The late detection of cardiotoxic side effects, such as electrocardiogram QT-interval prolongation, induced by compounds of pharmacological interest can dramatically impede drug research and development projects.
Frederic Sannajust reports.
The discovery and launch of new drugs with undetected cardiotoxic side effects could have hazardous consequences and trigger lethal cardiac dysrhythmias (ventricular arrhythmias or aTorsades de Pointe') in patients. It is therefore important to assess the potential cardiotoxic effects of new chemical entities (NCEs) at an early stage of drug development.
During the past three years, cellular-based assays (HERG-K+ channel) performed on cloned human ion channel cell-lines and electrophysiological (action potential duration measurement in mammalian Purkinje Fibres) systems have been developed.
Predicting drug potential
These in vitro test systems have become powerful tools for predicting the proarrhythmic potential of drugs prior to their entry into the clinic. However, they should not only enhance the speed and efficiency at which drugs will be developed in the future, but also complement the in vivo preclinical cardiovascular evaluation.
In addition, they can significantly reduce the number of animals used.
For the past seven years the development of numerous drugs (antiallergics, antibiotics, antidepressants, neuroleptics, antimalarials, antimycotics, antihypertensive and antitumoral drugs) has been aborted in late phases of drug development programmes because of undesirable effects on cardiac repolarisation.
For example, Sepracor's market capitalisation dropped by more than $2 billion in a single day because its allergy drug Soltarai was rejected by the FDA over questions concerning cardiac safety. This single incident defined the cardiac safety/QT issue and demonstrated that pharmaceutical manufacturers, physicians and regulatory authorities are greatly concerned about drugs inducing QT prolongation.
An additional ICH guideline aS7B' is currently in preparation (to be published in November 2002) to address the assessment of potential for QT prolongation. This guideline is complementary to the CPMP 986/96 recommendation (June 2001) indicating that cardiac action potential duration (APD) in mammalian Purkinje Fibres, cardiac HERG-K+ channel interactions and in vivo ECG (QTc-intervals) measurements in conscious unrestrained animals (via Telemetry) should be conducted prior to first administration to humans.
Abnormality
The long QT syndrome (LQT) is an abnormality of the excitation of the heart while its mechanical function is entirely normal.
QT refers to an interval measured on the electrocardiogram ECG and LQT has been correlated with defects in cardiac ion channels.
When cardiac cells are excited a complex sequence of voltage-dependent processes occurs as a function of time forming the specific shape of the cardiac action potential, AP (Fig. 1).
It has been shown that compounds which are associated with LQT predominantly have a secondary molecular pharmacological interaction with the rapidly activating delayed rectifier potassium channel named IKr.
The HERG-K+ channel assay focuses on the ionic potassium channel, whereas the Purkinje Fibre assay is a more integrative approach of ionic currents
(K+, Na+ and Ca2+) involved during conductance/depolarisation changes. u Whole-Cell Voltage Clamp (WCVC) HERG-K+ Channel Electrophysiology: The human aether-a-go-go-related gene' (HERG) encodes the major potassium channel that participates in the repolarisation current of the late phase of the cardiac AP. Huntingdon Life Sciences (UK Pharmacology group) recently acquired WCVC methodology and human embryonic kidney (HEK-293) and Chinese Hamster Ovary (CHO) cell-lines stably transfected with cDNA of HERG-K+ channel to allow electrophysiological examination of the effects of new NCEs on endogenous HERG-channel activity.
Experiments are performed with stimulation protocols mimicking the physiological conditions during a normal cardiac AP in the absence of overlapping currents that normally obscure its time course in native heart cells.
The effects of NCEs are compared with those of reference drugs (eg d,l-sotalol, terfenadine, astemizole, cisapride, verapamil, dofetilide,
E-4031).
It is well established that the greater the affinity of an NCE for HERG-K+ channels, the more likely it is to induce LQT. u Action Potential Duration (APD) measurement in Rabbit PURKINJE FIBRES: Cardiac cells express not one but many ion channels, any of which may be targets for drug action.
In vitro assays allowing precise control of drug concentration, at levels not easily attainable in vivo and at precise control of pacing rates, form a necessary adjunct to safety testing. Such design is provided by Purkinje Fibres which form a multi-cellular tissue that propagates the AP into the muscular walls of the ventricle which is highly sensitive to drugs that produce LQT.
This accurate AP measurement system validated at HLS/UK-Pharmacology group requires a conventional intracellular electrophysiological recording method to assess drug-effects on resting membrane potential, APD, amplitude and maximum rate-of-rise, under near-physiological conditions of ionic composition ([K+]=4 mM), pacing (0.2Hz1Hz2Hz) and temperature (37.0 ±0.5oC). u Complementary Assessment of In Vivo Cardiovascular Safety Pharmacology: Marmoset-Monkey-Dog ECG, BP and HR Telemetry measurements.
On occasions the QT activity is detected or confirmed only during an in vivo cardiovascular safety approach.
In vivo experiments investigating the cardiotoxicity of NCEs are typically performed in conscious or anaesthetised dogs and rarely, in rat, guinea-pig or rabbit arrhythmogenic models.
Effects of NCEs on mean blood pressure (MBP), heart rate (HR), lead II ECG (RR, PR, QT, QTc intervals, QRS duration, QT dispersion), body temperature and locomotor activity, can also be explored in conscious unrestrained marmosets, cynomolgus monkeys by using integrated telemetry devices (DataSciences International).
The major advantage of such experiments is that data are generated under physiological conditions which is essential for the pre-clinical characterisation of NCEs cardiotoxic effects.
However, it is necessary to take into account that the expression of cardiac ion channels can differ between humans and other species.
In addition, high cost and low throughput limit the application of this methodology which should be reserved for screening compounds that are advanced in the developmental process.
Conclusions
These complementary in vitro electrophysiological and in vivo pharmacological approaches are a step forward in the drug optimisation process and allow earlier screening of NCEs with more confidence and accuracy in the assessment of QT prolongation.
However, it remains difficult to predict the proarrhythmic potential of NCEs because predictability still depends on numerous factors, such as: plasma/cardiac concentration levels; pharmacokinetics; polypharmacology; repolarisation complexity; and extrapolation to clinical settings.
In addition, the early identification of the common adverse effect (ie LQT for NCEs) could significantly reduce the number of animals used (3 Rs) and the costs of pre-clinical development. u
ENQUIRY No 97
Dr. Frederic Sannajust is Head of Pharmacology, Huntingdon Life Sciences, Cambridgeshire, UK. www.huntingdon.com
Acknowledgement: We are very grateful to Drs Zayheda Amin and David Beard for their contribution and advice.
