Transcript observational assessments - Faculty of Pharmacy, Cairo University

Preclinical Assessment of Potential
Central Adverse Effects
Department of Pharmacology and Toxicology
Faculty of Pharmacy Cairo University
Biostatistics and Biological Standardization of Drugs (306)
Safety pharmacology
Cardiovascular
system
Central
nervous
system
Respiratory
system
Secondary
organ
systems
Safety
During the drug discovery phase, it is obligatory to assess potential
adverse effects of any drug candidate on the cardiovascular system,
the central nervous system, the respiratory system and some
secondary organ systems in experimental animals before the start of
clinical trials (humans)
Centrally acting drugs
…are capable of passing the blood brain barrier
The blood brain barrier is formed of capillary endothelial cells
connected by tight junctions that do not exist in normal circulation.
That is why the blood brain barrier is highly selective permeable
barrier that allows the diffusion of only water, some gases, lipid
soluble molecules, glucose and amino acids that are crucial to neural
function.
Centrally acting drugs cross the blood brain barrier
to exert …
Therapeutic benefits
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Antipsychotics
Antidepressants
Anticonvulsants
Sedatives
Hypnotics
Analgesics
Adverse effects
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Amnesia
Convulsion
Depression
Cognitive impairment
Insomnia
Involuntary movement
Motor incoordination
Nausea
…
Central adverse effects are due to
… changes in the structure and/or function of neurons or
glial cells in the nervous system
Interfering with synthesis, storage, metabolism,
release, degradation, uptake, or receptors of
metabolism
synthesis
storage
release
receptor
action
reuptake
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Glutamate
Acetylcholine
γ-aminobutyric acid
Glycine
Dopamine
Serotonin
Norepinephrine
Histamine
Opioid peptide and/or
Endocannabinoids
CNS safety pharmacology
Category A/core battery studies are obligatory to be performed at
the early stage of drug discovery. They are typically simple tests
that can be performed rapidly in a routine fashion. They include:
• Functional observational battery (FOB)
• Spontaneous motor activity
• Locomotor coordination
• Convulsive threshold
• Interaction with hypnotics
• Pain threshold and
• Body temperature
Category B/supplementary studies include cognitive function,
electrophysiology examination, drug dependence and other tests.
Category B tests are carried out when necessary.
Category A/core battery studies
In this practical session, we will screen for potential
adverse effects of some drugs on the CNS using modified
FOB, spontaneous motor activity and locomotor
coordination tests. Drugs are injected 15 min prior to
performing the tests and compared with a vehicle control
group
1- Functional observational battery (FOB)
FOB is used to characterize different neurological functions.
It includes observational assessments (innate behaviors, no
need to be taught, require little interaction between the
observer and the animal) and manipulative tests (require an
interaction between the observer and the animal).
Observational assessments include Manipulative tests include
• Spontaneous activity levels
• Neuromuscular tests
• Reactivity
• Sensory responses
• Gait
• Posture
• Involuntary motor movements
• Clinical signs
Observational assessments
I- Spontaneous activity levels Observe the animal’s spontaneous
activity in its home cage. Limit observation to 5 sec or less
Rank the level of spontaneous activity displayed in the cage
1 = No activity (animal may be asleep or sitting motionless)
2 = Slight (animal may move its head or body, just a very few times)
3 = Moderate (animal moves about some)
4 = Active (animal moves more actively around cage)
5 = High activity (animal moves about rapidly)
Observational assessments
II- Reactivity: Observe the level of excitability or resistance of the
animal in response to handling and/or removal from the home cage
Rank the level of reactivity/excitability using scoring criteria such as:
1= Low (no resistance, easy to hold or pick up)
2= Moderately low (slight resistance)
3= Moderately high (some squirming or moving around)
4= High (excited, squirming, twisting)
5= Very high (aggressive actions e.g. biting, tail and throat rattling)
Observational assessments
III- Gait: Look at the movement of all four limbs in relation to one
another and to the saggital plane of the body
Rank the degree of gait abnormality, using a scale such as:
1 = No abnormality
2 = Slight abnormality
3 = Moderate abnormality
4 = Severe abnormality
Describe the gait, for example:
Ataxia (uncoordinated, staggering, wobbly gait)
Hind limbs show exaggerated, overcompensated, or splayed movements
Feet (primarily hind feet) point outward from body
Forelimbs drag and/or show abnormal positioning
Walking on toes
Observational assessments
IV- Posture: Refers to the placement of the body and spinal
curvature
Rank the degree of posture abnormality, using a scale such as:
1 = No abnormality
2 = Slight abnormality
3 = Moderate abnormality
4 = Severe abnormality
Describe postural alterations using terms such as:
Completely flattened, pelvis flat on surface
Pelvis low, dragging somewhat
Hunched, back raised up
Observational assessments
V- Involuntary motor movements : Record the occurrence or
absence of
• Tremors (repetitive contractions/relaxations of major muscle
groups)
• Fasciculations/twitches (localized muscle contraction)
• Convulsions
• Sterotypy (repetition of specific gestures o movements) or
• Bizarre behaviors (behaviors not normally seen in the animal e.g.
straub tail (tail is stiff and is held in a vertical position).
Observational assessments
VI- Clinical signs : Examine the animal for
• Lacrimation
• Salivation
• Hair coat characteristics (color/staining, alopecia, and
piloerection)
• Ocular abnormalities (corneal opacity or cloudiness , eyeball
appears to bulge) and
• Muscle tone or mass (hypotonia or hypertonia)
Manipulative tests
VII- Neuromuscular tests
Indicate the presence or absence of the righting reaction.
Hold the animal in a supine position (lying with the face up) on a
surface. Quickly release the animal. The animal should
immediately flip to resume a normal standing posture with the
head turning over first, followed by the forelimbs, and then hind
limbs
Manipulative tests
VIII- Sensory responses
Visual approach response: Approach the animal at nose level with
the end of a blunt object, such as a pen or pencil. Hold the stimulus
∼3 cm from the face for ∼4 sec to give the animal time to make a
response.
Rank the magnitude of response to the stimulus:
1 = No reaction or response
2 = Slight or sluggish reaction (flinch or startle as evidence of
perception)
3 = Obvious reaction (locomotor orientation as evidence of
perception)
4= Clear reaction or response (more intense startle or locomotion)
5= Exaggerated reaction (may jump, bite, or attack
Manipulative tests
VIII- Sensory responses
Somatosensory response: Coming in from the side, touch the rump
of the rat gently with a blunt object, such as a pen, pencil, or brush.
Use a brief touch (1 to 2 sec) that is deliberate but not too forceful.
Rank the magnitude of response to the stimulus:
1 = No reaction or response
2 = Slight or sluggish reaction (flinch or startle as evidence of
perception)
3 = Obvious reaction (locomotor orientation as evidence of
perception)
4= Clear reaction or response (more intense startle or locomotion)
5= Exaggerated reaction (may jump, bite, or attack
Manipulative tests
VIII- Sensory responses
Auditory response: Position a sound stimulus e.g. metal clicker,
finger snap, or hand clap ∼5 cm above the back of the animal (i.e.
outside the field of view). Make a sudden sound.
Rank the magnitude of response to the stimulus:
1 = No reaction or response
2 = Slight or sluggish reaction (flinch or startle as evidence of
perception)
3 = Obvious reaction (locomotor orientation as evidence of
perception)
4= Clear reaction or response (more intense startle or locomotion)
5= Exaggerated reaction (may jump, bite, or attack
Manipulative tests
VIII- Sensory responses
Test tail-pinch response: Pinch the tail ∼1 cm from the tip. The very
tip of the tail is relatively insensitive. Pinch hard enough to produce a
reliable response in control animals, and be consistent in the
intensity of the pinch from test to test.
Rank the magnitude of response to the stimulus:
1 = No reaction or response
2 = Slight or sluggish reaction (flinch or startle as evidence of
perception)
3 = Obvious reaction (locomotor orientation as evidence of
perception)
4= Clear reaction or response (more intense startle or locomotion)
5= Exaggerated reaction (may jump, bite, or attack
2- Spontaneous motor activity
Can be quantified by manual counting of crossed squares in the open
field apparatus
The open-field apparatus consists of a
square arena of adequate size
surrounded by walls to prevent the
animal from escaping. The floor is
divided into equally spaced squares.
The open field apparatus should be
located in a quiet room with controlled
temperature and ventilation and lowlevel lighting to reduce anxiety. The
apparatus should be cleaned prior to the
test of each animal to reduce olfactory
confounding factors
2- Spontaneous motor activity
A. Administer the test drug or the vehicle at an appropriate time
prior to placing the mouse into the center of the open field.
B. Record the number of squares crossed by the mouse, the
number of rearings (when the front legs of the animal are
lifted completely off the surface, or when the animal places its
front legs on the side of the open-field apparatus) or any
stereotype or bizarre behavior within 10 min.
Test drug
Number of squares crossed
Number of rearings
Stereotype or bizarre behavior
Vehicle
3-Neuromuscular coordination
The ability of a rodent to maintain balance and keep pace with a
rotating rod has been used to assess motor function.
The rotarod consists of a circular rod about 75 cm long, divided
into 6 sections to allow simultaneous testing of 6 mice; the rod is
attached to a motor rotating the rod at a constant or increasing
speed. The rod is placed about 50 cm above a platform to
discourage the animals from jumping off the rod.
3-Neuromuscular coordination
A. Animals are prescreened; only those that are able to remain on
the revolving rod for at least 3 min are used for screening test
substances
B. Administer the test drug or the vehicle at an appropriate time
prior to placing the mouse on the rotarod and then start the
motor and timer.
C. Record the latency to fall from the rotarod for each mouse. It is
generally preferred to take the mean of at least two to three
measures from each animal.
D. Calculate the percent of animals falling from the rotarod within
3 min
Test drug
Latency to fall
Percent of animals falling within 3 min
Vehicle