# US 2019/0169670 A1 · Systems and methods for modeling disease and assessing adverse side effects of therapeutics therefor

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2019/0169670 A1
- Document ID: US20190169670A1
- Status: Published application
- Kind: A1
- Application number: 16/324,421
- Filed: 2017-08-11
- Published: 2019-06-06
- Earliest claimed: 2016-08-11
- Assignees: NovoHeart Limited
- Applicants: Novoheart Limited
- Inventors: Ronald A. Li
- Web page: https://patentgrep.com/patent/US20190169670A1

## CPC

- C12Q 1/025: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics (antimicrobial activity C12Q1/18)}
- A61K 31/496: Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
- A61K 31/519: ortho- or peri-condensed with heterocyclic rings
- A61P 9/06: Antiarrhythmics
- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- C12N 5/0657: {Cardiomyocytes; Heart cells}
- G01N 33/5014: {for testing toxicity}
- G01N 33/5044: {involving specific cell types}
- G01N 33/5061: {Muscle cells}
- C12Q 2600/142: Toxicological screening, e.g. expression profiles which identify toxicity
- G01N 2800/326: Arrhythmias, e.g. ventricular fibrillation, tachycardia, atrioventricular block, torsade de pointes
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The disclosure provides a substrate for the ordered growth and development of cardiomyocytes such as ventricular cardiomyocytes derived from pluripotent stem cells along with methods of culturing the cells on the substrates for use in assays such as cardiotoxicity assays. Further provided are methods for assessing cardiotoxicity using one or more criteria disclosed herein

## Claims

### Claim 1

A method of assessing the cardiotoxicity of a compound comprising:
(a) contacting an anisotropic layer of cardiac cells on a microfabricated substrate with a compound;
(b) stimulating the anisotropic layer of cells at one or more points;
(c) detecting an electrical signal propagation in the anisotropic layer of cells; and
(d) determining whether the compound exhibits cardiotoxicity.

### Claim 2 (depends on 1)

The method of claim 1 wherein the anisotropic layer of cells comprises patient- or disease-specific cardiac cells derived from a human.

### Claim 3 (depends on 1)

The method of claim 1 wherein the microfabricated substrate comprises grooves oriented along a single axis of the substrate.

### Claim 4 (depends on 3)

The method of claim 3 wherein the grooves have similar dimensions.

### Claim 5 (depends on 4)

The method of claim 4 wherein the grooves have a width of 1-30 μm.

### Claim 6 (depends on 5)

The method of claim 5 wherein the grooves have a width of 5-15 μm.

### Claim 7 (depends on 6)

The method of claim 6 wherein the grooves have a width of 15 μm.

### Claim 8 (depends on 3)

The method of claim 3 wherein the depth of the grooves is about 5 μm.

### Claim 9 (depends on 3)

The method of claim 3 wherein the spacing between grooves is about 5 μm.

### Claim 10 (depends on 1)

The method of claim 1 wherein the microfabricated substrate is polystyrene.

### Claim 11 (depends on 1)

The method of claim 1 wherein the anisotropic layer of cardiac cells is stimulated at one point.

### Claim 12 (depends on 11)

The method of claim 11 wherein the stimulation is 5-30 volts with a pulse duration of 5-30 milliseconds.

### Claim 13 (depends on 12)

The method of claim 12 wherein the stimulation is 10 volts with a pulse duration of 10 milliseconds.

### Claim 14 (depends on 1)

The method of claim 1 wherein a compound is determined to exhibit cardiotoxicity if the compound induces a spiral electrical propagation wave in the anisotropic layer of cardiac cells.

### Claim 15 (depends on 1)

The method of claim 1 wherein the compound is a Class 1A, Class 1B or Class IC anti-arrhythmic compound.

### Claim 16 (depends on 1)

The method of claim 1 wherein the anistropic layer of cardiac cells comprises cardiomyocytes that are at least 70% troponin T+.

### Claim 17 (depends on 1)

The method of claim 1 wherein the anisotropic layer of cardiac cells does not exhibit a reentrant event in the absence of exposure to a compound.

### Claim 18 (depends on 1)

The method of claim 1 wherein the anisotropic layer of cardiac cells has undergone a programmed electrical stimulation comprising a train of eight S1 stimuli delivered at 1.5 Hertz (Hz) followed by a premature extra stimulus (S2) with S1-S2 interval initiated at 550 milliseconds (ms) and consecutively shortened in steps of 50 ms until capture fails, at which point the. S1-S2 interval is increased by 20 ms, followed by 2-ms decrements until the effective refractory period (ERP) is achieved, wherein the ERP is defined as the maximum S1-S2 interval that failed to lead to AP propagation.

### Claim 19 (depends on 1)

The method of claim 1 wherein the anisotropic layer of cardiac cells exhibits a progressive dose-dependent slowing of APD50 conduction velocity as exposure to a class 1C anti-arrhythmic compound is progressively increased from 0.3-3 μM.

### Claim 20 (depends on 1)

The method of claim 1 wherein the anisotropic layer of cardiac cells exhibits a dose-dependent APD90 prolongation in response to a class 1A anti-arrhythmic compound.

### Claim 21 (depends on 1)

The method of claim 1 wherein the anisotropic layer of cardiac cells exhibits at least a two-fold increase in reentrant events in response to a class 1A anti-arrhythmic compound.

### Claim 22 (depends on 1)

The method of claim 1 wherein the anisotropic layer of cardiac cells does not exhibit a dose-dependent APD90 prolongation in response to a class 1B anti-arrhythmic compound.

### Claim 23 (depends on 1)

The method of claim 1 wherein the anisotropic layer of cardiac cells does not exhibit an increase in reentrant events in response to a class 1B anti-arrhythmic compound.

### Claim 24

A method of identifying cardiomyocytes suitable for use in an in vitro cardiotoxicity assay comprising:
(a) contacting a cardiomyocyte derived from a pluripotent stem cell with a microfabricated substrate; and
(b) identifying cardiomyocytes suitable for use in an in vitro cardiotoxicity assay as cardiomyocytes exhibiting at least two of the following properties:
(i) a radiating conduction pattern with an anisotropic ratio of at least 1.8 in response to electrical point stimulation;
(ii) at least 70% cardiac troponin T+;
(iii) a maximum capturing frequency of at least 1.5 Hz;
(iv) an absence of reentrant events in response to steady-state pacing from 0.5 Hz through to 3 Hz (10V, 10 ms) with 0.5 Hz increments until the loss of 1:1 capture; or
(v) an absence of induced spiral waves in response to programmed electrical stimulation comprising a first train of eight electrical pulses (S1) followed by a second train of electrical pulses (S2) with progressively shortened S1-S2 intervals.

### Claim 25 (depends on 24)

The method of claim 24 further comprising:
(a) exposing the cardiomyocytes to a therapeutically effective amount of a class IC anti-arrhythmic compound; and
(b) identifying the cardiomyocytes as suitable for us in an in vitro cardiotoxicity assay if the class IC anti-arrhythmic compound induced a dose-dependent slowing of signal conduction velocity.

### Claim 26 (depends on 25)

The method of claim 25 wherein the class IC anti-arrhythmic compound is flecainide.

### Claim 27 (depends on 24)

The method of claim 24 further comprising:
(a) exposing the cardiomyocytes to a therapeutically effective amount of a class 1A anti-arrhythmic compound; and
(b) identifying the cardiomyocytes as suitable for use in an in vitro cardiotoxicity assay if the class 1A anti-arrhythmic compound induces a dose-dependent slowing of conduction velocity.

### Claim 28 (depends on 27)

The method of claim 27 wherein the class 1A anti-arrhythmic compound is procainamide.

### Claim 29 (depends on 24)

The method of claim 24 further comprising:
(a) exposing the cardiomyocytes to a therapeutically effective amount of a class 1B anti-arrhythmic compound; and
(b) identifying the cardiomyocytes as suitable for us in an in vitro cardiotoxicity assay if the class 1B anti-arrhythmic compound does not induce a dose-dependent slowing of signal conduction velocity.

### Claim 30 (depends on 29)

The method of claim 29 wherein the class 1B anti-arrhythmic compound is tocainide.

### Claim 31 (depends on 24)

The method of claim 24 further comprising:
(a) exposing the cardiomyocytes to terfenadine; and
(b) identifying the cardiomyocytes as suitable for us in an in vitro cardiotoxicity assay if the terfenidine does not induce a reentrant event at 100 nM concentration but increases the likelihood of a reentrant event by at least 1.2-fold relative to exposure to a physiologically inert control compound when the terfenidine is administered at 300 nM concentration.

### Claim 32 (depends on 24)

The method of claim 24 further comprising:
(a) exposing the cardiomyocytes to cisapride; and
(b) identifying the cardiomyocytes as suitable for us in an in vitro cardiotoxicity assay if the cisapride increases the likelihood of a reentrant event by at least 2.7-fold relative to exposure to a physiologically inert control compound.

### Claim 33

A method of producing an anisotropic layer of cardiac cells comprising:
(a) dissociating pluripotent stem cell cultures of at least 80% confluence to yield individual cells;
(b) culturing individual cells in suspension under hypoxic conditions of about 5% O2 and about 5% CO2 for about 8 days to yield cultured cells, wherein the culturing comprises:
(i) incubating the cells in mTeSR™-1 medium supplemented with 10 μM Rho kinase inhibitor, 1 ng/mL bone morphogenetic protein-4 and 40 μg/mL matrigel for about 24 hours;
(ii) further incubating the cells in StemPro®-34 SFM complete medium comprising 50 μg/mL ascorbic acid, 2 mM GlutaMAX™, 10 ng/mL BMP4 and 10 ng/mL Activin-A for about 3 days;
(iii) continuing to incubate the cells in StemPro®-34 SFM complete medium comprising 50 μg/mL ascorbic acid, 2 mM GlutaMAX™, and 5 μM of a Wnt inhibitor for about 3 days;
(iv) transferring the cells to StemPro®-34 SFM complete medium comprising 50 μg/mL ascorbic acid under ambient O2, 5% CO2 conditions on day 8 to yield cardiospheres;
(c) maintaining the cardiospheres in StemPro®-34 SFM complete medium comprising 50 μg/mL ascorbic acid until day 15-22, with medium replenishment every 3-4 days;
(d) dissociating the cardiospheres by exposure to 1 mg/mL collagenase for 30 minutes, followed by exposure to 0.05% trypsin for 3 minutes;
(e) seeding the cardiospheres onto a matrigel-coated microfabricated substrate to form an anisotropic layer of cardiac cells;
(f) incubating the anisotropic layer of cardiac cells in high-glucose (4.5 g/L) DMEM medium comprising 10% heat-inactivated fetal bovine serum, 1× GlutaMAX™, and 1×MEM non-essential amino acids for 2 days; and
(g) replenishing the medium every 2 days with RPMI 1640 medium for at least 7 days, thereby producing an anisotropic layer of cardiac cells.

### Claim 34 (depends on 33)

The method of claim 33 wherein the pluripotent stem cell cultures are human pluripotent stem cell cultures.

### Claim 35 (depends on 33)

The method of claim 33 wherein the pluripotent stem cell cultures are dissociated with accutase.

### Claim 36 (depends on 33)

The method of claim 33 wherein the Wnt Inhibitor is IWR-1.

### Claim 37

A method of manufacturing a microfabricated substrate comprising:
(a) generating an elastomeric mold using photolithography to yield a mold having a series of channels oriented along the same axis in which each channel is 3-10 μm deep and 2-50 μm wide with a spacing between channels of 3-10 μm;
(b) embossing the features of step (a) onto plastic using heat to generate a substrate;
(c) exposing the substrate to UVO treatment for about 8 minutes; and
(d) producing the microfabricated substrate by sterilizing the substrate in a chemical sterilizing agent.

### Claim 38 (depends on 37)

The method of claim 37 wherein the elastomeric mold is a polydimethylsiloxane mold.

### Claim 39 (depends on 37)

The method of claim 37 wherein the depths of the channels are about the same.

### Claim 40 (depends on 39)

The method of claim 39 wherein the channel depth is about 5 μm.

### Claim 41 (depends on 37)

The method of claim 37 wherein the widths of the channels are about the same.

### Claim 42 (depends on 41)

The method of claim 41 wherein the channel width is about 10 μm or about 15 μm.

### Claim 43 (depends on 37)

The method of claim 37 wherein the spacings between channels is about the same.

### Claim 44 (depends on 43)

The method of claim 43 wherein the channel spacing is about 5 μm.

### Claim 45

A system that models a cardiac disease comprising a bio-hybrid material comprising: (a) human cardiomyocytes, and (b) a microfabricated substrate for cell attachment.

### Claim 46 (depends on 45)

The system of claim 45 wherein the cardiac disease is a cardiac arrhythmia.

### Claim 47 (depends on 46)

The system of claim 46 wherein the disease is Long QT syndrome, Brugada syndrome, genetic heart disease, amyloidosis, progeria, diabetic coma, jellyfish intoxication, hyperthyroidism, Yellow Fever, Chagas disease, aortic valve regurgitation, prescription drug abuse leading to arrhythmia, Rett syndrome, myocarditis, tricuspid atresia, Lyme disease, Churg-Strauss syndrome, forms of heart disease or failure involving arrhythmia, enlarged heart, broken heart syndrome, thyroid nodules, atrioventricular canal defect, cholera, mitral valve stenosis, multiple system atrophy (MSA), snoring, mitral valve prolapse, amniotic fluid embolism, gangrene, aplastic anemia, congenital heart disease in adults, heat exhaustion, Graves' disease, cardiomyopathy, premature ventricular contractions, Bradycardia, fatigue, tachycardia, dizziness, or shortness of breath.

### Claim 48

A method of screening for a therapeutic for cardiac disease comprising (a) incubating an anisotropic layer of cardiac cells under conditions maintaining cell viability; (b) contacting the cardiac cells with a candidate therapeutic for cardiac disease; and (c) measuring the effect of the candidate therapeutic for cardiac disease on the cardiac cells, wherein the candidate therapeutic for cardiac disease is identified as a therapeutic for cardiac disease if the candidate therapeutic for cardiac disease reduces a symptom of a cardiac disease in the cardiac cells.

### Claim 49 (depends on 48)

The method of claim 48 wherein the cardiac cells are attached to a microfabricated substrate.

### Claim 50 (depends on 48)

The method of claim 48 wherein the cardiac disease is an arrhythmia.

### Claim 51 (depends on 50)

The method of claim 50 wherein the cardiac disease is Long QT syndrome, Brugada syndrome, genetic heart disease, amyloidosis, progeria, diabetic coma, jellyfish intoxication, hyperthyroidism, Yellow Fever, Chagas disease, aortic valve regurgitation, prescription drug abuse leading to arrhythmia, Rett syndrome, myocarditis, tricuspid atresia, Lyme disease, Churg-Strauss syndrome, forms of heart disease or failure involving arrhythmia, enlarged heart, broken heart syndrome, thyroid nodules, atrioventricular canal defect, cholera, mitral valve stenosis, multiple system atrophy (MSA), snoring, mitral valve prolapse, amniotic fluid embolism, gangrene, aplastic anemia, congenital heart disease in adults, heat exhaustion, Graves' disease, cardiomyopathy, premature ventricular contractions, Bradycardia, fatigue, tachycardia, dizziness, or shortness of breath.

### Claim 52

A method for assessing the cardiotoxic effect of a compound comprising (a) contacting an anisotropic layer of cardiac cells with a compound; and (b) measuring the cardiotoxic effect of the compound on the cardiac cells.

### Claim 53 (depends on 52)

The method of claim 52 wherein the cardiac cells are attached to a microfabricated substrate.

### Claim 54 (depends on 52)

The method of claim 52 wherein the anisotropic layer of cardiac cells are healthy cells.

### Claim 55 (depends on 52)

The method of claim 52 wherein the anisotropic layer of cardiac cells are genetically, chemically, physically or electrically disposed to a cardiac disease.

### Claim 56 (depends on 55)

The method of claim 55 wherein the cardiac disease is Long QT syndrome, Brugada syndrome, genetic heart disease, amyloidosis, progeria, diabetic coma, jellyfish intoxication, hyperthyroidism, Yellow Fever, Chagas disease, aortic valve regurgitation, prescription drug abuse leading to arrhythmia, Rett syndrome, myocarditis, tricuspid atresia, Lyme disease, Churg-Strauss syndrome, forms of heart disease or failure involving arrhythmia, enlarged heart, broken heart syndrome, thyroid nodules, atrioventricular canal defect, cholera, mitral valve stenosis, multiple system atrophy (MSA), snoring, mitral valve prolapse, amniotic fluid embolism, gangrene, aplastic anemia, congenital heart disease in adults, heat exhaustion, Graves' disease, cardiomyopathy, premature ventricular contractions, Bradycardia, fatigue, tachycardia, dizziness, or shortness of breath.

### Claim 57 (depends on 52)

The method of claim 52 wherein the cardiotoxic effect is an arrhythmia.

### Claim 58 (depends on 1, 33, 48)

The method of any one of claim 1, 33, 48, or 52 wherein the cardiac cells are ventricular cardiac cells.

### Claim 59 (depends on 24, 45)

The method of any one of claim 24 or 45 wherein the cardiomyocytes are ventricular cardiomyocytes.