nanoemulsion as a potential ophthalmic delivery system for dorzolamide hydrochloride.pdf

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











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




























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



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




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)






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


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)






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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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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