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Preclinical Assessment of Potential Central Adverse Effects

Biostatistics and Biological Standardization of Drugs (306)

Department of Pharmacology and Toxicology

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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)

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tight junction
endothelium
basement membrane
astrocyte
O
O
blood
brain

The  blood brain barrier  is formed of capillary endothelial cells connected by  tight junctio ns  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.

…are capable of passing the  blood brain barrier

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Centrally acting drugs cross the blood brain barrier to exert …

Antipsychotics Antidepressants Anticonvulsants Sedatives Hypnotics  Analgesics

Amnesia Convulsion Depression Cognitive impairment Insomnia Involuntary movement Motor incoordination Nausea …

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… changes in the  structure and/or function  of neurons or glial cells in the nervous system

release reuptake receptor action synthesis metabolism storage

Interfering with synthesis, storage, metabolism, release, degradation, uptake, or receptors of

Glutamate Acetylcholine γ-aminobutyric acid  Glycine Dopamine

Serotonin Norepinephrine Histamine Opioid peptide and/or  Endocannabinoids

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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.

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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

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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).

1- Functional observational battery (FOB)

Observational assessments include Spontaneous activity levels Reactivity Gait Posture Involuntary motor movements Clinical signs

Manipulative tests include Neuromuscular tests Sensory responses

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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 activity  (animal may move its head or body, just a very few times) 3 = Moderate  activity(animal  moves about some) 4 = Active (animal moves more actively around cage) 5 =  High activity  (animal moves about rapidly)

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II- Reactivity:  Observe the level of  excitability  or  resistance  of the animal  to  handling  or removal  from the home cage

Rank the  reactivity/excitability through scoring the resistance to handling : 1= Low  reactivity/excitability ( no resistance to handling ,  easy to hold or pick up) 2= Moderately low  reactivity  (slight  resistance to handling) 3= Moderately high  reactivity ( some squirming or moving around) 4= High  reactivity ( excited, squirming, twisting) 5=  Very  high  reactivity  ( Very  high  resistance to handling /aggressive actions: biting , tail and throat rattling)

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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

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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

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V- Involuntary motor movements :  Record the  occurrence   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).

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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 changes  (hypotonia or hypertonia)

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Indicate the presence or absence of the  righting  reflex .  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

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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  response 2 = Slight or sluggish reaction or response (flinch or startle as evidence of perception) 3 = Obvious reaction or response (locomotor orientation as evidence of perception) 4= Clear reaction or response (more intense startle or locomotion) 5=  Exaggerated  response  (may  jump, bite, or  attack)

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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  response 2 = Slight or sluggish  response or reaction  (flinch or startle as evidence of perception) 3 = Obvious  response or reaction  (locomotor orientation as evidence of perception) 4= Clear  response or reaction ( more intense startle or locomotion) 5=  Exaggerated  response  (may  jump, bite, or  attack)

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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  response 2 = Slight or sluggish  response or reaction (flinch  or startle as evidence of perception) 3 = Obvious  response or reaction  (locomotor orientation as evidence of perception) 4= Clear response or  reaction ( more intense startle or locomotion) 5=  Exaggerated  response  (may  jump, bite, or  attack)

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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 response 2 = Slight or sluggish response or reaction (flinch or startle as evidence of perception) 3 = Obvious response or reaction (locomotor orientation as evidence of perception) 4= Clear response or reaction (more intense startle or locomotion) 5=  Exaggerated response  (may jump, bite, or attack)

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-The  open-field apparatus consists of  wooden box 60x60x40 cm.  -The walls are  to prevent the animal from  escaping. -The  floor is divided into  16 equal squares . -The apparatus  should be located in a quiet room with controlled temperature and ventilation and low-level lighting to reduce anxiety.  -The  apparatus should be cleaned prior to the test of each animal to reduce olfactory confounding  factors (to reduce odor bias)

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Open Field Test             to quantify the motor activity

Give the  test drug or the vehicle at an appropriate time prior to  placing the mouse into the center of the open  field  and record:  1-  The ambulation frequency :  number of squares crossed by the  animal by 4 limbs within 10 min. 2-  Rearing frequency : number of times the animal stand on the hind limbs with or without support of the fore limbs within 10 min. ( 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)  3- any  stereotype or bizarre behavior  within 10 min.

Test  drug(Test  gp ) Vehicle(Control  gp ) Number of squares crossed     Number of  rearings     Stereotype or bizarre behavior

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The ability of a  animal to  balance  and  walk on  rotating rod  has been used to assess  the neuromuscular coordination  (Rota rod test) . - The Rota rod  consists of a circular rod  ∼75  cm  long, divided into  6 sections  to allow simultaneous testing of 6  mice. - The  rod is placed  ∼50  cm  above a platform to discourage the animals from jumping off the rod.  -The  rod is attached to a motor rotating the rod at a constant or increasing speed.

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Animals are  pre-screened ; only those that are able to remain on the revolving rod for at least  3 min  are used for screening test  drugs. 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. 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. Calculate the  percent of falling animals  from  the rotarod within 3 min

Test  drug (Test  gp ) Vehicle (Control  gp ) Latency to fall     Percent of animals falling within 3 min

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Category A/core battery studies   (obligatory)

Category  B / supplementary studies  (when necessary)

-cognitive function -electrophysiology -drug  dependence

Observational  assessments -activity -Reactivity -Gait -Posture -Involuntary movements        Tremors                                                     Sterotypy                                                Bizarre behav.  -Clinical signs         Salivation                                    Lacrimation                                    Hair coat changes                                    Ocular abnormality                                    Muscle tone changes

Manipulative  tests -Neuromuscular tests          Righting reflex -Sensory responses          Visual response                                                Auditory response                                                Somatosensory response                                                Tail pinch response

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