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Research Article
Nanoemulsion as a Potential Ophthalmic Delivery System for
DorzolamideHydrochloride
Hussein O. Ammar,1,3 H. A. Salama,1 M. Ghorab,2 and A. A.
Mahmoud1
Received 24 July 2008; accepted 27 May 2009; published online 18
June 2009
Abstract. Dilutable nanoemulsions are potent drug delivery
vehicles for ophthalmic use due to theirnumerous advantages as
sustained effect and high ability of drug penetration into the
deeper layers of theocular structure and the aqueous humor. The aim
of this article was to formulate the antiglaucoma drugdorzolamide
hydrochloride as ocular nanoemulsion of high therapeutic efcacy and
prolonged effect.Thirty-six systems consisting of different oils,
surfactants, and cosurfactants were prepared and
theirpseudoternary-phase diagrams were constructed by water
titration method. Seventeen dorzolamidehydrochloride nanoemulsions
were prepared and evaluated for their physicochemical and drug
releaseproperties. These nanoemulsions showed acceptable
physicochemical properties and exhibited slow drugrelease. Draize
rabbit eye irritation test and histological examination were
carried out for thosepreparations exhibiting superior properties
and revealed that they were nonirritant. Biological evaluationof
dorzolamide hydrochloride nanoemulsions on normotensive albino
rabbits indicated that theseproducts had higher therapeutic efcacy,
faster onset of action, and prolonged effect relative to eitherdrug
solution or the market product. Formulation of dorzolamide
hydrochloride in a nanoemulsion formoffers, thus, a more intensive
treatment of glaucoma, a decrease in the number of applications per
day,and a better patient compliance compared to conventional eye
drops.
KEY WORDS: dorzolamide hydrochloride; glaucoma; nanoemulsion;
pharmacodynamic;physicochemical characterization.
INTRODUCTION
Ophthalmic drug delivery is one of the most interestingand
challenging endeavors facing the pharmaceutical scientist(1). It is
a common knowledge that the application of eyedrops as conventional
ophthalmic delivery systems result inpoor bioavailability and
therapeutic response because oflacrimal secretion and nasolacrimal
drainage in the eye(2,3). Most of the drug is drained away from the
precornealarea in few minutes. As a result, frequent instillation
ofconcentrated solutions is needed to achieve the
desiredtherapeutic effects (4). But, by the tear drainage, the
mainpart of the administered drug is transported via the
nasola-crimal duct to the gastric intestinal tract where it may
beabsorbed, sometimes causing side effects (5). In order toincrease
the effectiveness of the drug, a dosage form shouldbe chosen which
increases the contact time of the drug in theeye. This may then
increase the bioavailability, reducesystemic absorption, and reduce
the need for frequentadministration leading to improved patient
compliance.
To overcome these problems, various ophthalmic vehiclessuch as
suspensions, ointments, inserts, and aqueous gels havebeen
investigated to extend the ocular residence time ofmedications for
topical application to the eye (6). These oculardrug delivery
systems offer some improvement over conven-tional liquid dosage
forms but, because of blurred vision (e.g.,ointments) or lack of
patient compliance (e.g., inserts), theyhave not been universally
accepted. As a result, good ocularbioavailability following topical
delivery of a drug to the eyeremains a challenge yet to be resolved
satisfactorily (7).
Glaucoma is a serious eye disorder characterized by anincrease
in the intraocular pressure which leads gradually toloss of vision
due to damage of the optic disk, usually withoutsymptoms and is the
second leading cause of blindnessworldwide (810). It is believed
that glaucoma is a result ofan imbalance between aqueous humor
secretion and drainageprocesses within the ocular chamber (11).
Drugs used to treat glaucoma work broadly in one of twoways:
either to reduce the production or to increase thedrainage of
aqueous humor. Dorzolamide hydrochloride wassynthesized in the
1980s (12) and was shown to be about 20times more potent than the
carbonic anhydrase inhibitoracetazolamide with regard to the
inhibition of carbonicanhydrase isoenzyme II (13), which is thought
that thisisoenzyme plays a major role in aqueous humor
secretion(14). The pKa values of dorzolamide hydrochloride are
6.35and 8.5 (15) and its apparent partition coefcient is 1.96
forthe n-octanol/pH 7.4 buffer system (16).
1530-9932/09/0300-0808/0 # 2009 American Association of
Pharmaceutical Scientists 808
AAPS PharmSciTech, Vol. 10, No. 3, September 2009 (# 2009)DOI:
10.1208/s12249-009-9268-4
1 Department of Pharmaceutical Technology, National
ResearchCenter, Dokki, Cairo, Egypt.
2 Department of Pharmaceutics, Faculty of Pharmacy, Cairo
University,Cairo, Egypt.
3 To whom correspondence should be addressed. (e-mail:
[email protected])
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Topically effective aqueous dorzolamide eye drop solu-tion
(Trusopt) has become one of the most widely usedmedications for the
treatment of open-angle glaucoma since itbecame commercially
available in 1995 (17). The concentra-tion of dorzolamide HCl in
Trusopt is 2.2%, correspondingto 2.0% of the free base, pH 5.65.
Hydroxyethyl cellulose isused to increase the viscosity of Trusopt
eye drops to100 cps; this increased viscosity leads to increased
cornealcontact time and, consequently, to increased
bioavailability(15). However, the relatively low pH and high
viscosity havebeen shown to generate local irritation after topical
admin-istration of the eye drops (18).
The objective of our study was to formulate
dorzolamidehydrochloride as eye drops capable of delivering the
drug in asustained manner, thus avoiding frequent instillation of
thedrops which may induce toxic side effects and cellular damageat
the ocular surface (1921). In the meantime, the prepara-tion of
dorzolamide eye drops of high therapeutic efcacy andlacking the
undesirable effects of the market product(Trusopt) as irritation
and blurred vision (22) is anadditional aim of our study.
Nanoemulsions as a drug deliverysystem were utilized in our study
for formulating dorzolamidehydrochloride as ocular eye drops in
virtue of their distinctadvantages (2325). These include sustained
release of thedrug applied to the cornea, high penetration in the
deeperlayers of the ocular structure, and aqueous humor as well
asease of sterilization. Thus, these systems can achieve
thera-peutic action with a smaller dose and a fewer systemic
andocular side effects.
MATERIALS AND METHODS
Materials
Dorzolamide hydrochloride was obtained from HeteroDrugs Ltd.,
Hetero House, Erragadda, India. Tween 80(TW80), isopropyl myristate
(IPM), triacetin (glycerol triac-etate), and dialysis tubing
cellulose membrane (molecularweight cutoff 12,000 g/mol) were
obtained from Sigma-Aldrich Chemical Company, St. Louis, USA.
CremophorEL (CrEL, polyethoxylated castor oil), Miglyol 812
(Miglyol,caprylic/capric triglyceride), Transcutol P (Transcutol,
dieth-ylene glycol monoethyl ether), and Miranol C2M conc
NP(Miranol, disodium cocoamphodiacetate) were kindly sup-plied by
Seppic (Paris, France), Sasol Germany GmbH(Witten, Germany),
Gattefoss (Saint Priest, France), andRhodia, Inc. (CA, USA),
respectively. Propylene glycol (PG)was purchased from BDH
Laboratory Supplies, Poole,England. Dorzolamide hydrochloride
market product (Tru-sopt, 2.2% dorzolamide hydrochloride) was
provided byMerck Sharp and Dohme B.V. (Haarlem, Netherlands).Sodium
dihydrogen phosphate, disodium hydrogen phos-phate, and sodium
chloride were purchased from Siscoresearch laboratories Pvt. Ltd.,
Mumbai, India.
Methods
Construction of Pseudoternary-Phase Diagrams
The pseudoternary-phase diagrams of oil (isopropylmyristate,
Miglyol 812, and triacetin), surfactant (Tween 80
and Cremophor EL), and cosurfactant (propylene glycol,triacetin,
Transcutol P, and Miranol C2M conc NP) weredeveloped using water
titration method at 25C (26). Foreach combination of surfactant (S)
and cosurfactant (CoS),four phase diagrams were constructed with S
to CoS weightratios of 1:1, 2:1, 3:1, and 4:1. In case monophasic,
clear, andtransparent mixtures were visualized after stirring,
thesamples were marked as points in the phase diagram. Thearea
covered by these points represents the region wherenanoemulsion
exists which was calculated using AutoCADsoftware (Autodesk, Inc.,
San Rafael, CA, USA).
Preparation of Dorzolamide Hydrochloride Nanoemulsions
In order to mimic physiological dilution process afterocular
administration of the prepared nanoemulsions, nano-emulsions were
diluted 1:5 (v/v) with isotonic buffer solution(pH 7.4) and
assessed visually for transparency for a period ofat least 48 h
(27). Diluted systems that showed transparencyand no phase
separation were considered as true oil/water(o/w) nanoemulsions
maintaining their physical integrity andwere used for preparing
drug-loaded nanoemulsions.
Seventeen nanoemulsion vehicles were prepared at S toCoS weight
ratio of 3:1. Dorzolamide hydrochloride (2.22%w/w) was dissolved in
the prepared nanoemulsion sampleswith the aid of vortexing until
clear transparent systems wereobtained. Drug-loaded nanoemulsions
were prepared 48 hbefore investigation so that drug distribution
among the oil,water, and surfactant micelles attains thermodynamic
equi-librium and were stored at room temperature.
Benzalkoniumchloride was added as a preservative in all prepared
nano-emulsions in a concentration of 0.01% w/w.
Accelerated Physical Stability Studies
The prepared nanoemulsions were subjected to a seriesof
heatingcooling cycle (28), centrifugation (27), and freezethaw
cycle tests (28) and the nanoemulsions that were stablewere
considered for further studies.
Physicochemical Characterization of Nanoemulsions
Particle Size Analysis. A Photon Correlation Spectrom-eter
(Zetasizer 1000HS, Malvern Instruments Ltd., UK) wasemployed to
monitor the particle size of nanoemulsions.Light scattering was
monitored at 90 angle and 25C.
Rheological Measurements. Rheological measurementswere performed
at 250.1C using a Bohlin rheometer(Model CS 100, Bohlin
Instruments, UK) equipped with acone/plate apparatus 40 mm per 4.
For each sample,continuous variation of shear rate (80400 s1)
wasapplied and the resulting shear stress was measured.Viscosity of
dispersions with Newtonian ow propertieswas calculated according to
the relation: =/.
Refractive Index. Refractive index was determined at25C using
refractometer M46.17/63707 supplied by Higlerand Walts Ltd.,
England.
Surface Tension. Surface tension measurements werecarried out at
20C using a thermostatically controlledprocessor tensiometer K100
(Kruss GmbH, Germany) pro-
809Nanoemulsion as a Potential Ophthalmic Delivery System for
Dorzolamide Hydrochloride
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vided with a Du Nouy ring (ring radius 9.545 mm, wirediameter
0.37 mm).
pH and Osmotic Pressure. pH was measured at 25Cusing JENWAY
model 350 (JENWAY Ltd., UK) and theosmotic pressure was measured
using Micro Osmometermodel 3300, Advanced Instruments Inc.,
USA.
In Vitro Drug Release Studies
These studies were performed using US Pharmacopeiadissolution
apparatus type II (SR8 PLUS, Handson dissolu-tion tester, USA). The
release medium was 900 ml ofphosphate buffer (pH 7.4), the
temperature was set at 340.5C (the ocular surface temperature;
29,30) and the paddlerevolution speed was 50 rpm. Release
experiments wereconducted for 6 h as all tested preparations
attained 100%release within this time period.
A 0.5 ml of aqueous drug solution (pH 5.5), marketproduct, or
the drug-loaded nanoemulsion was instilled in thedialysis bag which
was secured with two clamps at each end.At denite time intervals, a
5-ml sample was withdrawn andreplaced by fresh buffer; these
samples were assayed fordorzolamide hydrochloride by Shimadzu UV
spectrophotom-eter (2401/PC), Japan at 252.6 nm. Triplicate
experimentswere carried out for each release study and the mean
value ofrelease efciency (RE) was calculated. The release
efciencywas calculated from the area under the release curve at
time t.It is expressed as a percentage of the area of the
rectanglecorresponding to 100% release, for the same total
time,according to the following equation (31):
RE
Rt
0y dt
y100 t 100
Where y is the percentage drug released at time t.
Ocular Irritation Studies
Six groups, each of six New Zealand albino rabbitsweighing 1.52
kg, were kept in an air-conditioned room at250.5C and fed a
standard pellet diet and water witharticial uorescent light
providing a cycle of night and day,12 h each. All animals were
healthy and free of clinicallyobservable abnormalities. The
experimental procedures con-form to the ethical principles of the
National ResearchCenter, Cairo (Egypt), on the use of animals.
The right eye received 50 l of the tested formulation,while the
left eye was used as a control. Application of thetested
formulation onto the rabbits cornea was repeatedevery 2.5 h through
a period of 7.5 h per day for threesuccessive days and once on the
fourth day. After 1 and 24 hfrom last instillation, eyes were
examined under generalanesthesia (35 mg/kg ketamine and 5 mg/kg
xylazine) utilizingDraize technique (32). The eyelids, cornea,
iris, conjunctiva,and anterior chamber were inspected for
inammation ortoxic reaction. Furthermore, both eyes were stained
withuorescein and examined under UV light to verify possiblecorneal
lesion.
After corneal examination, the corneas were separated,washed
with saline phosphate buffer (pH 7.4), and immedi-ately xed in
Bouins solution [85 ml picric acid, 10 mlformalin (3740%) and 5 ml
acetic acid] for 24 h. The corneaswere then dehydrated with an
ethyl alcohol gradient (70 90100%) and xylene, put in melted
parafn, and solidied inblock forms. Cross sections were cut,
stained with hematox-ylin and eosin, and microscopically examined
for pathologicalmodications (n=3).
Therapeutic Efficacy Studies
This study was of a single-dose crossover design and
wasperformed on aqueous drug solution (pH 5.5), the marketproduct
(Trusopt, as a reference standard) in addition to theselected
formulations. Sterility of formulations was achievedby ltration
through sterile 0.22-m pore size pyrogen-freecellulose lters.
Male albino rabbits weighing 22.5 kg were used; theanimals were
housed as previously described under OcularIrritation Studies and
the experimental procedures conformto the ethical principles of the
National Research Center,Cairo (Egypt), on the use of animals.
Intraocular pressure(IOP) measurements were performed with a Schitz
Tonom-eter (Rudolf Riester GmbH and Co. KG, Germany). Nomore than
three repeated readings for any eye wereperformed at each
measurement. Only measurements inwhich two consecutive readings
were identical were included.Animals which showed a consistent
difference of more than2 mmHg between IOP of both eyes, showed any
signs ofirritation, or were agitated during handling were
excluded.
Eyedrops were instilled topically into the upper quadrantof the
eye and the eye was manually blinked three times; oneeye received
50 l of the preparation and the other served ascontrol. IOP was
measured immediately prior to giving thedrug and at different time
intervals following the treatment.All measurements were done three
times at each interval andthe mean values were taken.
The percentage decrease in IOP was determined accord-ing to the
following equation:
%Decrease in IOP IOPcontrol eye IOPdosed eyeIOPcontrol eye
100
The pharmacodynamic parameters taken into considerationwere
maximum percentage decrease in IOP, time for maxi-mum response
(tmax), area under percentage decrease in IOPversus time curve
(AUC010h), and mean residence time(MRT). These parameters were
calculated using WinNonlinsoftware (Pharsight Co., CA, USA).
Statistical analysis of the results was performed usingone-way
analysis of variance followed by the least-signicantdifference
test. Statistical analysis was computed with theSPSS software (SPSS
Inc., Chicago, USA).
RESULTS AND DISCUSSION
Construction of Pseudoternary-Phase Diagrams
For the present study, one type of oil from differentcategories
such as long-chain triglycerides (isopropyl myris-
810 Ammar, Salama, Ghorab and Mahmoud
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tate), medium-chain triglycerides (Miglyol 812) as well
asshort-chain triglycerides (triacetin) were selected. These
oilsare well tolerated by the eye (25,3335).
Tween 80 and Cremophor EL were used as examples ofnonionic
surfactants. Tween 80 is widely used in ophthalmicpreparations due
to its safety. It is listed in US Pharmacopeia-National Formulary,
European Pharmacopeia, and the Japa-nese Pharmacopeia (36). An
ocular irritation evaluation testwas made by Alany et al. (37) and
classied Tween 80 aspractically nonirritant. On the other hand,
according toinformation provided by the manufacturer, the
instillation of0.05 ml of Cremophor EL in the rabbits conjunctival
saccaused only slight reddening of the conjunctiva, and
thisdisappeared within a few hours (38). The application of a50%
aqueous solution of this product caused slight irritationwith
lacrimation, which disappeared rapidly; 30% aqueoussolutions had no
irritant effect (38).
An additional important criterion for selection of
thesurfactants is their HLB values. The HLB value required toform
o/w nanoemulsions should be greater than 10 (39).Tween 80 and
Cremophor EL have HLB values of 15 and 1214, respectively, thus
fullling this requirement.
Propylene glycol, triacetin, Transcutol P, and MiranolC2M conc
NP were used as cosurfactants. Solutions of up to50% propylene
glycol caused no irritations to the rabbit eye,whereas the
undiluted application was associated with a weakconjunctival
redness (40,41). Triacetin, as reported by Hughes(33), is well
tolerated by the rabbit eye. Undiluted triacetinhas no or only
minor effect on the rabbit eye (34,35). On theother hand,
Transcutol P and the amphoteric surfactant,Miranol C2M conc NP, are
known as common emulsionexcipients suitable for dissolving or
dispersing lipophilic drugsin ocular preparations (42).
Thirty-six systems were prepared; the pseudoternary-phase
diagrams were mapped with the water titration methodat 25C to
identify the area of nanoemulsion regions.
As expected, the phase behavior was strongly inuencedby the
molecular volume of the oil incorporated within thenanoemulsion
(43). Depending on the chain length and onthe volume of the
molecule, penetration of the surfactant intothe hydrocarbon tails
will change the hydrocarbon chainvolume of the surfactant molecule
and, thus, the effectivecritical packing parameter (44). The
molecular volumes ofIPM, Miglyol, and triacetin are 529, 572, 188
3, respectively;accordingly, the nanoemulsion area was highest in
the case oftriacetin followed by IPM and then Miglyol, on using
PG,Transcutol, or Miranol as cosurfactants (Fig. 1).
On using triacetin as a cosurfactant, the highest nano-emulsion
area was found for IPM followed by Miglyol andthen triacetin (Fig.
1). This might be due to a decrease in thesolubilizing capacity of
the surfactants as triacetin acts here asan oil beside being a
cosurfactant. Oils, as triacetin, possess-ing a very small chain
length compared to the surfactanthydrophobe may be incorporated
into the nanoemulsiondroplet in a different manner to that
originally thought; inother words, an oil may be too small to act
as a cosurfactant,possibly preferring to locate more towards the
center of theaggregate (45).
The use of Cremophor EL surfactant resulted, in mostcases, in
smaller nanoemulsion existence areas compared toTween 80 (Fig. 1).
In order to increase the oil-solubilizing
capacity of Cremophor EL, a mixture of Cremophor EL andTween 80
(1:1 weight ratio) was tried. This resulted in anincrease in
nanoemulsion existence areas (Fig. 1). This mightbe due to the fact
that the exibility of surfactant layer and itsability to partition
at higher levels into the oilwater interfacemight be enhanced by
the combined surfactants; both ofwhich stabilized o/w nanoemulsion
formed (27,4649).Moreno et al. (27) reported that the combined use
of Tween80 and soybean lecithin was found to greatly increase the
oilcontent in microemulsions by threefold. Huibers and Shah(46)
also observed synergistic effects of surfactant combina-tions for
w/o microemulsions.
With respect to the cosurfactants used, Fig. 1 denotesthat the
use of the branched cosurfactant triacetin resulted,generally, in
the highest nanoemulsion existence area fol-lowed by PG and
Transcutol. Taha et al. (50) found thatoptimal w/o microemulsion
stability requires the cosurfactantmolecules to be branched and
short, such that they canoccupy sphere-like gaps between
interfacial surfactant mole-cules. On the other hand, Miranol
induced the least nano-emulsion area. This might be due to the
steric bulking ofMiranol which is expected to disturb the
interfacial packing ofthe nanoemulsion.
It is evident from Fig. 1 that for systems containing IPMor
Miglyol, increasing surfactant concentration in relation
tocosurfactant concentration led, generally, to an increase inthe
nanoemulsion existence area together with an increase inthe maximum
amount of oil that can be incorporated in thesystem. Kawakami et
al. (51) reported that increasing S toCoS ratio enhances micelle
formation, which consequentlyincreases the solubilization capacity
of the microemulsion(51). However, this trend was, generally,
reversed whentriacetin was used as the oil phase and PG or
Transcutol ascosurfactants since increasing S to CoS ratio from 1:1
to 4:1resulted in a decrease in the solubilizing capacity of
thenanoemulsion (Fig. 1). This might be due to the fact that, atthe
S to CoS ratio of 1:1, the cosurfactant was inserted intothe
cavities between the surfactant molecules exactly, and theformed
nanoemulsions had the maximum solubilizing capac-ity (51).
It is clear from the aforementioned results that thenanoemulsion
existence area can be modied according tothe type and amount of
oil, surfactant, and cosurfactant used.
It is noteworthy that the use of o/w nanoemulsions indrug
delivery is more straightforward than is the case withw/o
nanoemulsions. This is because the droplet structure ofo/w
nanoemulsions is often retained on dilution by abiological aqueous
phase, thereby permitting ocular adminis-tration (52). Our goal is
to formulate a dilutable ophthalmicnanoemulsion (o/w) having the
lowest possible surfactantcontent and optimal solubilization of the
hydrophilic andlipophilic components. Therefore, 17 dilutable
nanoemulsionswere formulated in which a S to CoS ratio of 3:1 and
watercontent of 77.78% w/w were used. The prepared nano-emulsions
were loaded with 2.22% w/w of dorzolamidehydrochloride (Table
I).
Accelerated Physical Stability Studies
Stability of nanoemulsions was studied using heatingcooling
cycles, centrifugation, and freezethaw cycle stress
811Nanoemulsion as a Potential Ophthalmic Delivery System for
Dorzolamide Hydrochloride
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tests. All nanoemulsions were stable after heatingcoolingcycles,
except NE 4 which displayed alteration in transparen-cy and was
thus excluded. The centrifugation test showed thatthe tested
nanoemulsions had good physical stability.Through freezethaw cycle
stress test, turbidity was observedwhen the nanoemulsions were
stored at 21C. Coagulationof the internal phase at low temperature
might have led tothis instability; however, these nanoemulsions
were easilyrecovered by storing at ambient temperature. Chen et al.
(53)reported that nanoemulsions should be kept above 15C
atleast.
Physicochemical Characterization of Nanoemulsions
Particle Size Analysis
The selected nanoemulsions showed a mean dropletdiameter of
8.412.7 nm (Table II). This small averagediameter was expected
since, in nanoemulsions, the cosurfac-tant molecules penetrate the
surfactant lm, lowering theuidity and surface viscosity of the
interfacial lm, decreasingthe radius of curvature of the
nanodroplets, and formingtransparent systems (54).
Fig. 1. Dependency of nanoemulsion existence area on types of
oil, surfactant, cosurfactant, andsurfactant to cosurfactant weight
ratio
812 Ammar, Salama, Ghorab and Mahmoud
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Rheological Measurements
It has been assumed that ophthalmic instillation of aformulation
should inuence the normal behavior of tears aslittle as possible.
Systems with low viscosity allow goodtolerance with little blinking
pain. In contrast, systems withenhanced viscosity, although less
tolerant, induce an increasein ocular contact time by reducing the
drainage rate and, as aconsequence, improve bioavailability (55).
Viscosity of eye-drops is required to be not higher than 20.0 mPa s
(56).
Dorzolamide hydrochloride nanoemulsions exhibited aNewtonian
behavior. The viscosity values of all dorzolamidehydrochloride
nanoemulsions were less than 10.0 mPa s(Table II).
It is obvious that increasing oil concentration of dorzo-lamide
hydrochloride nanoemulsions, on the expense of theSCoS mixture,
decreased the average viscosity of the nano-emulsion (NEs 6, 8, 11,
13, 15, and 17 versus NEs 5, 7, 10, 12,14, and 16, respectively,
Table II). It is also observed that theviscosity of the
nanoemulsions differed, generally, with thesurfactant used;
nanoemulsions containing Cremophor EL(NEs 1013) had higher
viscosity values relative to thosecontaining Tween 80 (NEs 59).
This might be becauseCremophor EL is semisolid while Tween 80 is
liquid at roomtemperature.
Refractive Index
Refractive index measurements detect possible impair-ment of
vision or discomfort to the patient after administra-tion of
eyedrops (57). Refractive index of tear uid is 1.340 to1.360 (58).
It is recommended that eye drops should haverefractive index values
not higher than 1.476 (59).
Table II depicts that dorzolamide hydrochloride nano-emulsions
had refractive index values ranging from 1.356 to1.358 which are
within the recommended values.
Surface Tension
The tear lm is destabilized when the surface tension ofeyedrops
is much lower than the surface tension of thelachrymal uid (60,61)
which ranges from 40 to 50 mN/m(62).
The surface tension of the prepared dorzolamide hydro-chloride
nanoemulsions ranged from 44.1 to 51.9 mN/m(Table II), which is
more or less similar to that of thelachrymal uid. Low surface
tension of nanoemulsionsguarantees good spreading effect on the
cornea and mixingwith the precorneal lm constituents, thus possibly
improvingthe contact between the drug and the corneal epithelium
(63).
pH
The ideal pH for maximum comfort when an ophthalmicpreparation
is instilled in the eye should be in the order of7.20.2 (64). In
some cases, the instillation of a solution witha pH different from
tears is irritant and causes a painfulsensation. This depends on
the volume instilled, the bufferingcapacity, composition of the
solution, and the contact timewith the eye surface (65). However,
different pH values canbe tolerated if the preparation is not or is
only very slightly
TableI.
Com
position
ofDorzolamideHydrochloride-LoadedNanoemulsions
Com
ponent
(%w/w)
Nanoemulsion
(NE)
12
34
56
78
910
1112
1314
1516
17
IPM
2.00
2.00
2.00
2.00
Triacetin
4.50
4.50
2.00
4.00
2.00
4.00
2.00
2.00
4.00
2.00
4.00
2.00
4.00
2.00
4.00
TW80
13.50
13.50
13.50
6.75
13.50
12.00
13.50
12.00
13.50
6.75
6.00
6.75
6.00
CrEL
6.75
13.50
12.00
13.50
12.00
6.75
6.00
6.75
6.00
PG
4.50
4.50
4.00
4.50
4.00
4.50
4.00
Transcutol
4.50
4.50
4.00
4.50
4.00
4.50
4.00
Miranol
4.50
Bidistilleddeionizedwater
was
used
astheaqueousphase(77.78%
w/w).Dorzolamidehydrochloridewas
used
inaconcentrationof
2.22%
w/w
IPM
(isopropylmyristate);
Tween80
(TW80);CremophorEL(CrEL);
PG
(Propylene
glycol)
813Nanoemulsion as a Potential Ophthalmic Delivery System for
Dorzolamide Hydrochloride
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Table II. Physicochemical Properties of Dorzolamide
Hydrochloride Nanoemulsions (meanSD)
NE
Physicochemical properties
Particle diameter (nm) Viscosity (mPa s) Refractive index
Surface tension (mN/m) pH Osmolality (mOsm/Kg)
1 11.80.8 4.630.32 1.3560.001 47.60.07 6.360.53 1,050312 12.70.9
4.500.28 1.3570.001 45.50.31 6.200.59 758353 12.50.7 4.460.24
1.3570.003 44.10.28 5.220.48 500365 9.20.8 4.540.29 1.3570.001
51.10.07 5.200.64 1,195276 9.70.6 4.190.29 1.3570.002 49.30.39
5.420.51 1,205307 8.80.7 5.190.56 1.3570.003 49.90.14 5.670.58
886258 9.20.7 4.220.26 1.3560.001 49.00.08 5.430.61 920259 8.40.4
4.680.26 1.3560.002 49.10.09 6.660.53 7091010 10.50.7 7.060.85
1.3570.002 51.30.13 4.340.47 1,2692511 10.50.8 5.560.22 1.3560.003
51.40.21 4.220.51 1,3203612 11.20.8 9.240.11 1.3580.003 51.70.16
4.340.31 1,0323113 11.10.8 6.650.13 1.3580.001 51.90.40 4.180.42
9692514 9.50.7 5.450.86 1.3570.001 49.60.14 4.550.47 1,2382615
10.10.6 4.790.23 1.3560.002 49.60.14 4.470.45 1,2472816 9.60.7
5.510.21 1.3570.003 50.30.13 4.520.48 9292517 9.80.5 4.960.26
1.3560.001 50.60.21 4.450.52 68729
Fig. 2. ad Release efciency for dorzolamide hydrochloride
solution, market product and nanoemulsionformulations
814 Ammar, Salama, Ghorab and Mahmoud
-
buffered because in this case the limited buffering capacity
ofthe tears is able to adjust the pH to physiologic levels
onadministration (57). The pH of therapeutic substances appliedas
eyedrops can vary from 3.5 to 8.5 (66).
The pH values of the prepared dorzolamide hydrochlo-ride
nanoemulsions were within the acceptable range (4.2 to6.7, Table
II).
Osmolality
The osmolality of lachrymal uid is between 280 and293 mOsm/kg on
waking. As a result of evaporation when theeyes are open,
osmolality may vary between 231 and446 mOsm/kg (67). Depending on
the drop size, solutionswith an osmolality lower than 100 mOsm/kg
or higher than640 mOsm/kg are irritant; however, the original
osmolality isrestored 1 or 2 min after instillation of the
nonisotonicsolution depending on the drop size (65).
The osmolality of the prepared dorzolamide hydrochlo-ride
nanoemulsions ranged from 500 to 1,320 mOsm/kg(Table II).
Nanoemulsions containing propylene glycol (NEs1, 5, 6, 10, 11, 14,
and 15) had higher osmolality compared tothose containing other
cosurfactants as Transcutol, triacetin,or Miranol (1,0501,320
versus 5001,032 mOsm/kg,Table II). Hasse and Keipert (63)
formulated ocular nano-emulsions with osmolality which ranged from
1,200 to2,400 mOsm/kg and found that they were nonirritant
usinghens egg test on the chorioallantoic membrane and Draizetest
on rabbits eyes.
In Vitro Drug Release Studies
Drug release from nanoemulsions containing IPM andTween 80
together with Transcutol or triacetin (NEs 2 and 3,respectively)
was lower (pNE 7>NE 5 (p0.05). There-fore, NE 5 (2%
TriacetinTW80PG) and NE 6 (4%TriacetinTW80PG) were selected for
subsequent studiesof the effect of oil concentration of the
nanoemulsion on thebioavailability of the drug.
Figure 2c comprises the results of the release study
fornanoemulsions containing triacetin, Cremophor EL, anddifferent
cosurfactants (NEs 1013). Comparing the releaseefciency of NE 10
with that of NE 12 containing the same oiland surfactant but
different cosurfactants (PG and Trans-
cutol, respectively) and of NE 11 with that of NE 12containing
the same oil and surfactant but different cosurfac-tants (PG and
Transcutol, respectively) reveals lack ofsignicant difference
(p>0.05). On the other hand, comparingthe release properties of
NE 10 with NE 11 and NE 12 withNE 13 which contain 2% and 4% w/w of
triacetin, respec-tively, denotes that nanoemulsions containing
higher contentsof triacetin had a higher release efciency (p
-
(70). This phenomenon could have signicant implications forthe
development of ocular systems for sustained delivery.
Based on the results of dorzolamide hydrochloriderelease
studies, NEs 3, 5, 6, 10, 12, and 14 were chosen forthe
bioavailability studies since these nanoemulsionsexhibited
relatively low release efciency and possiblesustained release of
the drug.
Ocular Irritation Studies
Clinical investigations revealed that the selected nano-emulsion
formulations (NEs 3, 5, 6, 10, 12, and 14) werenonirritant and
could be tolerated by the rabbit eye (averagetotal score
00.33).
Cross sections from the corneas of rabbits eye afterapplication
of the tested formulations together with a controlsection showed
that both corneal structure and integrity wereunaffected. Taking
into consideration that the rabbit eye ismore susceptible to
irritant substances than the human eye(71), this result would be
considered very promising.
Therapeutic Efficacy Studies
Figure 4 demonstrates the percentage decrease in IOP
ofnormotensive rabbits after administration of a single dose of
dorzolamide hydrochloride nanoemulsions (NEs 3, 5, 6, 10,12, and
14), drug solution, and the market product. It isobserved that
nanoemulsions, in contrast to the drug solutionor the market
product, induced a pronounced decrease inIOP already half an hour
postinstillation of the eyedrops. Thisindicates that formulation of
dorzolamide hydrochloride as ananoemulsion led to a faster onset of
drug action compared tothat of either drug solution or the market
product.
It is also observed that the mean maximum percentagedecrease in
IOP occurred 0.5 to 1.6 h after instillation ofnanoemulsions, drug
solution, or the market product (Fig. 4).In this respect,
nanoemulsions 3 (2% IPMTW80Triacetin)and 6 (4% TriacetinTW80PG) had
higher values (p0.05).
With respect to the duration of drug action, it is evidentthat
the effect of nanoemulsions was continued for up to 46 h, while
that of the drug solution and the market productlasted for only 3
and 4 h, respectively (Fig. 4). This wouldindicate that dorzolamide
hydrochloride nanoemulsionsexhibited a more prolonged effect
compared to either drugsolution or the market product.
Fig. 4. Percentage decrease in IOP after administration of
dorzolamide hydrochloride nanoemulsions, drug solution, andthe
market product
Table III. Pharmacodynamic Parameters after Administration of
Dorzolamide Hydrochloride Solution, Nanoemulsion Formulations, and
theMarket Product (meanSD)
Formula
Pharmacodynamic parameter
Max % decrease in IOP tmax (h) AUC010 h MRT
Drug solution 20.934.17 1.20.4 38.738.38 1.730.06NE 3 32.835.82
1.20.5 124.9823.35 2.410.08NE 5 26.687.54 1.10.6 85.4917.15
2.010.05NE 6 37.237.89 1.60.5 128.1416.32 2.550.15NE 10 25.716.59
0.90.2 130.5327.43 2.860.25NE 12 21.632.86 0.50.0 87.2414.99
2.290.14Market product 23.115.11 1.10.2 58.2010.90 2.060.06
AUC010h (area under the percentage decrease in IOP time curve);
MRT (mean residence time); NE (nanoemulsion)
816 Ammar, Salama, Ghorab and Mahmoud
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Table III demonstrates that the time for maximumpercentage
decrease in IOP (tmax) for the tested formulations,drug solution,
and the market product varied from 0.5 to1.6 h. Nanoemulsion 12 had
the least value for tmax.Dorzolamide hydrochloride nanoemulsions,
with the excep-tion of NEs 6 and 12, drug solution, and the market
productshowed similar values for tmax (p>0.05).
Concerning the parameter of area under the percentagedecrease in
IOP time curve (AUC010h, Table III), it isevident that the value of
this parameter, if compared to thatof the drug solution, follows
the order: NEs 3 (2% IPMTW80Triacetin), 6 (4% TriacetinTW80PG) and
10 (2%TriacetinCrELPG)>NEs 5 (2% TriacetinTW80PG), 12(2%
TriacetinCrELTranscutol), and 14 (2% TriacetinCrEL+TW80PG)>drug
solution (p NEs 3 (2% IPMTW80Triacetin) and 12 (2%
TriacetinCrELTranscutol) > NE 5 (2% TriacetinTW80PG) and
themarket product>NE 14 (2% TriacetinCrEL+TW80PG)and drug
solution (p
-
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819Nanoemulsion as a Potential Ophthalmic Delivery System for
Dorzolamide Hydrochloride
Nanoemulsion as a Potential Ophthalmic Delivery System for
Dorzolamide HydrochlorideAbstractINTRODUCTIONMATERIALS AND
METHODSMaterialsMethodsConstruction of Pseudoternary-Phase
DiagramsPreparation of Dorzolamide Hydrochloride
NanoemulsionsAccelerated Physical Stability StudiesPhysicochemical
Characterization of NanoemulsionsIn Vitro Drug Release
StudiesOcular Irritation StudiesTherapeutic Efficacy Studies
RESULTS AND DISCUSSIONConstruction of Pseudoternary-Phase
DiagramsAccelerated Physical Stability StudiesPhysicochemical
Characterization of NanoemulsionsParticle Size AnalysisRheological
MeasurementsRefractive IndexSurface TensionpHOsmolality
In Vitro Drug Release StudiesOcular Irritation
StudiesTherapeutic Efficacy Studies
CONCLUSIONReferences
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