Various drug screening methods in vivo for analgesic models like, Acute pain, Chronic pain and pain in cancer.

1. Drug screening methods of
Analgesic agents
Dr. Abhishek Y. Vyas
3rd year PG student
Department of Pharmacology
AMC MET Medical College

2. • Nociceptive component : disabling,
unpleasant sensation evoked by noxious
stimuli and conveyed to CNS by ascending
pathways
• Affective component : which is the
psychological response towards the pain
conveyed from CNS to dorsal horn by
descending pathways
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4. Drug screening methods of Analgesic agents
1. Animal Models of Acute Pain
1. Models using thermal stimulus
1. Hot plate method
2. The tail-flick method
1. Radiant heat
2. Immersing of the tail
3. Cold flick test
2. Models using electrical stimulus
1. Tooth pulp test
2. Monkey shock titration tesat
3. Models using chemical stimulus
1. Formalin test
2. Writhing test
3. Other chemical compounds
4. Models using mechanical stimulus
1. Haffner’s tail clip method
2. Randall salitto test
2. Animal models of Chronic Pain
1. Neuropathic pain models
2. Vincristine-induced neuropathy model
3. Diabetic neuropathy model
3. Models of Cancer Pain
1. Rat model of Bone cancer pain

5. Animal models of acute pain
• Animal test used in the search for new analgesics are designed as models for treatment of
pathological pain in man. They measure the power of the drug to increase the minimal stimulus
required to elicit pain or nociceptive response.
Hot Plate Method
•Widely used to evaluate opioid analgesics
Mice weighing ~20 g are used
Placed on the hot plate which maintained T 55-56 ͦC
Response as jumping, withdrawal of paws & licking of paws
are seen
Time period of response is recorded
Test compound are administered oral or S/C and latency
period recorded after 20,60 & 90 min
Values are compared before and after drug administration by
t-test7/15/2017 5

6. The Tail-Flick Test
• This test is widely and reliably used for reveling potency of opioid analgesics. In this test heat is
used as noxious stimuli and causes a simple nociceptive spinal reflex response in which the rat or
mouse flick its tail away the heat source. This test is very useful for discrimination between
centrally acting morphine like analgesics and non opioid analgesics.
The Tail Flick Test Using Radiant Heat
Mice weighing 18-22 g are used and placed in small cage leaving tail exposed
Tail held gently by observer. Light beam is focused to proximal third of tail
The mouse tries to pull the tail away and rotates the head. This reaction knows as
“escape reaction”
Reaction time of movement recorded
Test drug and standard are administered orally or s/c
Same procedure repeated and reaction time recorded after 30, 60
and 120 min
Lengthening time of reaction is interpreted as an analgesic action of drug7/15/2017 6

7. The Tail-Flick test using immersion of the tail
Wistar rats weight 170-210 g are used
Placed in separate cages in such a way that their tail hangs out freely
Distal 5cm part of tail of marked which is immersed in cup filled with warm water (Temp
55 ͦC)
A tail withdrawal reflex is seen within seconds
This reaction time is recorded. Normal reaction time was 1-5 sec
Test drugs are given orally or s/c and reaction time was recorded after 0.5, 1,2,3,and 6 hrs
Centrally acting analgesic drugs are capable of prolongation of tail withdrawal reflex and
hence withdrawal time of more than 6 sec in test animals denotes positive analgesic
response of test drug
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8. Models using electrical stimulus
Tooth pulp test
Rabbits 2-3 kg used and anesthetized with thiopental IV
Using dental drill, tooth pulp chambers are exposed close to the two front incisors
Clamping electrodes are placed into the drilled holes
After 30 min, electrical stimulus is applied by rectangular current 50Hz upto 1 sec
Current is started with 0.2 mA and increased until animal started licking. Threshold is
determined at least 3 times in each animal
Test compound is administered orally or IV
After 15,30, 60 and 120 min threshold current is measured with prior to drug administration
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9. Monkey shock titration test
• This method is used for final evaluation of a new
compound having analgesic potential
The monkeys are seated on restraining chairs
By A Coulbourn Instrument Programmable Shocker
electrical current is delivered through electrodes
which are coupled to shaved portion of tail
The current ranges from 0-4 mA through 29 progressive
steps
The monkey press the bar to interrupt the shock and for
each monkey, a stable baseline shock level is
recorded
After 24 hrs drug is administered and shock titration
activity is measured according to change in
maximum level of median shock intensity for drug as
compared to control levels
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10. Models Using Chemical stimulus
• This tests involve administration of irritant, algogenic chemicals as nociceptive stimulus.
Chemical stimulation induces slow form of stimulus.
Formalin Test
• 10% formalin is used as chemical noxious stimulus
• By injecting formalin solution into the paw of a rat/mouse a model of persistent
pain caused by peripheral tissue injuries and inflammation is created.
• In this test, animals show two phases of nociceptive behavior involving two
different stimuli:
1. Early phase starts immediately after injection and lasts for 3-5 min
2. Late phase starts after 10-15 min and lasts for 20-40 min
• This is due to combination of an inflammatory reaction in peripheral tissue and
functional changes in dorsal horn of spinal cord.
• Opioids are antinociceptive for both phases while NSAIDs are more effective in
second phase.
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11. Wistar rats weighing 180-300 g are used
Dorsum of front paw injected with 0.05 ml of 10% formalin s/c
Animals placed in separate cages for observation of pain response in both phases
Responses are elevation or favoring of the paw or licking and biting of the paw
Scoring of pain was recorded as per pain scale
After administration of test drug again scoring is done with 30, 60 min for comparison
If both paws are allowed to rest on the floor this is taken as positive analgesic
response
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12. Writhing Test
• This model projects visceral or peritoneal pain in animal and useful to detect peripheral
analgesic activity of test compound.
Mice weighing 20-25 gm are used
Phenylquinon (0.002 %) is suspended in 1% suspension
of carboxy methylcellulose
An aliquot of 0.25 ml of suspension is injected
intraperitonially in each animal
The animal reacts with characteristic stretching behavior
series of constrictions occur that travel along the abdominal wall and accompanied by turning
movement of the body and extension of the hind limbs. This response is called as writhing
A group of animals is used as test group in which test drugs are administered before
Phenylquinon
Mice are placed in glass chamber and number of writhes are recorded for 10 min in each animal
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13. Models using mechanical stimulus
Haffner’s Tail Clip Method
• This method is highly sensitive for centrally acting drugs
• Preferred sites for stimulus are hind paw and the tail
Mice are used and artery clip is placed at the root of the tail to apply noxious stimulus
A quick response is seen as biting the clip or tail
Time between application of clip and response is recorded
Test compounds are administered and same procedure is repeated after 15,30 and 60 min
and reaction time is measured
Reaction time of the test animals grater than the cut off time denotes a positive response
indicating analgesic activity
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14. Randall Selitto Test
• The principle of this test is that inflammation increases the sensitivity to pain and
thus decreases pain threshold
• In this test inflammation is induced by s/c administration of Brewer’s yeast and
then pressure is applied, which increases the intensity of pain.
Wistar rats are divided in two groups and in test group test agents are administered
After 15-30 min 0.1ml Brewer’s yeast (20% suspension) in distilled water is injected s/c
in planter surface of the left hind paw
After 3hrs using Randall Selitto apparatus, pressure is applied on planter surface of
foot at constant rate until animal struggles or squeals
In both group each animal tested for its control pain threshold and comparison is
made between groups
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15. Animal models of Chronic Pain
• Partial somatosensory nerve injury is the cause of causalgiform pain disorder in man.
• Causalgia is characterized by spontaneous burning pain combained with hyperalgesia
and allodynia
Sprague Dawley rats are used and anesthetized with halothane. Local incision is given and
sciatic nerve of both legs are exposed at level of mid thigh
A sutures are tide loosely around right sciatic nerve and left is just mobilized
During next days animal show a mild aversion of affected paw and foot drop
The thermal nociceptive threshold of hind paw is measured in each animal
Rats are placed beneath a transparent plastic cage upon a glass plate such that a halogen
projector lamp could be placed below it. Lamp is focused at planter area of one hind
paw
As soon as heat exposure of heat is applied a withdrawal response of hind paw is seen
The time interval between the exposure of heat and response is measured in each animal
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16. After 7-8 days test drug and vehicle are injected intrathecally in test and control group
respectively
Paw withdrawal latency (PWL) of hind paws is recorded before and after 5,15,30, 60 and
90 min of drug administration.
PWL which was the maximum during the first 30 min after drug or vehicle injection is
called as maximum PWL
To evaluate hyperesthesia, the difference score (DC) is calculated by maximum PWL of
control side/maximum PWL of the affected side
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17. Vincristine-induced neuropathy model
• this method provides a good model for study of a painful peripheral neuropathies
in patients receiving vincristin as a chemotherapeutic agents
Vincristine is administered daily for 2 weeks in rats
A decreased noceceptive threshold and hyperalgesia occurs after second day of
administration
Chronic lowered threshold and increased response to stimuli is seen during second
week of vincristine administration
Responses gradually return to baseline following discontinuation of treatment
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18. Diabetic Neuropathy Model
• Painful diabetic neuropathy is on of the most common complication of IDD in man
• Streptozotocin induced (STZ) diabetic rat has been used for chronic pain with signs of
hyperalgesia and allodynia
STZ is administered in rats to develop DM
Different types of stimuli like mechanical, thermal and cold applied
Decreased reaction threshold to noxious heat stimuli and non painful thermal stimuli is
observed. This serves as evidence of hyperalgesia and allodynia
This reactions is observed after 2 weeks of establishment if DM
Kiguchi S et al evaluated antinociceptive effect of oxcarbazepine in diabetic neuropathy
rat model and suggested that oxcarbazepine has analgesic action in neuropathy
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19. Model of cancer pain
• Bone metastasis is one of the major causes of cancer related pain.
• In present model, bone cancer is induced in the rat by syngeneic MRMT-1 mammary
tumor cell line
Sprague Dawley rats are given intra tibial injection of syngeneic MRMT-1 carcinoma cells
Control rats received heat-killed cells vehicle
Those who received syngeneic MRMT-1 carcinoma cells, develop behavioral sign indicative
of pain: allodynia, difference of weight bearing between hind paws
No tumor growth was observed in heat-killed MRMT-1 injected animals
The general activity of these animal is not changed
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