845 in response to 2 miUiMoles (mM) of sodium cyanide (NaCN) applied during time line 841 and 0.5 miUiMoles (mM) of Quinidine applied during time line

2_

842 and 20milliMoles (mM) of Barium ion (Ba ) at time line segment 843.

[0086] FIG. 8E is a bar graph that illustrates example effectiveness of various blockers on K⁺ currents induced by sodium cyanide, according to an embodiment. The horizontal axis 852 indicates the K⁺ channel blocker; and the vertical axis 854 indicates the normalized K⁺ current compared to NaCN induced K⁺ current. The number above each bar indicates the number of measurements averaged to obtain the value. The control bar 852a represents

2_

NaCN only with no blockers and achieves full current. The Zn bar 852b represents NaCN

2_

with Zn blocker and results in negligible current. The Quinidine bar 852c represents NaCN with Quinidine blocker and also results in negligible current. Such example K⁺ channel blockers are used in various embodiments of step 457 to measure the outward rectifying potassium current during method 450.

[0087] In other embodiments, the method 450 is directed to screening another candidate compound, other than ONO, based on the compound's ability to open a percentage of the outward rectifying potassium channels. Activating about 2% or more of the available outward rectifying sodium channels is sufficient. The two important characteristics of a screened compound is that when the compound is contacted to cardiomyocytes there is no significant change in the action potential duration of normal cardiomyocytes and when a set of conditions that produce a prolonged QT interval exist, the compound dramatically attenuates the increase in action potential duration. Support is provided by numerical simulations (in silico) of action potential based on a model of voltage gated ion channels in canine tissue.

[0088] FIG. 9A through FIG. 9C are graphs that illustrate example computed AP traces resulting from changes in ion channel currents based on a model of AP in canines, according to an embodiment. These graphs demonstrate the same two important properties in silico from the most up to date and accurate canine ventricular computer model to date, the modified Hund-Rudy model of the canine ventricular action potential as described in Lau, D.H., Clausen, C, Sosunov, E.A., Shlapakova, I.N., Anyukhovsky, E.P., Danilo, P., Jr., Rosen, T.S., Kelly, C, Duffy, H.S., Szabolcs, M.J., Chen, M., Robinson, R.B., Lu, J., Kumari, S., Cohen, I.S. and Rosen, M.R., "Epicardial border zone overexpression of skeletal muscle sodium channel skMl normalizes activation, preserves conduction, and suppresses ventricular arrhythmia," Circulation vl l9, ppl9-27. PMCID: PMC2731654, 2009.

[0089] FIG. 9A is a graph 900 that indicates AP model results for a control membrane and one in which additional outward rectifying potassium channels are opened, according to an embodiment. The horizontal axis is elapsed time with time scale indicated by scale bar 902 for 60 ms. The vertical axis is relative voltage with voltage scale indicated by scale bar 904 for 20 mV. For purposes of comparison, a zero voltage dashed line is drawn to emphasize correspondence to experimental measurements described above. The computed AP for a control membrane is shown by AP trace 911. The computed AP for a membrane with added outward K conductance representative of an opened K2P channel is shown by AP trace 913. Adding an outwardly rectifying current (K2P) does not alter APD. There is almost no change in the action potential duration. In this simulation, AP duration to a time 90% below maximum (APD₉₀) is 216 ms in control AP trace 911 and 213 ms in AP trace 913 in the presence of the additional K conductance. The permeability of the outward rectifier was 2.25xl0^"8 cm/s).

[0090] FIG. 9B is a graph 920 that indicates AP model results for a control membrane and one in which K channels are altered. The horizontal axis and vertical axis and dashed line and control AP trace 911 are as described above. An equivalent simulation with rapid potassium current (I^) removed is shown by AP trace 935 and a simulation in which the same outwardly rectifying (K2P) permeability as in FIG. 9A is added to the simulation in which /j_jj was removed is shown as AP trace 933. APD90, is 307 ms for AP trace 935 and APD90 is 239 ms for AP trace 933 Including an outwardly rectifying current (K2P) dramatically reduces APD lengthening induced by removal of

[0091] FIG. 9C is a graph 940 that indicates AP model results for a control membrane and one in which sodium and potassium channels are altered. The horizontal axis and vertical axis and dashed line and control AP trace 911 are as described above. An equivalent simulation with a 400% increased Na current (T^a) is shown by AP trace 955 and a simulation in which the same outwardly rectifying (K2P) permeability as in FIG. 9A is added to the simulation in which %a ^was increased is shown as AP trace 953. APD90 is 270 ms for AP trace 935 and APD90 is 232 ms for AP trace 953 Including an outwardly rectifying current (K2P) dramatically reduces APD lengthening induced by an increase in persistent Na current

[0092] There is a large reservoir of K2P channels that can be opened. Opening a small fraction (e.g., about 2%) of these channels should provide the outward current advantageous to re-polarize the cardiac action potential and prevent or terminate the appearance of a prolonged action potential duration leading to the arrhythmias caused by LQT syndrome.

[0093] At present a variety of drugs that prolong the QT interval are not sold commercially or are not even brought to full clinical development because of fear of the acquired LQT syndrome. In addition, a significant subset of patients with congenital LQT syndrome requires implantation of cardioverter/defibrillators. The proposed approach focuses on both these significant clinical problems. First, it provides a means for bringing therapeutic drugs of concern with regard to acquired LQT back to the market by using a K2P opener as an adjuvant. In this way, this method changes the way in which pharmaceutical manufacturers can develop new therapeutic agents. Second, it provides a completely fresh and novel approach to the congenital 1LQT syndrome.

3. Pharmaceutical formulations

[0094] The therapeutic agents may be present in the pharmaceutical compositions in the form of salts of pharmaceutically acceptable acids or in the form of bases. The therapeutic agents may be present in amorphous form or in crystalline forms, including hydrates and solvates. Preferably, the pharmaceutical compositions comprise a therapeutically effective amount.

[0095] Pharmaceutically acceptable salts of the therapeutic agents described herein include those salts derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate,

cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate,

hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate salts. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining pharmaceutically acceptable acid addition salts.

[0096] Salts derived from appropriate bases include alkali metal (e.g., sodium and potassium), alkaline earth metal (e.g., magnesium), ammonium and N⁺(Ci-4 alkyl)₄ salts. It is anticipated that some embodiment include the quaternization of any basic nitrogen-containing groups of the therapeutic agents disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization.

[0097] The therapeutic agents of some embodiments are also meant to include all stereochemical forms of the therapeutic agents (i.e., the R and S configurations for each asymmetric center). Therefore, single enantiomers, racemic mixtures, and diastereomers of the therapeutic agents are within the scope of the invention. Also within the scope of the invention are steric isomers and positional isomers of the therapeutic agents. The therapeutic agents of some embodiments are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, therapeutic agents in which one or more hydrogens are replaced by deuterium or tritium, or the replacement of one or more carbons by ¹³C- or ¹⁴C-enriched carbon are within the scope of this invention. [0098] In a preferred embodiment, the therapeutic agents of some embodiments are administered in a pharmaceutical composition that includes a pharmaceutically acceptable carrier, adjuvant, or vehicle. The term "pharmaceutically acceptable carrier, adjuvant, or vehicle" refers to a non-toxic carrier, adjuvant, or vehicle that does not destroy or significantly diminish the pharmacological activity of the therapeutic agent with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the

compositions of some embodiments encompass any of the standard pharmaceutically accepted liquid carriers, such as a phosphate-buffered saline solution, water, as well as emulsions such as an oil/water emulsion or a triglyceride emulsion. Solid carriers may include excipients such as starch, milk, sugar, certain types of clay, stearic acid, talc, gums, glycols, or other known excipients. Carriers may also include flavor and color additives or other ingredients. The formulations of the combination of some embodiments may be prepared by methods well- known in the pharmaceutical arts and described herein. Exemplary acceptable pharmaceutical carriers have been discussed above. An additional carrier, Cremophor.TM., may be useful, as it is a common vehicle for Taxol.

[0099] The pharmaceutical compositions of the some embodiments preferably administered orally, preferably as solid compositions. However, the pharmaceutical compositions may be administered parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. Sterile injectable forms of the pharmaceutical compositions may be aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3- butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

[0100] The pharmaceutical compositions employed in some embodiments may be orally administered in any orally acceptable dosage form, including, but not limited to, solid forms such as capsules and tablets. In the case of tablets for oral use, carriers commonly used include microcrystalline cellulose, lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. When aqueous suspensions are required for oral use, the active ingredient may be combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

[0101] The pharmaceutical compositions employed in the some embodiments may also be administered by nasal aerosol or inhalation. Such pharmaceutical compositions may be prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

[0102] Should topical administration be desired, it can be accomplished using any method commonly known to those skilled in the art and includes but is not limited to incorporation of the pharmaceutical composition into creams, ointments, or transdermal patches.

[0103] The passage of agents through the blood-brain barrier to the brain is not desired but can be enhanced by improving either the permeability of the agent itself or by altering the characteristics of the blood-brain barrier. Thus, the passage of the agent can be facilitated by increasing its lipid solubility through chemical modification, and/or by its coupling to a cationic carrier. The passage of the agent can also be facilitated by its covalent coupling to a peptide vector capable of transporting the agent through the blood-brain barrier. Peptide transport vectors known as blood-brain barrier permeabilizer compounds are disclosed in U.S. Patent No. 5,268,164. Site specific macromolecules with lipophilic characteristics useful for delivery to the brain are disclosed in U.S. Patent No. 6,005,004.

[0104] Examples of routes of administration comprise parenteral, e.g., intravenous, intradermal, subcutaneous, inhalation, transdermal (topical), transmucosal, and rectal administration; or oral. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can comprise the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as

ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions suitable for injection comprise sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers comprise physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the selected particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some cases, isotonic agents are included in the composition, for example, sugars, polyalcohols such as manitol, sorbitol, or sodium chloride. Prolonged absorption of an injectable composition can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0105] Sterile injectable solutions can be prepared by incorporating the active compound in the specified amount in an appropriate solvent with one or a combination of ingredients enumerated above, as needed, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other ingredients selected from those enumerated above or others known in the art. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation comprise vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0106] Oral compositions generally comprise an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be comprised as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0107] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0108] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and comprise, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0109] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

4. Processing Hardware Overview

[0110] FIG. 10 is a block diagram that illustrates a computer system 1000 upon which an embodiment of the invention may be implemented. Computer system 1000 includes a communication mechanism such as a bus 1010 for passing information between other internal and external components of the computer system 1000. Information is represented as physical signals of a measurable phenomenon, typically electric voltages, but including, in other embodiments, such phenomena as magnetic, electromagnetic, pressure, chemical, molecular atomic and quantum interactions. For example, north and south magnetic fields, or a zero and non-zero electric voltage, represent two states (0, 1) of a binary digit (bit). ). Other phenomena can represent digits of a higher base. A superposition of multiple simultaneous quantum states before measurement represents a quantum bit (qubit). A sequence of one or more digits constitutes digital data that is used to represent a number or code for a character. In some embodiments, information called analog data is represented by a near continuum of measurable values within a particular range. Computer system 1000, or a portion thereof, constitutes a means for performing one or more steps of one or more methods described herein.

[0111] A sequence of binary digits constitutes digital data that is used to represent a number or code for a character. A bus 1010 includes many parallel conductors of information so that information is transferred quickly among devices coupled to the bus 1010. One or more processors 1002 for processing information are coupled with the bus 1010. A processor 1002 performs a set of operations on information. The set of operations include bringing information in from the bus 1010 and placing information on the bus 1010. The set of operations also typically include comparing two or more units of information, shifting positions of units of information, and combining two or more units of information, such as by addition or multiplication. A sequence of operations to be executed by the processor 1002 constitute computer instructions.

[0112] Computer system 1000 also includes a memory 1004 coupled to bus 1010. The memory 1004, such as a random access memory (RAM) or other dynamic storage device, stores information including computer instructions. Dynamic memory allows information stored therein to be changed by the computer system 1000. RAM allows a unit of information stored at a location called a memory address to be stored and retrieved independently of information at neighboring addresses. The memory 1004 is also used by the processor 1002 to store temporary values during execution of computer instructions. The computer system 1000 also includes a read only memory (ROM) 1006 or other static storage device coupled to the bus 1010 for storing static information, including instructions, that is not changed by the computer system 1000. Also coupled to bus 1010 is a non-volatile (persistent) storage device 1008, such as a magnetic disk or optical disk, for storing information, including instructions, that persists even when the computer system 1000 is turned off or otherwise loses power.

[0113] Information, including instructions, is provided to the bus 1010 for use by the processor from an external input device 1012, such as a keyboard containing alphanumeric keys operated by a human user, or a sensor. A sensor detects conditions in its vicinity and transforms those detections into signals compatible with the signals used to represent information in computer system 1000. Other external devices coupled to bus 1010, used primarily for interacting with humans, include a display device 1014, such as a cathode ray tube (CRT) or a liquid crystal display (LCD), for presenting images, and a pointing device 1016, such as a mouse or a trackball or cursor direction keys, for controlling a position of a small cursor image presented on the display 1014 and issuing commands associated with graphical elements presented on the display 1014.

[0114] In the illustrated embodiment, special purpose hardware, such as an application specific integrated circuit (IC) 1020, is coupled to bus 1010. The special purpose hardware is configured to perform operations not performed by processor 1002 quickly enough for special purposes. Examples of application specific ICs include graphics accelerator cards for generating images for display 1014, cryptographic boards for encrypting and decrypting messages sent over a network, speech recognition, and interfaces to special external devices, such as robotic arms and medical scanning equipment that repeatedly perform some complex sequence of operations that are more efficiently implemented in hardware.

[0115] Computer system 1000 also includes one or more instances of a communications interface 1070 coupled to bus 1010. Communication interface 1070 provides a two-way communication coupling to a variety of external devices that operate with their own processors, such as printers, scanners and external disks. In general the coupling is with a network link 1078 that is connected to a local network 1080 to which a variety of external devices with their own processors are connected. For example, communication interface 1070 may be a parallel port or a serial port or a universal serial bus (USB) port on a personal computer. In some embodiments, communications interface 1070 is an integrated services digital network (ISDN) card or a digital subscriber line (DSL) card or a telephone modem that provides an information communication connection to a corresponding type of telephone line. In some embodiments, a communication interface 1070 is a cable modem that converts signals on bus 1010 into signals for a communication connection over a coaxial cable or into optical signals for a communication connection over a fiber optic cable. As another example, communications interface 1070 may be a local area network (LAN) card to provide a data communication connection to a compatible LAN, such as Ethernet. Wireless links may also be implemented. Carrier waves, such as acoustic waves and electromagnetic waves, including radio, optical and infrared waves travel through space without wires or cables. Signals include man-made variations in amplitude, frequency, phase, polarization or other physical properties of carrier waves. For wireless links, the communications interface 1070 sends and receives electrical, acoustic or electromagnetic signals, including infrared and optical signals, that carry information streams, such as digital data.

[0116] The term computer-readable medium is used herein to refer to any medium that participates in providing information to processor 1002, including instructions for execution. Such a medium may take many forms, including, but not limited to, non-volatile media, volatile media and transmission media. Non-volatile media include, for example, optical or magnetic disks, such as storage device 1008. Volatile media include, for example, dynamic memory 1004. Transmission media include, for example, coaxial cables, copper wire, fiber optic cables, and waves that travel through space without wires or cables, such as acoustic waves and electromagnetic waves, including radio, optical and infrared waves. The term computer-readable storage medium is used herein to refer to any medium that participates in providing information to processor 1002, except for transmission media.

[0117] Common forms of computer-readable media include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape, or any other magnetic medium, a compact disk ROM (CD-ROM), a digital video disk (DVD) or any other optical medium, punch cards, paper tape, or any other physical medium with patterns of holes, a RAM, a programmable ROM (PROM), an erasable PROM (EPROM), a FLASH-EPROM, or any other memory chip or cartridge, a carrier wave, or any other medium from which a computer can read. The term non-transitory computer-readable storage medium is used herein to refer to any medium that participates in providing information to processor 1002, except for carrier waves and other signals.

[0118] Logic encoded in one or more tangible media includes one or both of processor instructions on a computer-readable storage media and special purpose hardware, such as ASIC 1020.

[0119] Network link 1078 typically provides information communication through one or more networks to other devices that use or process the information. For example, network link 1078 may provide a connection through local network 1080 to a host computer 1082 or to equipment 1084 operated by an Internet Service Provider (ISP). ISP equipment 1084 in turn provides data communication services through the public, world-wide packet-switching communication network of networks now commonly referred to as the Internet 1090. A computer called a server 1092 connected to the Internet provides a service in response to information received over the Internet. For example, server 1092 provides information representing video data for presentation at display 1014.

[0120] The invention is related to the use of computer system 1000 for implementing the techniques described herein. According to one embodiment of the invention, those techniques are performed by computer system 1000 in response to processor 1002 executing one or more sequences of one or more instructions contained in memory 1004. Such instructions, also called software and program code, may be read into memory 1004 from another computer-readable medium such as storage device 1008. Execution of the sequences of instructions contained in memory 1004 causes processor 1002 to perform the method steps described herein. In alternative embodiments, hardware, such as application specific integrated circuit 1020, may be used in place of or in combination with software to implement the invention. Thus, embodiments of the invention are not limited to any specific combination of hardware and software.

[0121] The signals transmitted over network link 1078 and other networks through communications interface 1070, carry information to and from computer system 1000.

Computer system 1000 can send and receive information, including program code, through the networks 1080, 1090 among others, through network link 1078 and communications interface 1070. In an example using the Internet 1090, a server 1092 transmits program code for a particular application, requested by a message sent from computer 1000, through Internet 1090, ISP equipment 1084, local network 1080 and communications interface 1070. The received code may be executed by processor 1002 as it is received, or may be stored in storage device 1008 or other non- volatile storage for later execution, or both. In this manner, computer system 1000 may obtain application program code in the form of a signal on a carrier wave.

[0122] Various forms of computer readable media may be involved in carrying one or more sequence of instructions or data or both to processor 1002 for execution. For example, instructions and data may initially be carried on a magnetic disk of a remote computer such as host 1082. The remote computer loads the instructions and data into its dynamic memory and sends the instructions and data over a telephone line using a modem. A modem local to the computer system 1000 receives the instructions and data on a telephone line and uses an infra-red transmitter to convert the instructions and data to a signal on an infra-red a carrier wave serving as the network link 1078. An infrared detector serving as communications interface 1070 receives the instructions and data carried in the infrared signal and places information representing the instructions and data onto bus 1010. Bus 1010 carries the information to memory 1004 from which processor 1002 retrieves and executes the instructions using some of the data sent with the instructions. The instructions and data received in memory 1004 may optionally be stored on storage device 1008, either before or after execution by the processor 1002.

[0123] FIG. 11 illustrates a chip set 1100 upon which an embodiment of the invention may be implemented. Chip set 1100 is programmed to perform one or more steps of a method described herein and includes, for instance, the processor and memory components described with respect to FIG. 10 incorporated in one or more physical packages (e.g., chips). By way of example, a physical package includes an arrangement of one or more materials, components, and/or wires on a structural assembly (e.g., a baseboard) to provide one or more characteristics such as physical strength, conservation of size, and/or limitation of electrical interaction. It is contemplated that in certain embodiments the chip set can be implemented in a single chip. Chip set 1100, or a portion thereof, constitutes a means for performing one or more steps of a method described herein. [0124] In one embodiment, the chip set 1100 includes a communication mechanism such as a bus 1101 for passing information among the components of the chip set 1100. A processor 1103 has connectivity to the bus 1101 to execute instructions and process information stored in, for example, a memory 1105. The processor 1103 may include one or more processing cores with each core configured to perform independently. A multi-core processor enables multiprocessing within a single physical package. Examples of a multi-core processor include two, four, eight, or greater numbers of processing cores. Alternatively or in addition, the processor 1103 may include one or more microprocessors configured in tandem via the bus 1101 to enable independent execution of instructions, pipelining, and multithreading. The processor 1103 may also be accompanied with one or more specialized components to perform certain processing functions and tasks such as one or more digital signal processors (DSP) 1107, or one or more application- specific integrated circuits (ASIC) 1109. A DSP 1107 typically is configured to process real-world signals (e.g., sound) in real time independently of the processor 1103. Similarly, an ASIC 1109 can be configured to performed specialized functions not easily performed by a general purposed processor.

Other specialized components to aid in performing the inventive functions described herein include one or more field programmable gate arrays (FPGA) (not shown), one or more controllers (not shown), or one or more other special-purpose computer chips.

[0125] The processor 1103 and accompanying components have connectivity to the memory 1105 via the bus 1101. The memory 1105 includes both dynamic memory (e.g., RAM, magnetic disk, writable optical disk, etc.) and static memory (e.g., ROM, CD-ROM, etc.) for storing executable instructions that when executed perform one or more steps of a method described herein. The memory 1105 also stores the data associated with or generated by the execution of one or more steps of the methods described herein.

5. Alternatives and extensions

[0126] In the foregoing specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. Throughout this specification and the claims, unless the context requires otherwise, the word "comprise" and its variations, such as "comprises" and "comprising," will be understood to imply the inclusion of a stated item, element or step or group of items, elements or steps but not the exclusion of any other item, element or step or group of items, elements or steps. Furthermore, the indefinite article "a" or "an" is meant to indicate one or more of the item, element or step modified by the article.

6. References.

[0127] The entire contents of the following references are hereby incorporated by reference as if fully set forth herein, except for terminology that is inconsistent with the terminology used herein.

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Hoffman, et al., J. Cardiovasc. Electrophysiol. 7: 120, 1996 and 8:679, 1997.

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Lau, D.H., Clausen, C, Sosunov, E.A., Shlapakova, I.N., Anyukhovsky, E.P., Danilo, P., Jr., Rosen, T.S., Kelly, C, Duffy, H.S., Szabolcs, M.J., Chen, M., Robinson, R.B., Lu, J., Kumari, S., Cohen, I.S. and Rosen, M.R., "Epicardial border zone overexpression of skeletal muscle sodium channel skMl normalizes activation, preserves conduction, and suppresses ventricular arrhythmia," Circulation vl l9, ppl9-27. PMCJD:

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Claims

CLAIMS What is claimed is:

1. A method for screening for compounds capable of treating long-QT syndrome, the method comprising;

a. isolating cardiomyocytes and incubating the cardiomyocytes under conditions suitable for measuring ion currents;

b. providing a test population of one or more cardiomyocytes;

c. contacting a candidate compound to the test population;

d. measuring an outward potassium ion current from a cardiomyocyte in the test

population in response to applying a depolarizing voltage;

e. determining whether the outward potassium current is increased in the test population relative to an outward potassium current in a control population of one or more cardiomyocytes in response to applying the depolarizing voltage; and

f. determining that the candidate compound is capable of treating long-QT syndrome if the outward potassium current is increased in the test population relative to the control population.

2. A method for screening for compounds capable of treating long-QT syndrome, the method comprising;

a. isolating cardiomyocyte tissue and incubating the tissue under conditions suitable for measuring action potential;

b. providing a first test population of cardiomyocytes from the tissue;

c. contacting an agent that prolongs the action potential to the first test population;

d. contacting a candidate compound to the first test population;

e. measuring, after contacting the candidate compound, an action potential in the first test population in response to applying an excitation voltage;

f. determining whether a duration of the action potential of the first test population is reduced towards a control duration; and

g. determining that the candidate compound is capable of treating long-QT syndrome if the duration is substantively reduced.

3. A method as recited in claim 2, further comprising:

h. providing a second test population of cardiomyocytes from the tissue;

i. contacting the candidate compound to the second test population; and

j. measuring an action potential in the second test population in response to applying an excitation voltage,

wherein the step g of determining that the candidate compound is capable of treating long-QT syndrome further comprises determining that the candidate compound is capable of treating long-QT syndrome if the duration is substantively reduced and the action potential in the second test population is not substantively different from action potential in a control population of cardiomyocytes.