Screening of Analgesic Drug - NURSING ASSIGNMENT
INTRODUCTION
Pain is an unpleasant sensory and
emotional experience associated with actual and potential tissue damage.
Various types of pain are seen in humans, for example, somatic pain arising
from the skin, muscles, joints, ligaments and bones, visceral pain, referred
pain, neuropathic pain, cancer pain etc.
Chemical mediators of pain are
numerous. These mediators come from sources intrinsic to the neuron including
various neurotransmitters such as 5HT and substance P and extrinsic to the
nervous system, including substances from inflammatory/immune cells and red
blood cells such as prostaglandins, kinins, cytokines, chemokines and ATP that
are released following injury to the tissue.
Pain and Nociception
Pain
is produced by the excitation of particular receptors, the nociceptors or of
their afferent fibres. These remarkable cells respond to a broad spectrum of
physical (heat, cold and pressure) or chemical noxious stimuli. In general,
perception of noxious stimuli is termed as nociception. Nociception is not
exactly same as pain; pain is a subjective experience and includes a strong
effective component, whereas nociception lacks affective component. Most of the
afferent fibres that are excited by noxious stimuli are nonmyelinated C-fibres
with low conduction velocities (<1 m/s), known as C-polymodal nociceptors
(PMN), and other fibres are fine myelinated (Ad) fibres with rapid conduction.
Animal Models
Animal tests used in the search for
new analgesics are designed as models for the treatment of pathological pain in
man, but they usually differ from the original in that the test drug is given
before the noxious stimulus (mechanical, thermal, chemical and electrical and
electrical types of stimuli) in the animal models. Hence, these tests only measure
the power of a drug to increase the threshold of minimal required to elicit
pain or nociceptive response.
Haffner’s Tail Clip Method
In this method mechanical stimulus is
applied. This method is highly sensitive for centrally acting drugs.
Procedure
Male mice weighing 18 to 25 g are
used. An artery clip is placed at the root of the tail of the mice to apply
noxious stimulus. A quick response of the animal is seen as biting the clip or
tail, where clip has been placed. The time (reaction time) between application
of the clip and response is noted by a stopwatch. For testing analgesic
activity, test compounds are administered subcutaneously or orally. After 15,
30 or 60 min, same procedure is repeated and reaction time is measured. For
evaluations of analgesic drugs, cut off time is determined i.e. average
reaction time plus 3 times the standard deviation of the combined latencies of
the control mice at all time periods. Reaction time of the test animals,
greater than the cut off time denotes a positive response indicating analgesic
activity.
Hot Plate Method
Hot plate method has been widely used
to evaluate opioids analgesics.
Procedure
Mice weighing 18 to 22 g are used.
Animals are placed on the hot plate, which consists of electrically heated
surface. Temperature of the hot plate is maintained at 55-560C. Responses such as
jumping, withdrawal of the paws and licking of the paws are seen. The time
period (latency period) when animals are placed and until responses occur is
recorded by a stopwatch. Test compounds are administered orally or
subcutaneously and latency or latency period is recorded after 20, 60 and 90
min. These values are compared with the values before administration of the
test drug.
Radiant Heat Method
Mice weighing 18 to 22 g are used and
placed into small cages leaving the tail exposed mouse tail is held gently by
the observer. A light beam is focused (exerting radiant heat) to the proximal
third of the tail. The mouse tries to pull the tail away and rotates the head, this
reaction is known as escape reaction. The reaction is noted for 6 sec by the
observer. The test drug and standard are administered either orally and
subcutaneously. Same procedure is performed and reaction time is noted after
30, 60 and 120 min.
At each time interval those animals
that show higher reaction time than the time before drug administration are
regarded as positive. Percentages of positive animals are counted for each time
interval and each dose. ED values of test compounds can be calculated codeine,
pethidine and morphine are used as standard.
Tail Warm Water Immersion Method
Young female Wistar rats weighing 170
to 210 g are used. Rats are placed into separate cages and tail hangs out
freely. The distal 5 cm part of the tail is marked which is immersed (max upto
15 sec) in a cup filled with warm water (temp 550c). A tail
withdrawal reflex is seen within a few seconds. This reaction time is noted by
stopwatch. Normal reaction time is 1 to 5.5 sec. The test substances are given
orally or subcutaneously and reaction time is recorded after 0, 5, 1, 2, 3, 4
and 6 h.
A withdrawal time of more than 6 sec
in test animals denotes positive analgesic response of the test drug.
Tooth Pulp Electrical
Stimulation Method
Rabbits weighing 2 to 3 kg are used
and anesthesia is given with thiopental in the doses of 15mg/kg intravenously.
Using dental drill tooth pulp chambers
are exposed close to the two front upper incisors. Clamping electrodes
are placed into the drilled holes. After 30 min, electrical stimulus is applied
by rectangular current (frequency 50 Hz) upto 1 sec. Current is started with
0.2 mA and increased until animal starts licking and a threshold is determined
at least 3 times in each animal. Animal serves as its own control. Test compound
is administered orally or intravenously. After 15, 30, 60 and 120 min threshold
current is measured and compared with the threshold current prior to drug
administration.
Monkey Shock Titration Test
The monkey shock titration test is
used for final evaluation of a new compound as analgesic.
Procedure
The monkeys are seated in restraining
chairs, by a Coulbourn Instrument Programmable Shocker, electrical current is
delivered through electrodes, which are coupled to two test tube clamps
attached to a shaved portion of tail. The current ranges from 0-4 mP through 29
progressive steps. The monkey presses a bar to interrupt the shock. For each
monkey, a stable baseline shock level is recorded. After 24h drug is
administered and shock titration activity is measured according to a change in
maximum level of median shock intensity for drug as compared to control levels.
Formalin Test
This model is a chronic pain model and
highly sensitive for opioid-like drugs. Animals show two phases of nociceptive
behavior in formalin test involving 2 different stimuli. The first (early)
phase starts immediately after injection of formalin and last for 3-5 min. This
occurs due to chemical stimulation of
nociceptors causing C-fibre activation. Second (late) phase starts after 10-15
min of formalin injection and lasts for 20-40 min. This phase appears due to
combination of an inflammatory reaction in the peripheral tissue and functional
changes in the dorsal horn of the spinal cord. The C-fibre barrage initiates
functional changes during the early phase. Nonsteroidal anti-inflammatory drugs
are effective in second phase, while the first phase remains unaffected.
Procedure
Male Wistar rats weighing between 180
and 300g are used. In the dorsum of front paw of the animal 0.05ml of 10%
formalin is injected subcutaneously. Each animal is placed into separate cage
for observation of pain responses in early and late phases. These responses are
elevation or favouring of the paw or excessive licking and biting of the paw.
Scoring of these pain responses is done according to a pain scale. After
administration of the test drug, again scoring is done after 30, 60 min for
comparison. If both paws of the animal are allowed to rest on the floor with no
obvious of the injected paw, this is taken as a positive analgesic response.
Randall Selitto Test
Inflammation increases the sensitivity
to pain and decreases pain threshold. In Randall Selitto test, inflammation is
induced by Brewer’s yeast. After that, pressure is applied to inflamed area to
increase the intensity of pain.
Procedure
Male Wistar rats are used, divided
into two groups – test and control. Test agents an administered in test group
and vehicle is administered in control group animals. After 15 – 30 min, 0.1 ml
of Brewer’s yeast (20% suspension) in distilled water is injected
subcutaneously into the plantar surface of the left hind paw of the rat. After
3 h, using a special apparatus (Randall Selittol Apparatus) pressure is applied
to the plantar surface of the rat’s foot a constant rate until animal struggles
or squeals. In both the groups each animal is test for its control pain
threshold for comparison in between the groups. Animal, which is showing
control pain threshold greater than 80g is eliminated.
Neuropathic Pain Model
Male Sprague – Dawley rats are used
and anaesthetized with 4% halothane. A local incisic is given and sciatic
nerves of both legs are exposed at the level of midthigh. Chromic g sutures 4-0
are tied loosely with a square knot around the right sciatic nerve. Left scial
nerve is just mobilized. Incisions are closed layer to layer. During the next
days animal show a mild aversion of the affected paw and foot drop. The thermal
nociceptive threshold of hind paws is measured in each animal. For this, rats are
placed beneath a transpare plastic cage upon a raised glass plate such that a halogen
projector lamp could be placed below it. Lamp (source of radiant heat) is
focused at the planter area of one hind paw. As soon as heat is applied a
withdrawal response of hind paw is measured in each animal. 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 and vehicle administration. PWL which
was the maximum during the first 30 min after drug or vehicle injection is
called maximum PWL of the control side (left side) from the maximum PWL of the
affected side (right side). For evaluation of drug effects in hyperesthetic
rats, the dose is plotted against the change in DS (postdrug difference score
minus predrug difference scor).
A novel behavioural model of
neuropathic pain disorders has also been produced in rats by partial sciatic
nerve injury.
Some other models of pain include
electrical stimulation of tail and mechanical visceral pain model.
IN VITRO METHODS
Identification of several types of opioid receptors is the brain has allowed to perform
in vitro binding tests to study the action of central analgesics. Various new
receptors have been identified by using in vitro methods as therapeutic targets
for the treatment of pain, especially neuropathic and cancer pain such as
receptors for nociceptin, vasoactive intestinal peptide, cannabinoids and
vanilloid receptors. Agonists and antagonists for these receptors are being
evaluated for clinical use.
3H
– Naloxone Binding Assay
Opiate agonists and antagonists have
ability to displace radio labeled naloxone that is a potent narcotic
antagonist. 3H-Nsloxone- binding assay is developed to classify
analgesics as agonists, mixed agonist-antagonist and antagonists. The basic
principle of this assay is to determine IC50 values for 3H-Naloxone
in the presence of absence of Na+.
Procedure
Male Wistar rats are decapitated and
their brains are removed. Whole brains without cerebella are homogenized in 50
volumes of ice-cold 0.05 N tris buffer with a tissue homogenizer centrifugation
of homogenate is done at 40000g for 15min. Pellet is resuspended in buffer and
recentrifuged at 40000g. After this, the final pellet is resuspended in freshly
prepared 0.05 M tris buffer. Finally, tissue concentration in the assay becomes
10mg/ml.
In test tubes a mixture is prepared,
which consists of 310 ml H2O
20ml 5 mM dextrophan (total
binding) or 5mM levorphanol
(nonspecific binding), 50ml 2 M NaCl or
H2O, 50ml 0.05M Tris
buffer, pH7.7, 20ml drug or
vehicle, 50ml 3H-Naloxone
and 500ml tissue
suspension. The tubes are incubated for 30 min at 370c. Vacuum
filtration through Whatman GF B filters is done to stop the assay and washing
is performed at least 3 times with ice-cold 0.05 M Tris buffer, pH7.7. The
filters are then counted in 10ml of Liquiscint liquid scintillation cocktail.
Difference between binding in the presence of 0.1 mM dextrorphan
and 0.1mM levorphanol
is known as stereospecific binding.
Specific binding is around 1% of the
total added ligand and 50% of the total bound ligand in the absence of Na+
and 2% of the total added ligand and 65% of the total bound ligand in the
presence of Na+ (100mM). An increase in specific binding denotes an
increase in binding.
To evaluate analgesic activity, data
are converted into % stereospecific 3H-Naloxone binding displaced by
the test drug. Determination of IC50 is done by using computer
derived log probit analysis. IC50 is
used to calculate sodium shift. Opioid agonists show high sodium shifts,
antagonists show low shift and mixed opioid agaonists antagonists show medium
sift data are analyzed by a computer program.
Opiate Receptor Binding Assay
Opioid drugs exert their analgesic
effects mainly through m opioid
receptors.3H-dihydromorphine is highly selective for m receptors.
The compounds that inhibit binding of 3H-dihydromorphine in a
synaptic membrane preparation from rat brain can be identified by this assay.
Procedure
Male Wistar rats are used. Animals are
sacrificed by decapitation. Whole brains without cerebella are removed, weighed
and homogenized in 30 volumes of ice cold 0.05 M Tris buffer, pH 7.7.
Centrifugation of homogenate is performed at 48000g for 15 min and pellet is
resuspended in the same volume of buffer.
This homogenate is incubated to remove the endogenous opiate peptides
and centrifuged again. The final pellet is resuspended in 50 volumes of 0.05 M
Tris buffer.
In test tubes a mixture consisting of
1850 ml tissue
suspension, 80ml distilled
water, 20 ml vehicle or
levallorphan or appropriate concentration of drug and 50ml 3H-dihydromorphine
is prepared. Then incubation is performed for 30 min at 250c. The
assay is stopped by vacuum filtration through Whatman GF/B filters, which are
washed twice with 5ml of 0.05 M Tris buffer. Filters are placed into
scintillati vials with 10ml liquiscient scintillation cocktail and counted.
Specific binding is the difference between total binding and binding in the
presence of 0.1mM levallorphan. At each drug concentration, IC50
values are calculated from the percent specific binding.
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