Contemporary Scientific Principles of Neurotoxicity
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Dramatic
neuroscientific advances recently have led to an ever increasing rate of the
development of neuroactive drugs. The application neuroscientific advances
applied to safety screening practices however, has not consistently kept up
with their application for drug development itself. Modern understanding of
the pathology of neurotoxicity, its mechanisms and the tools available with
which to assess safety have advanced significantly in recent years resulting
in new capabilities for safety screening. However, the status quo of decades
old safety study designs continues to be practiced when conducting
neuropathological safety assessments. The execution of modern capabilities
in neurotoxicity screening is mandated to achieve compliance with regulatory
agencies, protects consumers from dangerous compounds and enables
researchers to accelerate drug development through the development of
predictive tools.
Below is a
discussion of three major elements for consideration in a contemporary
neurotoxicity assessment. The significance of each of these elements is
self-evident, however it is important to consider the nature and purpose of
the desired test when applying these principles. A screening protocol
designed to identify up to 50% of compounds that will exhibit a neurotoxic
effect is conducted by NTC at a cost of less than $1500 per compound tested
(or identify 80% of compounds for under $3000).
The goal of that type of study is to find neurotoxicity and the signature
timing of a typical response allows for this level of efficiency. For
comprehensive studies intended to rule out neurotoxicity, a more thorough
approach is required based on the principles below:
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When: Timing of
observations:
Although damage is permanent, the detectable pathology of
neurodegeneration is only detectable for a few days.
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Where: Adequacy of
Sampling:
Unlike other organs, the brain must be sampled more frequently to
independently assess damage to all major regions.
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What: Scope of detection:
The detection mechanism must be able to detect ALL elements that are
considered evidence of neurodegeneration.
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Where: Sampling
Current Scientific
Principles
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The brain’s 600+ neuronal populations do not
have homogeneous vulnerability.
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Some compounds characteristically affect
only one structure in the brain and have no effect on others.
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Conversely, other compounds affect not just
one particular structure, but have an effect on many others. This is to
say, although a compound may have been designed to have a specific effect
on a given structure of the brain, e.g. hippocampus, other structures may
also be affected and perhaps in a deleterious manner.
Sampling represents the simplest and easiest
application of modern science to apply. The concept, simply put, is that you
won’t find neurodegeneration unless you look in the ‘correct’ location in
the CNS. The ‘correct’ location in this case is not necessarily where an
effect may be anticipated, rather wherever it happens to exist.
With our current scientific databases and
tools, neither the potential of off target effects (such as
neurodegeneration) to occur, nor the location of the effect can be
anticipated. Compounds that are neuroactive by design, as well as compounds
not capable of directly crossing the blood brain barrier have the potential
to cause neurodegeneration. In either case, both the effect and location may
be unpredictable.
Past practices and sampling of the brain were
conducted in a manner similar to more homogenous tissues like liver and
kidney. It was thought that a handful of sections (4-5) sampled through the
brain would provide adequate representative pathology. In that approach,
only the structures that are present within those few cross sections would
have the capability of being analyzed for neurodegeneration. This approach
would prevent the researcher from analyzing results from the ‘correct’
location if that location didn’t happen to be one that was sampled.
We now know that analyzing one area of the
brain and extrapolating the results to another area is no more appropriate
than analyzing liver tissue and extrapolating its results to provide
conclusions about the kidney or heart. The significance or severity of
neurotoxicity is not necessarily defined by the volume of neurodegeneration
that occurs (translating to easy detection), rather by the severity of
effect to one or multiple of the 600+ populations of the brain. It is
important then to design a sampling approach that allows the opportunity to
view neurodegeneration to be witnessed in any of the populations of the
brain where it may exist.
Selective assessment, therefore is neither
adequate nor does it meet the requirements specified by the FDA. For any
species, sampling at a rate that generates at least 25 evenly spaced coronal
sections ensures representative sampling of all major elements of the brain.
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When: Timeline of
neurodegeneration
Current Scientific
Principles
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Cells vulnerable to a particular agent of destruction tend to die in a
consistent timeline.
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The window of time for detecting debris from neuronal cell disintegration
is typically 2- 3 days, after which the debris from the disintegrated cell
has been removed.
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Multiple mechanisms can be responsible for cell death, but once
disintegration begins, cells tend to follow a similar pathway of
destruction.
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Neuronal elements tend to die/disintegrate in a characteristic overlapping
sequence (synaptic terminals, dendrites, cell bodies, axons).
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The collective window of time for detecting the debris from the
disintegration of all neuronal elements is typically 6+ days.
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The beginning of detectable cell death from an acute dose can range from
2-12 days.
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Neurotoxicity may be caused by a single event (acute dose), or by repeated
exposure (subchronic or chronic dosing).
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Subsequent
exposures to an agent of destruction may not cause additional cell death.
Looking for neurodegeneration at the ‘correct’
time is just as important as looking in the ‘correct’ place. It is known
that affirmative detection of neurodegeneration of any single cell is
possible only for a window of 3-6 days. The point in time that the 3-6 day
window begins from an acute dose or from multiple doses varies from one
compound to another, but all cells vulnerable to that compound tend to
follow the same pattern. Just as sampling requires all structures to be
analyzed, timing requires that multiple time points be viewed to create the
best opportunity to witness the neurotoxic effects.
Timing of detection is a crucial
consideration in neurodegeneration assessment because although the effects
of neurodegeneration are permanent, evidence of the damage is ‘relatively’
transient. Pathological detection of neurodegeneration requires witnessing
the disintegrative destruction of the neuron or observing the absence of a
(disintegrated) neuron within a field of surviving neurons. The latter is
most feasible when overt neurotoxicity causes entire populations to be
destroyed and is less practical in less overt cases of neurotoxicity when
only representative cells are destroyed. In either case, the affirmative
detection of degenerating cells provides the most conclusive evidence of
neurodegeneration. Timing is a key consideration in affirmative detection.
The disintegrative event of a neuron lasts
between 2-3 days during which various stains can be applied to reveal
evidence of the disintegration. Stained cells appear normal prior to the
disintegration and the debris of the cell has been completely removed after
2-3 days, so nothing is left to stain.
Cells within a population that are vulnerable
to cell death from a specific drug tend to disintegrate at the same time.
For short term acute effects, the timing of cell death within a population
“in close step”, while for subchronic and chronic effects, the timing begins
to have a wider distribution.
One of the most common pitfalls in acute and
subchronic study designs is timing the sacrifice times to be too late. For
acute toxicity, the most likely time point for neurodegeneration to be
detected is from 3-5 days, however it can occur during any 2-3 day window up
until 12 days after exposure. It is not uncommon for a study design to
schedule a pathologic review only at the end of a 14 day or 28 day testing
period. This practice virtually eliminates the opportunity for the detection
of neurodegeneration that occurred at any point prior to that sacrifice date
(less ~2-3 days).
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What: Scope of
neurodegeneration (all elements)
Current Scientific
Principles
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In the absence of cell death itself, the
disintegration of other neuronal elements can render a cell just as
ineffective as when cell death occurs.
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The scope of neurodegeneration includes the
disintegration of neuronal cells AND other neuronal elements.
The final consideration afforded by modern
science deals with what structures should be considered in assessing for
neurodegeneration. Degenerating neurons have always been considered to be a
significant adverse effect, but there are other neuronal elements to
consider as well. The destruction of the remaining neuronal elements (axon
terminals, dendrites, and axons) are each in isolation capable of rendering
even an intact nucleus ineffective (as good as dead). Most often, however,
the destruction of any of these elements is part of the degenerative
progression of elements discussed in “timing” (above) that indicates not
only the destruction of that element, but the likely destruction of the
neuron as well. In either case, regulatory guidelines clearly define the
destruction of these elements to be adverse effects and thus should be
considered during a neurotoxicity screen. Traditional staining mechanisms
for toxicity (such as H&E) are incomplete solutions for addressing
neurotoxicity.
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