Screening methods of antiglaucoma drugs

Sign of well Being
Department of Pharmacology, Govt. Medical College, Bhavnagar.
Screening Methods for Anti Glaucoma Drugs
Presented By:
Dr. Jhanvi J. Vaghela,
Second year resident,
Department of Pharmacology,
Government Medical College,
Bhavnagar- 364001, Gujarat (India).
Email Id- drjhanvivaghela91@gmail.com
30 June 2017

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OUTLINE
■ Introduction
■ Models for screening of antiglaucoma drugs
❖ Ocular injections
❖ Reducing serum osmolarity
❖ Application of lasers
❖ Steroids
■ References
30 June 2017

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Introduction
■ Second leading cause for blindness globally.
■ Third leading cause of blindness in India.
■ “Glaucoma is a progressive optic neuropathy characterized
by visual field changes and cupping of optic disc.”
■ Raised intraocular pressure (IOP) is one of important risk
factor for glaucoma.
■ The rise in IOP is due to
❖ ↑ aqueous formation
❖ ↓ rate of outflow
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■ IOP of normal individual is 10-20 mm Hg.
■ It can rise up to 60 mm Hg in glaucoma patients.
■ Due to raised IOP there is blockage of axonal flow of
cytoplasm from neuronal cell bodies.
■ It results in lack of nutrition of fibers and ultimately it
causes death of neurons.
■ Also compression of retinal artery increases neuronal
damage.
30 June 2017

Sign of well Being
Department of Pharmacology, Govt. Medical College, Bhavnagar.30 June 2017

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■ Studies of glaucoma are carried out in animal having close
resemblance of eye structure with human eye.
■ The methods of increasing the IOP should be
❖ Easy to carried out,
❖ Produce a reliable increase in IOP,
❖ Allow tonometric monitoring in experimental
animals,
❖ Minimize secondary ocular changes like
inflammation.
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Tonometry
Schiotz tonometer:
Eye is anesthetized and a small handy instrument is
placed on cornea and pressed lightly.
Applanation Method:
A flouresein stained strip is touched to the side of the eye.
The dye is washed out along with lacrimation. The
instrument is fitted with slit lamp. The probe of tonometer is
made to touch with cornea by moving instrument forward
Non contact Method:
The IOP is measured without contact with eye. A puff of air
is released when eye is focused which indents the eye. The
force required for indenting eye is measured.

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Ocular
injections
Reducing
serum
osmolarity
Application
of lasers
Steroids
The IOP elevated beyond limits by following methods

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Ocular Injections
■ There are reports of production of glaucoma in the
experimental animal by injection of a variety of agents.
30 June 2017
Posterior chamber injection
• Alpha chymotrypsin
Anterior chamber injection
• Methylcellulose
• Autologous ghost red blood cells,
• Kelloin,
• Prostaglandin,
• Alkali

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Alpha chymotrypsin induced experimental
glaucoma
■ It is a proteolytic enzyme secreted by pancreas.
■ Intravitreal injection of alpha chymotrypsin into owl
monkey or rabbit eye elevates IOP by 5-25 mm Hg with in
2-3 days.
■ And suggests that rise in IOP is due to predominant
changes in trabecular meshwork caused by Alpha
chymotrypsin.
■ The proteolytic action of alpha chymotrypsin on zonular
material and the debris of the tissue thus formed blocks
aqueous outflow channels and elevates the IOP.
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■ Rabbits of 1.5-2 kg under Nembutal
anesthesia
■ One of eye is anesthetized with 2%
xylocaine
■ Eyeball is fixed with forceps
■ 30G sterile needle, connected with
tubing to a 1 ml Hamilton syringe is
inserted carefully for intra vitrial
injection
■ 150 units of α chymotrypsin dissolved in
sterile saline (0.5 ml) is injected in
posterior chamber
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■ Care is taken to prevent contact of
α chymotrypsin with corneal
stroma
■ After 2 days IOP measured at
regular intervals
■ Stable rise in IOP achieved within
15 days
■ Sustain raised IOP 28-45 mm Hg is
observed till 50 weeks
■ To evaluate anti glaucoma activity
the test agent is administered
topically or orally once glaucoma is
established.
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Chronic Rat Model of Glaucoma
■ Morrison, et al. developed this model of glaucoma
■ Aqueous humor escapes eye through trabecular
meshwork and canal of schlemm’s into a vascular
plexus which connects general circulation through
episcleral veins.
■ Injecting mild sclerosants (hypertonic saline) through
one of veins resulting in scarring of trabecular
meshwork by which decreasing outflow and increasing
IOP occur.
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■ Male Brown Norway rats (Weight 300-400 gm)
■ Kept in light for 3 days
■ Anesthetized by intraperitoneal injection of 1
ml/kg solution containing 5ml ketamine, 2.5ml
xylazine, 1ml acepromazine & 0.5 ml sterile
water.
■ A volume of 50µl micro filtered 1.75M
hypertonic saline solution is injected in to
limbal vascular plexus.
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Following injection IOP measured twice a week .
Mean IOP elevation ranged from 7-28 mm Hg.

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Methylcellulose induced
glaucoma
■ Methylcellulose (0.5-4%) made in 0.9% saline,
free if air bubbles
■ Injected into anterior chamber of rabbit eyes
following evacuation of aqueous humor (150-190 µl)
■ 32g needle is inserted into anterior chamber just
above iris apparatus.
■ IOP increase from 20 to approximately 30 mm hg in
next two hours.
■ Zhu & Cai produced reliable and about 8 wks intra
ocular hypertension by series of 4 intra anterior
chamber injection of 1-2% methylcellulose in
rabbits.30 June 2017

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Autologus Ghost Red Blood Cells induced
Glaucoma
■Fixed red blood cells or fixed ghost red blood
cells (GBCs) when injected into anterior chamber
of rabbit or monkey produce chronic glaucoma.
■GBCs have relatively rigid membrane and do not
possess flexibility by which normal blood cells
exit trabecular meshwork.
■ GBCs cause trabecular obstruction in eyes with
intraocular hemorrhage resulting in secondary
glaucoma.
■IOP elevation last for 7 to 36 days in rabbit and
2-42 days in monkey.
30 June 2017

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Hyaluronic Acid Induced Glaucoma
■ Male wistar rats
■ (160-200gm weight)
■ Anesthetized with i.p. injection of
■ ketamine (50mg/kg) & xylazine (0.5mg/
kg)
■ Hyaluronic acid (25µl) injected in one eye
and contralateral eye is injected with same
volume of vehicle.
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■ Chronic intracameral injection of hyaluronic
acid in rats induced significant histological
alteration in retina and optic nerve showing
similarity with some characteristics of open
angle glaucoma.
■ Raised IOP last for 70 hrs in rabbits and
24 hrs in owl monkeys
■ Single administration of HA
injection raised IOP for 8 days.
■ weekly injection of HA induce
a sustained elevation of
intraocular pressure for 10 wks
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Reduced Serum Osmolarity
■ Animals are subjected to forced ingestion of water or
intravenous injection of 5% glucose solution or
hypertonic saline.
■ This reduces serum osmolarity and a temporary change
in the IOP observed.
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Water loaded Rabbit Model of Glaucoma
■ Albino rabbits of 1.5 -2 kg body weight,
either sex are required
■ Kept fasting overnight
■ On day of experiment anaesthetized with
30mg/kg phenobarbitone, 45 min before
experiment.
■ Maintenance dose 4 mg/kg given, 10 min
before water loading of animal
■ Baseline IOP measured
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■ Tap water 100 ml/kg administered orally
through intra gastric infant feeding tube
■ IOP measured at baseline, 15, 30, 45, 60
min after water loading
■ For evaluating anti glaucoma activity, test
drug is administered as eye drops in one
eye.
■ While vehicle is instilled in contralateral
eye (control).
■ Santafe et al. measured effect of topical
diltiazem in rabbits water loaded with
60ml/kg water
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IOP Recovery Model
30 June 2017
■ Intra venous injection of hypertonic saline in marginal ear vein
results in fall in IOP
■ It comes back normal in 2hrs.
■ If the test drug is a potential anti-glaucoma agent, it will extend the
time required for recovery of normotensive pressure.

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Perfused excised eye model system
■ Novel glaucoma therapy includes modulation of
cytoskeleton protein actin & tubulin in the trabecular
meshwork.
■ Due to induced changes in the cell shape and attachment,
cytoskeleton of aqueous outflow pathway cells can
influence aqueous humor outflow
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Application of Lasers
■ Chronic experimental models are produced using laser
treatment.
■ Researcher have successfully induced glaucoma in
monkeys, rats and mice.
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Laser induced glaucoma in monkeys
■ Adult cynomologus monkey with normal
anterior chamber, normal IOP & normal optic
disk is selected.
■ Anaesthetized with i.m. injection of ketamine
(9 mg/kg) & i.v. injection of phenobarbitone
(11 mg/kg)
■ Placed in front of slit lamp of argon laser
delivery system.
■ Eyes are treated with Oxybuprocaine &
200-400 circumferencial laser burn made with
small gonioscopic lens aiming middle of
meshwork.
■ Beam diameter 50-100 µm for 0.1-0.5 second
at 600-800 mW.
30 June 2017

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■ They are treated same every 1-2
weeks.
■ Once stable IOP obtained test
drug is instilled and compared
with control.
■ Serle et al. carried out
c o m p a r a t i v e s t u d y o f
latanoprost & isopropyl
unoprost by this method.
30 June 2017

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Laser induced glaucoma in Rats
■ Wistar rats 350-450 gm anaesthetized
w i t h k e t a m i n e + x y l a z i n e +
acepromazine.
■ Blue green argon laser treatment given
on episcleral veins within 0.5-0.8 mm
from limbus.
■ Treatment is given in two parts with gap
of 1 week.
■ Amount of energy used is 1 W for 0.2
sec for delivering 130-150 spots.
■ IOP measured with tonometer before &
after treatment.
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■ IOP value raises twelve folds from
baseline by laser treatment and it is
maintained for two months.
■ Drug treatment is given either at time of
or 10 days after IOP elevation.
■ Unilateral experimental glaucoma in
wistar rats is also induced using a diod
laser (532 nm wave length) aimed at
trabecular meshwork.
■ Laser induced experimental glaucoma is
induced in rats after intra cameral
injection of india ink

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Laser induced glaucoma in mouse
■ Alihara et al. established this mouse model of
laser induced glaucoma.
■ 30 min before one eye is dilated with topical
administration of 4 µl of mixture of atropine,
tropicamide, phenylephrin & cyclopentolate
■ Mouse is anesthetized and placed under
stereomicroscope.
■ Fabricated micro needle connected to 1 µl
syringe was mounted on platform of a bio-
microscope with diod laser system.
■ Laser beam 532 nm applied to corneal
limbus. (power 100mW, duration 0.05 sec,
spot size 200µm)
30 June 2017
Laser photocoagulation with flattening of
anterior chamber successfully induces 30%
elevation in IOP for at least 6 weeks.

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Steroid glaucoma
30 June 2017
■ Experimental glaucoma induced by
steroids is reported in young rabbits and
cats.
■ Dexamethasone sodium phosphate
injection diluted to desire concentration
of 0.5 or 1% with normal saline.
■ This solution applied over cornea in a
volume of 10 µl 2-3 times a day.
■ A gradual rise in IOP observed which
become significant in 2-3 weeks.
■ Prednisolone acetate 1% also used
instead of dexamethasone

Sign of well Being
■ Ocular hypertension model in rats generated by instillation
of topical dexamethasone to rat eye 4 times daily for
1,2&4weeks
■ Galassi et al.(2006) induce glaucoma by
administration of 1 % dexamethasone drops in rabbit
eye.
■ Bonomi et al. administered weekly subconjunctival
injection of 4 mg repository betamethasone
suspension in rabbits for three weeks and produce
sustained increase in IOP in 96% animals.
■ Haster et al. studied effect of three subconjunctival
injection of steroids betamethasone, cortisone &
triamcinolone in rabbits. And found elevation of IOP by
all three drugs & most consistent elevation with
triamcinolone.
30 June 2017

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Autoimmune Glaucoma
30 June 2017
Autoimmune response possibly having role in RGC degeneration in
normal pressure glaucoma.
Serum samples of glaucomatous patients have found to have increased
levels of heat shock protein 27 (HSP 27) & 60 (HSP 60).
An in vivo model was established to elicit the auto immune response
through immunization with HSPs.
HSP 27 & HSP 60 immunization in lewis rats induce RGC degeneration &
axonal loss 1-4 months later in pattern similar to human glaucoma.

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Genetic Models
■ Family history and
genetic factors have
an important role in
glaucoma as it is
progressive disease
and affects elderly.
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DBA/2J Mouse Model
■ This is genetic mouse model of glaucoma charecterised
by chang et al. (1999) in which IOP increase with age due
to pigment dispersion from iris & obstruction of
trabecular meshwork.
■ They spontaneously develop complex ocular
abnormalities such as glaucomatous loss of retinal
ganglion cells.
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Buphthalmic Rabbits, JWHR bu/bu
■ Inoue et al. evaluated effect of topical CS-088, an
angiotensin (AT) 1 receptor antagonist, on IOP in
hereditary ocular hypertensive rabbits.
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Human Trabecular Meshwork (HTM)
Culture
■ Human donor eyes enucleated
within 48-96 hours of death.
■ Tr a b e c u l a r m e s h w o r k i s
separated and cells are cultured at
37˚C in a 7% CO2 medium .
■ Cells are placed onto glass cover
slips coated with 2% sterile
gelatin solution.
■ On reaching confluence, the
cells treated with test drug
30 June 2017

Sign of well Being
Department of Pharmacology, Govt. Medical College Bhavnagar.
References
1. Vogel HG, editor. Drug discovery and evaluation: pharmacological assays.
Springer Science & Business Media; 2002 Jun 13.
2. Tripathi KD. Essentials of medical pharmacology. JP Medical Ltd; 2013 Sep
30.
3. Gupta SK. Drug screening methods. Jaypee Brothers; 2004.
4. Zhu MD, Cai FY. Development of experimental chronic intraocular
hypertension in the rabbit. Australian and New Zealand journal of
ophthalmology. 1992 Aug 1;20(3):225-34.
5. Melena J, Santafé J, Segarra J. Betamethasone-induced ocular hypertension in
rabbits. Methods and findings in experimental and clinical pharmacology.
1997 Oct;19(8):553-8.
6. Anderson DR. Experimental alpha chymotrypsin glaucoma studied by
scanning electron microscopy. American journal of ophthalmology. 1971 Feb
1;71(2):470-6.
7. Quigley HA, Addicks EM. Chronic experimental glaucoma in primates. II.
Effect of extended intraocular pressure elevation on optic nerve head and
axonal transport. Investigative ophthalmology & visual science. 1980 Feb
1;19(2):137-52.
6/30/2017 $41

Sign of well Being
Department of Pharmacology, Govt. Medical College,
Bhavnagar19/03/2016 $42Government Medical College, Bhavnagar
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