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Evaluating Drugs for Glaucoma and Cataract
1. MALIBA PHARMACY COLLEGE
Guided
By:-
Dr. Bhavin
Evaluation Of drugs used in
Glaucoma &
Cataract
Prepared By:-
Hiren Monapara
2nd Sem. M.Pharm
Enrollment No:- 1
4. GLAUCOMA
Glaucoma is an eye
disease of damage to
optic nerve which carries
information from the eye
to the brain.
It is second Leading
cause of blindness in the
world.
This usually occurs in the
presence of high
intraocular pressure.
NORMAL
VISION
Vision as it might be
affected by glaucoma
4
5. PHYSIOLOGY OF GLAUCOMA
Clear liquid called aqueous humor circulates inside the front portion of
the eye.
To maintain a healthy level of pressure within the eye, a small amount
of aqueous humor is produced constantly, while an equal amount flows
out of the eye through a microscopic drainage system—the trabecular
meshwork.
5
6. An increased IOP
reading indicates a
problem with the amount
of aqueous humor
(fluid) in the eye: either
the eye is producing
too much, or it's not
draining properly.
This increase in
intraocular pressure
cause damage to the
optic nerve and leading
to vision loss.
6
7. TYPES OF GLAUCOMA
Open-angle glaucoma: the
most common form of glaucoma.
It is often called "the silent thief of
sight" because you have no
warning sign, no hint that anything
is wrong.
Glaucoma gradually reduces your
peripheral vision, but by the time
you notice it, permanent damage
has done.
Closed-angle glaucoma: a less
common and more urgent form of
glaucoma.
It is a medical emergency. Optic
nerve damage and vision loss will
occur within hours.
Two main categories of
glaucoma:
7
9. 1.ALPHA-CHYMOTRYPSIN INDUCED GLAUCOMA
Rationale:
Alpha chymotrypsin is a proteolytic enzyme secreted
by pancreas.
Intravitreal injection of alpha chymotrypsin into rabbits
elevates the ocular pressure by 5 to more than 25
mmHg with in 2 to 3 days.
That raise in IOP is due to predominant change 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 the aqueous outflow channels and
elevates the IOP.
9
10. PROCEDURE:
Male New Zealand rabbits weighing 2-3 kg are used.
The animal are lightly anaesthetised with pentobarbitone
to avoid any nystagmus and treated with 10 mg/kg of
indomethacin intraperitoneally to prevent the onset of any
inflammatory reaction.
The right eye is anasthetised by topical application of 2%
lidocaine.
The anterior chamber is cannulated with a 30 gauge
needle attached to a reservoir set at a pressure of 25
mmHg.
10
11. Then a second cannula,32-gauge, is introduced into
the anterior chamber near the limbus and directed to
the posterior chamber through the pupil.
A sterile isotonic saline solution (0.5 ml) containing
150 units of alpha-chymotrypsin is irrigated through
the cannula into the posterior chamber.
Both cannulae are then removed without significant
loss of aqueous humor.
11
12. The eyes are examined at daily intervals for the first week, then
on alternate days for the second week, and then weekly for the
duration of the experiments.
Intraocular pressure is measured with a tonometer adapted for
rabbit eyes.
Treatment with drugs is performed after alpha chymotrypsin
injection.
EVALUATION:
Intraocular pressure of animals treated with alpha-chymotrypsin
and those treated additionally with drugs is expressed as mean
±SEM and compared statistically with untreated controls using
ANOVA followed by Student’s t-test.
12
13. 2.METHYL CELLULOSE INDUCED GLAUCOMA:
Rationale:
Glaucoma can be induced by injecting 0.5-4% methylcellulose in
the anterior chamber of Rabbits
The particulate matter of methylcellulose which may obstruct the
trabecular mesh-work, this account for its potential for elevated
IOP.
Procedure:
0.5% methylcellulose made in 0.9% saline, free of air bubbles is
injected into the anterior chamber of right rabbit eyes following
evacuation of aqueous humor.Which serve as control.
The left eye is additionally treated with test drug, which serve as
test.
The needle is inserted into anterior chamber just above the
aperture of iris and aqueous humor is evacuated slowly.
13
14. The volume of the methylcellulose is equal to the
volume of aqueous humor removed ( 150 µl to 190
µl).
Same needle of 0.032 cm diameter is used for both
evacuation and refilling along with three-way
stopcock
In this model the IOP increase from 20 to
approximately 30 mmHg in next 2 hours.
Evaluation:
The IOP of both eye is compared to evaluate efficacy
of test drug.
14
15. 3.PHOTOCOAGULATION INDUCED GLAUCOMA IN
MONKEY:
Purpose and Rationale:
Lee etal.(1985), Woodward etal. (2003) used
a method originally described by Gaasterland
and Kupfer (1974) for the production of
sustained, elevated IOP in the rhesus
monkey by repeated, circumferential argon
laser photocoagulation of the mid-trabecular
meshwork.
15
16. PROCEDURE:
Rhesus monkeys are anaesthetized with systemic
pentobarbital and placed at the slit lamp delivery
system of the argon laser.
Both eye are treated with tropical proparacaine 0.5%
and photocoagulated using a modified koeppe-type
gonioscopy lens with hydroxy propyl methyl cellulose
as the filling fluid.
Approximately 200 laser applications are made to
each eye, aimed at the middle of the trabecular
meshwork,using 50 µbeam diameter, 0.2 to 0.5
second duration and 0.4 to 0.8 watts of power.
16
17. Drugs are applied locally as solution with a drop size
of 50 microL.
IOP is recorded in 6- hour diurnal curves at 9:30
am, 10:00 am, 10:30 am, 11:30 am, 12:30 pm, 1:30
pm, 2:30pm, 3:30 pm.
The baseline diurnal curve serves as control and is
made 1 or 2 days before the experimental diurnal
curve.
On the experimental day, baseline IOP is measured
at 9:30 am.
The test drugs are administered to both eye of the
monkey immediately after the 9:30
am, measurement. 17
18. EVALUATION:
The IOP on the treated day are compared to
that of the control day.
Alternatively, the differences in IOP at
intervals after therapy between the treated
and control day measerments are compared
to the initial (0 hour) differences between the
treated and control day values.
18
20. 1.APPLANTATION PNEUMOTONOMETRY:
Applanation tonometry measures intraocular
pressure either by the force required to
flatten a constant area of the cornea or by
the area flattened by a constant force.
20
21. PROCEDURE:
New Zealand white rabbits, weighing 2.0–2.5 kg, are
acclimated to the laboratory environment at least one
day prior to experimentation.
Intraocular pressure (IOP) measurements consist of
topically administering one drop (approximately 50 μl)
of a short-acting local anesthetic (Ophthaine 0.5%)
directly on the eyes of an unrestrained rabbit.
Approximately 40–45 s following local anesthetic
administration, an IOP reading for each eye is taken
for 10 s and recorded as mm Hg. IOP is determined
by applanation pneumotonometry.
21
22. On the day of the experiment, two baseline IOP
measurements are determined and designated
as P1 and P2, respectively.
The P1 measurement is recorded 30 min prior
to drug administration while the P2
measurement occurs just prior drug or placebo
administration.
The P2 measurement is designated as IOP at
zero time and is used as the baseline control
value for determinationof percent reduction in
pressure. 22
23. Immediately following the P2 measurement, the
appropriate concentration of drug or vehicle is
administered topically to the right eye of 5 rabbits.
This is done by placing three successive 20 μl
additions directly onto the eye at two- or three-minute
intervals.
. Simultaneously, the left eye receives vehicle with
the same regimen as the drug-treated right eye.
The vehicle (control) treatment is to monitor potential
contralateral ocular effects. Following drug or vehicle
treatment, IOP measurement are taken at the
following time periods: 0.5; 1.0; 1.5; 2.0; 3.0; 4.0; 5.0
and 6.0 h. 23
24. EVALUATION
The IOP of each rabbit eye for each time period is initially recorded in
mm Hg.
However, to account for initial IOP differences among rabbits, the
changes in mm Hg are normalized by converting to percent reduction in
outflow pressure (ROP).The ROP is calculated according to an equation
proposed by Mishima (1981):
% ROP = (Po – Pe / Po – Ep) × 100
Where,
Po - the IOP reading in mm Hg at zero time,
Pe - the experimental IOP reading at the appropriatetime following drug
or administration
Ep - the episcleral venous pressure taken as a constant of 10 mm Hg.
24
25. The percent ROP for each rabbit at each time period is
recorded and grouped to determine a mean percent ROP
for each time period as well as theduration of action.
The overall activity of the drug based on the mean
percent ROP for five rabbits is categorized as follows:
% reduction in outflow
pressure
• 0-10.0
• 10.1-20.0
• 20.1-40.0
• >40.0
Activity
• None
• Slight
• Moderate
• Marked
25
26. 2.TELEMETRY:
In this method pressure transducers are implanted in the eye of rabbits
weighing 3 to 3.5 kg using aseptic conditions under anesthesia induced
by 50 mg/kg of ketamine.
This transducers are a part of implantable pressure monitors which are
cylindrical, measuring 25 mm in length and 15 mm in diameter and
weight approximately 9 g.
They contain a pressure transducer, a short-range amplitude modulation
radiotransmitter, and a battery in a biocompatible case.
Pressure is conducted to the transducer through a fluid-filled
polyurethane catheter that is 15 cm long and 0.7 mm in diameter.
The 3-mm distal tip of the catheter couples the transducer to the
surrounding fluid; it is modified with a thin flexible wall, and the end is
filled with a biocompatible gel.
The thin wall conducts transient pressure changes, whereas the gel
prevents mixing of the fluid in the catheter with the surrounding
biological fluid and conducts slow pressure changes.
26
29. CATARACT:
Cataract is the clouding of
lens inside the eye which leads
to a decrease in vision.
Cataract is main leading cause of
blindness.
Globally, approximately 42% of
blindness is due to cataract.
Several factors like age, genes,
diabetes, malnutrition, myopia,
renal failure, hypertension,
sunlight exposure, smoking,
steroid, etc. are responsible for
the development of cataract.
NORMAL
VISION
Vision as it might
be affected by 29
30. When light passes through the
pupil, it is focused by the lens
to produce clear, sharp
images on the retina, the light-
sensitive membrane on the
back of the eye that functions
like the film of a camera.
When this arrangement is
disturbed in any way, the
transparency is lost .
This results in scattering of
light, blurring, and blocking of
the image.
30
31. PHYSIOLOGY OF CATARACT
The lens is made mostly of water and protein fibers
The protein fibers are arranged in a precise manner
that makes the lens clear and allows light to pass
through without interference
With aging, the composition of the lens undergoes
changes and the structure of the protein fibers
breaks down
Some of the fibers begin to clump
together, clouding areas of the lens, and leading to
the loss of transparency
31
32. EXPERIMENTAL MODEL
In Vivo:
1. Selenite- induced cataract.
2. Galactose induced cataract.
3. Streptozotocin cataract.
4. Naphthalene induced cataract.
In Vitro:
1. Selenite- induced cataract in incubated rat lenses.
2. Hydrogen Peroxide induced cataract in incubated lenses.
3. Steroid induced cataract in incubated lenses.
4. Naphthalene induced cataract in incubated lenses.
5. Ca+² induced cataract in incubated lenses.
32
33. 1.SELENITE- INDUCED CATARACT:
Rationale:
Altered epithelial metabolism
(oxidises critical sulfhydryl groups)
inactivation of Ca+²–ATPase or opening of ion channels in the
epithelial membranes
Calcium accumulation
Activation of m-calpain (calpain II)
Degrades Crystallins
Insolubilization of proteolyzed ß- and α-
crystallins, co-precipitation of γ-crystallins
Light scatter and cataract
33
34. Procedure:
Selenite cataract is usually produced by a single subcutaneous injection of 19-
30 µmoles/kg body weight of sodium selenite (Na2SeO3) into suckling rats of 10-
14 days of age.
Young rats are housed together with their mother.
Mother is fed normal diet and water ad libitum and she suckles the pups.
The young rats of same age are grouped into three.
One group is left as normal group. Rest of the two groups, control and treated
are given subcutaneous injections of sodium selenite in the scruff of the
neck/abdomen of young rat.
Treatment group is given the anticataract agent intraperitoneally four hours prior
to the sodium selenite administration.
Cataracts in the sucklings are visible to the naked eye after they open their eye
on 15th or 16th day of life.
The opacity appears yellowish white and is localized in the center of the lens,
hence called the neclear cataract.
34
35. Evaluation:
The eye of the treated group can be compared with
the control group to evaluate the efficacy of the drug.
35
36. 2.GALACTOSE INDUCED CATARACT:
Rationale:
The galactose-induced cataract is the activation of
the polyol pathway, with conversion of galactose into
galactitol by aldose reductase.
Because the cellular membranes of the lens are
impermeable to galactitol.
They accumulate to high levels and produce osmotic
effects. Increase in intralenticular tonicity draws water
into the lens fibres causing them to swell.
which produces scattering of light and diminished
lens transparency.
36
37. Procedure:
Rats of either sex, weighing 50 to 60 g are fed 30%
galactose in diet.
Diet and water are given ad libitum.
The rats are divided into two groups, control and treated.
The test agent is administered orally or topically in the
treatment group.
The eye of the rats are examined weekly by using a slit
lamp to see the cataractogenic changes.
The vacuoles start appearing at the lens within a week’s
period,the vacuoles gradually increase in number and
size. This takes about 14 days.All these changes are not
visible through unaided eye.
During the third week, opalescence of the lens is visible
and it becomes totally opaque in 30 days time. 37
38. Normal: clear lens;
stage I: peripheral opacity in the
lens;
stage II: irregular peripheral opacity
and slight involvement of the lens;
stage III: irregular opacity involving
entire lens;
stage IV: pronounced opacity.
Evaluation:
Morphological changes in the lens are compared in all the groups to
evaluate the efficacy of the drug.
•Different stages of cataract are graded as given below.
38
39. 3.STREPTOZOTOCIN CATARACT:
Rationale:
Diabetes related cataractogenic changes are seen in the animal
injected with streptozocin.
The chemical structure of streptozocin(STZ) has a glucose
molecule with a highly reactive nitrosourea side chain, which
supposedly initiates its cytotoxic action.
The glucose moiety directs this agent to the pancreatic β cells.
There it binds to β cells demage it, this alter the sugar metabolism
leading to diabetes.
The enzyme aldose reductase (AR) catalyzes the reduction of
glucose to sorbitol.
It has been shown that the intracellular accumulation of sorbitol
leads to osmotic changes resulting in hydropic lens fibers that
degenerate and form sugar cataracts.
39
40. Procedure:
Albino rats of 150 – 200 gm body weight are used.
Diabetes is induced by intraperitoneal injection of
streptozocin 50 to 70 mg/kg body weight.
Streptozocin is dissolved in 0.02 M sodium citrate
buffer. The solution is filtered through 0.22 millipore
filter into a sterilized container placed on ice. The
solution thus prepared is used within 10 min of
preparation.
The test drugs can be either administered
systemically or applied twice daily as eye drops to
test group.
Progression of cataract stages is observed through a
slit lamp. 40
42. 4.NAPHTHALENE INDUCED CATARACT
Rationale:
Because the morphology as well as the toxic manifestations of
naphthalene-induced cataract is reported to be similar to that of age-
related cataract, naphthalene cataractogenesis in rats has been used as
a valuable animal model to study the etiology of agerelated cataract in
humans.
Ingested naphthalene is metabolized First in liver to an epoxide and then
is converted into naphthalene dihydrodiol.
This stable compound on reaching the eye gets converted enzymatically
to dihydroxynaphthalene.
Being unstable at physiological pH 1,2-dihydroxynaphthalene
spontaneously autoxidises to 1,2-naphthoquinone.
Which has ability quickly react with glutathione, protein sulfhydryl
groups, amino acids and causes its alkylation.
This lead to precipitation of high molecular weight protein, hence cause
opalescence in the lens.
42
43. Procedure:
The Wistar rats weighing between 180-200 g were
randomly divided into two groups of six each.
Naphthalene is dissolved in warm paraffin oil in a
conc. of 10g/100ml and administered orally in a dose
of 1g/kg every second day for 6 weeks.
Test compound may be administered orally every day
or applied as eye drops 1-4 times daily to test group.
On 42nd day cataract was examined under slit lamp.
On the 43rd day lenses will be removed from the
eyes of all the animals.
43
44. Evaluation:
Lenses are estimated for lens
glutathione, lens soluble protein and the lens
water content.
The value of treated animal are compared
with those of control.
44
45. REFERENCES:
H. Gerhard Vogel. Drug Discovery and Evaluation. Second Edition:1295-
301
SK Gupta. Drug Screening Methods, First Edition,2004:347-55, 333-44
N.S.Parmar, Shiv Prakash. Screening Methods in Pharmacology:313-
16, 316-20
Kiritsinh R Vaghela. DEPARTMENT OF PHARMACOLOGY AND
TOXICOLOGY,S.K. PATEL COLLEGE OF PHARMACEUTICAL EDUCATION
AND RESEARCH, KHERVA -382711.GUJARAT, INDIA. OCTOBER, 2005.
45
46. Thomas R. Shearer,1* Hong Ma,1 Chiho Fukiage,2 and Mitsuyoshi Azuma2. Selenite
Nuclear Cataract: Review of the Model. Molecular Vision 3: 8, 1997
Joe Prasad Mathew, V.C.Thomas and Issac Thomas. Protein profiles in the pre-
cataractous and cataractous stages of selenite cataract in rats. Indian J. Environ. Toxicol.
10(1) : 26-29, 2000
Jasmina B. Mackic, Fred N. Ross-Cisnero. Galactose-Induced Cataract Formation in
Guinea Pigs:Morphologic Changes and Accumulation of Galactitol.
Andreas Pollreisz and Ursula Schmidt-Erfurth. Diabetic Cataract—
Pathogenesis, Epidemiology and Treatment, Journal of
Ophthalmology.Volume 2010 (2010), Article ID 608751,
doi:10.1155/2010/608751
Kothadia AD1, Shenoy AM1*, Shabaraya AR2, Rajan MS1, Viradia UM1, Patel NH1.
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