Clinical & Experimental

Ophthalmology Ince et al., J Clin Exp Ophthalmol 2015, 6:2

http://dx.doi.org/10.4172/2155-9570.1000408

Research Article Open Access

A Novel Pilocarpine Microemulsion as an Ocular Delivery System: In Vitro and

In Vivo Studies
Iskender Ince1*, Ercument Karasulu1,2, Halil Ates3, Altug Yavasoglu4 and Levent Kirilmaz2
1Center for Drug R&D and Pharmacokinetic Applications, 35100, Bornova, Ege University, Izmir, Turkey
2Department of Biopharmaceutics and Pharmacokinetics, Ege University, 35100, Bornova, Izmir, Turkey
3Department of Ophthalmology, Ege University, 35100, Bornova, Izmir, Turkey
4Department of Histology & Embryology, Ege University, Faculty of Medicine, 35100, Bornova, Izmir, Turkey 35100
*Corresponding author: Iskender Ince, Ph.D, Center for Drug R&D and Pharmacokinetic Appl., 35100, Bornova, Ege University, Izmir, Turkey, Tel: +902323392754; E-
mail: iskender.ince@ege.edu.tr
Received date: Feb 09, 2015, Accepted date: Mar 25, 2015, Published date: Mar 30, 2015
Copyright: 2015 Ince I, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Despite several disadvantages like a rapid wash out and dilution of the formulation leading to a low bioavailability,
eye drops are the most commonly used dosage form for the ocular route. Due to their properties and numerous
benefits, microemulsions are promising systems for topical drug delivery.

The purpose of this work is to develop a suitable ocular microemulsion formulation with adequate
physicochemical stability for enhancing the bioavailability of pilocarpine. Physicochemical characteristics of the
microemulsion including the drug content, refractive index, conductivity, viscosity, intraocular pressure (IOP) and
ocular tolerance were investigated. The ocular irritation test and the IOP lowering activity of the microemulsion were
studied in New Zealand white rabbits.

The developed formulation showed good physicochemical properties and a beneficial stability for six months.
After microemulsion instillation into the rabbit eyes, the intraocular pressure was reduced significantly. The ocular
irritation test used suggested that microemulsion formulation did not cause any significant allergies to the eye.

Keywords: Pilocarpine; Intraocular; Microemulsion; Glaucoma; a co-surfactant. Moreover, since they are composed of aqueous and
Bioavailability; Rabbit oily components, they can accommodate both hydrophilic as well as
lipophilic drugs [5]. Microemulsions of many ocular drugs like
Introduction ofloxacin, timolol and prednisolone were successfully prepared with
sustained effect and better bioavailability [6-8].
Standard treatment of ocular diseases is mainly performed by
topical application (90%), consisting of eye drops in the form of Glaucoma, which is the increased intraocular pressure, exhibits a
aqueous solutions. Due to the natural precorneal cleaning of the eye group of eye diseases leading to the damage of optic nerves, leading to
and high tear fluid turnover, these facts produce the major problems blindness as a worst-case scenario. Decreasing high IOP is the only
related to topical drug application to the eye. Only 1-5% of the applied effective approach that is currently available for treating this disease
drug penetrates into the cornea and reaches therapeutic and protecting the eye from later consequences [9].
concentrations in intraocular tissues. The challenging objective aimed A hygroscopic, odorless, bitter tasting drug in the form of white
at dealing with these problems is to develop topical drug delivery crystals or powder, pilocarpine hydrochloride (PHCl) is soluble in
systems with improved ocular retention, increased corneal drug water and alcohol but virtually insoluble in most non-polar solvents.
absorption and reduced systemic side effects [1]. In conventional PHCl {(IUPAC: (3S-cis)-2(3H)-furanone-3-ethyldihydro-4-[(1-
ophthalmic dosage forms, water-soluble drugs are available as aqueous methyl-1H-imidazol-5-yl) methyl] monohydrochloride, Mr=244}.
solutions, while water-insoluble drugs are available as suspensions, PHCl is a miotic drug used to control and reduce the IOP, if necessary.
ointments or gels. Low corneal bioavailability and lack of efficiency in Ocular bioavailability of topically applied PHCl amounts to 0.1-3% if
the posterior segment of ocular tissue are some of the serious the drug is administered three to four times per day as eye-drops; and
handicaps related to these ocular systems. Recent research efforts have that impairs patient compliance. The poor bioavailability is attributed
focused on the development of new and more effective drug delivery to the low lipophilicity of pilocarpine, to loss of the drug from the
systems such as nanotechnology-based formulations like precorneal area via drainage and to very fast dilution of the
nanoemulsion/microemulsion, nanosuspension, solid lipid formulation [10].
nanoparticle etc [2-4].
The aim of this work was to develop a novel microemulsion for eye
Microemulsions were preferred owing to their easy and cheap drops for topical ocular administration, by using PHCl as a model
production and excellent stability. Microemulsions have emerged as a drug and to evaluate its physicochemical characteristics, such as
promising application form for ocular usage. They are clear, isotropic stability, permeation, ocular irritation and IOP lowering activity.
mixtures of oil, water and a surfactant frequently in combination with

J Clin Exp Ophthalmol Volume 6 Issue 2 1000408

ISSN:2155-9570 JCEO, an open access journal
Citation: Ince I, Karasulu E, Ates H, Yavasoglu A, Kirilmaz L (2015) A Novel Pilocarpine Microemulsion as an Ocular Delivery System: In Vitro
and In Vivo Studies. J Clin Exp Ophthalmol 6: 408. doi:10.4172/2155-9570.1000408

Page 2 of 6

Experimental Characterization of the microemulsions

PHCl was kindly supplied by Bilim Pharmaceuticals (Turkey). Brij The microemulsions were analyzed for various physicochemical
35P, Span 80, and soybean oil were purchased from Fluca attributes. The average droplet size and polydispersity index (PDI) of
(Switzerland). 1-Butanol and sodium chloride were purchased from microemulsions in the presence or absence of PHCl were measured by
Riedel-de Han (Germany), -tocopherol was kindly provided by photon correlation spectroscopy (Zetasizer Nano ZS, Malvern
Roche (Turkey). Phenol and cyclohexane were purchased from J. Instruments, UK). The viscosities of microemulsions were measured at
T.Baker (The Netherlands). Almond oil was purchased from Cagdas 25 2C using a viscosimeter (ULA spindle, equipped with a model
Laboratories (Turkey). All other chemicals used were of analytical ULA-40Y water jacket, DV-II-Pro Brookfield, USA). The refractive
grade. index of microemulsions was evaluated at 25 2C using a
refractometer (Abbe Refractometer, Atago, Japan). Electrical
Preparation of the microemulsions conductivity of the microemulsions was studied at 25 2C using a
conductometer and conductometer probe (Mettler Toledo,
The microemulsion was prepared following a procedure called the Switzerland). Experiments were carried out three times for each
titration method. Soybean oil was used as the oil phase, Brij 35P and sample, and the results were presented as a mean SD.
Span 80 were the surfactants (S) and 1-butanol was used as the co-
surfactant (CoS). Brij 35P/Span 80 ratio was 1:200 (m/m). First, Brij Stability of microemulsions
35P was dissolved in 1-butanol at 25C and then mixed with Span 80.
Then, this mixture was added to an appropriate amount of soybean Microemulsion formulation was stored at 4 1, 25 2 and 40 2C
oil. The microemulsion formulation studies were carried out by in a dark setting for 6 months. The physical stability of
titrating slowly with PHCl solution while stirring the mixture with a microemulsions containing PHCl was determined via clarity, phase
bar using a magnetic stirrer (IKA, Germany) (100 rpm) until a separation observation, droplet size, refractive index, viscosity and
turbidity was observed. The final concentration of PHCl in our electrical conductivity. For the chemical stabilities, concentration of
microemulsion was 2%. PHCl in the formulations was also investigated by HPLC analysis at 4
1, 25 2 and 40 2C for up to 6 months.
Pseudo-ternary phase diagrams were constructed to obtain the
concentration range of the components for the existing range of
microemulsions. For each phase diagram, mixtures of soybean oil and
In vitro permeation studies
surfactant/co-surfactant concentrations were prepared at mass ratios Diffusion cells were used for the permeability studies of PHCl.
of 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, and 8:2. These mixtures were titrated with Synthetic membrane (cellulose, Mr 12.000) was mounted on a glass
water drop-by-drop, with continued stirring at 25 2C until the diffusion cell. Before starting the experiment, the cellulose membrane
mixture became clear. The mixtures were assessed by visual was first hydrated in an isotonic phosphate buffer solution (PBS) with
characterization after being equilibrated. Typical microemulsion pH 7.4 at room temperature for 30 min. These cells provided a
vehicles were selected and prepared at different component ratios after diffusion area of 1.326 cm2. PBS pH 7.4 (10 mL, 600 rpm, 37C) was
the microemulsion regions in the phase diagram were detected with used in the receptor compartment. The donor compartment contained
the aid of phase diagrams drawn using a computer program developed 1 mL microemulsion (M) (containing 2% (m/m) PHCl) or commercial
by Ege et al. [11]. The composition of the microemulsion formulations gel (G) (containing 4%, (m/m), PHCl) formulation. Approximately 10
is given in (Table 1). PHCl was dissolved in water and slowly mL of the receptor medium was withdrawn at predetermined intervals
incorporated into the microemulsion under stirring. After PHCl was (after 30, 60, 90, 120, 180, 40, 300, 360, 420 and 480 min), and replaced
entirely dissolved in the microemulsion, the clear microemulsion- immediately with an equal volume of receptor solution to maintain a
based formulation was obtained. The final concentration of PHCl in constant volume. All samples were filtered through a membrane filter
microemulsion was 2% (m/m). PHCl microemulsions were sterilized (0.2 micron, 25 mm Nylon, Millipore Millex-GN), and immediately
using an aseptic membrane filtration technique. injected into an HPLC system. Three replicates of each experiment
were performed. All experiments were performed at 25 2C. Sink
Best formulation components conditions were maintained in the receptor compartment during in
% (w/w) (w/w) /10 g vitro permeation studies.

Soybean oil 37.12 3.64 HPLC analysis of pilocarpine hydrochloride

Span 80 29.54 2.9 The samples were analyzed using the HPLC (HP Agilent 1100
Brij 35P 0.15 0.02
series) system that included a quaternary pump, 100 L loop,
automatic electronic degasser, automatic thermostatic column and UV
1-butanol 29.69 2.91 detector. The column was a Supelco-C18 column (4.6 mm 150 mm,
5 m). The mobile phase contained acetonitrile and phosphate buffer
Water 3.50 0.33
(50+50 (V/V) (pH 7.4)). The flow rate was adjusted to 0.3 mL min-1,
Pilocarpine hydrchloride - 0.2 and the injection volume was 100 L.

Table 1: Percentage weight and batch composition of microemulsion In vivo studies

formulation in the presence or absence of PHCl. Experimental glaucoma studies (n=20) and the tolerance test (n=6)
were conducted using both male and female New Zealand albino
rabbits weighing 1.5-2.5 kg. All animals were healthy and free of

J Clin Exp Ophthalmol Volume 6 Issue 2 1000408

ISSN:2155-9570 JCEO, an open access journal
Citation: Ince I, Karasulu E, Ates H, Yavasoglu A, Kirilmaz L (2015) A Novel Pilocarpine Microemulsion as an Ocular Delivery System: In Vitro
and In Vivo Studies. J Clin Exp Ophthalmol 6: 408. doi:10.4172/2155-9570.1000408

Page 3 of 6

clinical symptoms. The study was approved by the Animal Ethical Histological studies
Committee of Ege University, Turkey. Animals were housed in a room
maintained at 22 1C with an alternating 12 h light-dark cycle. Then the rabbits were sacrificed at the end of ocular tolerance test
Animals were orally fed daily with a normal diet in a standard and the eyes were enucleated. Each eyeball was fixed in a
laboratory chow. They were fed on balanced diet pellets. Tap water glutaraldehyde-formalin solution for approximately 24 h, and washed
was also available ad libitum. The animals were transported to a quiet with tap water for a night. Calcium deposits or ossified region of
laboratory at least 1 h before the experiment. The rabbits were kept in eyeballs were decalcified with a decalcification solution (20% sodium
restraining boxes throughout the course of each experiment. All tests citrate+45% formic acid, 1:1, V/V) for 3 days, and washed with tap
were performed in an air-conditioned, illumination-controlled room water during night. Otherwise, each globe of the eye was dehydrated
(22 1C). through an increasing ethanol series, immersed in xylene and was
finally fixed in paraffin wax at 56C. Paraffin blocks were cut serially in
5 m slices using a rotary microtome (RM 2145, Leica Co., Germany).
IOP lowering activity studies Sections were stained with haematoxylin and eosin (H&E) and
In the rabbit eyes, the glaucoma was induced experimentally by examined by a light microscope (Olympus BX-51, Japan).
means of subconjunctival injection of a sclerosing solution of 5%
(m/V) phenol in almond oil, which caused an increase in IOP, but no Statistical analysis
apparent macroscopic or microscopic damages occurred in the eye.
Glaucoma in rabbits is caused artificially by subconjunctival Repeated measure analysis of variance was used for the evaluation
application of phenol (5%) in almond oil 3 times every 15 days [12,13]. of the pharmacodynamics response with regard to treatment groups.
When a measurement of the intraocular pressure of the right rabbit Difference between the groups was determined as statistically
eye was repeated twice and the results were identical, the experiments significant for 0.05 significant level. Tukeyss LSD procedure was used
were started. for post-hoc analyses.

The rabbits with induced glaucoma were divided into two groups Results and Discussion
each with ten rabbits. Each group was designated to receive one of the
formulations: microemulsion (ME), commercial collyrium (C), or
commercial gel (G). M, C and G contain 2% (m/V), 2% (m/m) and 4% Preparation of microemulsions
(m/m) PHCl, respectively. M, C and G formulations were applied as a The aim of the construction of pseudoternary phase diagram was to
single dose (25 L) into the right eyes of rabbits. No drug was find out the existence range of microemulsions. Pseudoternary phase
administered to the contra-lateral eye (left), and they were considered diagrams were with various mass ratios of soybean oil, Span 80, Brij
as controls. The initial IOP (zero reading) of both eyes for each rabbit 35P, 1-butanol and water. Optimum formulation was found surfactant
was measured. After the instillation of different formulation samples, to the co-surfactant ratio 1:8 (m/m) for W/O microemulsion (Figure
the ocular bioavailability of PHCl was assessed by measuring IOP in 1).
both eyes after 15, 30, 45, 60, 90, 120, 180, 240, 300, 360, 420 and 480
min. IOP of rabbits eye was measured using a standardized Schiotz
tonometer (Oculus, Optikgerate GmbH, Germany)

Ocular tolerance test

This test was carried out in the eyes of six rabbits for both
microemulsion without drug and physiological saline (NaCl) 0.9%
(m/V). A volume of 25 L of the microemulsion was instilled in to the
conjunctival sac of the right eye and 25 L of the physiological saline
was instilled in to the conjunctival sac of the left eye of each rabbit.
During the ocular irritation test on rabbit eyes, two times applications
per day of one drop were carried out for 5 days for group one. On the
other hand, eight-times applications of one drop every hour for 8
hours were carried out for 5 days for group two, resulting in a
decreased reaction on the eye.
Pre-exposure and post-exposure evaluations of the eye lids,
conjunctiva, cornea and iris were performed by external observation
with proper illumination. Additional information was provided by slit-
lamp biomicroscopy [14]. The evaluations were made 72 and 120 h
after exposure to the sample. A scale of weighted scores for grading the
severity of ocular lesions was used. Afterwards, one drop of 2% m/V
fluorescein sodium (in water) was applied to the treated eyes of the
rabbits and the surface of the cornea was observed by using the slit- Figure 1: Pseudoternary phase diagram of soybean oil. Brij 35P,
lamp. Span 80 and water.

J Clin Exp Ophthalmol Volume 6 Issue 2 1000408

ISSN:2155-9570 JCEO, an open access journal
Citation: Ince I, Karasulu E, Ates H, Yavasoglu A, Kirilmaz L (2015) A Novel Pilocarpine Microemulsion as an Ocular Delivery System: In Vitro
and In Vivo Studies. J Clin Exp Ophthalmol 6: 408. doi:10.4172/2155-9570.1000408

Page 4 of 6

The area of W/O ME became enlarged and the optimum conditions for preparing PHCl containing eye drops were pH of 4-5.5,
formulation was obtained at its highest level. The exact composition concerning its chemical stability. The microemulsion used in this
according to oil, S, coS and aqueous phases was shown (Table 1). study was prepared at a pH of 4.6.

Temperature
Characterization of the microemulsions Parameter Time
The characterization parameters of microemulsions are listed in 4C 25C 40C

(Table 2). In the absence of PHCl, the average droplet size of 1 month 99.68 0.15 99.34 0.13 97.01 0.17
microemulsion was 0.709 nm PDI 0.219. However, in the presence of
2% m/m PHCl, the average microemulsion droplet size was 0.695 nm Drug content (%) 2 months 99.37 0.21 98.64 0.10 95.23 0.20
PDI 0.432. These findings support a recent study that found the mean
3 months 98.85 0.19 97.37 0.19 92.92 0.27
droplet size was significantly decreased after loading the drug [15].
The PDI value described the homogeneous of the droplet size. All PDI 1 month 4.51 0.08 4.52 0.07 4.50 0.06
values were smaller than 0.5 pointing to the fact that droplets were Electrical
homogeneous. On the other hand, viscosity of microemulsions was 3 months 4.52 0.08 4.52 0.07 4.51 0.09
conductivity (mS)
found to be 2,184.10-3 1.10-5 cP. The average refractive index of 6 months 4.51 0.06 4.52 0.08 4.50 0.10
microemulsions was 1.442 0.002. Incorporation of PHCl into
microemulsion increased refractive index. 1 month
1.442
1.441 0.001
1.442
0.001 0.000

Parameter Microemulsion (mean SD) 1.442 1.442

Refractive index 3 months 1.442 0.003
0.002 0.001
Refractive index 1.442 0.002
1.441 1.442
6 months 1.442 0.001
Conductivity (mS) 4.50 0.08 0.002 0.001

Viscosity (mPas) 21.84 0.01 Viscosity (mPas) 1 month 22.23 0.06 21.66 0.23 21.94 0.02

Particle size PDI 0.695 nm pdi; 0.432 3 months 22.05 0.09 22.11 0.06 22.17 0.08

6 months 22.43 0.08 22.45 0.10 22.60 0.06

Table 2: Physicochemical properties of the developed microemulsion
(n=3). Considering the initial concentration of 2% w/v as 100%.

The electrical conductivity of microemulsions was found to be 4.50 Table 3: Stability results of the monitored parameters for the
0.008. There was a strong correlation between the specific structure developed microemulsion (mean SD n=3).
of the microemulsions and their electrical conductivity behavior [16].
According to the conductivity measurements, the investigated In vitro permeation studies
microemulsions could be divided into W/O and O/W. The
conductivity of microemulsion samples showed that PHCl The permeation of M, and G, both containing 4% (m/m) PHCl,
microemulsion is of (W/O) type. If there was no significant difference were compared in vitro using a cellulose membrane. 4% PHCl
in the conductivity after storage, it indicated that the external phase is containing gel had a higher and faster drug release compared to that of
oily and that this phase did not change during the storage. microemulsion containing 4% PHCl (Figures 2 and 3).
Incorporating the co-surfactant into the microemulsion resulted in
a significant reduction in the viscosity of the formulations, with the
flow changing to a simple Newtonian flow. Because of a direct relation
between the shear stress and shear rate, it could be proven that our
microemulsion is a Newtonian fluid. The Newtonian property of PHCl
microemulsions is compatible with reports in the literature [17].
The refractive index of a substance at a certain temperature and
density reflects its purity. A microemulsion stored at three different
temperatures up to 6 months showed no significant differences of the
refractive index, indicating a constant structure of the microemulsion
(Table 3). The refractive index of the tear fluid is about 1.34-1.36 [7].
PHCl containing microemulsion had a refractive index of 1.441-1.442.
Both refractive indices were similar, which ensured an ocular
application without adverse effects to the eye capacity.
Zurowska et al. [18] have reported about the good chemical stability Figure 2: Comparative in vitro release profile of PHCl through
of a submicron emulsion containing PHCl prepared at pH 5 and synthetic membrane for microemulsion (M) (PHCl content 2%)
stored at 4C for 6 months. In this study, it was found that PHCl and commercial gel (G) (PHCl content 4%).
degraded with first-order kinetics. With the help of the Arrhenius
equation t90 values at three different temperatures were calculated as
455 days for 4C, 227 days for 25C and 152 days for 40C. The best

J Clin Exp Ophthalmol Volume 6 Issue 2 1000408

ISSN:2155-9570 JCEO, an open access journal
Citation: Ince I, Karasulu E, Ates H, Yavasoglu A, Kirilmaz L (2015) A Novel Pilocarpine Microemulsion as an Ocular Delivery System: In Vitro
and In Vivo Studies. J Clin Exp Ophthalmol 6: 408. doi:10.4172/2155-9570.1000408

Page 5 of 6

The method of diffusion cell is not representative of the real In vivo studies
situation in vivo because cellulose membrane cannot mimic the
barriers of corneal membrane and the constant volume of diffusion There are several anatomic and physiological similarities between
cell will not be able to eliminate the drug released by tear fluid human and rabbit eye. The results of this investigation were expressed
turnover. by decreases in percent IOP (Figure 4). Each time point results were
calculated in relation to the IOP before the therapy started (time 0) for
prevents or minimizes errors caused by intra-day deviations.
Considering pharmacodynamic properties of PHCl containing
commercial C, G and ME formulations, it reveals that the maximum
effect of C was achieved after 90 min, in contrast to a G and ME with
the maximum effect observed after 120 min. After the application of C
to the eye, the IOP reverts 6 hours later to the original value, whereas
using G or ME that time accounts two hours longer, namely 8 hours.
That means that the effect of ME and G lasts much longer compared to
commercial collyrium, in contrast to the microemulsion with 2%
(m/m) PHCl.
Sznitowska et al. [10] prepared submicron emulsions containing
pilocarpic acid mono-and di-ester as a prodrug. They determined the
miotic effect on rabbit eyes, and compared them with solutions of 0.5
and 2% PHCl. Using the solutions, Cmax was achieved after 30 min.
Figure 3: Intraocular pressure lowering activity of containing PHCl and for the submicron emulsion with the prodrug after 90 min. The
commercial (C; PHCl content 2%), microemulsion (M; PHCl miotic effect of the two solutions decreased after 6 hours while the
content 2%), commercial gel (G; PHCl content 4%). same effect of the submicron emulsion was for the same time. Cmax
observed for PHCl microemulsion after 120 min. After the application
of microemulsion PHCl to the eye, the IOP reverts 8 hours later to the
original value. The obtained results of PHCl microemulsion were very
HPLC analysis of pilocarpine hydrochloride
similar to the results of the submicron emulsion [13].
The detection wavelength was 215 nm and the retention time was
The pharmacological effects of ME and commercial gel were found
6.6 min. No interference of the other formulation components was
delayed and lasted longer than commercial collyrium because both of
observed. The peak area was correlated linearly with PHCl
them could improve retention and prolonged release of incorporated
concentration in the range of 0.2-2.0 mg mL-1 with the limit of
drug.
detection (LOD) at 0.008 mg mL-1, and the average correlation
coefficient was 0.999.

Figure 4: Light microscopic photographs of the histological study (H&E 100X) A: Physiological saline; B: Microemulsion (applied twice daily);
C: Microemulsion (applied eight times daily).

Ocular tolerance test Histological studies

Physiological saline served as control and caused no irritation for There were no histopathological observations on the eyes treated
both doses. Surfactants often cause a local irritation resulting from an with physiological saline as the control group (Figure 4A). When the
ocular application. Due to these facts, the surfactants must be chosen prepared microemulsion formulation was applied twice a day, every
carefully. Furrer et al. [19] examined the potential ocular irritation of morning and evening (low dose) for 5 days (Figure 4B), just like on the
several surfactants on rabbit and rat eyes in comparison to control eyes treated with physiological saline, there were no
physiological serum. They showed that 0.5% Brij 35P caused almost no histological pathology so this application is non-irritant . However,
irritation, just like physiological saline. Thats why it was decided that when the same formulation was applied 8 times with 1-hour intervals
it could be used as a suitable surfactant. for 5 days (high dose) (Figure 4C), edema, bleeding and related
flattening were observed on the pigment epithelium cells at the retina.

J Clin Exp Ophthalmol Volume 6 Issue 2 1000408

ISSN:2155-9570 JCEO, an open access journal
Citation: Ince I, Karasulu E, Ates H, Yavasoglu A, Kirilmaz L (2015) A Novel Pilocarpine Microemulsion as an Ocular Delivery System: In Vitro
and In Vivo Studies. J Clin Exp Ophthalmol 6: 408. doi:10.4172/2155-9570.1000408

Page 6 of 6

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