IJRPC 2017, 7(2), 162-170 Jyothirmai et al.

ISSN: 22312781

INTERNATIONAL JOURNAL OF RESEARCH IN PHARMACY AND CHEMISTRY

Available online at www.ijrpc.com Research Article

FORMULATION AND EVALUATION OF OCULAR

INSITU HYDROGELS OF ACYCLOVIR
KSL. Jyothirmai*, M. Manasa, D. Sravani, GT. Sundari devi,
M. Mounika, N. Santhi Priya, M. Santhosh Aruna and N. Rama Rao
Department of Pharmaceutics, Chalapathi Institute of Pharmaceutical Sciences,
Lam, Guntur, Andhra Pradesh, India.

ABSTRACT
The present research work deals with the formulation and evaluation of in-situ gelling system
based on sol-to-gel transition for ophthalmic delivery of an antiviral agent acyclovir , to overcome the
problems of poor bioavailability and therapeutic response exhibited by conventional formulations.
Aciclovir an antiviral agent preferentially used in the treatment of infections caused by herpes
simplex virus, which is the main cause of viral conjunctivitis.so in the present study ocular insitu
hydogels of acyclovir were prepared to treat viral conjunctivitis.Carbopol 940 was used as the gelling
agent in combination with HPMCK100M which acted as a viscosity enhancing agent.Poly vinyl
alcohol was used as crosslinker. The prepared formulations were evaluated for pH, viscosity, clarity,
drug content, gelling capacity, Invitro drug release, Exvivo diffusion studies, antimicrobial efficacy
,ocular irritancy, The clarity, pH, and drug content of the developed formulations were found in range
6.0-6.8, , 82-95% respectively. The gel provided sustained drug release over an 6 hour period. The
developed formulation can be used as an in-situ gelling vehicle to enhance ocular bioavailability and
helped in the reduction in the frequency of instillation thereby resulting in better patient compliance.

Keywords: In-situ gelation; acyclovir; Carbopol 940; HPMC K100M, HPMCK4M ,HPMC E50LV.

INTRODUCTION polymers is the approach to overcome the

Ophthalmic drug delivery is one of the most drawbacks of conventional eye preparations.
attractive and challenging field facing the Acyclovir is preferentially taken up by virus
pharmaceutical scientist. A significant infected cells because of selective generation
challenge to the formulator is to circumvent the of active inhibitor in the virus infected cell and
(4).
protective barriers of the eye without causing its inhibitory effect on viral DNA synthesis
(8).
permanent tissue damage Most of the Carbopols are mainly used in liquid or
ocular treatments call for the topi-cal semisolid pharmaceutical formulations as
administration of ophthalmically active drugs to suspending or viscosity increasing agents.
(5) .
the tissues around the ocular cavity The Formulations containing carbopol include
most conventional ocular dosage forms for the creams, gels and ointments for use in
delivery of drugs are eye drops (solution, ophthalmic, rectal and topical preparations.
suspension) and ophthalmic ointments. Short HPMC is widely used in oral and top-ical
residence time, pulsed dosing of drug, pharmaceutical preparations as coating agent,
frequent in-siltation, and large drainage factor film formers, rate controlling polymers for
are the limitation associated with conventional sustained release, stabilizing agents,
ocular do-sage form. Newer ocular drug viscosifier
delivery systems are being explored to The aim of present study is to formulate and
develop extended duration and controlled evaluate ph triggered in situ ophthalmic gel
release strategy. forming solution of acyclovir by using
Formulation of in-situ ocular gel of acyclovir’s combination of hydroxyl propyl methyl
1.
an antiviral drug was to treat viral cellulose HPMC K4M K100M , E50LV HPMC
conjunctivitis of the eye, using biodegradable was used as viscosifying agent and

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carbopol940 as gelling agent. To achieve the PROCEDURE FOR THE PREPARATION OF

objective, independent formulation variables IN SITU GELLING SYSTEMS
2
such as polymer to polymer ratio and different A 3 full factorial design was adopted to
viscosity grades of HPMC (K4M K100M optimize the variables and 9 experiments were
E50LV) were examined to identify the best conducted in total. In this design, two factors
(7).
formulation were using three square full were evaluated each at 4 levels The
factorial design. polymer-to-polymer (HPMC, HPMC K15M)
(X1) and the amount of bioadhesive polymer
MATERIALS AND METHODS (Carbopol 940) (X) were chosen as
Acyclovir was obtained as a gift sample from independent variables and Y as de-pendent
hetero pharmaceuticals pvt. ltd.,. variables (viscosity, drug content, and in-vitro
hydroxypropylmethyl cellulose (HPMC) and drug release). The levels of independent
HPMC k15m were obtained from colorocon variables are shown in Table 1.
asia pvt. ltd, Mumbai, and CARBOPOL 940 The dispersions of HPMC and carbopol were
was obtained from sd fine pvt. ltd., Mumbai, prepared .Carbopol940 was sprinkled and
India. all other chemicals/reagents used were allowed to hydrate over night the solution was
of analytical grade, available commercially and stirred under an overhead stirrer and buffer
used as such without further processing. a salts were added to the above solution.
uv/vis spectrophotometer (systronics, double Polyvinyl alcohol was added to the above
beam uv-vis spectrophotometer) was used for polymer solution . Acyclovir was added to the
drug analysis. polymer solution .Purified water was then
added to make up the volume to 100ml and
the prepared formulations was sterilized 120°C
for 15 min.

Table 1: Factorial design of insitu

gel forming systems
CARBOPLO 940 HPMCK100M HPMCE50LV
1 1 1
1 2 1
1 3 1

Table 2: Formulation table of insitu hydrogels

POLYVINLY
CARBOPOL HPMC HPMCE50LV
FORMULATIONS DRUG (g) HPMC K4M(g) ALCOHOL
940(g) K100M(g) (g)
(PVA)
F1 0.5 0.1 0.1 - - 1ml
F2 0.5 0.1 0.2 - - 1ml
F3 0.5 0.1 0.3 0.2 - 1ml
F4 0.5 0.3 - 0.1 - 3ml
F5 0.5 0.3 - 0.2 0.2 3ml
F6 0.5 0.3 - 0.3 0.3 3ml
F7 0.5 0.5 - 0.1 0.2 5ml
F8 0.5 0.5 - 0.2 0.4 5ml
F9 0.5 0.5 - 0.3 0.6 5ml

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EVALUATION STUDIES volume of the receptor medium. The aliquots

PHYSICAL APPEARANCE were diluted with receptor medium and
The physical appearance of formulations was absorbance was measured at 275 nm %
observed visually under clariy test apparatus. cumulative release was calculated for all the
The clarity of formulations was observed formulations.
against white and black back ground
respectively . STABILITY STUDIES
The 30 days stability studies were carried out
pH DETERMINATION for optimized formulations. sterile gel forming
0.3g of gel was dissolved in 100ml of distilled ophthalmic solution were filed in glass vials
water and ph was measured using pHmeter closed with gray butyl rubber closures and
sealed with aluminum caps the formulation
DETERMINATION OF VISCOSITY vials kept in stability chamber maintained at
Viscosity of instilled formulation is an important 40 2 C and relative humidity 75 5% RH for
factor in determining residence time of drug in one month samples are with drawn at
the eye. Viscosity of samples was determines 0,7,15,30 days interval and evaluated for drug
using Brookfield digital viscometer with content, pH, appearance, clarity .
spindle3 at an angular velocity run from 10 to
100 rpm per minute. EX VIVO DIFFUSION STUDY
Ex vivo permeation study was carried out
GELLING CAPACITY using franz diffusion chamber and goat
Determination of invitro gelling capacity was corneal membrane used to separate donor
done by visual method. Colored solutions and receptor compartment the whole eyeball
(1%amaranth solution in water) of in situ gel of goat were procured from a slaughter house
forming drug delivery systems were prepared. and carried to laboratory in cold condition in
The in vitro gelling capacity of prepared normal saline maintained at 4 c. The corneal
formulation was measured by placing 5ml of membrane was washed and placed in pH 7.4
artificial tear fluid in a glass test tube phosphate buffer and then it was mounted on
maintained at 37 1 c temperature .1ml of by sand-witching between the clamped donor
colored solution was added with the help and receptor compartment prior to application
pipette as the solution comes in contact with of formulations the membrane was allowed to
gelation solution, it was immediately converted equilibrate for 30 minutes. Accurately weighed
in to a stiff gel like structure the invitro gelling 1ml of gel was spread uniformly on corneal
capacity was graded in three categories on the membrane which was in contact receptor
basis of gelation time and time period for medium. The receptor compartment was filled
which the formed gel as remained as such. with phosphate buffer at ph 7.4 at 37 0.5 c
+ gelation slowly and dissolve . and stirred it continuously at 20 rpm to
++ gelation immediate and remains for few simulate blinking action of eye lids whole
hours assembly adjusted to magnetic stirrer and pre
+++ gelation immediate and remained for determined intervals (30min,1hr, 2hrs up to
extended period of time 6hrs). 1ml sample was with drawn from
receptor compartment replacing the sampled
IN VITRO RELEASE STUDIES volume with phosphate buffer solution pH7.4
The in-vitro release of aciclovir from the after each sampling for a period of 6hrs the
prepared formulations was studied through samples withdrawn were analyzed by UV-
cellophane membrane using a modified USP visible spectrophotometer at 275nm
XXIII dissolution testing apparatus. The
dissolution medium used was pH 7.4 buffer. DRUG CONTENT
Cellophane membrane was previously soaked Drug content was determined by taking 1ml of
overnight in the dissolution medium and was formulation and diluting it to 100ml distilled
tied to one end of a specifically designed glass water aliquot of 5ml was with drawn and
cylinder (open at both ends of 5 cm diameter). diluted to 25ml with distilled water .Acyclovir
A 2ml volume of the formulation was concentration was determined at 275nm by uv
accurately pipetted into this assembly. The visible spectrophotometer .
cylinder was attached to the metallic drive
shaft and suspended in 100ml of dissolution STERILITY STUDIES
medium maintained at 37±1°C so that the ANTI MICROBIAL ACTIVITY
membrane just touched the receptor medium Antimicrobial efficiency studies were carried
surface. The shafts was rotated at 50 rpm/min. out to ascertain the biological activity of sol-to-
Aliquots each of 1ml volume, were withdrawn gel systems against micro organisms .This
at hourly intervals and replaced by an equal was determined in agar diffusion medium

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employing cup plate technique. Sterile (viscosity and gelation capacity) nine
solutions of marketed acyclovir eye ointment formulations (F1,F2,F3, F4, F5, F6, F7, F8 and
was used as standard. The standard and F9) were selected and evaluated for drug
developed test solutions were taken into content, and in-vitro dissolution . The drug
separate cups bored into sterile nutrient agar content of all the formulations was in range
medium previously seeded with organisms (82-95%).
pseudomonas aeruginosa. After allowing the The Exvivo diffussion studies were carried
diffusion of solutions for 2hrs, plates were out for optimizing the in situ gelling systems,
incubated for 24hrs at 37 c .The zone of namely F4, F5,F6, F1, F2, F3,F4 ,F5,F6,F7,F8
inhibition was compared with that of standard and F9. The release profiles are shown in the
.each samples were tested . figure 7.8 . FormulationF3, F4 and F5
containing only Carbopol940 released around
OCULAR IRRITATION STUDIES(DRAIZE 80% of the drug in the first 3-4 hrs. Hence it
TEST) was necessary to use additional release
Albino rabbit are used as test species one eye retardant to obtain the desired release profile.
is designated the test eye was done under the So further studies were carried out using
guidance of institutional animal ethics Methocel (HPMC K 4M , K 100M , E50 LV as
commitee.The contra lateral eye severs as a a release retarding polymers.
matched control and is usually left untreated Anti microbial studies revealed that zone of
.Single drop approximately 0.04ml is instilled inhibition(ZOI) was not observed in all
in to lower conjunctival cul-de-sac; normal formulations .Hence formulations were treated
blinking is allowed ,although the eyelids can as sterile preparations.The results of the
be held together for several seconds after ocular irritation studies indicate that the
instillation .observations was done at developed formulation was non-irritant.
1,24,48,72 hours one week after exposure. Excellent ocular tolerance was noted. No
Ocular changes was graded by a scoring ocular or abnormal clinical signs to the cornea,
system that includes rating any alterations to iris or conjunctiva were visible.The formulated
the eyelids, conjunctiva, cornea, and iris. insitu gelling system F1,F2,F3, F4,F5,
F6,F6,F7,F8, F9 batch were subjected to
RESULTS AND DISCUSSION stability study which indicates No significant
Physical appearance of the formulations were change in physical parameters and drug
light white in color and clear. The pH value of content of the final formulation stored at
all the prepared formulations ranged from 6.0 stressed condition (40ºC with 75% relative
to 6.8, which is considered acceptable to avoid humidity) after one month. Therefore it can be
the risk of irritation upon application to the eye. concluded that drug is stable in final
The two main fundamentals of gelling system formulation at room temperature and at 40ºC
are viscosity and gelling capacity. The for one month.
viscosity of the different formulations was
compared as shown in Table 4. The viscosity CONCLUSION
was directly dependent on indicated that the Acyclovir is a antiviral drug used in the
viscosity increased with increase in treatment of herpes infections of the eye was
concentration of HPMC K15M and carbopol successfully formulated as an ion activated in
940 (1 to 4%). F7, showed the maximum situ gel forming ophthalmic solution using
viscosity whereas the minimum viscosity at sodium alginate in combination with HPMC as
100 rpm was shown by F1. Except for the a viscosity enhancer . Acyclovir entrapped in
formulations F1, F2, F5, F6, All the an in situ gel forming systems was formulated
formulations gelled instantaneously on in a solution form such that the acyclovir drops
addition to the simulated tear fluid and when instilled into the eye undergo a solution-
extended for few hours. The in-situ formed gel gel transition in cul- de -sac.the loss of drug is
should preserve its integrity without dissolving overcome due to the immediate gel formation.
or eroding for prolonged period to facilitate Buy considering the results of all the
sustained release of drugs locally. evaluation parameters F4,F5,F6 were
On the basis of physicochemical properties considered as ideal formulations.

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Table 3: Clarity Testing

Of All Formulations
FORMULATION CLARITY
F1 Clear
F2 Clear
F3 Clear
F4 Clear
F5 Clear
F6 Clear
F7 Clear
F8 Clear
F9 Clear

Table 4: Gelling Capacity

of All Formulations
FORMULATION GELLING CAPACITY
F1 +
F2 +
F3 ++
F4 ++
F5 ++
F6 +++
F7 +++
F8 ++
F9 +

Table 5: Viscosity of Different Formulation

VISCOSITY @ pH 6.1
At different shear rates
Formulation code
2 4 6 8 10
F1 5389.2 4592 3832 3265 2721
F2 7322 6392 5449 4539 3679
F3 9481 8298 7090 5741 4621
F4 832 729 620 519 420
F5 953 830 714 582 479
F6 1167 1031 897 752 591
F7 2778 2489 2142 1825 1420
F8 3554 3107 2821 2341 1788
F9 4121 3581 3121 2621 2061

Table 6: Invitro Drug Release of All Formulations

Time Cumulative % release of drug
(hr) F1 F2 F3 F4 F5 F6 F7 F8 F9
0 0 0 0 0 0 0 0 0 0
1 9.21 8.39 11.89 17.35 15.22 22.60 17.95 18.40 12.67
2 14.73 15.24 15.22 29.89 38.72 39.32 29.02 33.88 29.23
3 21.32 24.27 19.29 38.81 55.31 61.01 44.83 45.12 42.59
4 28.19 29.18 27.44 53.22 67.27 76.43 52.93 54.24 53.82
5 35.34 32.09 34.53 67.53 79.01 84.32 59.87 59.82 60.05
6 47.76 41.35 40.21 79.87 86.32 91.36 67.50 62.85 69.78
7 55.79 52.54 51.67 85.34 75.43 69.72 75.73
8 63.62 61.53 59.32 79.04 77.03 80.06
9 72.13 72.37 68.01 82.71 82.14 81.12
10 80.33 76.89 75.98 85.92 83.14

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Table 7: Cumulative% Release

of All Formulations
Cumulative % release of drug
Cumulative %
Time (hr) release of drug
F4 F5 F6
0 0 0 0
1 29 29 24
2 38 41 42
3 67 63 59
4 91 86 71
5 -- -- 88

Table 8: Zone Of Inhibition Observed

For Different Formulations
Formulation code ZOI (cm) Test organism
Marketed formulation Nill staphylococcus aureus
F1 Nill staphylococcus aureus
F2 Nill staphylococcus aureus
F3 Nill staphylococcus aureus
F4 Nill staphylococcus aureus
F5 Nill staphylococcus aureus
F6 1.2 staphylococcus aureus
F7 1.4 staphylococcus aureus
F8 Nill staphylococcus aureus
F9 Nill staphylococcus aureus

Table 9: Ocular Irritation Testing

S.NO PREPARATION RESULTS
1 F1 NIL
2 F2 NIL
SIGN OF
3 F3
LACHRYMATION
4 F4 NIL
5 F5 NIL
6 F6 NIL
7 F7 NIL
8 F8 NIL
9 F9 NIL
10 MARKETED FORMULATION NIL
0.9% NACL NEGATIVE
11 NIL
CONTROL

Table 10: Stability Studies of All Formulations

TIME PHYSICAL APPERANCE pH DRUG CONTENT
0 + 6.7 85.33 ±0.41
2 + 6.7 84.95 ±0.06
3 + 6.7 84.95 ±0.06
7 + 6.7 83.65 ±0.19
10 + 6.7 83.50 ±0.53
15 + 6.7 82.39 ±0.62
30 + 6.7 82.39 ±0.62

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Fig. 1: Clarity Testing of All Formulations

Fig. 2: Gelling Capacity of All Formulations

40000
VISCOSTY

30000
20000
10000
0
Formulatio…

F1
F2
F3
F4
F5
F6
F7
F8
F9

VISCOSITY @ pH 6.1

Fig. 3: Viscosity of All Formulations

Fig. 4: Exvivo Diffusion Studies

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100
80
60
40
20
0
0 1 2 3 4 5 6
Fig. 5: Cumulative %Release of All Formulations

100%
80%
60%
40%
20%
0%
1 2 3 4 5 6 7 8 9 10 11 12

Time (hr) Cumulative % release of drug F1

Cumulative % release of drug F2 Cumulative % release of drug F3
Cumulative % release of drug F4 Cumulative % release of drug F 5
Cumulative % release of drug F 6 Cumulative % release of drug F 7
Cumulative % release of drug F 8 Cumulative % release of drug F 9
Fig. 6: Invitro Release of All Formulations

Fig. 7: Zone Of Inhibition of All Formulations

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Fig. 8: ocular irritancy of all formulations

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