656 Chiang Mai J. Sci. 2013; 40(4)

Chiang Mai J. Sci. 2013; 40(4) : 656-668http://it.science.cmu.ac.th/ejournal/Contributed Paper

Development of a Stable Latanoprost Solution forUse as Eye DropsSomchai Sawatdee [a], Hirihattaya Phetmung [b] and Teerapol Srichana*[a,c][a] Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand.[b] Inorganic and Materials Chemistry Research Unit, Department of Chemistry, Faculty of Sciences,

Thaksin University, Songkhla 90000, Thailand.[c] Nanotec-PSU Excellence Center on Drug Delivery System, Faculty of Pharmaceutical Sciences,

Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand.*Author for correspondence; e-mail: teerapol.s@psu.ac.th

Received: 7 December 2012Accepted: 23 February 2013

ABSTRACTThe aim of this study was to develop latanoprost as a sterile eye drop solution using

hydroxypropyl--cyclodextrin (HP--CD) as a solubilizing and stabilizing agent.Latanoprost eye drops were prepared according to the requirements for eye drop preparations.The inclusion complex of latanoprost/HP--CD was characterized by NMR spectroscopy.The efficacy of latanoprost eye drops prepared by using the complex with HP--CD wasevaluated by measuring the intraocular pressure in a rabbit model. The stability of latanoprosteye drops was determined after storage at 5C and 25C/60%RH. The NMR results showthat latanoprost was incorporated into the cavity of cyclodextrin. The dosage form of thenew eye drop solution was stable at 25C/60%RH for at least 6 months. The decrease ofintraocular pressure observed was similar to that for Xalatan.

Keywords: latanoprost, hydroxypropyl--cyclodextrin, intraocular pressure reduction,glaucoma

1. INTRODUCTIONLatanoprost is the F2 analogue of

prostaglandins used for the treatment ofopen angle glaucoma. Its chemical nameis isopropyl-(Z)-7[(1R, 2R, 3R, 5S) 3,5 - d i h y d r o x y - 2 - [ ( 3 R ) - 3 - h y d r o x y - 5 -phenylpentyl] cyclopentyl]-5-heptenoate [1]and its molecular weight is 432.58 g/mol.The chemical structure is shown in Figure 1.It is a selective agonist of the prostaglandinF prostanoid receptor that reduces theintraocular pressure and increases the outflow

of the aqueous humor. The main mechanismof action of this agent is an increaseduveoscleral outflow [2]. Latanoprost is aprodrug that is well absorbed via the cornea,and is then activated by hydrolysis to the activeform of latanoprost acid (see Figure 1).Latanoprost is a colorless or slightly yellowoily viscous liquid and practically insolublein water [3]. The solubility of latanoprostwas reported to be 12.9 g/mL in water[4] but latanoprost eye drops under the

Chiang Mai J. Sci. 2013; 40(4) 657

trade name of Xalatan was manufacturedby Pfizer Inc. (USA) contains 50 g/mLof latanoprost in an aqueous preparation.

The manufacturer recommends thatXalatan to be stored in the refrigerator.

The development of latanoprost as anaqueous preparation is important since it maythen act more quickly and have betterbiological activity. Latanoprost has beendeveloped as an eye-drop preparation byforming a complex micelle [5]. In that formit can be stored at room temperature toimprove its usability and solubility. Oculardrug excipients are able to enhance thesolubility of eye-drops by using cosolventssuch as PEG300 and glycerol [6], a surfactant/chitosan micelle system [7], a complex micellesystem (non-ionic surfactants, polyethyleneglycol monostearate 25 and polyethyleneglycol monostearate 40) [8] and cyclodextrinas solubilizing agents [9-11].

Cyclodextrins are a group of structurallyrelated natural products formed duringbacterial digestion of cellulose. These cyclicoligosaccharides consist of (-1, 4)-linked-D-glucopyranose units having a lipophiliccentral cavity and a hydrophilic outer surface[9]. In aqueous solutions cyclodextrins areable to form inclusion complexes with manydrugs by taking up a drug molecule or morefrequently some lipophilic moiety of themolecule, into its central cavity. Cyclodextrinderivatives used in ophthalmology includethe hydroxypropyl--cyclodextrin (HP--CD)for ocular drug delivery [10, 11]. Due to thelow solubility of latanoprost in water,cyclodextrins has therefore been chosen to

enhance its solubility [3, 12] and to formulateas an aqueous solution and maintain its stability[10-11]. HP--CD is not only highly solublein water (>600 mg/mL) but also has lowtoxicity and is a common additive in eyepreparations. The advantages of HP--CDin latanoprost eye drops are in enhancingdrug stability. In addition, cyclodextrin mayenhance corneal permeability [13].

The aim of this study was to prepareand investigate an inclusion complex oflatanoprost with HP--CD as an eye-droppreparation for improving latanoprostsolubility and stability.

2. MATERIALS AND METHODS2.1 Materials

Latanoprost (lot no. PCDINT01909)was obtained from Precise ChemiPharma Put. Ltd. (India). HP--CD orCavasol W7 HP Pharma (lot no. 73B033)was from ISP Pharmaceuticals, USA.Sodium hyaluronate (lot no. A04A) was fromShiseido Co., Ltd., Japan. All other excipientswere pharmaceutical grades and otherreagents were reagent grades. Fluoresceinsodium was obtained from BDH Limited(England) and xylazine hydrochloride wasfrom Farvet, Netherlands. Pentobarbitalwas obtained from Ceva, France. Alpha-chymotrypsin was from Merck, Germany.Xalatan (lot no. S08720) was used as a

Figure 1. Chemical structure of latanoprost and its active metabolite-latanoprost acid.

658 Chiang Mai J. Sci. 2013; 40(4)

reference formulation. A Tonometer(Tonovet Type TV01, Denmark) wasemployed to measure intraocular pressure.

2.2 AnimalsMale New Zealand white rabbits, hybrid

strain weighing about 3 kg were purchasedfrom the Department of Animal Science,Faculty of Agriculture, Kasetsart University,Thailand. All animal experiments werereviewed and approved by the Office of theNational Research Council of Thailand(approved no.PS-54006). Rabbits wereindividually housed in stainless-steel cagesand in a 12 h light/dark cycle in a controlledroom temperature, and were allowed freeaccess to food and tap water for a minimumof a week before the experiments.

2.3 The One-dimensional 1H, 13C NMRSpectrum Experiment

Latanoprost and HP--CD weredissolved in CD3OD and D2O respectively.Complexes were prepared by dissolvingHP--CD and latanoprost (same molar ratioin the eye drop preparations) in purifiedwater until a clear solution was obtained.The dry complexed powder was reconstitutedin D2O. The 1H-NMR and 13C-NMRspectra were performed on a FourierTransform NMR Spectrometer 500 MHz,Model UNITY (Varian, Germany). 1H-NMR

chemical shifts were referenced to an internalstandard TMS; for the 13C-NMR spectra, theresonance line of CDCl3 at 77.00 ppm wasused as a reference.

2.4 Preparation of Model LatanoprostEye Drop Formulations

The formulation was designed as a clearophthalmic solution, free from particulatematter. The ingredients and sodium chlorideequivalent values are shown in Tables 1 and 2.The formulations were adjusted to a pH 6.7with an osmolarity of 294 mOsmol/L.This is able to comfort the eyes [14].Benzalkonium chloride in a solution of50% was added as a preservative. HP--CDwas used as a solubilizing agent for thelipophilic drug at an optimized concentrationof 0.359 %w/v. The finished productspecifications were set as standardguidelines for an eye drop preparation.The details of manufacturing followed thestandard method for a sterile eye preparation.This product was filtered through a 0.45 mpre-filter membrane, and then sterilized byfiltration through a 0.2 m polysulfonemembrane using an aseptic technique. Threepilot batches manufacturing (10 L) for thestability study were prepared and dispensedinto a 2.5 mL natural color LDPE eye dropbottle with an LDPE inner plug and PPscrew on a pilfer proof cap.

Table 1. Formulation for the latanoprost eye drops used in this study.Ingredients

LatanoprostHydroxypropyl--cyclodextrinMonobasic sodium phosphate (monohydrate)Dibasic sodium phosphate (anhydrous)Sodium hyaluronateSodium chlorideBenzalkonium chloride (50% solution)Water for injection qs to

Composition (mg)0.1258.97517.50015.0004.50010.0001.000

2.5 mL

Chiang Mai J. Sci. 2013; 40(4) 659

Table 2. The amount of sodium chloride equivalent of ingredients in the formulation.Ingredients

LatanoprostHydroxypropyl--cyclodextrinMonobasic sodium phosphate (monohydrate)Dibasic sodium phosphate (anhydrous)Sodium hyaluronateBenzalkonium chloride (50% solution)

Equivalent to sodium chloride(mg in 1 mL of water)

insignificant0.152.032.520.020.03

Sodium chloride should be added in the formulation = 9 mg - 4.75 mg = 4.25 mg in 1 mLof water.

2.5 Analysis of Latanoprost andValidation Method

The latanoprost concentration of theeye drops was determined by highperformance liquid chromatographic (HPLC)system (Ultimate, USA). A HPLC systemconsisted of solvent delivery pump (SR-3000),injector with an injection volume of 20 L(WPS 3000SL), a UV-detector at a wavelengthof 210 nm and a reversed phase column(C18, 2504.6 mm) were kept at 25C.The mobile phase consisted of acetonitrile:0.05 M phosphate buffer (60:40 v/v) andadjusted to pH 3.0 and a flow rate wasmaintained at 1.0 mL/min. The system metthe requirements according to USP [15] andhad a tailing factor of a standard solutionchromatograph of not more than 2.0 anda %RSD standard response of 6 replicateinjections of not more than 2.0%.

The method was validated for 8parameters: specificity, range, linearity,precision, accuracy, limit of detection (LOD),limit of quantitation (LOQ), and robustness.In addition the systems suitability parameterwas also calculated. To demonstrate specificityin the presence of other excipients used inthe formulation, latanoprost was spiked(approximately 25 g/mL) into the drugproduct. A chromatogram was run, andobserved result and compared with thestandard solution to confirm the same

retention time and also that the product usedwas capable of separating the analyte peakfrom its excipients. To evaluate the linearity,the standard solutions corresponding to50-150 g/mL of latanoprost concentrationwere prepared. The slope, intercept andcorrelation coefficient were obtained fromthe linear regression analysis. To determinethe repeatability and intermediate precision,six replicated samples of latanoprost wereanalyzed three times within the same day(intra-day) and three other days (inter-dayvariation). The LOD and LOQ values werecalculated as the signal-to-noise ratio of 3:1and 10:1 respectively.

2.6 Sterility TestThe stability test of latanoprost eye drops

was performed as method described understerility tests <71> specified in USP [15].

2.7 Stability StudyThe accelerated and long-term stability

studies were performed following theASEAN Harmonized on stability of drugproducts guidance [16]. Three differenceproduct lots were kept at the followingstorage condition and sampling times weretaken into account. Those conditions were5C and 25C with 60% relative humidityfor 1, 3, and 6 months, respectively.

The manufacturer of Xalatan claims that

660 Chiang Mai J. Sci. 2013; 40(4)

“Once opened the container may be storedat room temperature for up to 30C for6 weeks”. It means that the products meetthe specification after opening eye bottle.Therefore in-use stability was performed fortest the specification especially the sterilityof the products. The protocol was developedto simultaneously simulate the real lifeapplication of eye drops by samplinglatanoprost eye drops and storing them at30C, then the eye drop bottles wereopened and the content used for testingsamples daily. Sufficient amounts of eye dropsremained for testing every week for 6 weeks.

2.8 Eye Irritation TestThis test method followed the Acute

Eye Irritation/Corrosion method no. 405in the OECD Guidelines for animal Testingof Chemicals [17]. The eye irritationwas performed by using 3 rabbits inthis experiment. Both eyes of each rabbitprovisionally selected for testing wereexamined within 24 h before testing started.Only one drop of fluorescein sodiumophthalmic solution (2%w/v) was droppedinto both eyes of the test rabbits. After1 minute the rabbits eyes were washedwith normal saline solution and the eyeirritation determined using a hand slit lamp.Animals showing eye irritation, oculardefects, or pre-existing corneal injury werenot used. For testing the latanoprost eye drops,a dose of 0.1 mL was used. The duration ofthe observation period was sufficient to fullyevaluate the magnitude and reversibility ofthe observed effects. The eyes were examinedat 1, 24, 48, and 72 h after the test samplewas applied. To determine the reversibility ofthe effects of eye irritation, the animals wereobserved for 21 days post administration ofthe latanoprost fluid. If reversibility of theeye irritation was seen before 21 days,

the experiment was stopped at that time.The grades of ocular reactions (conjunctivae,cornea and iris) were recorded at eachexamination (for details see grading asdescribed in the full reference). Any otherlesions in the eye (e.g. pannus, staining) oradverse systemic effects were reported.

2.9 Intraocular Pressure Reduction inRabbits

The 18 rabbits were fasted 12-16 hbefore experiments and separated into 3groups (6 rabbits per group) which restrainedin a box-type fixation. The intraocularpressure of each rabbit was measured usingthe tonometer while they were conscious.Animals were anaesthetized using 2 mg/kgof xylazine hydrochloride intramuscularlyfollowed by an intravenous injection of25 mg/kg of pentobarbital. Fifty mL (10 U)of -chymotrypsin was injected into theposterior ocular chamber of both eyesusing a 30-gauge needle for reduction ofintraocular pressure in the eyes [18-19].After injection, rabbit’s eyes were treatedwith chloramphenicol and betamethasoneeye drops twice a day for 10 days. After 21days, the intraocular pressures of therabbits were measured and recorded forthe experiment when the intraocularpressure exceeded 25 mmHg. In this study,3 groups of rabbits were administered with50 L of a 0.9% sodium chloride solution,50 L of latanoprost eye drops and50 L of Xalatan eye drops, respectively. Theintraocular pressures of both eyes weremeasured immediately prior to administrationof the drug and 1, 2 and 3 h after drugadministration, respectively. The differencebetween the multiple groups was assessed bythe student’s t-test. Values of p less than0.05 were considered to be significantlydifferent.

Chiang Mai J. Sci. 2013; 40(4) 661

3. RESULTS3.1 Interaction of Latanoprost withCyclodextrins

1H, 13C NMR were employed toinvestigate the free latanoprost andcomplex latanoprost formation. The atomicnumbering of latanoprost is shown inFigure 1. Latanoprost was analyzed in D2O

and the complex was analyzed in CD3OD.Table 3 shows that the chemical shifts offree latanoprost are in good agreement witha previous report [20]. The chemical shiftdisplacements on the solutions containinglatanoprost/HP--CD complex are alsolisted in Table 3.

The 13C NMR spectra showed a2-propyl (-CH(CH3)2) peak at 22.098 ppmand benzene ring peaks at 129.405, 130.108,130.818, 143.744 ppm with an attachedproton of about 7 ppm. This group is thenon-polar which formed a complex withinthe cavity of cyclodextrin.

The chemical shift of the 13C and 1H ofthe latanoprost was affected by thecyclodextrin environment. The signal of the

C-atom number C1-C7 and C13-C17disappeared. This refers to the side chain oflatanoprost (including 2-propyl and thebenzene ring). The most significant changesof the latanoprost complex proton chemicalshifts were observed for the CH, CH2 protonof C1-C7, C13-C17, and CH3 of the 2-propylend and also the CH of the benzene ring.All signals disappeared from the NMR spectra.This determined that there was an insertion

Table 3 Proton and carbon chemical shifts of free latanoprost and a solution containing thelatanoprost/hydroxypropyl--cyclodextrin complex.

Atom number

1234567

8910111213

14

151617

2-propyl (CH-O)2-propyl (2 x CH3)

ipso2 x ortho2 x meta

para

Free latanoprost13C

175.01934.91726.08327.416129.313126.66527.585

51.60873.83643.82178.44452.44330.130

36.247

72.05240.42233.075

68.89522.098143.744130.108130.818129.405

1H---

2.262 (t, J = 14.87 Hz, 2 H)1.244 (m, 2 H)2.107 (m, 2 H)5.390 (m, 1 H)5.520 (m, 1 H)2.083, (m, 1 H)2.236 (m, 1 H)1.392 (m, 1 H)

4.061 (bdd, J = 4.63, 4.63, 1 H)1.502 (dd, J = , 2 H)

3.834 (bdd, J = 7.56, 8.05, 1 H)1.329 (m, 1 H)1.551 (m, 1 H)1.662 (m, 1 H)1.707 (m, 1 H)1.768 (m, 1 H)3.548 (m, 1 H)1.976 (m, 1 H)

2.624 (ddd, J = 9.76, 9.51, 9.76 Hz, 1 H)2.766 (ddd, J = 9.76, 1.46, 9.76 Hz, 1 H)

4.941 (h, J = 37.56 Hz 1 H)1.192 (d, J = 7.31 Hz, 6 H)

---7.190 (bd, J = 8.29 Hz, 2 H)7.232 (bt, J = 14.87 Hz, 2 H)7.123 (bt, J = 17.07 Hz, 1 H)

Latanoprost complex13C*******

62.29174.32257.69878.42866.788

*

*

***

******

1H—-************

1.0094.6301.0193.7861.009

**

**

******

****---***

Chemical shift () ppm

* Signals disappeared** Affected proton chemical shift

662 Chiang Mai J. Sci. 2013; 40(4)

Figure 2. Proposed scheme for the inclusion complex of latanoprost inside hydroxypropyl--cyclodextrin.

of the latanoprost side chain into the HP--CD. In addition, the dihydroxy cyclopentaneprotons (OH, CH, and CH2 at C8-C12)exhibited minor changes in chemical shiftdownfield of the complexation with HP--CD. However, the inclusion of the dihydroxycyclopentane moiety into the HP--CD cavitywas not complete, and there was only a partialinsertion of the latanoprost into the cavity ofthe cyclodextrin.

We concluded that the latanoprostmolecules interacted with HP--CDas illustrated in the proposed scheme(Figure 2). This shows that the OH group ofcyclopentane is outside the cavity and thebenzene ring and dimethyl side chain aredeeply enclosed within the cavity. It is highlypossible that the hydroxyl group is restrictedby hydrogen bonding on the rim of the cavityor with the aqueous solution.

3.2 Formulation Development ofLatanoprost Eye Drops

Latanoprost does not dissolve in wateror phosphate buffer solution when preparedat a concentration of 50 g/mL. After HP--CD was used as a solubilizing agent, a clearsolution was obtained.

The latanoprost complexed with HP--CD formulations included a phosphatebuffering agent adjusted to a pH of 6.7 byusing monobasic sodium phosphatemonohydrate and anhydrous dibasic sodiumphosphate. The amount of the buffering agentwas calculated by the Henderson-Hasselbalchequation according to equation (1).

pH = pKa + log (1)

=6.72This formulation (Table 1) was

developed for its tonicity that was calculatedby the sodium chloride equivalent methodby using the E-value. The E-value oflatanoprost does not appear in any literatureso the theoretical osmolarity was calculatedaccording to equation (2) [14].

mOsmol =

= 0.12 mOsmol/LDue to the low concentration of

latanoprost in the eye drop formulation itsosmolarity value was low resulting in a lowcontribution to the calculated sodiumchloride equivalent value. The excipientsequivalent to amount of sodium chloride (mg)which calculated from equation (2) is shownin Table 2. All excipients equivalent to totalamount of sodium chloride is 4.75 mg. Thisis not sufficient to produce isotonicity of eyedrop formulation (0.9% w/v of sodiumchloride). Therefore we added sodiumchloride to about 0.4% w/v for isotonicadjustment and the osmolarity was re-evaluated experimentally.

The composition of the formulaincludes: 50 g/mL latanoprost, 3.59 mg/mLHP--CD as solubilizing agent, 7 mg/mLmonobasic sodium phosphate monohydrateand 6 mg/mL of anhydrous dibasic sodiumphosphate as buffering agent, 1.8 mg/mLsodium hyaluronate for adjustment

[salt][acid]

LgLg

gmols

gosmol

osmol1000mOsmol

(2)

Chiang Mai J. Sci. 2013; 40(4) 663

of viscosity, 4 mg/mL sodium chloridefor isotonic adjustment, 0.2 mg/mLbenzalkonium chloride as preservative andwater for injection added to 1 mL. Theamount of the working formula (per 1 bottle)is presented in Table 1. The calculated valueof osmolarity is close to the obtained value(294 vs 275 mOsmol/L).

3.3 Method ValidationThe method for validation of

the latanoprost assay was performedbecause latanoprost is not officially inany pharmacopoeia. The latanoprostchromatogram revealed a specificity of themethod. The retention time of latanoprostwas about 4 min and the peak was notinterfered with any excipient in eye dropformulations. The linear plots betweenconcentrations and peak areas produced agood correlation with r2 = 1. The systemprecision was determined from the resultsof six replicate injections. The %RSD of theassay for latanoprost within one day wasnot more than 2% (0.22 - 0.82%) and %RSDbetween 3 days was 0.62%. The recoveryexperiment to study the accuracy was carriedout by the spiking placebo method. A knownquantity of drug corresponding to 75, 100,and 125% of the label claim of the drug wereadded. The %recovery of all concentrationwas 99.3%. The results indicate that themethod is highly accurate for determinationof latanoprost. The results of the othervalidation parameters were well within theacceptance criteria [15]. For results of the

validation, the assay method was ensuredduring the stability study.

3.4 Stability StudyA stability study of an ophthalmic

solution of latanoprost was performed andthe result is shown in Table 4. The solutionwas found to be clear and found to be freefrom particulate matter. The assay content oflatanoprost was between 98.9 and 102.7 %LAafter storage at 5C for a period of 6 months.The latanoprost content after storage at25C was between 99.4 and 101.7 %LA fora period of 6 months. The latanoprost contentwas stable at 5C and room temperature(the content changes were less than 2% ofthe initial). The pH and viscosity of theproducts also remained constant throughoutthe period of the stability studies.Determination of the pH and viscosity ofthe latanoprost eye drops were about 6.7 and15.5 cps, respectively and these did notchange after storage for 6 months at both 5Cand 25C. The osmolarity of the latanoprosteye drops for both storage conditions was274-280 mOsmol/L. In addition, the solutionremained sterile after storage for 6 months.The results indicate that this formulation isstable in the packaging of the eye drops.

The in-use stability study results are shownin Table 5. The assay results during the studyperiod (6 weeks) were 100.3 and 99.2 %LA.The pH value was constant at 6.7 and theviscosity slightly increased by about 15 to 17cps. This product was sterile after repeatedopening the eye drops during the in-use study.

664 Chiang Mai J. Sci. 2013; 40(4)

Table 4. Stability results of latanoprost eye drops (mean SD, n = 6) from 3 differentbatches.

101.6 1.35101.6 1.35

6.7 0.006.7 0.00

15.5 0.5615.5 0.56

275 6.08275 6.08

98.9 1.5599.4 1.95

6.7 0.006.6 0.06

15.3 0.4615.2 0.51

274 3.21277 4.16

99.5 1.05101.7 1.49

6.7 0.006.7 0.06

15.6 0.3615.5 0.50

275 4.16279 6.80

102.7 0.76100.7 0.89

6.7 0.066.6 0.00

15.5 0.4415.3 0.68

280 8.50278 6.43

Clear, colorless liquid, free from visible particlesClear, colorless liquid, free from visible particles

MonthsTest

Appearance

Assay(90-110% LA)

pH(6.0-8.0)

Viscosity(10-20 cps.)

Osmolarity(270-328mOsmol/L)

Stabilitycondition5C25C/60%RH

5C25C/60%RH

5C25C/60%RH

5C25C/60%RH

5C25C/60%RH

0 1 3 6

Table 5. In-use stability results of latanoprost eye drops (3 difference batches). Mean SD,n= 6

3.5 Eye Irritation Test and IntraocularPressure Reduction

The eye irritation test of the latanoprosteye drops was determined by observing therabbit’s eyes. No eye irritation of any rabbitwas found after 72 h.

The intraocular pressure reduction afteradministration of normal saline as control,latanoprost eye drops and Xalataneye dropsthat were free from HP--CD formulationare shown in Figure 3. The intraocular pressureafter administration of normal saline solutiondid not change after 1 h and decreased to

0.330.33 and 1.160.87 mmHg after 2 and3 h, respectively. The intraocular pressurereduction after administration of latanoprosteye drops decreased to 6.002.62, 7.832.52,8.832.49 mmHg after 1, 2 and 3 h,respectively. The intraocular pressure reductionafter administration of Xalatan eye dropsdecreased to 6.661.11, 8.001.86, 9.501.76mmHg after 1, 2 and 3 h, respectively. Thestatistics showed that latanoprost eye dropsand Xalatan eye drops significantly changedthe intraocular pressure in comparison withnormal saline (p < 0.01).

100.3 1.206.71 0.0015.38 0.65

Sterile

97.2 1.406.71 0.0014.98 0.45

Sterile

97.9 1.356.67 0.0616.65 0.56

Sterile

99.2 1.706.70 0.0016.96 0.66

Sterile

99.2 1.556.71 0.0616.94 0.58

Sterile

Stored at 30C with drops removed from the eye bottles everyday

Clear, colorless liquid, free from visible particlesInitial 2nd week 4th week 5th week 6th week

Test

AppearanceAssay (90-110% LA)pH (6.0-8.0)Viscosity (10-20 cps.)Sterility

Chiang Mai J. Sci. 2013; 40(4) 665

4. DISCUSSIONAlthough increasing solubility of

latanoprost by introducing alcohol ispossible, it is not practical in eyes’ preparations.The solubility of latanoprost in aqueoussolution is limited (12.9 and 50 g/mL inwater and PBS pH 7.2, respectively). In-houseexperiment revealed that latanoprost did notcompletely dissolve in water (pH 7.0) or PBS(pH 7.2) when prepared at a concentrationof 50 g/mL. The clear solution was obtainedafter adding the HP--CD as solubilizingagent at wide range of pH (5.0 to 7.2).

The inclusion of latanoprost into HP--CD was formed as host-guest complex.The lipophilic cavity of the cyclodextrinsprovides solubility of latanoprost while thehydrophilic surface contributes to itssolubility in water. The proposed schematicrepresentation of latanoprost complex withcyclodextrin is presented in Figure 2. The twoside chains of latanoprost (benzene ring and2-propyl) were completely incorporated intothe cavity of the cylodextrin because of itshydrophobic properties. The dihydroxycyclopentane stays outside the cyclodextrincavity because of the hydrophilic propertiesof its hydroxyl group. Complexes betweencyclodextrins and numerous guest moleculeshave been studied by the NMR method

[21-22]. Changes in the chemical shifts areemployed to identify interactions betweencyclodextrins and drug molecules and todetermine binding constants. The solubility oflatanoprost was enhanced as a result of itforming a complex with cyclodextrin.

The latanoprost solution eye dropformulation was adjusted to pH 6.7 becauselatanoprost was more susceptible fromhydrolysis at less than pH 4 and at morethan pH 6.7 [23]. In addition, the previouswork reported that latanoprost ophthalmicat pH 5-6 was more stable than that at pH7.0 [24]. Therefore formulation adjustmentto pH 6.7 was done. Benzalkonium chloridewas employed as preservative in thisstudy that is similar to other ophthalmicformulations [25]. Ochiai et al. [26] foundthat latanoprost-stabilizing micelles were notinfluenced by the presence of benzalkoniumchloride 0.02%. The viscosity increasingagent in eye drop formulation needs toretain drug in the eyes. Normally,hydroxypropylmethylcellulose (HPMC) waschosen as viscosity increasing agent due toits safety. In addition, chitosan can be usedfor increasing viscosity in artificial tear[27]. Sodium hyaluronate was used in thisformulation for adjustment of the viscosity[28]. The results indicated that sodium

Figure 3. Intraocular pressure (IOP) changes in rabbits (n = 6) after administration of 0.9 %NaCl ( ), Latanoprost eye drops (O) and Xalatan eye drops (). The data represent thechanges in IOP from baseline values (mean SE, n = 6).

666 Chiang Mai J. Sci. 2013; 40(4)

hyaluronate solution was better toleratedthan that of the hydroxyethylcellulose solution.The concentration of sodium hyaluronatewas 0.18% according to the concentrationfor dry eye treatment [29]. The viscosity oflatanoprost eye drops was about 15 cpsfrom the addition of sodium hyaluronate.Eye drops were able to be dropped out ofthe bottle but be able to retain in the eyesfor their pharmacological action.

The osmolarity of the products didnot change significantly versus time (0-6months) and temperature (5C and 25C).Latanoprost eye drops conformed withintheir specifications. In addition, the complexformation increased the stability of latanoprostby the insertion of easily hydrolyzed part intothe cavity of the HP--CD.

After the eye-drop bottles were opened,all stability parameters were within acceptablerange including their sterility. This packagingof latanoprost eye drops containing 2.5 mL(about 80 drops) was sufficient for 6 weekstreatment.

After injection of alpha-chymotrypsininto rabbits’ eyes for 21 days, it inducedtheir intraocular pressure in all rabbits by morethan 25 mmHg. The ocular hypertensiverabbit model prepared by intravitrealinjection may cause inflammation in the eyeand may block the trabecular meshworkresulting in an irregular release of aqueoushumor. The effect of latanoprost (both thenew developed formulation and Xalatan)lowered the intraocular pressure comparedwith normal saline solution. The HP--CDcomplex formation with latanoprost did notchange the efficacy of latanoprost in itsformulation when compared with Xalatan.Both products decreased the intraocularpressure by about 10 mmHg within 3 h.The normal value of the intraocular pressurein humans is between 10 and 20 mmHg(average is 15.5 mmHg). The latanoprost/

HP--CD eye drops did not alter the efficacyof latanoprost because latanoprost wasincorporated in the cavity and did not changethe form of this drug such as ionization andsalt formation. The latanoprost eye dropsreduced the intraocular pressure after drugadministration to a normal value (about 15mmHg) within 3 h (intraocular pressuredecreased from 25 mmHg to 15 mmHg orreduced by about 10 mmHg from thebaseline). There were no differences inintraocular pressure after being treated withlatanoprost eye drops and Xalatan (p>0.05).From the specification of the finished product,the stability study and the efficacy of the eyedrops we can claim that this developedformulation using HP--CD as complexingagent is suitable for an eye drop preparationof latanoprost.

Gonzalez et al. [10] compared clinicaland stability studies of latanoprost withcyclodextrin-containing eye drops (notknown formulation) and Xalatan eye drops.The results showed intraocular pressurereduction in human was about 9 mmHg inboth groups and exhibited similar adverseeffects.

5. CONCLUSIONThe formation of the complex between

latanoprost and HP--CD was confirmed by1H-NMR and 13C-NMR. When latanoprostHP--CD inclusion complex was developedas a sterile eye drop solution, the formulationwas a clear solution and suitable for use aseye drops. This product was stable whenstored at 5C and 25C for at least 6 months.

ACKNOWLEDGEMENTSThis work was supported in part by (1)

the Higher Education Research Promotionand National Research University Project ofThailand, Office of the Higher EducationCommission, (2) Graduate School, Prince of

Chiang Mai J. Sci. 2013; 40(4) 667

Songkla University, (3) Nanotec-PSUExcellence Center on Drug Delivery System,Faculty of Pharmaceutical Sciences, Princeof Songkla University. The author wouldlike to thanks Interthai PharmaceuticalManufacturing Limited (IPML) for supportall of chemicals and facilities. We thankto Thailand Institute of Scientific andTechnological Research (TISTR) for animalexperimental part. The author would like tothank Dr. Brian Hodgson for assistancewith the English.

DECLARATION OF INTERESTThe author declares no conflict of

interest.

REFERENCES

[1] Rele, R.V., Mhatre V.V., Parab, J.M. andWarkar, C.B., Simultaneous RP HPLCdetermination of latanoprost and timololmaleate in combined pharmaceuticaldosage form, J. Chem. Pharm. Res., 2011;3(1): 138-144.

[2] Widomski, P., Baran, P., Go biewski, P.,Surowiec, I. and Bielejewska, A.,Validated liquid chromatographicmethod for analysis of the isomers oflatanoprost, Acta Pharm., 2008; 20(2):157-164.

[3] Jimenez-Bayardo, A., Tornero-Monta o,J.R., Lopez-Sanchez, M.I. and Cruz-Olmos, E. 2011. US Patent No.US2011/0105605 A1 (2011).

[4] Drug bank database. Available oninternet at http://www.drugbank.ca/drug/DB00654.

[5] Ochiai, A., Iida, K., Takabe, H.,Kawamura, E., Sato, Y., Kato, Y.,Ohkuma, M. and Danjo, K., Formulationdesign of latanoprost eye drops toimprove the stability at roomtemperature, J. Pharm. Sci. Technol., 2010;

70: 324-332.

[6] Jithan, A.V., Mohan, C.K. andVimaladevi, M., Development andevaluation of a chloramphenicolhypertonic ophthalmic solution, Indian J.Pharm. Sci., 2008; 70(1): 66-70.

[7] Pepi , I., Hafner, A., Lovri , J., Pirki , B.and Filipovi -Gr i , J., A nonionicsurfactant/chitosan micelle system in aninnovative eye drop formulation, J.Pharm. Sci., 2010; 99(10): 4317-4325.

[8] Ochiai, A. and Danjo, K., Thestabilization mechanism of latanoprost,Int. J. Pharm., 2011; 410: 23-30

[9] Brewster, M.E. and Loftsson, T.,Cyclodextrin as pharmaceuticalsolubilizers, Adv. Drug Deliv. Rev., 2007;59: 645-666.

[10] Gonzalez, J.R., Baiza-Duran, L.,Quintann-Haa, J., Tornero-Montano, R.,Castaneda-Hernandez, G., Ortiz,M., Alarcon-Oceguera, F., Beltran-Loustaunau, M., Cortez-Gastelum,M., Garciduenas-Mejia, J., Gomez-Bastar, P., Jimenez-Roman, J., Korder-Ortega, V., Paczka-Zapata, J., Torres-Segura, M. and Velasco-Gallegos, G.,Comparison of the stability, efficacy, andadverse effect profile of the innovator0.005% latanoprost ophthalmic solutionand a novel cyclodextrin-containingformulation, J. Clin. Pharmacol., 2007; 47:121-126.

[11] Bayado, J. and Guadalajara, A., Methodof preparing a latanoprost ophthalmicsolution and solution thus produced,EP Patent no. EP1759702A1, 2007.

[12] Ono, N., Rytting, J. and Pipkin, J., Effectsof sulfobutylether -cyclodextrin(Captisol) on the solubility and stabilityof latanoprost in aqueous formulations,The AAPS J. 2007; 002571.

[13] Rajewski, R.A. and Stella, V.J.,Pharmaceutical applications of

668 Chiang Mai J. Sci. 2013; 40(4)

cyclodextrin 2. In vivo drug delivery, J.Pharm. Sci., 1996; 85(11): 1142-1169.

[14] Troy, D.B., Remington: The Science andPractice of Pharmacy, 21st edition,Lippincott Williams & Wilkins, 2005.

[15] US Pharmacopeial Convention, USP33-NF28, Rockville, MD, 2010.

[16] ACCSQ-PPWG meeting, Philippines,ASEAN guideline on stability study ofdrug product. 22 February 2005.

[17] OECD/OCDE, OECD Guideline forthe testing of chemicals, acute eyeirritation/corrosion, 24 April 2002.

[18] Konno, T., Maruichi, M., Takai, S., Oku,H., Sugiyama, T., Uchibori, T., Nagai, A.,Kogi, K., Ikeda, T. and Miyazaki, M.,Effect of chymase on intraocularpressure in rabbits, Eur. J. Pharmacol.,2005; 524: 132-137.

[19] Orihashi, M., Shima, Y., Tsuneki, H. andKimura, I., Potent reduction in intraocularpressure by nipranidol plus latanoprostin ocular hypertensive rabbits, Biol. Pharm.Bull., 2005; 28(1): 65-68.

[20] Martynow, J.G., J wik, J., Szelejewski,W., Achmatowicz, O., Kutner, A.,Wi niewski, K, Winiarski, J., Zegrocka-Stendel, O. and Go biewski, P., A newsynthetic approach to high-purity (15R)-latanopros, Eur. J. Org. Chem., 2007;689-703.

[21] Fernades, C.M., Carvalho, R.A., Costa,S.P. and Veiga, F.J.B., Multimodelmolecular encapsulation of nicardipinehydrochloride by -cyclodextrin,hydroxypropyl--cyclodextrin andtriacyl--cyclodextrin in solution,Structure studies by 1H NMR and

ROESY experiments, Eur. J. Pharm. Sci.,2003; 18: 285-296.

[22] Misuik, W. and Zalewska, M.,Spectroscopic investigations on theinclusion interaction betweenhydroxypropyl-b-cyclodextrin andbupropion, J. Mol. Liq., 2011; 159:220-225.

[23] Kimura, A., Asada, H., Tuchimoto, N.and Shimazaki, A., Stabilization oflatanoprost ophthalmic solution at roomtemperature, The AAPS J. 2004; 000873.

[24] Sakai, Y., Yasueda, S. and Ohtori, A.,Stability of latanoprost in an ophthalmiclipid emulsion using polyvinyl alcohol, Int.J. Pharm., 2005; 305: 176-179.

[25] Rowe, R.C., Sheskey, P.J., and Quinn,M.E., Handbook of PharmaceuticalExcipients, 6th edition. PharmaceuticalPress, London, UK, 2009.

[26] Ochiai, A., Ohkuma, M. and Danjo, K.,Investigation of surfactant suitable forstabilizing of latanoprost, Int. J. Pharm.,2012; 436: 732-737.

[27] Leesawat, P., Vearnsilp, K. Yanasarn, N.and Thanawattanawanich, P., Artificial tearformulation from chitosan, Chiang Mai J.Sci., 2005; 32(3): 501-505.

[28] Ludwig, A. and Van, O.M., Evaluationof sodium hyaluronate as viscous vehiclefor eye drops, J. Pharm. Belg., 1989; 44(6):391-397.

[29] Vogel, R., Crockett, R.S., Oden, N.,Laliberte, T.W. and Molina, L.,Demonstration of efficacy in thetreatment of dry eye disease with 0.18%sodium hyaluronate ophthalmic solution,Am. J. Opthalmol., 2010; 149(4): 594-601.