A Computerized Test Battery to Study Pharmacodynamic Effects on the Central Nervous System of Cholinergic Drugs in Early Phase Drug Development

With human life expectancy steadily increasing over the last century the prevalence and incidence of diseases of the aging brain, such as dementia and other neurodegenerative processes, also grow. In parallel, the development of new drugs to treat these diseases is therefore expanding. However, many new drugs intended to be active in the CNS fail to reach the market due to lack of central effects or unwanted side effects in later phases of drug development¹. In traditional phase 1 studies the objectives are to gain information on the pharmacokinetics, that is, the effect that the human body has on the drug (for example by metabolizing), as well as safety and tolerability of the new drug. Early proof of pharmacodynamic effect (the effect that the drug has on the body), however, may be even more important in decisions on moving forward in the clinical development of a new compound and may help avoid erroneous decision making with consequences at later phases of the development process².

In the past two decades, the Centre for Human Drug Research (CHDR) has developed a computerized test battery of neuropsychological and neurophysiological measurements sensitive to CNS effects of drugs. This test battery is used repeatedly over the day to measure pharmacodynamic effects of a new compound. It thereby provides evidence of the drug's ability to have the desired effect, to penetrate the blood-brain barrier and enter the brain, or the lack thereof³. Also, outcomes of the test battery could provide information on the mechanism of action of a compound as the individual tests correspond to specific drug-responsive CNS domains. For example, if effects of the new drug are seen on the maze learning test, which is a test for visuospatial working memory, this could indicate that the drug acts on receptors in parts of the brain involved in visuospatial working memory. In addition, the test battery is used to screen for CNS side effects for compounds that are not designed to work in the CNS, and where CNS activation needs to be ruled out.

The test battery is made up of a large number of cognitive and neurophysiological tests, which have been shown to be sensitive to detect pharmacodynamic effects of CNS active drugs^3,4,5,6. The core test battery comprises six neuropsychological domains: executive functioning, attention, memory, visuomotor functioning or coordination, motor skills, and subjective drug effects. The core tests are: saccadic eye movement⁷, smooth pursuit eye movement⁸, the Bowdle VAS⁹, the Bond and Lader VAS¹⁰, body sway, adaptive tracking¹¹, visual verbal learning¹², and qEEG, which cover the main cognitive and neurophysiological domains mentioned earlier. These tests have been shown to be able to measure changes in CNS functions as a result of administration of several types and classes of drugs (see below). The battery can be repeatedly administered (up to 12 times following drug administration) due to the 30-min total administration time, which is essential to characterize the concentration-effect profile of a drug. The test battery can be expanded and adjusted with different tests fit to investigate specific drug classes, or even specific receptors. The test battery has been validated in a wide range of drugs acting on different CNS systems (e.g., benzodiazepines, antipsychotics, ethanol, and cannabis^{12,13,14,15,16,17,18,19,20,21}) to be able to reliably demonstrate drug related CNS effects.

While other computerized test batteries exist (described for example in Egerhazi et al.²² and Underwood et al.²³), and are widely used in clinical trials, the test battery described in this paper stands out as it not only includes neuropsychological tests such as the VVLT and the VASs, but also neurophysiological measurements (e.g., EEG, eye movement tests), thereby combining different aspects of brain functioning in one test battery, and better reflecting the multimodal nature of cognitive behavior. Furthermore, as the test battery is computerized, the test results are generated electronically. This results in outcome values that are the same when used in different studies by different research staff, allowing for standardization of results, as well as values that are less error prone compared to scoring by hand. The outcome files can be easily uploaded into electronic database systems and can be used to generate interim reports of the pharmacodynamic effects of new drugs within a day.

There is at least one class of drugs where early proof of pharmacological effect in the brain has been difficult; the (pro)cholinergic drugs. Acetylcholine is one of the main neurotransmitters of the CNS and has been shown to play a key role in cognition, specifically in processes such as learning and memory^24,25. Consequently, cholinergic dysfunction is indicated to underlie neurodegenerative processes such as Alzheimer's disease²⁶. New compounds designed to enhance cognitive functioning, such as muscarinic and nicotinic receptor specific agonists, are now entering clinical studies.

As early phase studies are usually performed in healthy, often young subjects who cognitively perform at a normal level, it is difficult to study or even show proof of pharmacodynamic effect of a new drug intended to treat cognitive decline in patients with a disease of the brain.

Our group has therefore developed a tool that can be used for demonstrating early proof of pharmacology of a new drug: the pharmacological challenge model. An already approved and marketed drug is used to induce temporary and reversible disease-like symptoms in healthy subjects, via a pharmacological mechanism related to the disease that is targeted as indication for the new compound. In most cases this effect is an unwanted side effect of the drug, resulting from activation of receptors at a different location in the human body compared to the site where the drug is intended to work. For example, the muscarinic acetylcholine receptor antagonist scopolamine is used for the treatment of nausea and vomiting due to motion sickness. Side-effects resulting from antagonizing muscarinic acetylcholine receptors in the brain are the anti-cognitive effects such as reduced attention and memory resembling the deficits seen in Alzheimer's disease²⁷.

Since scopolamine is used as a muscarinic acetylcholine challenge model to induce Alzheimer-like, yet temporary, cognitive effect in healthy subjects²⁷, CHDR has developed and validated a pharmacological challenge model with mecamylamine. Mecamylamine is a non-competitive nicotinic acetylcholine receptor antagonist²⁸ which results in cholinergic dysfunction, i.e., transient cognitive deficits, in healthy young males^29,30.

The above mentioned computerized test battery has been used to investigate the potential of different dose levels of mecamylamine to show effects on the neurophysiological and cognitive tests. The expectation was that with increasing dose, the effects on the different tests would also increase. Subsequently these effects were related to the plasma concentrations of the drug, resulting in the plasma concentration-effects (pharmacokinetic-pharmacodynamic) relationship of mecamylamine²⁹.

The tests incorporated in the design of this study were chosen based on the expected effects known from the literature and the pharmacological mechanism of action of mecamylamine on the nicotinic receptors:

Adaptive Tracking Test:

This is a pursuit-tracking task, for the measurement of visuomotor coordination and sustained attention. A circle of known dimensions moves randomly about a screen. The subject must try to keep a dot inside the moving circle by operating a joystick. If this effort is successful, the speed of the moving circle increases. Conversely, the velocity decreases if the test subject cannot maintain the dot inside the circle. In contrast to non-adaptive tracking methods, this leads to a constant and individually adapted challenge throughout the procedure. The adaptive tracking test used was developed by Hobbs & Strutt, according to specifications of Borland and Nicholson¹¹.

Smooth Pursuit and Saccadic Eye Movement Tests:

The use of a computer for measurement of saccadic eye movements and smooth pursuit was originally described by Baloh et al.⁷, and for smooth pursuit by Bittencourt et al.⁸, and has been extensively validated at CHDR by Van Steveninck et al.^19,20,21 The subject is required to follow a light source with the eyes, which moves horizontally on a screen at 58 cm distance. The light source moves continuously for measurement of smooth pursuit and jumps from side to side for measurement of saccadic eye movements.

VASs:

Assessment of subjective feelings of alertness, mood, and calmness was performed using a set of 16 visual analog lines as described by Norris (1971) and Bond and Lader¹⁰. Visual analog scores rely on the ability of subjects to semi-quantify a subjective state. Visual analog lines consist of 10-cm line segments. The subject is presented with 16 lines, 1 at a time, on the computer screen. At the two ends of the line, two opposing words representing states of mind (e.g., happy – sad, tense – relaxed) are presented. Subjects put a mark on a point on the line that best represents their subjective state corresponding to the condition tested. The result is a distance (mm) calculated from the mark on the line.

Body Sway:

A string originating from a potentiometer, which is incorporated into the test battery computer, is used to measure postural stability in a single plane while the subject stands still with the eyes closed (described in de Haas et al.¹²).

VVLT:

The VVLT is a word learning and memory test, described in more detail in de Haas et al.¹² Subjects are presented with a series of 30 words, one by one on the computer screen. The words need to be pronounced and remembered. There are three immediate recall trials, one delayed free recall trial (i.e., without presentation of the words) after approximately 20 min and a recognition trial.

Pharmaco-EEG:

For the standard pharmaco-EEG, electrodes are limited to the midsagittal leads (Fz, Cz, Pz and Oz), two electrodes for recording eye movements (outer canthi), and a ground electrode placed 2 cm above the nasion. Changes in the amplitude of the following frequency bands are quantified by spectrum-analysis (i.e., fast Fourier transformation): ß-band (13.5-35 Hz), γ-band (35-48.9 Hz), α-band (7.5-13.5 Hz), and θ-and δ-bands (7.5 Hz or less).