The African Journal of Pharmaceutical

Sciences and Pharmacy

The African Journal of Pharmaceutical
AJPSP 2:1, 91-103, APRIL 30, 2011
Sciences and Pharmacy

Effect of Formulation Factors on In Vitro Corneal Permeation

of Fluconazole through Excised Sheep Cornea
Sunil Thakral*1, Munish Ahuja2
1
Gurukul College of Pharmacy, Suratgarh, Rajasthan, India
2
Department of Pharmaceutical Sciences, Guru Jambheshwar
University of Science and Technology, Hisar, Haryana, India
*
Corresponding author’s email: sunil.thakral@gmail.com

ABSTRACT
Fungal keratitis is a corneal inflammation with blurred vision and painful eye, which can be treated
with fluconazole eye drops. The aim of the study was to find out effect of various formulation
parameters on in vitro corneal permeation of fluconazole eye drops. The corneal permeation studies
were conducted using freshly excised sheep cornea, mounted between donor and receptor chambers
of an all glass modified Franz diffusion cell, containing 11 ml of ringer bicarbonate (pH 7.4,
34o±1oC). At the end of the experiment, each cornea (freed from sclera) was weighed, soaked in 1 m
methanol, dried overnight at 90 °C and reweighed. From the differences in weights corneal
hydration was calculated. The formulation with pH 6 and concentration 2% containing phosphate
buffer and mannitol as tonicity modifier, butyl alcohol as preservative, and hydroxypropyl methyl
cellulose (HPMC) as viscosity modifier showed maximum permeation.

KEYWORDS: Fungal kerititis, Fluconazole, In Vitro Permeation, Corneal hydration, Preservative,

Viscosity modifier

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INTRODUCTION its tendency to mimic other types of stromal

inflammation, and because its management is
restricted by the availability of effective
Pharmaceutical preparations are
antifungal agents and the extent to which they can
applied topically to the eye to treat surface
penetrate into corneal tissue (4).
or intraocular conditions. These may
include conditions such as infections of the
The synthetic bistriazole antifungal
eye or eyelid, secondary to bacterial, fungal
AJPSP: African Journal of Pharmaceutical Sciences and Pharmacy

compound fluconazole exhibits outstanding

or viral pathogens, allergic or infectious
physical and pharmacokinetic properties.
conjunctivitis, inflammations, elevated
Fluconazole is a stable, water-soluble, bis-triazole
intraocular pressure and glaucoma, as well
antifungal that has low molecular weight, high
as dry-eye due to an inadequate production
bioavailability, good ocular penetration when
of fluids bathing the eye (1).
used either systemically or topically, and low
toxicity. It is potentially useful as a topical ocular
Upon topical administration of
agent. It is quite effective against candida species
ophthalmic drugs to the eye, most of the
(5).
administered amount is rapidly eliminated
from the pre-corneal area due to drainage
Azoles bind to a cytochrome P-450 fungal
via the nasolacrimal duct and dilution by
enzyme involved in the 14 demethylation of
tear turnover. The cornea, considered a
either lanosterol or 25-
major pathway for ocular penetration of
methylenedihydrolanosterol, resulting in a
topically applied drugs, is an effective
decrease in ergosterol synthesis and an
barrier to drug penetration because the
accumulation of 14- -methylated sterols. This
corneal epithelium has annular tight
leads to increased permeability of the fungal cell
junctions (zonula occludens), which
membrane, alteration of membrane enzymes,
completely surround and effectively seal the
inhibition of growth, and ultimate death of the
superficial epithelial cells. Thus topical
fungal cell. All azoles, except fluconazole, appear
drug administration to the eye results in low
to compromise the function of immune system
bioavailability due to the short residence
cells, especially lymphocytes. This lessens the
time as well as the limited corneal
degree of tissue damage occurring with the
absorption of drugs (2). Improvement of
inflammatory reaction but also affects the efficacy
ocular bioavailability can permit reduction
of the azoles in vivo. Since azoles, with the
of the instillation frequency or of the dose,
exception of fluconazole, achieve only limited
with a consequent decease in undesired side
concentrations in the eye, they are to be
effects. As a result of this,, various studies
considered fungistatic when used in ocular fungal
have been carried out with the aim of
infections (6).
improving the ocular bioavailability (3).
Thus the aim of the study was determine
Fungal infections of the cornea
the effect of various formulation parameters on in
(mycotic or fungal keratitis, keratomycosis)
vitro corneal permeation of fluconazole eye drops.
present as suppurative, usually ulcerative,
lesions. Fungal keratitis is recognizable by
the presence of a coarse granular infiltration
of the corneal epithelium and the anterior
stroma. Such a corneal infection poses a
challenge to the ophthalmologist because of

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MATERIALS AND METHODS
Fluconazole was obtained as a gift
sample from APL research Center, Mandal
(A.P.), India. Hydroxypropyl cellulose–high
viscosity grade (HPC-H) was obtained as a
AJPSP: African Journal of Pharmaceutical Sciences and Pharmacy
gift sample from Jubilant Organosys Ltd.,
Noida, India. Hydroxypropyl methyl
cellulose (HPMC E50LV Premium) was
purchased from Loba Chemie Pvt. Ltd.,
Bombay, India. Sodium chloride, potassium
chloride, magnesium chloride, calcium
chloride, sodium bicarbonate, sodium
dihidrogen orthophosphate and dextrose
were purchased from Qualigens fine
chemicals (Mumbai, India). All other
chemicals purchased were of analytical
grade and were used as received. Fresh
whole eye ball of sheep were obtained from
local butcher shop (Hisar, India), within
one-half hour of the animal being
slaughtered. The apparatus used in
permeation studies was same as published
elsewhere (7).
PREPARATIONS OF TEST FORMULATIONS:
Fluconazole ophthalmic solutions of
different pH
Fluconazole (0.3% w/v) ophthalmic
solutions, pH 4.0, 5.0, 6.0 and 7.2 were
prepared in distilled water and made
isotonic with sodium chloride (0.9% w/v).
The pH was adjusted with 0.01N HCl and
sodium bicarbonate.
Fluconazole ophthalmic solutions of
increasing concentration of pH 6.0
Required quantity of fluconazole was
dissolved in 25 ml distilled water to achieve
a concentration of 0.2, 0.3 and 0.5% w/v
and pH, adjusted to 6.0 and made isotonic
with sodium chloride.
AJPSP 2011; 2:1
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Fluconazole ophthalmic solution (0.2%w/v, pH-
6.0) of different buffers
Fluconazole (0.2g) was dissolved in 100 ml of
Sorenson phosphate buffer (pH 6.0, 0.0667M
USP) and citrate buffer (pH 6.0, 0.0667M USP),
and the solution were made isotonic by adding 0.5
g and 0.346 g of sodium chloride respectively.
Fluconazole ophthalmic solutions (0.2% w/v, pH
6.0) containing different tonicity modifiers
Fluconazole 0.2% w/v ophthalmic solution in
0.0667M phosphate buffer (pH-6.0), made
isotonic with either sodium chloride, glucose or
mannitol were prepared.
Fluconazole ophthalmic solutions (0.2%w/v, pH
6.0) containing preservatives
Fluconazole 0.2% w/v solutions in isotonic
phosphate buffer (0.0667 M, pH 6.0), containing
benzalkonium chloride (BAC 0.01% w/v), phenyl
mercuric nitrate (PMN 0.001%w/v), phenyl
mercuric acetate (PMA 0.001% w/v), benzyl
alcohol (BA 0. 5%v/v) or thiomersal (TM
0.05%w/v) were prepared.
Fluconazole Ophthalmic solutions (0.2% w/v,
pH 6.0) containing different viscolizing agent
Fluconazole 0.2% w/v solution in 0.0667M
isotonic phosphate buffer (pH 6.0), containing
either hydroxypropyl methylcellulose (HPMC) or
hydroxypropyl cellulose-high viscosity grade
(HPC-H) or polyvinyl alcohol (PVA 1.4% w/v)
were prepared.
In Vitro transcorneal permeation study (8-10)
Corneal preparation
Whole eye balls of the sheep were obtained from
the local butcher’s shop within half one-half of
the animal being slaughtered, then immediately
Thakral and Ahuja
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transported to the laboratory in cold (4°C)
normal saline (0.9%). The cornea were
carefully excised along with 2-4 mm of
surrounding sclera tissues and washed with
cold normal saline till free from proteins.
PERMEATION EXPERIMENT
AJPSP: African Journal of Pharmaceutical Sciences and Pharmacy
Fresh cornea was mounted by
sandwiching the surrounding scleral tissue
between clamped donor and receptor cells
of modified version of Franz diffusion cell
in such a way that its epithelial surface
(apical) faced the donor compartment and
endothelial surface faced to receptor
compartment. Cell was placed on magnetic
stirrer in holding position. The receptor
compartment was filled with 11 ml. of
freshly prepared bicarbonate ringer solution
(pH 7.4) and stirred using Teflon coated
magnetic stir bar. Drug solution (1 ml) was
placed to the epithelial side of cornea in
donor cell and stirring of the receptor fluid
(jacketed with water at 34±1°C) was
started. At appropriate intervals, 2 ml
samples were withdrawn from the receptor
compartment and withdrawn sample
volume was replaced with equal volume of
fresh bicarbonate ringer solution to ensure
sink conditions. Withdrawn samples were
analyzed spectrophotometrically (Varian-
Cary 5000 UV-VIS-NIR) by measuring
absorbance at λmax. of 260 nm. Each
experiment was continued for about 2.0
hours and was performed at least in
triplicate.
At the end of the experiment, each
cornea (freed from sclera) was weighed,
soaked in 1 ml methanol, dried overnight at
90 °C and reweighed. From the difference
in weights, corneal hydration was
calculated.
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Calculation of Apparent permeability coefficient
The apparent permeability coefficient was
calculated using following equation:
Papp. = ΔQ/Δt × 1/ (A.C0.60)
Where ΔQ/Δt (μg/min.) is the flux across
corneal tissue, A is the exposed surface area of
corneal tissue (0.786cm2), C0 is the initial drug
concentration (μg/ml) in the donor compartment
and 60 is included to convert minutes to seconds.
The flux across the cornea was determined from
the slop of the regression line obtained from the
linear part of the curve, between the cumulative
amount permeated (Q) vs. time (t) plot.
Statistical analysis
Statistical calculation were done by one-
way analysis of variance (ANOVA) followed by
Dunnett’s test. A p value < 0.05 was considered
significant.
RESULTS AND DISCUSSION
EFFECT OF PH ON IN VITRO TRANSCORNEAL
PERMEATION OF FLUCONAZOLE
Table 1 and Figure 1 compare the effect of
pH on corneal permeation of fluconazole.
Fluconazole is a weak acidic with a pKa of 2.0
(11). Thus, reducing the pH of the formulation
should theoretically increase the corneal
permeability by providing the higher unionized
lipid soluble fraction of the drug. The results show
a similar trend, as the pH of the formulation was
reduced from 7.2 to 4.0, there was an increase in
apparent corneal permeability of fluconazole. The
highest apparent corneal permeability of
fluconazole was obtained at pH 6.0. The
unusually higher permeation of fluconazole at pH
6.0 indicates that apart from passive diffusion,
some other pH- specific transport mechanisms are
also involved in corneal permeation of
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fluconazole and needs to be investigated
further. The normal pH of lacrimal fluid is
7.2 and eyes can tolerate pH between 6.0 to
8.5 without much discomfort (12). Upon
instillation of eye drops of different pH into
the eye, the lachrymal secretion owing to its
buffering action will bring back the pH to
AJPSP: African Journal of Pharmaceutical Sciences and Pharmacy
4.7 fold greater amount of lacrimal function
would be required to bring the pH of tears to
normal. The titre value also reveasl that the
irritation potential of formulations will be in the
order of pH 6.0 < pH 5.0 < pH 4.0. Taking
corneal permeability and irritation potential into
account, fluconazole ophthalmic solutions of pH

normal value of lacrimal fluid i.e. 7.2. The 6.0 were found to be optimum and thus selected
results of titration of ophthalmic for further study.
formulations with 0.01N NaOH to pH 7.2
indicate that during in vivo use 1.7 fold and

TABLE 1: Comparative corneal permeation of fluconazole from formulations of different pH

% Cumulative Permeation* Titre

Papp.× 106 Relative % Corneal
pH Value#
(cm./sec) Papp. Hydration
60 90 120 (ml)
4.0 15.32±1.12 17.26±0.78 19.95±0.62 1.19±0.01† 1.25 0.056 80.72±0.77

5.0 10.63±1.79 13.54±2.51 15.74±2.83 0.95±0.17 1.40 0.020 81.51±0.79

6.0 16.23±4.25 19.17±4.02 21.26±3.05 1.29±0.19† 1.89 0.012 80.14±0.94

7.2 7.84±2.16 10.08±2.07 11.24±2.83 0.68±0.16 1.0 -- 80.22±0.98

* Values are mean±S.D. (n=3), Vol. of 0.01N NaOH required to titrate 1.0 ml of the formulation. † Significantly
#

different (p < 0.05) as compared to formulation of pH 7.2 as determined by one-way ANOVA followed by Dunnett’s
test.

FIGURE 1: Effect of pH on In Vitro Transcorneal Permeation of Fluconazole

Cumulative Permeation (µg)

800
700
600 pH - 4.0
500 pH - 5.0
400 pH - 6.0
300 pH - 7.2
200
100
0
0 50 100 150
Time (min.)

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EFFECT OF CONCENTRATION ON IN VITRO The results show that there was a

TRANSCORNEAL PERMEATION OF significantly decrease in apparent corneal
FLUCONAZOLE permeability (Papp.) of fluconazole as its
concentration was increased from 0.2% to 0.5%.
Table 2 and Figure 2 compare the Similar results of decrease in corneal permeability
effect of concentration on corneal of ketorlac and diclofenac (15) with increase in
permeation of fluconazole. Fluconazole concentration of drug have been observed. Thus,
AJPSP: African Journal of Pharmaceutical Sciences and Pharmacy

ophthalmic solutions are commercially
available as 0.3% w/v solution. In some
studies 0.5% w/v (13) and 0.2% w/v (14)
solutions of fluconazole have been used
successfully in therapeutic management of
fungal keratitis.
keeping in view the higher loss of drug at higher
conc. (0.5%) and therapeutic effectiveness of
fluconazole at lower concentrations (0.2%), a
fluconazole ophthalmic solution of 0.2% w/v
concentration was selected for further studies.

TABLE 2: Comparative corneal permeation of fluconazole from formulations of different

concentrations.
% Cumulative Permeation*
Conc. Papp×106 Relative % Corneal
(% w/v) (cm./sec) Papp. Hydration
60 90 120
0.2% 22.18±1.27 26.51±2.91 29.44±3.56 2.68±0.33 2.07 81.34±0.16

0.3% 16.23±4.25 19.17±4.02 21.26±3.05 1.29±0.19 1.0 80.14±0.94

0.5% 9.52±3.19 10.72±3.75 12.02±3.90 0.44±0.14† 0.34 81.62±0.82

†
*Values are mean±S.D. (n=3). Significantly different (p < 0.05) as compared to formulation of 0.2% w/v
conc. as determined by one-way ANOVA followed by Dunnett’s test.

FIGURE 2: Effect of Concentration on In Vitro Transcorneal Permeation of Fluconazole.

900
Cumulative Permeation (µg)

800
700
600
0.20%
500
400 0.30%

300 0.50%

200
100
0
0 50 100 150
Time (Min.)

EFFECT OF BUFFERS ON IN VITRO

TRANSCORNEAL PERMEATION OF
FLUCONAZOLE

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the pH shift during storage of the product may

Figure 3 and table 3 compare the have adverse impact on the corneal permeation.
effect of different buffers on corneal
permeation of fluconazole. The results The results of titre value of fluconazole
show that buffering the formulation with ophthalmic solution show that phosphate buffer
either citrate or phosphate caused a has slightly more buffer capacity than citrate
significant decrease in corneal permeation buffer and thus would be more able to resist
AJPSP: African Journal of Pharmaceutical Sciences and Pharmacy

of fluconazole. There was no significant change in pH during storage. So, it will also have
difference between the apparent corneal somewhat more irritation potential as compared to
permeability (Papp.) of fluconazole from citrate buffer. Thus, phosphate buffer was selected
citrate or phosphate buffered formulation. as the vehicle for further study.
Since permeation of fluconazole is pH
dependent, if buffered vehicle is not used,

TABLE 3: Comparative corneal permeation of fluconazole from 0.2% ophthalmic formulations

of pH 6.0 containing different buffer

% Cumulative Permeation* Titre %

Papp×106 Relative
Buffer Value Corneal
(cm./sec) Papp.
60 90 120 (ml) Hydration
Unbuffered 22.18±1.27 26.51±2.91 29.44±3.56 2.68±0.33 1.0 0.04 81.34±0.16
Phosphate 14.54±0.63 18.59±1.82 19.76±2.64 1.83±0.24† 0.68 3.50 80.11±0.79
Citrate 19.33±3.41 20.14±1.60 21.98±2.22 1.99±0.19† 0.74 2.34 80.17±1.22
*Values are mean±S.D. (n=3). † Significantly different (p < 0.05) as compared to unbuffered formulation as
determined by one-way ANOVA followed by Dunnett’s test.

FIGURE 3: Effect of buffer on In Vitro Transcorneal Permeation of Fluconazole.

Cumulative Permeation (µg)

700
600
500 Unbuffered
400 Phosphate
300 Citrate
200
100
0
0 50 100 150
Time (Min.)

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EFFECT OF TONICITY MODIFIERS ON IN followed by glucose containing formulation and

VITRO TRANSCORNEAL PERMEATION OF the least corneal permeation was provided by
FLUCONAZOLE fluconazole ophthalmic solutions containing
sodium chloride as tonicity modifier. There was a
Table 4 and Figure 4 compare the effect of significant increase in apparent corneal
different tonicity modifiers on corneal permeability of fluconazole when mannitol was
permeation of fluconazole. Ophthalmic employed as tonicity modifier as compared to
AJPSP: African Journal of Pharmaceutical Sciences and Pharmacy

solution containing mannitol as tonicity sodium chloride. Thus, mannitol was selected for
modifier provided maximum permeation further study.

TABLE 4: Comparative corneal permeation of fluconazole from 0.2% ophthalmic formulations of

pH 6.0 containing different tonicity modifier
% Cumulative Permeation*
Papp.×106 Relative % Corneal
pH
(cm./sec) Papp. Hydration
60 90 120
Sodium
14.54±0.63 18.59±1.82 19.76±2.64 1.83±0.24† 1.0 80.11±0.79
Chloride
Glucose 16.41±1.45 17.86±0.58 19.89±1.60 1.80±0.09† 0.98 80.50±0.26
Mannitol 21.11±0.45 24.03±0.95 25.95±1.05 2.37±0.09 1.29 80.70±0.37
†
*Values are mean±S.D. (n=3). Significantly different (p<0.05) as compared to formulation containing mannitol as
determined by one-way ANOVA followed by Dunnett’s test.

FIGURE 4: Effect of Tonicity Modifier on In Vitro Transcorneal Permeation of Fluconazole

600
Cumulative Permeation (µg)

500
400 Glucose
300 Sodium Chloride
Mannitol
200
100
0
0 50 100 150
Time (min.)

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EFFECT OF PRESERVATIVES ON IN VITRO fluconazole was provided by benzyl alcohol. The

TRANSCORNEAL PERMEATION OF results show a significant increase in corneal
FLUCONAZOLE permeation of fluconazole when BA was
employed as preservative. So, no significant
Table 5 and Figure 5 compare the difference in apparent corneal permeability was
effect of different antimicrobial observed when BAC or TM or PMN or PMA
preservatives on corneal permeation of were employed as preservatives in fluconazole
AJPSP: African Journal of Pharmaceutical Sciences and Pharmacy

fluconazole. The corneal permeation of ophthalmic formulations. Thus BA was selected

fluconazole was found to be in the order of to be used as preservatives in model formulation.
BA > PMN > BAC > PMA > TM. The
maximum corneal permeability of

TABLE 5: Comparative corneal permeation of fluconazole from 0.2% fluconazole formulations

containing different preservative in phosphate buffer (pH-6.0, 0.0667M)

% Cumulative Permeation*
Papp.×106 Relative % Corneal
pH
(cm./sec) Papp. Hydration
60 90 120
BAC 17.63±3.83 23.99±5.05 31.93±5.12 2.82±0.48 1.18 81.23±1.71

TM 17.68±2.64 21.89±3.74 29.01±2.98 2.55±0.30 1.07 80.21±0.64

PMN 28.95±1.69 34.22±2.02 40.41±0.61 3.62±0.09 1.52 79.97±1.30
PMA 19.54±13.03 23.86±13.59 31.14±11.73 2.74±1.11 1.15 79.73±1.33
BA 43.02±4.20 61.12±5.14 74.29±4.27 6.71±0.42† 2.83 79.75±0.81
Control
21.11±0.45 24.03±0.95 25.95±1.05 2.37±0.09 1.0 80.70±0.37
(Unpreserved)
* Values are mean±S.D. (n=3). † Significantly different (p<0.05) as compared to control formulation as determined by
one-way ANOVA followed by Dunnett’s test.

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FIGURE 5: Effect of Preservative on In Vitro Transcorneal Permeation of Fluconazole

1800

Cumulative Permeation (µg)

1600
1400 TM (0.05%)
1200 PMN (0.001%)
1000 PMA (0.001%)
800
AJPSP: African Journal of Pharmaceutical Sciences and Pharmacy

BA (0.01%)
600 BAC (0.01%)
400 Control (Unpreserved)
200
0
0 50 100 150
Time (min.)

Effect of Viscosity modifiers on in vitro no viscolizer. Viscolizers are employed to

transcorneal permeation of Fluconazole increase the pre-corneal residence time of the drug
so that a higher and prolonged ocular availability
Table 6 and Figure 6 compare the is achieved. It is expected that during in vivo use
effect of different viscolizers on corneal HPMC containing formulation having viscosity of
permeation of fluconazole with the control 8.24 cps will provide a greater pre-corneal
formulation. The results reveal that there residence and thus greater ocular availability.
was no significant difference between the Thus, HPMC was selected to be used as viscosity
apparent corneal permeability (Papp.) of modifier in model formulation.
viscolizer containing preparation as
compared to control formulation containing

TABLE 6: Comparative corneal permeation of fluconazole from 0.2% ophthalmic formulations

containing different viscolizer in phosphate buffer (pH-6.0, 0.0667M)

Viscosity % Cumulative Permeation*

%
Modifier Viscosity Papp. ×106 Relative
Corneal
(cps) (cm./sec) Papp.
60 90 120 Hydration

PVA 3.46±0.03 35.11±12.60 45.63±16.99 52.76±18.32 4.79±1.69 2.02 79.20±1.19

HPMC 8.24±0.20 23.91±3.34 29.82 ±5.04 37.89±10.90 3.36±0.89 1.41 79.46±1.08

HPC-H 5.77±0.12 37.81±8.83 42.69±9.58 48.34±11.18 4.36±1.01 1.83 80.20±0.63

Control 1.09±0.24 21.11±0.45 24.03±0.95 25.95±1.05 2.37±0.09 1.0 80.70±0.37

*Values are mean±S.D. (n=3)

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FIGURE 6: Effect of Viscolizing Agents on In Vitro Transcorneal Permeation of Fluconazole

1600
Cumulative Permeation (µg)
1400
1200
PVA (1.4%)
1000
AJPSP: African Journal of Pharmaceutical Sciences and Pharmacy

HPMC (1.0%)
800
HPC-H (0.25%)
600
Control
400
200
0
0 50 100 150
Time (min.)

CONCLUSION hydroxypropyl cellulose-high viscosity grade

(0.25% w/v) provided an almost equivalent
decrease in in vitro diffusion of fluconazole across
In the present investigation, the
the cornea.
effect of formulation parameters, on
fluconazole corneal permeation, were
In in vivo use HPMC (1.0% w/v) having
characterized. Transcorneal permeation
higher viscosity of all the tested formulations is
studies were conducted using isolated sheep
expected to provide maximum pre-corneal
cornea. On the basis of these studies
residence and thus would be most suitable.
following conclusions can be drawn:
Fluconazole solution demonstrates the maximum
corneal permeation at pH 6.0. Further
1). The use of Sorenson phosphate buffer
investigations in in vivo models are warranted in
as a vehicle (0.0667 M, pH 6.0) favors the
order to adequately assess the ocular
permeation of fluconazole. 2). Tonicity
bioavailability of in vitro optimized model
adjustments with mannitol provide better
formulations.
permeation than glucose, followed by NaCl.

Fluconazole (0.2% w/v), ophthalmic ACKNOWLEDGMENTS

solution (pH 6.0) containing benzyl alcohol
(0.5% w/v) provided maximum in vitro The authors are grateful to Professors and
ocular availability through sheep cornea Head of Department and Librarian of Department
followed in turn by ophthalmic solution of Pharmaceutical Sciences, Guru Jambhswar
containing phenyl mercuric nitrate, University of Science and Technology, Hissar,
benzalkonium chloride, phenyl mercuric Haryana, for providing laboratory and library
acetate and thiomersal. Polyvinyl alcohol facility. Also, the authors wish to thank Gurukul
(1.4% w/v) or hydroxypropyl College of Pharmacy, Suratgarh for providing
methylcellulose (1.0% w/v) or guidance and library facilities.

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