International Journal of Pharmaceutics 495 (2015) 783–791

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Influence of drug loading and type of ointment base on the in vitro

performance of acyclovir ophthalmic ointment
Manar Al-Ghabeish, Xiaoming Xu, Yellela S.R. Krishnaiah, Ziyaur Rahman, Yang Yang,
Mansoor A. Khan*
Food and Drug Administration, CDER/OPQ/OTR/DPQR, 10903 New Hampshire Avenue, Silver Spring, MD 20993, USA

A R T I C L E I N F O A B S T R A C T

Article history: The availability of in vitro performance tests such as in vitro drug release testing (IVRT) and in vitro
Received 10 July 2015 permeation testing (IVPT) are critical to comprehensively assure consistent delivery of the active
Received in revised form 24 August 2015 component(s) from semisolid ophthalmic drug products. The objective was to study the impact of drug
Accepted 27 August 2015
loading and type of ointment base on the in vitro performance (IVRT and IVPT) of ophthalmic ointments
Available online 4 September 2015
using acyclovir as a model drug candidate. The in vitro drug release for the ointments was evaluated using
a modified USP apparatus 2 with Enhancer cells. The transcorneal permeation was carried out using
Keywords:
rabbit cornea on modified vertical Franz cells. The drug retention in cornea (DRC) was also determined at
Ophthalmic ointment
Performance tests
the end of transcorneal drug permeation study. The in vitro drug release, transcorneal drug permeation as
Acyclovir well as DRC exhibited a proportional increase with increasing drug loading in the ointment. On
Transcorneal permeation comparing the in vitro drug release profile with transcorneal permeation profile, it appears that drug
Semisolid dosage form release from the ointment is controlling acyclovir transport through the cornea. Furthermore, enhanced
Drug retention in cornea in vitro transcorneal permeation relative to the in vitro drug release underscores the importance of the
Drug release interplay between the physiology of the ocular tissue and ointment formulation. The results indicated
that IVRT and IVPT could be used to discriminate the impact of changes in drug load and formulation
composition of ophthalmic ointments.
ã 2015 Published by Elsevier B.V.

1. Introduction performance) under certain scale-up and post-approval changes

Semisolid pharmaceutical dosage forms including creams and
ointments are common topical drug delivery systems applied for
local action in the skin or cornea. However, in certain cases these
semisolids can also deliver the drug across the topical barrier to
reach systemic circulation (Calpena et al., 1999; Gonzalez-Mira
et al., 2012). The semisolids account for 11% of approved
ophthalmic dosage forms ranking second after the ophthalmic
solutions (Brigden et al., 1981). Ophthalmic semisolids offer some
unique advantages over the ophthalmic solutions such as
increased pre-corneal residence time, sustained release properties
and eye dryness relief (Hardberger et al., 1975).
The key parameter for any drug product is its efficacy as
demonstrated in controlled clinical trials. The time and expense
associated with such trials make them unsuitable for quality and
performance evaluation. Therefore in vitro surrogate tests have
been recommended to test the product sameness (quality and
* Corresponding author. Fax: +1 301 796 9816.
E-mail address: Mansoor.Khan@fda.hhs.gov (M.A. Khan).
(SUPAC) as it is believed to collectively reflect any differences due
to several physicochemical properties such as solubility, particle
size of drug and rheological properties of the dosage form (FDA,
1997). A variety of physical and chemical tests commonly
performed on semisolid products and their components (e.g.,
solubility, particle size and crystalline form of the active
component, viscosity, and homogeneity of the product) have
historically provided reasonable evidence of consistent perfor-
mance (FDA, 1997).
The in vitro performance tests such as in vitro drug release
testing (IVRT) and in vitro permeation testing (IVPT) are
increasingly becoming integral parts of the overall quality
assessment to ensure consistent delivery of the active compo-
nent(s) from semisolid products. There are well established in vitro
drug release testing methods (compendial or non-compendial) for
performance evaluation of oral solid dosage forms, but no
standardized release testing methods exist for ointments. Several
methods have been reported in literature such as the use of
diffusion bags or diffusion cells and measuring the drug
release from ointment thin layer placed in a bottle or a beaker,
none of which can be standardized easily (Baranowski et al., 2014;

http://dx.doi.org/10.1016/j.ijpharm.2015.08.096
0378-5173/ ã 2015 Published by Elsevier B.V.
784 M. Al-Ghabeish et al. / International Journal of Pharmaceutics 495 (2015) 783–791
Olejnik et al., 2012). In the context of this information, studies have
been carried to understand the kinetics of drug release from
acyclovir oleaginous ointments (Xu et al., 2015). Formulation
factors and process variable influencing the in vitro drug release
from oleaginous ointment bases have been identified. However,
the impact of formulation differences on transcorneal drug
permeation of acyclovir ointments remains unknown. Based on
this report, the present study was carried out to find the influence
of formulation factors (type of ointment base and drug loading)
that can discriminate the in vitro performance (IVRT and IVPT) of
ophthalmic ointments using acyclovir as a model drug candidate.
The availability of such discriminatory in vitro performance tests
for ophthalmic ointments would be useful to assess product
sameness between pre-and post-change products as well as
assessing in vitro equivalence of generic drug products.
Given the limited ocular surface residence time of ophthalmic
ointments and the fact that most ophthalmic drugs are intended to
act locally over the ocular surface, the rate and extent of drug
release can influence the performance of the drug product.
Furthermore, some viral and bacterial infections may develop
within the cornea and into the iris, lens, or anterior chamber of the
eye (Grant, 1987; Liesegang et al., 1989; Sanitato et al., 1984). In
such cases, an effective treatment may require targeting beyond
the surface into inner corneal tissues and interior side of the cornea
(Hill et al., 2014; Wilhelmus, 2000, 2015). Therefore, in addition to
measuring the drug release from ointment to the ocular surface,
evaluating the rate and extent of drug permeation through the
cornea, as well as the amount of drug retention in the cornea (DRC)
would be beneficial.
The United States Pharmacopeia classified ointments based on
the type of ointment base such as oleaginous, absorption, water-
removable, and water-soluble bases, as per U.S. Pharmacopeia
(USP38-NF33, 2015). Most of the approved ophthalmic ointments
are of the oleaginous type containing petrolatum and mineral oil.
Less than 15% of the approved ophthalmic ointments products
have lanolin alcohol in addition to the petrolatum and mineral oil
(absorption base) (Brigden et al., 1981). Even though it has been
reported that water soluble bases (e.g., polyethylene glycol
ointments) may provide better release profile for polar drugs
(Gurol et al., 1996; Konur-Hekimog ^lu et al., 1983; Mutimer et al.,
1956; Shigeyama et al., 1999; USP38-NF33, 2015), the use of water
soluble base in ophthalmic ointments is limited due to concern of
possible discomfort from osmotic effect of the dissolved base
material. However, water-soluble base was still included in the
current study to understand the impact of changing drug release
mechanism on ocular permeation. In this study, the influence of
three ointment bases (oleaginous, absorption and water soluble
bases) on the in vitro performance (IVRT and IVPT) was studied. The
ingredients used for the bases are approved for ophthalmic use and
showed minimal irritation when applied on rabbit eyes (CIR-
ExpertPanel, 2006; FDA, 2015; Fruijtier-Polloth, 2005; Tucker et al.,
1983). Water washable bases were eliminated from the study
because of the possible eye irritation of hydrophilic surfactants
such as lauryl sulfate that are required for the formulation.
Table 1
Formulation composition of acyclovir ointments.
Ointment base Composition (g), 100 g total
Acyclovir Mineral oil (heavy)
Oleaginous 2.00 14.70
Oleaginous 4.00 14.40
Oleaginous 6.00 14.10
Absorption 4.00 14.11
Water-soluble 4.00 – –
Acyclovir was selected as a model drug for the current study. It
is a synthetic analogue of thymidine that has a selective antiviral
activity against herpes viruses such as herpes simplex virus (HSV)
and varicella-zoster virus (VZV) (Brigden et al., 1981; Elion, 1993).
The prevalence of ocular HSV infection was estimated to be
150 cases per 10,000 individuals in developed countries, (Hill et al.,
2014) and about 10–20% of people who had VZV infection such as
chickenpox may develop corneal complications. (Sanjay et al.,
2011) Epithelial keratitis (superficial layer) is the most common
presentation of ocular infection of herpes viruses. However,
depending on the stage of the infection, herpes viruses can also
grow deeper into cornea leading to sight threatening scaring and
vascularization of the cornea (Hill et al., 2014; Wilhelmus, 2000,
2015). Therefore, it is of critical importance to understand not only
the rate and extent of drug release from ointment, but also the rate,
extent as well as the drug retention in the cornea. The present
report describes the details on the impact of drug loading and type
of ointment base on the in vitro performance of acyclovir
ophthalmic ointments.
2. Materials and methods
2.1. Materials
Acyclovir USP (>99%) was purchased from RIA International LLC
(East Hanover, NJ, USA). Polyethylene glycol 400 (PEG-400),
polyethylene glycol 3350 (PEG-3350), white petrolatum USP,
heavy mineral oil USP, phosphate buffer saline (PBS, 10X) and
glacial acetic acid USP were purchased from Fischer Scientific,
Norcross, GA. Lanolin alcohol (Ceralan1) was obtained from
Lubrizol (Cleveland, Ohio). Unless otherwise specified, all materi-
als were of analytical grade.
2.2. High performance liquid chromatography (HPLC) analysis of
acyclovir
The HPLC system is consisted of an Agilent 1260 Series (Agilent
Technologies, Wilmington, DE, US) equipped with a binary pump,
online degasser, column heater, autosampler and photodiode array
UV/vis detector. Data collection and analysis were performed using
ChemStation (Agilent Technologies). The current method was
adopted from literature (Parry et al., 1992), and validated as per the
USP <1225> and ICH Q2R1guideline. Briefly, the separation was
achieved on a SunFireTM C18 column (5 mm, 4.6 mm
150 mm).
The elution was isocratic at 1.2 mL/min with a mobile phase of 0.5%
(v/v) acetic acid (pH 2.8). The column temperature was maintained
at 25  C. The injection volume was 5 mL and detection was by UV at
254 nm. Linearity was established in the concentration range of
0.04–10 mg/mL (r2 = 0.999), and the method was precise (<1%
relative standard deviation for both intra- and inter- day variation)
and accurate (99.8% recovery). The limit of detection (LOD) and
limit of quantitation (LOQ) were determined to be 0.01 mg/mL and
0.03 mg/mL, respectively. The standard curve, constructed as
described above, was used for determining the acyclovir quantity
White petrolatum Lanolin alcohol PEG-400 PEG-3350
83.30 – – –
81.60 – – –
79.90 – – –
79.97 1.92 – –
– 57.60 38.40
 M. Al-Ghabeish et al. / International Journal of Pharmaceutics 495 (2015) 783–791
in the samples of content uniformity, in vitro drug release,
transcorneal drug permeation and DRC studies.
2.3. Preparation of acyclovir ointments
The formulation composition of ointments used in this study is
shown in Table 1, and prepared by melt fusion method. Briefly,
weighed quantity of formulation ingredients were melted at
temperature above 50  C but not exceeding 70  C in a water bath,
after which acyclovir added to the molten mixture under 1500 rpm
stirring. Agitation was continued for at least 1 h at same
temperature to obtain a homogeneous mixture. Ointment was
then allowed to cool down to 30  C (1.5  0.3  C/min), while
maintaining a constant stirring. Once cooled, the ointment
formulations were packed in multiple-dose aluminum tubes
(approximately 5 g each tube) and stored at 25  C/60%RH until
further use.
2.4. In vitro transcorneal drug permeation study across rabbit cornea
Fresh corneas from male New Zealand white rabbits, harvested
within 24 h, were supplied by InVision BioResources (Seattle, WA).
Each cornea was individually stored in Optisol-GS (Chiron Intra-
optics, Irvine, CA) within a contact lenses case and maintained at
4  C both during shipment and upon receiving. All corneas were
used within 3 days of evisceration to reduce possible damage of
corneal epithelium during storage. (Greenbaum et al., 2004; Means
et al., 1996). The morphology of the cornea including its
transparency was examined immediately upon receiving, before
starting the experiment and at the end of the experiment. Only
clear cornea was used for the experiment, and no changes were
observed in the cornea at the end of experiment.
Vertical Franz diffusion cells with spherical joint (PremeGear,
Hellertown, PA) were used to study the in vitro transcorneal drug
permeation (Fig. 1). The cornea was washed thrice with PBS (pH
7.4, RT) before mounting onto the spherical joint in between the
donor and receptor chamber. Depending on the size of the
diffusion cell (0.20 cm2 and 0.64 cm2), 5 or 10 mL of pre-warmed
PBS (pH 7.4, 37  C) was placed in the receiver chamber and 200 or
400 mL of PBS placed in the donor chamber for equilibrating the
cornea with PBS. After at least 10 min, the PBS was removed from
the donor chamber and excess PBS, if any, removed by KimWipe.
The donor chamber of the diffusion cell was filled in excess
quantity of acyclovir ointment samples, and spread uniformly with
a close-end capillary tube. The transcorneal permeation of
acyclovir solutions (250, 500 and 1000 mg/mL in PBS) was also
studied. The donor chambers were then covered with parafilm to
reduce evaporation during the experiment. The diffusion cells
were thermostatically controlled at 37.0  0.5  C, and receptor
Fig. 1. In vitro transcorneal drug permeation study with cornea mounted on the vertical Franz cell with a spherical joint.
785
medium constantly stirred at 400 rpm. Samples (200 mL) were
withdrawn from the receiver chamber at 0, 15, 30, 60, 120, 180 and
240 min, and immediately replaced with warm PBS. The samples
were analyzed by HPLC to determine the cumulative amount of
drug permeated per unit surface area.
2.5. Drug retention in cornea
At the end of transcorneal drug permeation study, the ointment
was carefully and completely removed from the corneal surface
using cotton-tipped applicators (once with dry Q-tips and thrice
with PBS-impregnated Q-tips). Care was taken not to damage the
corneal epithelium during the removal of applied ointment.
Cornea were removed from the diffusion cells, and briefly rinsed
thrice for 10 s in PBS to remove traces of ointment from corneal
surface, if any. The drug exposed cornea was punched out using
circular die cutter (11 mm in diameter) and add to a polypropylene
conical tube (Becton Dickinson Labware, Franklin Lanes, NJ, USA)
containing 3 mL of drug extraction solvent (0.5% v/v acetic acid in
H2O). The samples were screw-capped and sonicated in a bath
sonicator (Branson 8210, Branson Ultrasonics, Danbury, CT, USA)
for 99 min at 37  C. At the end of the sonication cycle, samples were
centrifuged at 1000 rpm for 5 min at R.T. to spin down the tissue
debris. The supernatant liquid was filtered through a nylon syringe
filter (0.45 mm), and 400 mL of the filtrate transferred to a 100 kDa
cut-off insert and centrifuged in an ultracentrifuge tube at
14,000
g for 10 min to remove water-soluble proteins. The
filtered samples were injected (50 mL) into HPLC to determine
acyclovir concentrations. The values of acyclovir concentration
were used to calculate the amount of DRC (Krishnaiah et al., 2014).
In a preliminary study, the amount of drug remained in acyclovir
solution before and after cornea exposure was obtained so as to
calculate the theoretical amount of drug retained in cornea at 4 h.
This was compared to the amount of drug extracted from cornea to
determine the drug extraction efficiency/recovery. The extraction
efficiency was not less than 95% indicating that the extraction
method used in DRC studies was able to recover maximum
possible drug from the cornea.
2.6. In vitro drug release testing
A modified USP apparatus 2 was used with 200 mL flat bottom
vessels and mini paddles (Agilent Technologies, Santa Clara, CA).
The samples were placed in a special holder for semisolids;
Enhancer cellsTM (Agilent Technologies, Santa Clara, CA) with an
exposed area of 4 cm2. About 0.6 g of the ointment was placed in
the cell and the surface was flattened using a spatula. Pre-wetted
0.45 mm pore size nylon filter membrane (circle shaped, 3 cm in
diameter; Millipore, Billerica, MA) was placed on ointment surface
786 M. Al-Ghabeish et al. / International Journal of Pharmaceutics 495 (2015) 783–791
and held tightly in place with a screw cap. Excess water was
removed from the surface of the filter membranes and samples
equilibrated for at least 10 min before placing them in the
dissolution vessel. The release test was performed using PBS
(pH 7.4, 37  C) as the release medium at stirring rate of 200 rpm.
Given that the solubility of acyclovir at this condition (pH 7.4,
37  C) is 2.5 mg/mL (Shojaei et al., 1998), the violation of sink
conditions was not expected, i.e., the release medium volume
(200 mL) was more than 10 times the 14.4 mL volume required to
dissolve the amount of acyclovir in the highest loaded ointment
(30 mg of acyclovir presented in 0.6 g of the 6% load ointment).
Samples (0.5 mL) were withdrawn at 5, 15, 30, 60, 120 and 240 min
and analyzed using high performance liquid chromatography
(HPLC) to determine the amount of drug released per unit surface
area.
2.7. Statistical analysis
The significance of the observed differences in the permeation
rate constant, DRC and release constant was tested by t-test
[GraphPad Prism for Windows Version 6.0, GraphPad Software Inc.,
USA]. A value of P < 0.05 was considered statistically significant.
3. Results and discussion
3.1. Transcorneal drug permeation from acyclovir solution
To eliminate the interference of formulation on transcorneal
permeation of acyclovir, aqueous drug solutions were used as a
control. Transcorneal permeation study was conducted using
acyclovir in solutions at three different concentrations (250,
500 and 1000 mg/mL). The concentration of acyclovir at the
receiver chamber (highest reached was 0.008 mg/mL) was less
than 10% of the solubility of acyclovir (2.5 mg/mL) indicated that
the sink conditions were maintained throughout the experiment.
The permeation of acyclovir was quantified by plotting the
cumulative amount permeated per area (mg/cm2) vs. time (h)
for up to 4 h. The steady state flux (J, mg/cm2 h) was obtained from
the slope using linear regression (Fig. 2A), and the lag time (tlag,
min) was calculated from the x-intercept. Permeability coefficient
(P) of acyclovir across rabbit cornea was calculated as the ratio of
steady state flux (J) and donor chamber drug concentration (C).
The transport of acyclovir across rabbit cornea exhibited zero
order kinetics (Fig. 2A), and the steady state flux was positively
correlated to drug concentration in donor chamber (Fig. 2B). The
Fig. 2. Transcorneal drug permeation from acyclovir solutions (250, 500 and 1000 mg/mL) prepared in pH 7.4 PBS (n = 6); (A) Cumulative amount of acyclovir permeated as a
function of time; (B) steady state flux as a function of acyclovir concentration in donor chamber.
lag time for acyclovir permeation across the cornea was
14.10  3.97 min, and the permeability coefficient was 7.29  1.31

106 cm/sec. Both of these values were in agreement with those
reported earlier (lag time of less than 30 min and permeability
coefficient between 3.25
106 cm/sec and 7.5
106 cm/sec)
(Anand and Mitra, 2002; Majumdar et al., 2003; Majumdar et al.,
2009; Majumdar et al., 2008).
Acyclovir is a relatively small (225.21 Da) polar compound with
partition coefficient (LogP) of 1.56 and its aqueous solubility (pH
7.4) is 1.2–1.6 mg/mL (NCBI; Shojaei et al., 1998). Because of these
properties, acyclovir transport across biological membrane occurs
mostly by passive diffusion (Shojaei et al., 1998). Furthermore,
other studies suggested that acyclovir does not traverse the rabbit
cornea via typical nucleoside transporter pathway (Majumdar
et al., 2003). Therefore, it is highly likely that the transport
mechanism of acyclovir molecule across rabbit cornea is a result of
passive diffusion of the drug.
3.2. Transcorneal permeation of acyclovir from ointments
As shown in Fig. 3A, the amount of acyclovir permeated across
the cornea was increasing with time from oleaginous ointment.
However, the profile differed from those observed for solution
samples in that the kinetics was not zero order (i.e., linear with
respect to time). This suggests that the acyclovir concentration
gradient across the cornea is not at a steady state. One reason could
be that the amount of molecular acyclovir form was altering with
time due to its release from the oleaginous ointment.
In acyclovir ointment formulation, the drug is present in solid
state as dispersed particles in the oleaginous ointment base.
Hence, overall transcorneal permeation of acyclovir relies on two
distinct yet highly related processes: (1) release or dissolution of
drug from ointment to aqueous medium; and (2) diffusion of
acyclovir molecule across rabbits’ cornea. Depending on the rate of
these processes, either of these two processes could become the
rate limiting step in the overall transport of acyclovir from
oleaginous ointment through the cornea. For instance, in the early
phase of the transcorneal drug permeation, establishment of
acyclovir concentration gradient across cornea may take longer
time (e.g., lag time) relative to the rapid release of drug from the
ointment. As the rate of permeation increases, drug concentration
at the donor chamber may start to decline and trigger further drug
release. During this latter phase, drug release process rather than
drug permeation may become the rate-limiting step. According to
Fig. 3A, even though the cumulative amount was increasing with
 M. Al-Ghabeish et al. / International Journal of Pharmaceutics 495 (2015) 783–791 787

Fig. 3. Transcorneal permeation of acyclovir (4%) from oleaginous ointment base (A) in linear time scale; (B) fitted in square root of time (Higuchi model).

time, the rate of acyclovir permeation was actually reducing with

time. Given that the transcorneal flux of acyclovir was concentra-
tion dependent (Fig. 2A) and that drug presented in excess amount
in the ointment (4%), the decline in the permeation is likely caused
by the reduction of molecular acyclovir in the donor chamber.
Consequently, the overall transcorneal transport of acyclovir was
controlled by drug release.
In terms of permeation kinetics, it was found that transcorneal
permeation of acyclovir from oleaginous ointment better fitted to
square root time dependency on based on Higuchi model rather
than zero-order model (Fig. 3 and Table 2). Therefore, acyclovir
transport across the cornea from the oleaginous ointment could be
expressed by the following equation:
pffiffi
Q=A ¼ K t þ y Intercept
where Q/A is the cumulative amount permeated per unit area, K is
Fig. 4. Transcorneal permeation of acyclovir from oleaginous ointment base with 2,
the transcorneal permeation rate constant and t is the time. It is 4, and 6% w/w of drug loading (n = 6).
worth noting that there was a lag time in transporting acyclovir
from the ointment across the cornea as indicated by the x- transcorneal permeation of acyclovir. Increasing the loading of
intercept, with a value of approximately 13 min. This coincides acyclovir in the oleaginous bases promoted drug release from
with the observation earlier for acyclovir solutions (Fig. 2), ointment into the aqueous media (Table 3) which further enhanced
confirming that the drug release from the oleaginous ointment the rate of permeation through cornea. (Table 3).
started almost instantly.
3.2.2. Effect of ointment bases
3.2.1. Impact of drug loading The use of lanolin alcohol in the absorption ointment base
With oleaginous ointment, it was found that transcorneal produced a profound effect on the transcorneal permeation of
permeation rate constant (Higuchi rate constant) was linearly acyclovir from the ointment resulting in a significantly lower
proportional to the amount of drug present in the ointment base transcorneal permeation rate compared to that from the oleagi-
(Table 2 and Fig. 4). Given the earlier findings that the extent of nous base (Fig. 5A). Lanolin alcohol is derived from the
drug release from oleaginous ointment into aqueous media was saponification of wool wax (lanolin) and composed of aliphatic
dependent on the drug loading (Xu et al., 2015) and that the flux of mono alcohol, 1, 2 diols, triterpene alcohols, cholesterol and small
acyclovir in solution was also concentration dependent (Fig. 2B), it amounts of hydrocarbons. (Motiuk, 1979) It is widely used in
is evident that both release behavior of the drug from ointment and cosmetics, personal care products and pharmaceutical ointments
the diffusion of drug through cornea played a role in the where it is incorporated with petrolatum and mineral oil in
ointment vehicles to work as an emollient.
The surface active property of the lanolin alcohol is expected to
Table 2
Permeation rate constant of acyclovir from oleaginous and absorption bases (n = 6).
promote wetting of the drug and hence release/permeation of the
drug, surprisingly the opposite of what was observed. To
Formulation Zero Order Square root of time

K0 r2 K r2
(mg/cm2 h) (mg/cm2 h1/ Table 3
2
) Drug release constant from oleaginous bases containing 2, 4 and 6 % w/w of
acyclovir using modified USP apparatus 2 with Enhancer cells (pH 7.4 PBS, 37  C,
Oleaginous, 6.30  1.45 0.9302  0.050 16.35  3.25 0.9806  0.0318 n = 6).
2%
Oleaginous, 14.42  2.23 0.9760  0.019 36.75  5.30 0.9969  0.0078 Drug loading in oleaginous ointment Release constant
4%
Kln (mg/cm2) r2
Oleaginous, 24.53  4.89 0.9782  0.037 62.46  11.37 0.9972  0.0055
6% 2% 0.712  0.037 0.9815  0.0089
Absorption, 7.74  2.07 0.9784  0.0028 19.72  5.16 0.9980  0.0036 4% 1.356  0.142 0.9902  0.0066
4% 6% 2.815  0.699 0.9925  0.0056
788 M. Al-Ghabeish et al. / International Journal of Pharmaceutics 495 (2015) 783–791
Fig. 5. Comparison of transcorneal permeation of acyclovir from ointment bases containing 4% w/w of acyclovir and acyclovir solutions (n = 6).
understand the root cause of this phenomenon, a look at the
unique physical constrain of the ointment/cornea interface is
needed. It is well known that the outmost layer of cornea is
epithelium which is lipophilic in nature. This surface is always
covered by a thin layer of aqueous fluids, e.g., tear fluid for in vivo
(approximately 10–100 mL) or in the case of in vitro transcorneal
permeation experiment covered with hydration fluids. The
common feature in both oleaginous and absorption base cases is
the limited aqueous fluids available for drug dissolution/release.
Due to its surface active nature, addition of lanolin alcohol to the
oleaginous base helps the wetting and incorporation of water into
the base. Therefore, presence of lanolin alcohol at the cornea
surface may compete with acyclovir particle for the limited
available aqueous fluids reducing the amount of drug dissolved/
released, and subsequently decreasing the rate of drug permeation.
Overall, the transcorneal permeation of acyclovir from the
water soluble ointment exhibited a bi-phasic drug permeation
(Fig. 5B). In the first phase (up to 1 h), the cumulative amount
permeated was comparable to that permeated from other
ointment bases. In the second phase, the rate of acyclovir
permeation increased significantly. However, the maximum
concentration at the receiver chamber (0.02 mg/ml) was still less
than 10% of the solubility of acyclovir, and hence the desired sink
condition was not violated. Beyond 1 h of study, the volume of
receptor compartment decreased by about 0.2 mL with an equal
increase in the volume of donor compartment contents, and hence
receptor chamber was replenished with equal volume at the time
of sampling (2 and 3 h). It appears that water molecules back-
diffused into the donor compartment across the cornea due to high
osmotic pressure generated by solubilized PEG. The PEGs are fully
miscible with water which means that the ointment base would
dissolve slowly with water, releasing acyclovir as the erosion
proceeds. Higher degree of acyclovir transcorneal permeation from
water-soluble base compared to that from acyclovir solution
suggests that PEG might have enhanced the drug solubility as well
as the drug permeation as low molecular weight PEG are known to
act as cosolvents with water. However, in light of the observed
osmotic effect, use of PEGs as ointment base for in vivo ocular drug
delivery may be questionable due to the possible physical
discomfort and irritation in the eye.
3.3. In vitro release of acyclovir and relation to transcorneal drug
permeation
The in vitro release testing (IVRT) of acyclovir from ointment
formulations was tested using a modified USP apparatus 2. The
modifications include: the samples were placed in especial
semisolid holders, i.e., Enhancer cells, and the use of 200 mL
vessels and mini paddles. The test conditions were chosen to
mimic the in vivo ocular conditions in terms of temperature (37  C)
and pH (PBS, pH 7.4).
For oleaginous base, the release profile of acyclovir from the
ointment showed an increased pattern with an increase in the
acyclovir loading (Fig. 6). The rate of drug release was rapid at the
beginning yet slowed down with time. In an attempt to explain the
underlying mechanism of drug release, cumulative amount
released was fitted with a logarithmic time model, where the
kinetics of the drug release from oleaginous ointment was
attributed to an expanding boundary at the interface of ointment
and aqueous media (Xu et al., 2015). However, on close
examination of the IVRT results with the in vitro permeation
testing (IVPT) data, it was noticed that the amount permeated was
significantly higher than possibly being released during the same
time period (Fig. 7). The high amount of acyclovir transported from
the oleaginous ointment base across the cornea could possibly be
attributed to: (1) an enhancement in the permeability of the
cornea; and/or (2) an increase in the acyclovir release from
ointment leading to availability of more soluble drug for
permeation. In the first scenario, the lipophilic nature of
petrolatum in the oleaginous base might have facilitated its
interaction with the lipophilic corneal epithelium and reduce the
barrier for drug diffusion. In the case of the second situation,
similar interaction between petrolatum and epithelium might
have resulted in further expansion of the transient-boundary,
making more drug amount available for dissolution and subse-
quent release. Indeed, this might explain the difference in kinetics
Fig. 6. Release profiles of acyclovir from oleaginous bases with different drug
loading using modified USP apparatus 2 with Enhancer cells (pH 7.4 PBS, 37  C,
n = 6).
 M. Al-Ghabeish et al. / International Journal of Pharmaceutics 495 (2015) 783–791 789

Fig. 7. Comparison of in vitro release testing (IVRT) and in vitro corneal permeation (IVPT) of acyclovir from oleaginous (A), absorption (B) and water soluble (C) bases (4% of
acyclovir loading, pH 7.4 PBS, 37  C, n = 6). The IVRT was conducted using modified USP apparatus 2 with Enhancer cells, while the IVPT study was conducted across rabbit
cornea.

observed between the release (logarithmic time model) and the
permeation (square root time model). Under IVRT condition, the
ointment base (petrolatum and mineral oil) is not readily miscible
with water, and consequently the boundary between ointment
base and water is moving in the same direction as the drug
diffusion (release). This might have caused a rapid depletion in the
amount of drug available for dissolution with time, leading to
logarithmic change in the drug release rate (Xu et al., 2015). Under
the IVPT setting, the presence of the interaction between
petrolatum and cornea as well as the initial spreading of the
ointment might have helped in the penetration of the aqueous
phase (i.e., opposite moving boundary relative to the drug
diffusion). Such physical conditions are perfectly in line with
those outlined by Higuchi, and consequently it is not surprising
that overall permeation kinetics followed square root of time.
The amount of acyclovir permeated at 4 h from absorption base
was also higher than the amount of drug release (Fig. 7B), possibly
caused by similar factors as oleaginous ointment. However, the
increase in the permeation was not as high as in oleaginous base,
likely a result of reduced drug release by water-competing lanolin
as discussed earlier. Lastly, the amount of acyclovir permeated
through the cornea from the water soluble base was significantly
(P < 0.05) lower than the amount of drug released during similar
time period (Fig. 7C). The volume of aqueous medium available in

Fig. 8. Amount of the drug retained in the cornea (mg/cm2) after 4 h of acyclovir ointment application of the ointment; (A) oleaginous ointments of 2, 4, and 6% acyclovir
loading (B) water soluble, oleaginous, absorption ointments with 4% acyclovir loading.
790 M. Al-Ghabeish et al. / International Journal of Pharmaceutics 495 (2015) 783–791
IVRT is far more than that available in IVPT studies. This might have
resulted in high degree of solubilization of the PEG ointment base
leading to enhanced drug release. Additionally, the permeability of
cornea is much lower than that of synthetic membrane used in
IVRT study resulting in reduced drug permeation.
3.4. Drug retention in cornea
For the treatment of severe viral infection and/or preventing
virus spread in the eye, it is highly important to examine the
amount of DRC in addition to determine the amount of drug
permeated across the cornea. This is because virus infecting the
eye can spread from the upper epithelial layer of the cornea to
deeper layers and even interior parts of the eyes.
Increased drug loading in the oleaginous ointment resulted in
almost exact proportional rise in the DRC values after 4 h (Fig. 8A).
For different ointment bases with identical drug loading, DRC
values exhibited similar trend as those observed for the IVPT:
water soluble > oleaginous > absorption base (Fig. 8B). This could
be explained based on the unique interplay between the
physiology of the eye and physicochemical properties of the drug.
The structural anatomy and the cellular components of the cornea
help in creating a protective barrier that prevent exogenous
substances including drug molecules to permeate into the eye. The
human cornea measures approximately 12 mm in diameter and
520 mm in thickness, and consists of five layers, including the
epithelium, basement membrane (Bowman’s layer), stroma,
Descemet’s membrane and endothelium. Among them, the corneal
epithelium is 4–6 cell layers thick and possesses tight lateral
intercellular junctions that limit paracellular drug transport. The
stroma consists of highly organized fine collagen fibers and
glycosaminoglycan that helps in holding substantial large amount
of water, and it forms the bulk structure of the cornea (about 80–
85% of the cornea thickness). Endothelium is a porous monolayer
contains ions pump that regulates the cornea water content, and is
generally much more permeable. Depending on the properties of
the drug (e.g., LogP), different portions of the cornea may become
barriers to permeation. For polar drugs like acyclovir, lipophilic
epithelium may present a greater barrier for permeation. On the
other hand, the stroma may exert a diffusional barrier to lipophilic
drugs due to its hydrophilic nature (Malhotra and Majumdar, 2001;
Sosnova-Netukova et al., 2007). Consequently, for a hydrophilic
drug like acyclovir the DRC values observed in this study were
closely related to IVRT and IVPT.
4. Conclusions
The in vitro performance of acyclovir ophthalmic ointments
prepared with different bases and drug loadings was studied. The
in vitro drug release, transcorneal drug permeation, and drug
retention in cornea were found to be closely related, and highly
influenced by the drug loading as well as the type of ointment base.
The results indicated that increased drug loading in oleaginous
ointment bases promoted drug release into the aqueous media
which further enhanced the rate of permeation through cornea. On
comparing the in vitro drug release profile with the transcorneal
permeation profile, it was determined that the drug release from
the ointment may be the overall controlling process for acyclovir
transport through the cornea. The findings in the current study can
help in understanding the relationship of in vitro drug release and
transcorneal drug permeation from acyclovir ointments. The in
vitro performance evaluation provided an insight on the possible
impact of drug loading and type of ointment base on the in vivo
performance of acyclovir ophthalmic ointment. The results
indicated that IVRT and IVPT could be used to discriminate the
impact of changes in drug load and formulation design of
ophthalmic ointments.
Disclaimer
The findings and conclusions in this article have not been
formally disseminated by the Food and Drug Administration and
should not be construed to represent any Agency determination or
policy.
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