CARDIOVASCULAR

PRECLINICAL TESTING

Ass. Prof. Omaima Ahmedy

[email protected]
 Cardiovascular safety CVS pharmacology

❖ The aim of cardiovascular safety pharmacology is to evaluate

the effects of test substances on the CVS in order to detect
potentially “undesirable” effects before engaging in clinical
trials.
First: Basic Cardiovascular Testing:
An escalating dose method is used to determine the minimum
dose necessary to see any changes in cardiovascular function.

Components of CVS Preclinical Testing Include:

Hemodynamics (HR, BP, CO)
Electrophysiology (ECG)→ QT prolongation
Cardiac Safety by ECG Findings:
Focus on QTc Duration
❖ Many «non cardiovascular drugs» have
been removed from the market or severely
Restricted
In April 2007, U.S. Food and Drug Administration (FDA) not to recommend
the approval of etoricoxib based on the drug's cardiovascular (CV) risks.

❖ Most common cause of withdrawal (or restriction of the use)

of these drugs is prolongation of QT interval in association
with polymorphic ventricular tachycardia or Torsade de
Pointes
A listing of classes of drugs with torsades de pointes risk
(http://www.torsades.org)
Class A Antiarryhthmics Procainamide, quinidine, disopyramide
Class III Antiarryhthmic Amiodarone, dofetilide, sotalol
Vasodilators/antiischemic Bepridil ,Lidoflazine ,Prenylamine ,Papaverine
agents (intracoronary)
Ca Antagonists Piperidil
Antidepressants Amitryptiline, citalopram, clomipramine,
desipramine, doxepin, imipramine, maproptiline
Antifungal agents Fluconazole, itraconazole, ketoconazole,
micoconazole
Antihistamine agents Astemizole, dephenhydramine, hydroxizine,
terfenadine
Anticancer drugs doxorubicine, zorubicine

Antimicrobial Agents amphotericin, clarithromycin, clindamycin,

cotrimoxazole, erythromycin, sparfloxacin,
trimethoprin-sulphamethoxazole
Antimalarials Chloroquine, halofantrine, quinine
Antipsychotics Chlorpromazine, haloperidol, flufenazine, lithium,
pimozide, prochlorperazine,risperidone,
thioridazine, trifluoperazine
Miscellaneous Cisapride, probucol, indapamide, thiazide/loop
diuretics
Action potential AP
❖ The function of the heart is to pump blood through the body.
To do this, the atria and ventricles must contract in a precise
sequence in response to a conducted electrical signal, the
action potential (AP).

❖ AP indicates the “electrical activity” of the cell during the

contraction and relaxation of the heart. Specific ionic
currents contribute to each phase of the cardiac action
potential.
❖ An electrical stimulus is generated by
the “sinus node” (also called the
sinoatrial node, or SA node)in the
right atria of the heart.
❖ The electrical impulse travels from
the sinus node to the
“atrioventricular node” (also called
AV node). The impulse continues
down the conduction pathway via the
“bundle of His” into the ventricles.
The bundle of His divides into right
and left pathways, called “bundle
branches”, to stimulate the right
and left ventricles.

❖ The electrical system of the heart causes the heart muscle to

contract and send blood to either the organs of the body (via
the left ventricle) or to the lungs (via the right ventricle).
❖ Electrocardiogram (ECG) is used to
see how the heart is functioning as it
records how often the heart beats
(heart rate) and how regularly it
beats (heart rhythm)

❖ If the heart is beating steadily, it will

produce the typical ECG pattern: )
The first peak (P wave) shows how the
electrical impulse (excitation) spreads
across the two atria of the heart. The atria
contract pumping blood into the ventricles, and
then immediately relax.
❖ The electrical impulse then reaches the ventricles. This can
be seen in the Q, R and S waves of the ECG, which is called
the “QRS complex”. The ventricles contract. Then the “T
wave” shows that the electrical impulse has stopped
spreading, and the ventricles relax once again.
Normal sinus rythm
❖ “QT INTERVAL”: represents ventricular action potential
(ventricular depolarization + ventricular repolarization).

❖ Rate-Corrected QT Interval (QTc)

❖ Measured QT interval decreases when the heart beats more

rapidly; therefore the QT interval should be corrected for
heart rate

❖ QT Interval corrected for heart rate = QTc (Bazett correction)

= QT / √RR
❖ General Population Average QTc = 380-400 msec
❖ Bazett correction has major limitations (used between 50 and
90 bpm)

❖ Increased risk of “torsades de pointes” (potentially fatal

polymorphic ventricular tachycardia) as QTc interval increases
QTc >500 msec
❖ The ventricular action potential is composed of four phases:
phase 0 is depolarization, phase 1 is early repolarization,
phase 2 is a plateau, phase 3 is repolarization and phase 4 is
the resting potential. The different phases depend on the
opening and/or closure of specific ion channels.
 Mechanisms Of Drug-Induced QT Prolongation and Tdp

❖ Block of repolarizing K+ currents

❖ Stimulation of ICa
❖ Stimulation of INa

❖ “Excessive intracellular positive ion”s →

delayed ventricular repolarization
and QTc interval prolongation

❖ QTc prolongation → early afterdepolarizations (EAD) → re-

entry → torsade de pointes (TdP) and fatal ventricular
arrhythmias [sudden cardiac death].

❖ TdP( twisting of points); irregular QRS complexes twisting

around ECG baseline.
EADs can disrupt normal repolarization, leading to areas with different
action potential durations (APDs). These differences in APD can create
a repolarization potential difference boundary. Difference in refractory
periods among layers of the myocardium
❖ Tdp can cease spontaneously or induce fatal ventricular
fibrillation.diagram of arrythmias.docx
 Risk Factors for QT prolongation

1. Congenital long QT syndrome (LQTS)

2. Female Sex

3- Bradycardia

4- Hypokalemia

5- Hypomagnesemia

6- Atrial Fibrillation (AF)

 1. Congenital long QT syndrome (LQTS)

•Mutations in channel subunits or genes that code for regulatory

proteins involved in channel functions, trafficking and kinetics
•The most prevalent dominant subtypes are LQT1, LQT2 and
LQT3
•LQT1 and LQT2, due to mutations in the “K+ channel”
•LQT3, due to mutations in the “Na+ channel”
•LQT1, LQT2 events are usually triggered by exercise or
emotional stress
•LQT3 events most often during sleep or at rest and have the
highest fatality rates.
 2. Female Sex:
•Longer QTc in women than men and twice the risk of drug-
induced TdP (2-3:1)

•67-70% of drug-associated TdP occurs in women

•Based upon published clinical observations, QT shortens after

puberty in men but not women.

•Sex hormones modulate repolarization:

1- estrogen prolongs the QT interval and
2- downregulates potassium channel expression
whereas testosterone activates IKr and shortens QTc → lowers
risk of TdP in men.

•Therefore, androgens are protective against drug-induced

prolongation of repolarization, whereas estrogen seems to be
proarrhythmic.
 TdP - Gender Distribution

Two clinical trials showing gender distribution in drug-

induced TdP
Different values of QTc in both adult males and
females
 3- Bradycardia:

❖ Bradycardia is a commonly observed risk factor for drug-

induced long QT interval syndrome and TdP.

❖ Bradycardia is mostly induced by

1- a slow sinus rhythm,
2- hypothermia,
3- hypothyroidism.

❖ This can physiologically prolong ventricular repolarization and

QT resulting in a higher risk of TdP.
 4- Hypokalemia

❖ Lowering extracellular potassium decreases IKr that may

cause QT interval prolongation in hypokalemic patients.
Unpredicted???
❖ Simple electrochemical considerations predict an increase in
outward potassium current with hypokalemia
❖ An explanation for this paradoxical behaviour:
❖ Sodium and potassium ions compete for access to
extracellular binding sites on the channel
❖ Sodium is a potent blocker of the current.
❖ Inhibitory effect of sodium on IKr becomes more apparent on
hypokalemia
❖ very fast inactivation that IKr undergoes after opening
 5- Hypomagnesemia

❖ Hypomagnesemia increases TdP risk, possibly by modulating

the L-type calcium channel function → EADs.

❖ Another possible mechanism of prolonged QT caused by

hypomagnesemia could be through its effect on membrane Na
K ATPase, which provides the energy for transport of sodium
out of the cell and potassium into the cell.

❖ Thus, hypomagnesemia changes the membrane potential .

 6- Atrial Fibrillation (AF)

❖ Many antiarrhythmic drugs used in treatment of AF block IKr

as a major mechanism of action → marked QT prolongation
and TdP

❖ Sotalol is a class III anti-arrhythmic (K channel blocker)which

prolongs action potential in atria, ventricles and nodal
tissues.

❖ As a non cardio- selective β blocker and k channel blocker ,

sotalol blocks potassium channel in phase 3 it leads to QT
prolongation and TdP.
 Drug-induced additive QTc prolongation

❖ Pharmacodynamic interactions
o 2 drugs with QT prolonging effects e.g sotalol, a Class III
antiarrhythmic with moxifloxacin, a fluoroquinolone with mild
QTc-prolonging effects
o Drugs with QT prolonging effect in presence of other risk
factor e.g. hypokalemia or hypomagnesmia

❖ Pharmacokinetic interactions “ADME”

o Increment of drug absorption or Inhibition of drug metabolism
or clearance ex:(terfenadine with ketoconazole)
o Patient taking terfenadine, an antihistamine with potent Ikr-
blocking activity, and then prescribed ketoconazole, a potent
CYP3A4 inhibitor, which significantly increases terfenadine
levels)
❖ High concentrations of the drugs due to overdose or rapid
infusion
 Management of Tdp

❖ Patients often respond to magnesium, usually

magnesium sulfate 2 g IV over 1 to 2 minutes.
If this treatment is unsuccessful, a 2nd bolus is
given in 5 to 10 minutes, and a magnesium infusion of
3 to 20 mg/minute may be started in patients without “renal
insufficiency.”

❖Isoprenaline (isoproterenol,
non selective β-agonist) for
bradycardia prior to Tdp

❖Cardioversion for unstable cases

electrical or pharmacological
 Preclinical screening to examine drug-
induced QT prolongation

A) In vitro ion channels effects (isolated cells)

IKr - cloned HERG channels
ICa & INa
B) In vitro action potential duration effects
Purkinje fibers
Papillary muscles
Isolated intact hearts
C) In vivo approaches
A) In vitro ion channels effects

❖ Patch-clamp techniques using isolated cells expressing K

channels (ventricular myocytes or HEK293 cells )

❖ Single glass micropipette electrode onto surface of cell

membrane allow recording inward or outward currents from a
single ion channel.
 K+
channel

Cell-attached patch clamp uses a micropipette attached to

the cell membrane to allow recording from a single ion
channel.
Patch clamp recording reveals transitions between two
conductance states of a single ion channel: closed (at
top) and open (at bottom)
hERG (human ether-a-go-go-related gene)

• HEK293 cells expressing hERG (gene that codes K channel)

• hERG channel mediates repolarizing IKr current (conducts K+ ions
out of cell)
• Blockade of these channels → long QT syndrome → pro-
arrhythmic state → risk of TdP, VF and sudden death
• Adding test drugs and calculate IC50 and compare to therapeutic
plasma level of drug

• The half maximal inhibitory concentration (IC50) is a measure of

the potency of a substance in inhibiting a specific biological or
biochemical function. IC50 is a quantitative measure that indicates
how much of a particular inhibitory substance (e.g. drug) is needed to
inhibit, in vitro, a given biological process or biological component by
50%.
Membrane repolarisation via HERG potassium channel *

hERG expressed K channel

* B) In vitro action potential duration effects

*Potent blockade of hERG channel does not necessarily

lead to a QT prolongation
*Myocardial action potential is net result of numerous ion
channels effects
*Action of a given channel can be masked by activities of
other competing channels
*K current inhibition and APD prolongation both provide
early risk assessment with better correlation to in vivo
result
❖ An example of a drug demonstrating that hERG liability is
insufficient to lead to TdP is the vasodilator verapamil, which
acts therapeutically as a calcium channel antagonist.

❖ verapamil also inhibits hERG channels at clinically relevant

concentrations, but it has no known risk for TdP .
❖ Verapamil is able to inhibit hERG without inducing
arrhythmogenesis because its calcium channel blocking
activity bypasses the downstream cellular mechanism by
which hERG blockade initiates torsadogenesis.

❖ TdP is thought to be initiated by cellular calcium excess that

leads to pathological electrical interruptions in the action
potential (EADs).

❖ Verapamil's calcium channel blocking action prevents the

increased Ca2+ current (and the consequent EADs).
❖ The MAP technique is useful for assessing the local electrical
activity of the myocardium in contact with the depolarizing
electrode.

Simultaneous recordings of intracellular transmembrane action potential (TAP) and

extracellular monophasic action potential (MAP) show high-fidelity correlation.
Relationship between cardiac membrane currents, *
action potential duration and the QT interval of the
ECG

Brown A W. – Cell Calcium - 2004; 35 : 543–547

Record of MAP obtained in a polygraph
* Purkinje fiber

*Non contractile tissue, which facilitates electrode

positioning and stability (no interfering factors)
*Too sensitive for drug-induced effects on repolarization
Biphasic effect of cisapride on dog *
Purkinje fiber AP

Brown A W. – Cell Calcium - 2004; 35 : 543–547

* Papillary muscle

*Papillary muscle is contractile tissue → allowing

measurement of contractile force (inotropic state) with
APD

Heart showing papillary muscle

* Isolated Langendorff heart

*Most sophisticated in vitro approaches for determining

drug-induced effects on repolarization
*Local action potentials across ventricular wall
*Transmural electrocardiogram which measures
transmembrane action potential by placing electrodes near
epicardial and endocardial surfaces of preparation

Isolated Langendorff heart

C) In Vivo Approaches *

Using Anesthetized Animal Models

*Anaesthetized rats/dogs are often used to screen effect
of tested drugs on basic cardiovascular functions.
*QT interval from ECG in addition to arterial blood
pressure, heart rate and ventricular contractility

ECG

BP

Spirometer
Advantages
▪ In-depth evaluation of drug effects on heart and vascular
function in a variety of perfusion beds (aorta, coronary
arteries,…)
▪ A highly stable hemodynamic state (animal anesthetized) with
very low variability of parameters measured

Disadvantages:
▪ Possible effects of anesthesia on ventricular repolarization →
affects QT interval duration
▪ Possible limitation of use of iv administration for drugs
intended for oral route.
Cardiovascular Safety Studies in Conscious Dog and Other
Rodent Species
▪ Radiotelemetric implants to record a number of
cardiovascular parameters in awake, freely moving animals.

▪ After surgery, animal is allowed to recover from anesthesia

and returned to its home cage.
▪ A receiver unit is placed next to cage to pick up radio signals
emitted by the telemetry device.
▪ Measurements of BP, HR, body temperature and ECGs are
routinely taken this way.
Advantages
•Conscious free moving animals in its home cage throughout
measurement and testing procedures
•Elimination of external influences (all recordings can be
performed from a site remote or hidden from animal)
•Drugs can be applied orally, iv, sc, or through inhalation

Radiography of an implanted rat showing the

implantation of the transmitter as well as the
electrodes for ECG recording
Disadvantages
▪ Surgery for full-implant telemetry systems
▪ Wires placed in surgery provide sites for infection
Evaluation of QT/TdP Signal:
Two main categories of signals and associated levels of concerns:
❖ A strong signal is an observation that supports a linkage to a
possible TdP risk.
❖ Examples of strong signals include treatment-evoked
ventricular arrhythmias in animals or humans or association
with a chemical or pharmacological class of agents known or
suspected to elicit TdP arrhythmias.
❖ A weak signal is a possible cause for concern.
❖ Examples of weak signals include an average increase in
QT interval (compared with baseline or placebo) outside
the normal range
❖ Significant change from baseline in rate-corrected QT
interval (QTc, using an species-appropriate method)
❖ Current uncertainties in extrapolation of nonclinical
findings to humans
❖ Factors for Evaluation of TdP Risk:
❖ Magnitude: is the magnitude sufficient to have clinical impact
from the background variability of the model being used
(∆QTc/baseline >60 msec→ high risk)
❖ Dose-response: are dose- or concentration-response
relationships apparent in the data? Evidence of dose-response
can help distinguish treatment-evoked effects from
experimental variation?
❖ Reversibility and reproducibility: is the effect reversible
upon removal of exposure; can the effect be reproduced
following re-exposure (e.g., treatment-evoked)?
❖ Therapeutic index (TI) (LD50/ED50)
Higher risk for drugs with low TI and affecting QT interval
❖ Cardiac safety index(CSI), ratio between the level where
there is clear therapeutic effects (ED90) and that at which
there is an indication of minimal HERG activity (such as an
EC10).
❖ i.e. lowest concentration to cause QT prolongation (lowest
effect level, LOEL), compared with measured unbound plasma
concentration (ED90) associated with dosing regimen
❖ Obviously margins should reflect disease severity and medical
need.
❖ For example, one could predict that a 10-fold margin might
be acceptable for drugs used in diseases which are lethal if
untreated (e.g. cancer, AIDS, some other infections, etc.)
❖ A 30-fold margin may be acceptable for drug treatments for
serious debilitating diseases (e.g. stroke, Parkinson's disease,
schizophrenia, epilepsy, asthma, arthritis, etc.).
❖ A margin of 100-fold or even higher might be required in the
case of less serious diseases (e.g. Raynaud's, seasonal rhinitis,
eczema, etc.). These higher margins would also be
appropriate for drugs prescribed to psychiatric patients at risk
from suicidal overdose. Furthermore, one has to consider
changes to therapeutic target, patient population, and route
of administration.
❖ Tissue distribution
▪ Drugs that prolong ventricular repolarization in a fashion
unrelated to its pharmacokinetic profile should be
evaluated for tissue accumulation or for presence of
long-lived metabolites.
▪ Is drug accumulated or sequestered in cardiac tissues?
▪ Drugs that accumulate in cardiac tissue may impact risk
that is not reflected by either total or unbound
circulating drug concentrations.
❖ Metabolites whether major metabolites are human-specific,
or present in sufficient concentrations to pose an arrhythmia
risk.
❖ Species specificity
➢The absence of a signal in multiple species at similar
exposures may lessen concern that a particular signal will
be observed in man, or indicate the presence of a species-
specific active metabolite.
Torsades de pointes (TdP) : risk stratification *

CYP = cytochrome P450; IKr = rapid component of the delayed rectifier potassium
current
Center for Education Research and Therapeutics – University of Arizona