BIOAVAILABILITY AND OCULAR DISPOSITION OF KETOROLAC ...

Bull. Pharm. Sci., Assiut University, Vol. 30, Part 2, 2007, pp. 275-297.

ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــReceived in 10/12/2007, Received in revised form in 29/12/2007 & Accepted in 30/12/2007

BIOAVAILABILITY AND OCULAR DISPOSITION OFKETOROLAC TROMETHAMINE FROM VARIOUSOPHTHALMIC PREPARATIONS

Hamdy M. Abd El-Aleem1, Farouk M. Sakr1, Osama A. Soliman1,Hatem, El-Awady2 and Germeen N. Salama1

1Department of Pharmaceutics, Faculty of Pharmacy, MansouraUniversity, Mansoura, Egypt

2Ophthalmic Center, Faculty of Medicine, Mansoura University,Mansoura 35516, Egypt

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Ketorolac tromethamine was formulated in different ophthalmicpreparations namely; eye drops, gels, ocuserts and ointments usingthe following carriers; sodium carboxymethyl cellulose (sod.CMC), polyvinyl alcohol (PVA), hydroxypropylmethyl cellulose(HPMC), absorption base and gelatin. The ophthalmicformulations were prepared containing 0.5% of the drug. Allprepared formulae containing the drug were subjected to stabilitystudy and release characteristics. Also, the effect of these drugcarriers on the uptake and ocular disposition of ketorolactromethamine by the eye tissues of rabbits (conjunctiva, cornea,iris-ciliary body and aqueous humor) was studied.

The obtained results revealed that, the released amounts fromthe ophthalmic preparations after six hours can be arranged in thefollowing order; Eye drops > ocuserts > gel > ointments. Sod CMCeye drops exhibited the higher rate of release than sod CMC gel,PVA and absorption base ointments. Gelatin ocuserts exhibited thehigher rate of release than HPMC ocuserts and carbopol 934 &sod CMC ocuserts. The release rate of the drug from eye drops and

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PVA ointment obeys first order kinetics, while, other preparationsobey Higuchi diffusion model with non-Fickian kinetics.

Most formulations (including eye drops) could be stored for 6months at 25°C, 35°C, 45°C without physical or chemicaldegradations of the drug. However, PVA and absorption basesshowed some changes in the drug content (t90 was 2.84 months forboth at 45°C). The decomposition rate of ketorolac tromethaminefollowed the first-order degradation kinetic. The highest stableformulae are, sod CMC eye drops and gel (t90 values were, 151.9and 91.18 months, respectively at 45°C). The highest concentrationof the drug (Cmax) from all tested formulations is provided inconjunctiva followed by cornea, iris-ciliary body, then aqueoushumor. The peak time for maximum drug concentration (Tmax)from sod CMC eye drops was two hours. While, that for sod CMCgel, PVA ointment and gelatin ocuserts was three hours in alltissues after the application of the tested formulations. In addition,the total availability of the drug from the tested formulations was inthe following order: gelatin ocuserts > sod CMC gel > PVAointment > sod. CMC eye drops.

INTRODUCTION

The topical application ofophthalmic preparations is consideredthe preferred way to achieve thetherapeutic levels of drug used to treatthe ocular diseases. Ocularbioavailability of drugs from eyedrops is poor, due to pre-corneal lossfactors, including tear dynamics, non-productive absorption, transientresidence time in the cul-de-sac, andthe relative impermeability of thecorneal epithelial membrane. Only, asmall fraction of a topically applieddose reaches the inner eye with theactual amount dependent on thephysicochemical properties of thedrug and its vehicle1

.

An ideal ocular drug-deliverysystem should be able to control the

delivery of drugs of varyingphysicochemical properties andprovide sustained therapeutic action.In order to overcome the largedrainage factor imposed by the eyeand compensate, at least in part, forthe low permeability of cornealtissues, several strategies have beenexplored to develop extended-duration drug delivery systems. Theseinclude, boosting the eye dropviscosity or using ointments and gelsas conventional improvements on eyedrops. Also, controlled/continuousdrug delivery systems includingocuserts was developed and underdeveloped2.

Various types of vehicles wereinvestigated in the rabbits eye tissues,for their ability to maintain ocularresidence time of some drugs such as


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sodium cromoglycate. Drugdisposition in various ocular tissuesand fluid compartments is discussedfor each type of vehicle. Theoleaginous base was found toconcentrate the drug in theconjunctiva more than other eyetissues3.

ketorolac tromethamine is a nonsteroidal anti-inflammatory agent thatinhibits the activity of cyclo-oxygenase, one of the two majorenzymes responsible for theconversion of arachidonic acid toinflammatory substances. Also, it iseffective in relieving ocular itchingand the hallmark symptoms ofallergic conjunctivitis4.

Madhu, et al.5, evaluated theeffect of benzalkonium chloride/ethylene diamine tetraacetic acid(BAK/EDTA) on the ocular bio-availability of ketorolac tromethaminefollowing ocular instillation to normaland de-epithelized cornea of rabbitseyes. They concluded that, the ocularbioavailability was not altered byBAK/EDTA in rabbits with intactcornea, but it was decreased byBAK/EDTA in rabbits with de-epithelized cornea.

Ling & Combs6, studied the ocularbioavailability and tissue distributionof ketorolac tromethamine in rabbitsfollowing the instillation ofradiolabeled ophthalmic solutions[14C]. Also, Malhotra & Majumdar7,studied in vivo ocular availability ofketorolac following ocular instillationof aqueous, oil, and ointmentformulations to normal corneas ofrabbits.

The aim of this work was toformulate ketorolac tromethamine invarious ophthalmic preparations(drops, gel, ointments and ocuserts).All the prepared formulae weresubjected to the study of the releasecharacteristics and stability of thetested drug at different storageconditions.

Also, the determination of thedrug concentration in different eyetissues (conjunctiva, cornea, iris-ciliary body, and aqueous humor)using certain selected preparations atdifferent time intervals were be of ourgoal.

EXPERIMENTAL

Materials• Ketorolac tromethamine (Ranbaxy

Laboratories, Ltd., New Delhi,India).

• Sodium carboxymethyl cellulose,propylene glycol, potassiumdihydrogen phosphate and gelatin(S.D. fine chemicals, Mumbai,India).

• Sodium dibasic phosphate (Prolabo,Paris, France).

• Hydroxypropylmethyl cellulose(Dow Chemical Company, U.S.A).

• Carbopol 934, polyvinyl alcohol,lanolin (B.P.), yellow softpetrolatum, benzalkonium chloride,disodium edetate, sodiummetabisulphite (BDH ChemicalLtd, G.B. Liverpool, England).

• Ethyl acetate, N-hexane, methanol(Adwic, El Nasr pharmaceuticalchemicals, Co., Egypt).

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• Cellulose membrane, spectrapore,M.W., cut off 12000-14000 (FisherSci. Co., Pittsburgh, U.S.A).

• Experimental animals: albinorabbits of 1.8-2 kg.

• All other chemicals and solventswere of analytical reagent grade.

Equipment• U.V. spectrophotometer (Shimadzu,

UV-150-02, Sersakusho, Ltd,Kyoto, Japan).

• pH-meter (Beckman Instrumentsfullerton, CA 92634, U.S.A).

• Rotary viscometer (Haake Inc.,Germany).

• MSE minor centrifuge (MSEscientific instruments, ManorRoyal, Crawley RH/0200 Sussex,England).

• Thermostatically controlled shakingwater bath (Grant instrumentCambridge Ltd., BarringtonCambridge B2, 5002, England).

• Ovens (Gering model SPA-Gelman,Instrument No. 16414, Germany)

MethodologyPreparation of sodium carboxy-methyl cellulose eye drops

Ketorolac tromethamine wasdissolved in double distilled water ina concentration of 0.5% w/v in which0.01% benzalkonium chloride, 0.03%sodium metabisulphite and 0.1%EDTA were previously dissolved.This was done by the aid of a highspeed stirrer until completedissolution. Then, sod CMC polymerwas added in a concentration of

0.25% on the surface of the previoussolution. The mixture was stirred untilcomplete dissolution (Table 1).

Preparation of sodium carboxy-methyl cellulose gel

Ketorolac tromethamine wasdissolved in water in which 0.01%benzalkonium chloride, 0.03%sodium metabisulphite and 0.1%EDTA were previously dissolved, bythe aid of a high speed stirrer, until asolution is formed, then sod CMCwas added to the surface of theprevious solution in the concentrationof 2.1% which gave a gel with goodrheological properties (Table 1).

Preparation of ointments

Preparation of polyvinyl alcoholointment

Ketorolac tromethamine wasdissolved in the water firstly by theaid of a high speed stirrer, until asolution was formed; then, PVA wasadded with gentle heating in a waterbath, until the required ointment wasobtained (Table 1).

Preparation of absorption baseointment

The ointment was prepared firstlyby fusion method using 10% lanolin,90% petrolatum. Then, the drug wasadded to the melted base usinggeometrical mixing at lowtemperature with continuous stirringon cold until obtaining a homogenousdistribution of the drug in theointment (Table 1).


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Table 1: The composition of ketorolac tromethamine ophthalmic preparations ina percent of w/w.

Drops Gel Ointment OcusertsFormulation

IngredientsSod

CMCSod

CMCPVA

Lanolin-petrolatum

HPMC GelatinCarbopol934 sodCMC

Ketorolactromethamine

0.5 0.5 0.5 0.5 0.5 0.5 0.5

Sod CMC 0.25 2.1 1.2

PVA 15

Yellow softparaffin

89.36

Lanolin 10HPMC 5Propyleneglycol

20 20 20

Gelatin 5

Carbopol 934 0.5

Distilled water 99.11 97.26 84.36 74.36 74.36 77.66

Where;

Sod CMC = sodium carboxymethyl cellulose

PVA = polyvinyl alcohol

HPMC = hydroxypropylmethyl cellulose

Benzalkonium chloride, sodium metabisulphite and EDTA were added inconcentrations of 0.01, 0.03 and 0.1%, respectively.

Preparation of ketorolac trom-ethamine ocuserts

Ketorolac tromethamine 0.5% wasdissolved in 20 ml distilled water.This solution was added to solutionsof HPMC (5% w/v), mixture ofCarbopol 934 0.5% w/v & 1.2%(w/v) of sod CMC or gelatin (5%w/v). These solutions contain 0.01%benzalkonium chloride, 0.03%

sodium metabisulphite and 0.1%EDTA. Then 20 ml of propyleneglycol was added to the samesolution. Equal volumes of theprepared solutions were transferredinto polytetrafluorethylene (PTFF)moulds. Each mould was coveredwith an inverted funnel (stem orificediameter 6.9 mm) to control solventevaporation. The solvent was

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permitted to evaporate for 48 hours atambient temperature. The formedfilms were transferred to desiccatorscontaining silica gel, where it wasstored for another 24 hours beforeuse8. The prepared ocuserts 0.4-0.5mm thickness) were cut in the form ofcircular discs, each of one cmdiameters. The ocuserts wereindividually sealed in foil sachetsuntil used (Table 1).

Physicochemical properties of theophthalmic preparations

Determination of the viscosity ofprepared ophthalmic preparations

The viscosity values of differentpreparations (eye drops, gel andointments) were determined usingrotary viscometer which has beencalibrated before use. Thetemperature was maintained at37°C±0.5°C. One gram of eachformulation containing the drug wasplaced on the plate of viscometer (2.9cm in diameter) and Cone (2.8 cm indiameter). The torque values (S) weredetermined for each "N" value(speed). Then, viscosity is calculatedusing the following equation:

NG.S=

Where;η = Viscosity in mpa.s (mpa. s = 1

centipoise)G = Instrumental factor = 14200

(mpa.s/scala grad. minute)S = Torque (scale grad.)

N =. Speed (rpm)

Determination of pH of theprepared formulations

One gram of each formula wasdispersed in 30 ml of distilled water.The pH was determined using pHmeter. For ocuserts, one ocusert ofeach preparation was used.

Determination of ketorolactromethamine content in theprepared formulations

One gram from each formula wasaccurately weighed and placed in atightly closed volumetric flaskcontaining phosphate buffer pH 7.4and was heated at 37°C±0.5°C on athermostatically controlled waterbath. Then, the volume wascompleted to 100 ml with the bufferand the contents of each flask wereshaken for 15 minutes. Then, 10 mlwas taken, and centrifuged. Then thesupernatant was filtered and measuredat λmax 322 nm. For ocuserts, oneocusert of each preparation was usedto determine its drug content.

In-vitro release study of ketorolactromethamine from differentophthalmic preparations

The release of the drug fromophthalmic preparations was carriedout using the dialysis method9. Fivegrams of ketorolac tromethamine eyedrops, gel and ointments wereaccurately weighed and spread on acellophane membrane to occupy acircle of 5 cm in diameter (or threeocuserts). The loaded membrane waspreviously soaked in a sufficientamount of the dissolution mediumwhich is phosphate buffer of pH 7.4.


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The loaded membrane with the eyedrops, gel, ointments or ocuserts wasstretched over one end of a dialysisglass tube which was made watertight by a rubber band. The invertedtubes were suspended so that, themembrane was just below the surfaceof 100 ml of phosphate buffer of pH7.4 at 37°C±0.5°C contained in abeaker of 250 ml capacity.

Samples each of one ml werewithdrawn from the dialysis glassbeakers after time intervals: 5, 10, 20,30, 60, 120, 180, and 240, 300 and360 minutes. The volume of thedissolution medium in the beaker wascompensated by adding an equalvolume of phosphate buffer pH 7.4directly after withdrawal of eachsample. The samples were dilutedwith buffer solution pH 7.4, and thenmeasured spectrophotometrically atλmax 322 nm. The concentrations werecalculated on the basis of the standardcalibration curve. Blank experimentswere carried out using non-medicatedformulations and served as control.The experiments were triplicated andthe average of three determinationsfor the amount of drug released wasgraphically illustrated as a function ofthe time.

Stability studyThe ophthalmic formulations were

subjected to accelerated stabilitystudy at 25, 35 and 45°C for sixmonths. Samples were withdrawn atpredetermined time intervals; 1, 2, 3,4, 5, and 6 months, then examined forphysical characteristics (appearance,pH and viscosity), release profiles and

drug content which were measuredspectrophotometrically at λ max 322nm. Also, the kinetic study of thedrug in the pharmaceuticalpreparations was investigated10.

In-vivo study of ketorolactromethamine from differentophthalmic preparations in rabbitseyes

Tested formulationsThe following preparations were

selected according to the drugreleased and stability of ketorolactromethamine at different conditions.1- 0.5% (w/v) ketorolac trom-

ethamine in sod. CMC eye drops.2- 0.5% (w/w) ketorolac trom-

ethamine in sod. CMC gel.3- 0.5% (w/w) ketorolac trom-

ethamine in PVA ointment.4- 0.5% (w/w) ketorolac trom-

ethamine in gelatin ocusert.

Construction of calibration curveof ketorolac tromethamine inrabbits eyes tissues extract

Fifty mg of the drug was dissolvedin 100 ml filtered extract of separatedrabbits’ eye tissues and aqueoushumor to give a concentration of 500µg/ml. This rabbits eye extract wasprepared by extracting the groundrabbits eye tissues twice with 5 ml ofmixture composed of ethyl acetateand n- hexane in a ratio of 30:70,respectively. These combined extractswere evaporated, then the residueswere reconstituted in one ml ofmethanol. The solutions werecentrifuged and filtered. Differentvolumes of this prepared stock

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solution were transferred tovolumetric flasks, each completed to25 ml with the extract to giveconcentrations of 2, 4, 6, 8, 10, 12,14, 16, 18 and 20 µg/ml. Ultravioletabsorbance of the differentconcentrations were measured at thepredetermined λmax 322 nm. Theobtained results showed a directrelationship between theconcentrations and absorbancevalues11.

Application of ketorolactromethamine formulations intorabbits eyes

Individual doses of 100 mg ofeach preparation were accuratelyweighed (eye drops, gel, ointment orone gelatin ocusert), then immediatelyand carefully transferred by micro-spatula into the center of the lower lid(cul-de-sac) of each eye of the albinorabbits weighed 1.8-2 kg. The lowereyelid was gently moved to spread thedose on corneal surface. Duringdosing, care should be taken not toirritate the eye or touch the cornealsurface. All rabbits were kept in up-right position in restraining boxes.Four rabbits were used fordetermination of the amount of drugdisposed in different eye tissues andaqueous humor at each time interval.

Ketorolac tromethamine ocularconcentration was determined at 1, 2,3, and 5 hours after drug application.Therefore, four eyes which receivedthe medicament in each testedformula were used for determinationof drug concentration. For eachanimal, one eye was loaded with the

tested formulation, while, the otherwas loaded with the plain vehiclesand served as a control.

Separation of eye tissuesFollowing the killing of the rabbits

and separation of their conjunctivalsurfaces, one ml of aqueous humorwas aspirated from the anteriorchamber using micrometer syringe.Then, a single incision was made witha scalpel at the corneal margin andthe entire cornea was excised. Thewhole cornea and conjunctivalsurfaces were rinsed with the leastamount of normal saline. The anteriorsegment tissues; conjunctiva, cornea,iris-ciliray body and aqueous humorwas obtained in that order. Thesurgical procedures on each eye werecompleted within 10 minutes ofsacrificing the animal. So that, anyerror due to redistribution of the drugduring the time required obtainingocular tissue samples wereminimized. Each individual tissuewas transferred into scintillation vialand the net weight of tissue wasdetermined using an analyticalbalance.

Extraction of ketorolac trom-ethamine from different tissues andfluid of rabbits eyes

The concentration of ketorolactromethamine in different eye tissuesand fluid was determined after 1, 2, 3and 5 hours of the application of eachformulation. At each time interval,conjunctiva, cornea, and iris-ciliarybody of each eye were separatedimmediately, rinsed with isotonicsaline solution weighed and ground


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with powdered glass. The groundtissues were extracted twice with 5 mlof mixture composed of ethyl acetateand n- hexane in a ratio of 30:70,respectively6. On the other hand, anaqueous humor was mixed twice with5 ml of the extracting solvent. Thesecombined extracts were evaporated,and then the residues werereconstituted in one ml of methanol.

The solutions were centrifuged at9000 rpm for 30 minutes. After that,filtered using 0.45 µm milliporefilters. This solution was used for thedetermination of ketorolactromethamine concentration in eachrabbit’s eye tissues at each timeinterval.

Determination of ketorolactromethamine in rabbit’s eyetissues and fluid from differentophthalmic preparations

The determination of drugconcentrations in different eye tissuesand fluid had been done by taking thesupernatant and measuring the drug atλmax 322 nm using UV spectro-photometer. Each sample was run intriplicate. The drug concentration wascalculated from the calibration curve.The spectrophotometeric methodreported by Bushby and Hitching12.

Soliman11, determined the oculardisposition of sulfamethoxazole andtrimethoprine mixture in rabbit’s eyesusing spectrophotometeric method.Recently, El-Dahan13, also, provedthat, the spectrophotometeric methodused for determination ofenrofloxacin in rabbit’s eyes tissuesand fluids was in agreement with the

results obtained from the micro-biological method.

RESULTS AND DISCUSSION

Physicochemical properties ofketorolac tromethamine preparations.

The viscosity of prepared eyedrops, gel and ointments

The values of viscosity of eye gel,drops and ointments were illustratedin Table (2). It was noted that, sodCMC gel showed a higher viscosityvalue than sod CMC eye drops whichare 1256, 1019.8 m pa.s, respectively.Also, for ointments, PVA showed thehighest viscosity value (2745.75 mpa.s). While, absorption baseointment showed the lowest viscosityvalue (784.5 m pa.s).

pH of the prepared formulationsThe pH values of prepared

formulae were shown in Table (2).These values of all formulations werein the range from 6.13 to 6.93. So, theeye can tolerate these formulationswith little discomfort. It was provedthat, alkaline pH induced greaterlacrimation than acidic pH in albinorabbits14.

Drug contentThe obtained results are presented

in Table (2). The percentage ofketorolac tromethamine content in allpreparations was in the range of98.5% to 100% of the drug contentwhich complies with the officialrequirements15.

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Table 2: Physical evaluation of different ophthalmic preparations containingketorolac tromethamine (0.5% w/w).

Parameter

FormulaepH

Drugcontent

(% W/V)

Viscosity(m pa.s)

Drops Sod. CMC 6.22 100 1019.8Gel Sod. CMC 6.58 100 1256

PVA 6.2 99.8 2745.75Ointments Absorption base 6.87 98.5 784.5

HPMC 6.5 100 -Gelatin 6.13 100 -OcusertsCarbopol 934 & sod CMC 6.93 99.9 -

In-vitro release characteristics ofketorolac tromethamine fromophthalmic preparations

Figures (1&2) illustrate the releasecharacteristics of ketorolac trom-ethamine from different ophthalmiceye drops, gel, ointments andocuserts. The maximum amountsreleased of the drug after six hoursfrom sod CMC drops and gel were;23.12, 13.27 mg, respectively. While,the amounts released of the drug fromophthalmic ointments were; 10.29,0.296 for PVA and absorption baseointments, respectively. Also, theamounts released from ocuserts were14.27, 18.48 and 8.97 for HPMC,gelatin and carbopol 934 & sod CMCocuserts, respectively.

The obtained results showed that,sod CMC eye drops exhibited ahigher rate of drug release than sodCMC gel. This may be due to the lowviscosity of the eye drops comparedto gel which permits the rapid release

of the drug from the preparation16. Inaddition, sod CMC eye drops showeda higher release rate than PVAointment, this may be due to the highviscosity value of PVA ointmentcompared to sod CMC eye drops,which may retard the release of thedrug from the base. This finding wasin agreement with Hirano et al.17,who studied the effect of the viscosityon the release profile of ibuprofenfrom different bases. They concludedthat, the higher the viscosity of thebase, the slower the drug release.Also, they reported that, the increaseof viscosity of the vehicle isaccompanied by a reduction in theamount of the drug released, andadded that, the increase in theviscosity of the base will impart amore closed barrier resisting drugtransfer to the outer surface and thiswill retard the movement of the drugmolecules to the external releasemedium.


286

0 100 200 300 4000

5

10

15

20

25S o d C M C e ye d ro p sS o d C M C g e lP VA o in tm e n tAb so rp tio n b ase

T im e ( m in )

Con

cent

rati

on(m

g/1 0

0 m

l)

Fig. 1: In-Vitro release profiles ofketorolac tromethamine fromvarious ophthalmic preparationsat 37°C in phosphate buffer ofpH 7.4.

0 1 0 0 2 0 0 3 0 0 4 0 00 .0

2 .5

5 .0

7 .5

1 0 .0

1 2 .5

1 5 .0

1 7 .5

2 0 .0H P M CG e la t inC a r b o p o l 9 3 4 & S o d C M C

T im e ( m in )

Con

cent

rati

on(m

g/1 0

0 m

l)

Fig. 2: In-Vitro release profiles ofketorolac tromethamine fromvarious ophthalmic ocuserts at37°C in phosphate buffer of pH7.4.

On the other hand, the absorptionbase ointment showed the leastrelease rate compared to the allprepared formulations, despite ofhaving lower viscosity value. Thismay be due to higher water solubilityof the eye drops, gel and ocuserts andthe higher lipophilicity of theointment base. This finding was inagreement with that of El-Shaboury,et al.18, who proved that, sod CMCgel exhibited the highest rate of drugrelease, while, hydrocarbon baseshowed the lowest one. The authorattributed this result to the higherwater solubility of sod CMC gel andthe higher lipophilicity as well as thepoor water miscibility of thehydrocarbon base. So, the amounts ofthe drug released from ophthalmicdrops, gel, ointments can be arrangedin the following order; Eye drops >gel > PVA ointment > absorptionbase ointment.

The amount of ketorolactromethamine released from eyedrops was higher than that releasedfrom ocuserts, then followed by eyegel and finally, eye ointments whichshowed the lowest amounts of thedrug released. This finding was inagreement with that of El-Dahan13,who proved that, the amount ofnaproxen released from eye dropswas higher than that released fromocuserts and followed by that releasedfrom eye gels.

The results of the drug releasefrom ocuserts showed that, theamount of the drug released fromgelatin ocuserts was higher than thatreleased from HPMC ocuserts, then

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finally carbopol 934 & sod CMCocuserts which showed the lowestamount of the drug released fromocuserts. So the ocusert bases couldbe arranged in this order according tothe higher drug release; Gelatin >HPMC > Carbopol 934 & sod. CMC.

Shaker19 proved that, phenyl-ephrine hydrochloride could beformulated in the form of ocusertsusing different polymers, and thehighest amount of drug released wasshown by HPMC followed by MCocular film then Eudragit. This maybe due to the hydrophilic nature aswell as water solubility of HPMC andMC films in contrast to the waterinsolubility of Eudragit ocular films.

Manvi et. al.20, exerted manyefforts to prepare ocuserts ofketorolac tromethamine using gelatin.Their results indicated that, gelatinpolymeric ocuserts containingketorolac tromethamine achieved theobjectives of increased contact time,prolonged release, decreasedfrequency of administration and thusimprove the patient compliance.

Kinetic analysis of the release dataIn order to determine the

mechanism of ketorolac trom-ethamine release from differentophthalmic preparations, the in-vitrorelease data were analyzedmathematically according to: zero-order, first order and Higuchidiffusion model. The results wererepresented in Table (3).

From the kinetic estimation of therelease data, it was found that, thebest fitting linear relations with the

highest correlation coefficient werefound with Higuchi diffusionequation for all formulations (0.998,0.9877, 0.963, 0.961, and 0.992 forsod CMC gel, absorption baseointment, HPMC ocuserts, gelatinocuserts and carbopol 934 & sodCMC ocuserts, respectively, excepttwo formulations were found to obeyfirst-order release kinetics which aresod CMC eye drops and PVAointment with correlation coefficientsof (0.994, 0.999), respectively.

The slopes of lines when log Q/Awas plotted versus log t was morethan 0.5. So, the release pattern canbe described by non-Fickian diffusionkinetics. From the obtained results, itwas found that, sod CMC eye dropsshow the highest diffusion coefficientand permeability coefficient (10x10-4,0.019 ug. cm-2. min-1

, respectively).While, the lowest values for diffusioncoefficient and permeabilitycoefficients were obtained fromabsorption base and carbopol 934 &sod CMC ocuserts. The lowest valueof partition coefficients was shown bysod CMC drops (0.095), while, thehighest value was shown by theabsorption base ointment (9.9).

Stability studyThe different formulae were found

to be physically stable as they did notshow any color or odor changes orsyneresis after the storage. While, forgelatin ocuserts, it was noted that,after the storage period it washardened and cracked. The pH of theprepared formulae remainedunchanged during all the storageperiod at different temperatures.


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Table 3: Kinetic modeling of ketorolac tromethamine released from different preparations through cellulose membranebarrier.

Drops Gel Ointments OcusertsFormulation

Parameter Sod CMC Sod CMC PVAAbsorption

baseHPMC Gelatin

Carbopol934

Sod CMC

ro 0.935 0.9863 0.9962 0.9692 0.879 0.906 0.97Zeroorder Ko (mg.min-1) 0.0675 0.0341 0.0274 0.0007 0.04 0.04 0.024

r1 0.9949 0.9974 0.9994 0.9695 0.0949 0.96 0.98

K1 0.0075 0.0018 0.0013 0.0011 0.0035 0.004 0.0013Firstorder

t ½ (min) 92.4 376.25 501.6 602 2.13 173.25 533

rh 0.985 0.998 0.9789 0.9877 0.963 0.961 0.992Q/A vs.t ½ D (ug. cm-2.

min-1)

10 x 10-4 5 x 10-4 2 x 10-4 1 x 10-4 4 x 10-4 5 x 10-4 1 x 10-4

rh 0.9937 0.9985 0.9891 0.9879 0.95 0.88 0.994

Higuchidiffusionequation Log

Q/A vs.log t Slope 0.755 0.6191 0.6665 0.4661 0.92 0.6 0.8

Pm x10-4 (ug. cm-2min-1) 0.009 0.0009 0.0007 0.0001 0.001 0.001 0.0006

Kp 0.095 0.096 0.19 9.90 0.14 0.113 0.339

Where;K = Rate constantKp = Partition coefficient.R = Correlation coefficient.D = Diffusion coefficient.Pm = Permeability coefficient

289

The U.V scanning revealed theabsence of any interaction ordecomposition products of the drug inthe tested preparations. With regardto ketorolac tromethamine content,sod CMC eye drops containing0.25%, showed drug content of99.7% after their storage for sixmonths at 25°C, while those stored at35°C and 45°C showed drug contentof 99.65% and 99.6%, respectively.So, there are small or nearly nochanges in the drug content of theprepared eye drops throughout thestudy period. This was in accordancewith the results obtained bySoliman11, who proved that, atropinesulfate eye drops containing 0.5% sodCMC and 10% PVP K25 could bestored for long time (two years)without degradation of the drug orcomplexation of their components.

In addition, the tested gel of sodCMC, which was subjected toaccelerated stability tests, showed alittle decrease of the drug contentpercentage to 99.5% of its initialcontent after storage for 6 months at45°C. So, the drug content in this geldid not significantly changed whenstored at 25°C, 35°C and 45°C for 6months. In another word, the drugwas nearly equally stable in the testedophthalmic gel.

The ocuserts containing 5%HPMC, showed drug content of96.8% after storage for six months at25°C, while, those stored at 35°C and45°C showed drug contents of 96.1%and 95.2%, respectively. Also, forgelatin ocuserts, it showed drugcontent of 97.8% after the storage for

six months at 25°C, while, thosestored at 35°C and 45°C showed drugcontents of 97.5% and 97%,respectively. Finally, for carbopol 934& sod CMC, it showed drug contentof 99.1% after storage for six monthsat 25°C. While, those stored at 35°Cand 45°C showed drug contents of99% and 98.5%. So, there was aslight change or nearly no changes inthe drug content in the preparedocuserts throughout the study period.

The percentage of ketorolactromethamine content in thesepreparations was in the range whichcomplies with the officialrequirements (USP XXIV). This wasin agreement with El-Dahan13, whoproved that, naproxen could beformulated in ophthalmic drops, gelsand ocuserts using sod. CMC andHPMC. In addition, the preparedformulae were stable after storage forsix months at the temperatures of25°C, 35°C and 45°C.

On the other hand, the ointmentsshowed great changes in thepercentages of the drug content.These results proved that, after sixmonths, the percentages of the drugcontents were 81% (for PVA base)and 78.65% (for absorption base).The degradation may affect thebiological activity and causes adecrease in the initial concentration ofthe product which may be permittedwithin a range of not more than10%19.

The kinetic analysis of the data todetermine the mechanism ofdegradation, t50 and t90 was illustratedin Table (4). Analysis of data


290

Table 4: Degradation rate constant (K), half life (t50) and expiration date (t90) of ketorolac tromethamine ophthalmicpreparations after storage for six months at different temperatures.

Temperature ofstorage 250C 350C 450C

Parameter

Formulae

KMonth-1

t50

Montht90

MonthK

Month-1t50

Montht90

MonthK

Month-1t50

Montht90

Month

Sod CMC eye drops 0.6×10-3 1003 151.9 0.6×10-3 1003 151.9 0.6×10-3 1003 151.97

Sod CMC gel 0.6×10-3 1003 151.9 1.1×10-3 601.8 91.18 1.1×10-3 601.8 91.18

PVA base ointment 27×10-3 25.07 3.7 34×10-3 20.06 3.03 36×10-3 18.8 2.84

Absorption baseointment

25×10-3 27.3 4.1 32×10-3 21.4 3.25 36×10-3 18.8 2.84

HPMC ocuserts 4×10-3 150.4 22.7 6×10-3 100.3 15.19 7×10-3 91.18 13.8

Gelatin ocuserts 3×10-3 188.0 28.4 4×10-3 167.1 25.3 4×10-3 150.4 22.7

Carbopol 934 & SodCMC ocuserts

1×10-3 50.15 7.5 1×10-3 376.1 56.9 2×10-3 300.9 45.59

Where;K = Degradation rate constant.t 50 = Time after which 50% of ketorolac tromethamine content

Remained.t90 = Time after which 90% of ketorolac tromethamine content

remainedSod CMC = Sodium carboxymethyl cellulose.PVA = Polyvinyl alcohol.

291

revealed that, the first-orderdegradation kinetics of ketorolactromethamine was fitted for allinvestigated ophthalmic preparations.It was noted that, the degradation rateconstants of ketorolac tromethaminehave low values at low temperatures(25°C), while, it has higher values athigh temperatures (45°C). Thedegradation rate constants (K) at25°C for the preparations were;0.0006, 0.0006, 0.027, 0.025, 0.004,0.003 and 0.001 month-1 for sodCMC eye drops, sod CMC gel, PVAbase, absorption base, HPMCocuserts, gelatin ocuserts andcarbopol 934 & sod CMC ocuserts,respectively.

The degradation rate constants at45°C were; 0.0006, 0.0011, 0.036,0.036, 0.007, 0.004 and 0.002 month-

1 using the same above preparationsin the same arrangement. The half-lifeperiod for ketorolac tromethamineformulations stored at 45°C were;1003.04, 601.8, 18.8, 18.8, 91.18,150.4 and 300.9 months using sodCMC eye drops, sod CMC gel, PVAbase, absorption base, HPMCocuserts, gelatin ocuserts andcarbopol 934 & sod CMC ocuserts,respectively.

The results indicated that, theformulae containing drug in bothPVA ointment and absorption baseointment showed, the highestdegradation rate constant (0.036month-1 at 45°C) and the shortesthalf-life period (18.8 months).

The expiration dates of ketorolactromethamine at 25°C were 151.97,151.97, 3.79, 4.1, 22.7, 28.4 and 7.5months using sod CMC eye drops,sod CMC gel, PVA ointment,

absorption base ointment, HPMCocuserts, gelatin ocuserts andcarbopol 934 & sod CMC ocuserts,respectively.

In-vivo studyThe concentration of ketorolac

tromethamine uptaken by eye tissuesand aqueous humor from ophthalmiceye drops, gel, ointment and ocusertswere graphically illustrated in Figures(3-7).

The maximum concentration ofketorolac tromethamine in differentrabbits eye tissues (Cmax) were; 26.1,64.33, 35.08 and 78.45 µg /gm inconjunctiva, 12.49, 36.77, 28.21 and37.08 µg /gm in cornea, 10.52, 32.28,17.4 and 33.85 µg /gm in iris-ciliarybody and 3.29, 9.19, 7.01 and 10.52,µg /gm in aqueous humor for sod.CMC eye drops, sod CMC gel, PVAointment and gelatin ocuserts,respectively (Table 5). Theseobtained results showed that, thehigher concentration of the drug wasuptaken in conjunctiva followed bycornea, iris-ciliary body, then aqueoushumor from all tested preparations.This may be due to the direct contactof these tissues with the tear poolwhich housing the drug.

This finding was in agreementwith the results obtained by El-Shaboury et al.18, who found thesame results on studying the ocularavailability of bromhexine fromophthalmic solution and gels. Also,Shaker19 stated that, the highestconcentration of ciprofloxacinhydrochloride found in conjunctivaltissue may be interpreted in term ofdrug protein binding in the tissuesrich in protein content.


292

0 1 2 3 4 5 60

10

20

30

40

50

60

70

80S o d C M C e y e d r o p s

S o d C M C g e l

P V A o in tm e n t

G e la t in o c u s e r t

T im e (h o u r s )

Con

cent

rati

on o

f ke

toro

lac

trom

itha

min

e (

g/g

m)

Fig. 3: Availability of ketorolactromethamine in conjunctivafrom various ophthalmicpreparations.

0 1 2 3 4 5 60

10

20

30

40S o d C M C e y e d r o p s

S o d C M C g e l

P V A o in t m e n t

G e la t in o c u s e r t

T im e ( h o u r s )

Con

cent

rati

on o

f k e

toro

lac

trom

itha

min

e (

g/g

m)

Fig. 4: Availability of ketorolactromethamine in cornea fromvarious ophthalmic preparations.

0 1 2 3 4 5 60

10

20

30

40So d C M C e ye d r o p s

So d C M C g e l

PV A o in tm e n t

G e la t in o cu s e r t

T im e (h o u r s)

Con

cent

rati

on o

f ke

toro

lac

trom

itha

min

e (

g/g

m)

Fig. 5: Availability of ketorolactromethamine in iris-ciliary bodyfrom various ophthalmicpreparations.

0 1 2 3 4 5 60

1

2

3

4

5

6

7

8

9

10

11 Sod CM C e ye dr ops

Sod CM C ge l

PV A o in tm e nt

Ge latin ocus e r t

Time (hours)

Con

cent

ratio

n of

ket

orol

ac tr

omith

amin

e (

g/g

m)

Fig. 6: Availability of ketorolactromethamine in aqueous humorfrom various ophthalmicpreparations.

293

0 1 2 3 4 5 60

2 0

4 0

6 0

8 0

1 0 0

1 2 0

1 4 0

1 6 0S o d C M C e y e d r o p s

S o d C M C g e l

P V A o i n t m e n t

G e l a t i n o c u s e r t

T im e ( h o u r s )

Co

nc

en

tra

tio

n o

f k

eto

r ola

c t

r om

ith

am

ine

( g

/gm

)

Fig. 7: Total ocular availability ofketorolac tromethamine indifferent rabbit’s eye tissues andaqueous humor from variousophthalmic preparations.

However, in deeper tissues (irisciliary body and aqueous humor), thedrug was found to be in a higherconcentration in iris- ciliary bodystructure than in aqueous humor. Thisis because the drug that appears inaqueous humor comes from irisciliary body or endothelial surface ofthe cornea as reported by Habib etal.8.

The obtained results showed that,the peak time for maximum drugconcentration (Tmax) from Sod CMCeye drops was two hours and thatfrom Sod CMC gel, PVA ointmentand gelatin ocuserts was three hoursin all tissues after application of theseformulations (Table 5). From theobtained results, Sod CMC gel hadlonger peak time than Sod CMC eyedrops. This result was in agreementwith Shaker19 who proved that, thegels containing ciprofloxacin hydro-

chloride have a longer peak time (2hours) than the ophthalmic solution (1hour). This may be due to retardationof the drug release from the gels ascompared to ophthalmic solutions,which have less controlling effect onthe drug release.

Also, PVA ointment and gelatinocusert have the same peak time asSod CMC gel, which is longer peaktime than that of Sod CMC eye drops.This could be explained by theprolongation of the drug contact timewith the eye tissues from theseformulations.

Soliman11 reported that, thesemisolid preparations (gels, andointments) containing atropine sulfatehad the longer peak time (3 hours)than ophthalmic solutions (2 hours).This is because; gels and ointmentsretard the release of the drugcompared with the ophthalmicsolutions which have less controllingeffect on the drug release. Also, itwas reported by Sieg & Robinson21,that the peak concentration offluorometholone ointment into rabbitseyes not reached until 3 hours aftertopical dosing.

The values of area under time-concentration curve of ketorolactromethamine in different rabbits eyetissues (AUC) were; 95.16, 385.45,127.97 and 257.44 µg.hr/gm inconjunctiva, 36.48, 135.69, 105.02and 101.8 µg.hr/gm in cornea, 27.97,103.98, 63.68 and 89.65 µg.hr/gm iniris-ciliary body, and 9.45, 26.81,24.42 and 38.22 µg.hr/gm in aqueoushumor for sod. CMC eye drops, sodCMC gel, PVA ointment and gelatinocuserts, respectively (Table 6).


294

Table 5: Maximum peak concentration and peak time of ketorolac tromethamineophthalmic preparations.

Peak concentration and peak time of ketorolac tromethamineophthalmic formulations

Sod CMC eyedrops

Sod CMC gel PVA ointment Gelatin ocusert

Formulae

Tissue Cmax

µg/gmTmax

(hr)Cmax

µg/gmTmax

(hr)Cmax

µg/gmTmax

(hr)Cmax

µg/gmTmax

(hr)Conjunctiva 26.1 2 64.33 3 35.08 3 78.45 3

Cornea 12.49 2 36.77 3 28.21 3 37.08 3Iris-ciliaryBody

10.52 2 32.28 3 17.4 3 33.85 3

Aqueoushumor

3.29 2 9.19 3 7.01 3 10.52 3

Table 6: Area under the curve of ketorolac tromethamine ophthalmicpreparations for each tissue.

AUC (µg.hr / gm )Formulae

Tissue

Sod CMCeye drops

Sod CMCgel

PVAointment

Gelatinocusert

Conjunctiva 95.16 385.45 127.97 257.44

Cornea 36.48 135.69 105.02 101.8

Iris-ciliarybody

27.97 103.98 63.68 89.65

Aqueoushumor

9.45 26.81 24.42 38.22

Where;PVA = polyvinyl alcoholSod. CMC = sodium carboxymethylcellulose

295

These obtained results showed that,the higher value of the drug wasobtained in conjunctiva followed bycornea, iris-ciliary body, then aqueoushumor from all tested preparations.This may be due to the direct contactof these tissues with the tear poolwhich housing the drug.

The total availability of the drugin rabbits eye tissues after two hourswas presented in Table (7) andillustrated in Figure (7). It was 52.4µg /gm for sod. CMC eye drops.While, the total availability of thedrug in eye tissues after three hourswas 142.57, 87.7 and 159.9 µg /gmfor sod CMC gel, PVA ointment andgelatin ocuserts, respectively. Thus,the total availability and AUC of thedrug from the tested formulations wasin the following order: gelatinocuserts > sod CMC gel > PVA

ointments > sod. CMC eye drops.This was in agreement with Shaker19

who proved that the total availabilityof ciprofloxacin hydrochloride fromthe tested formulations was in thefollowing order: ocuserts >ophthalmic gel > ophthalmicsolutions.

Also, this was in agreement withMalhotra & Majumdar7 who studiedthe in-vivo ocular availability ofketorolac tromethamine following theocular instillation of aqueous, oil, andointment formulations to normalrabbit’s eyes tissues. They found that,ointment formulation provided higherCmax and AUC values compared withaqueous drops, although the drugcontained in the dose of ointment was50% of that contained in a dose ofaqueous drops.

Table 7: Total ocular availability of ketorolac tromethamine from variousophthalmic preparations in rabbits eyes tissues and aqueous humor.

Total ocular availability of ketorolac tromethaminein µg /gm

Formulae

Time(in hours)

Sod CMCeye drops

Sod CMCgel

PVAointment

Gelatinocusert

1 35.63 71.66 a 68.64 ab 87.84 abc

2 52.4 93.46 a 77.87 ab 121.95 abc

3 37.32 142.57 a 87.7 ab 159.8 abc

5 26.11 101.51 a 43.04 ab 99.48 abc

Data are expressed as meana- Significantly different at P< 0.0001 compared with Sod CMC eye dropsb- Significantly different at P< 0.0001 compared with Sod CMC gelc- Significantly different at P< 0.0001 compared with PVA ointment.In all above-mentioned statistical comparisons, one way analysis of variance(ANOVA) followed by Tukey-Kramer Multiple comparisons test was adapted.


296

ConclusionFrom the obtained results, it could

be concluded that:• According to the released amounts

from these ophthalmic preparationsafter six hours, they can bearranged in the following order;Eye drops > ocuserts > gel >ointments.

• Such formulations could be storedfor 6 months at 25°C, 35°C, 45°C,without physical or chemicaldegradations of the drug in theselected formulations, except forointments containing PVA andabsorption base which showedsome changes in the drug content.

• The decomposition rate of ketorolactromethamine followed the first-order degradation kinetics. Themost stable formula is sod CMCeye drops and gel, meanwhile, theless stable formulations are PVAand absorption base ointments.

• Ketorolac tromethamine wasrapidly uptaken from ophthalmicsolution than from gels, ointmentand ocuserts. While, the ocularavailability was improved by gelsand ocuserts.

• The peak time of maximumketorolac tromethamineconcentration in rabbit’s eye tissuesand aqueous humor was two hoursfor sod CMC ophthalmic solutionand three hours for sod CMCophthalmic gel, PVA ointment andgelatin ocuserts.

• All tested formulations provided thehighest Cmax of the drug inconjunctiva followed by cornea,iris-ciliary body, then aqueous

humor and it could arranged asfollows; gelatin ocuserts > sodCMC gel > PVA ointment > sod.CMC eye drops.

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14- J. M. Conard, W. A. Reay, E.Polcyn and J. R. Robinson, J.Parent. Drug Assoc., 32, 149(1978).

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