http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–12 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.920432

RESEARCH ARTICLE

Delivery of gatifloxacin using microemulsion as vehicle: formulation, evaluation, transcorneal permeation and aqueous humor drug determination Mohd Abul Kalam1, Aws Alshamsan1,2, Ibrahim A. Aljuffali1, Anil K. Mishra3, and Yasmin Sultana4

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Department of Pharmaceutics, Nanomedicine Research Unit, College of Pharmacy, King Saud University, Riyadh, KSA, 2Prince Salman Bin Abdulaziz Chair for Kidney Disease, King Saud University, Riyadh 11451, Saudi Arabia, 3Department of Radiopharmaceuticals, Institute of Nuclear Medicine and Allied Sciences (INMAS), Delhi, India, and 4Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi, India Abstract

Keywords

The successful ophthalmic delivery system is reliant on the diminution in the precorneal loss of drugs by increasing the corneal contact time and increasing the transcorneal permeability, which may enhance the bioavailability of drug to the eyes. The objective of this investigation was to develop and evaluate the potential of microemulsions of gatifloxacin with respect to the conventional eye drops of gatifloxacin. Oil-in-water microemulsions were prepared with different concentrations of oil, surfactant and co-surfactant using aqueous titration method. All formulations showed circular shape droplets, displayed an average droplet size ranged between 51 and 74 nm and absolute zeta potential values ranged from 15 to 24 mV, with optimum physicochemical characteristics suitable for eye. The optimized microemulsion possessed good stability, showed greater adherence to corneal surface and good permeation of gatifloxacin in the anterior chamber of the eye, resulting in a twofold increase in gatifloxacin concentration than the conventional dosage form. Hence, the optimized microemulsions showed increased intraocular penetration and enhance ocular bioavailability of gatifloxacin.

Isopropyl myristate, ocular irritation, ternary phase diagram, transcutol-P, Tween-80

Introduction Intraocular drug delivery is extraordinarily obstructed due to the impermeability of strong defensive barriers of the eye. The specific anatomy and physiology of the eye prevents the absorption and permeation of most of the active moieties (Shen et al., 2010). The eye drops are convenient to use, but majority of the drug is diluted by tear and rapidly drained from corneal surface, cul-de-sac and nasolachrymal drainage by constant tear turnover, which can overcome using microemulsions by which the therapeutic availability and efficacy of ophthalmic drug can be enhanced to a greater extent (Sultana et al., 2005; Silva et al., 2013; Verma et al., 2013). Due to limited ocular residence time of conventional eye drops, frequent instillation of eye drops is essential to maintain a prolonged continuous level of drug, but this causes the eye a massive and unpredictable dose of drugs, and a larger fraction of the drug may be lost by nasolachrymal drainage and the drained drug subsequently absorbed into systemic circulation, which may lead to undesirable systemic side effects (Mathiowitz, 1999). A number of formulation

Address for correspondence: Dr. Mohd Abul Kalam, Department of Pharmaceutics, Nanomedicine Research Unit, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, KSA. Email: [email protected]; [email protected]

History Received 5 April 2014 Revised 29 April 2014 Accepted 29 April 2014

scientists attempted to enhance the ocular bioavailability of drugs by overcoming pre-corneal constraints by manipulating the corneal permeation and prolonging pre-corneal retention of the formulation (Kaur & Smitha, 2002). Microemulsions are promising alternative with improved ocular retention, increased corneal drug absorption and reduced systemic side effects and maintain the simplicity and convenience of the dosage form as eye drops. Microemulsions are a useful vehicle to increase ocular availability of a many drugs like, pilocarpine hydrochloride (Chan et al., 2007), dexamethasone (Fialho & da Silva-Cunha, 2004; Kesavan et al., 2013), chloramphenicol (Lv et al., 2006), cyclosporine (Gan et al., 2009), etc. Microemulsions are the combination of three to five components: oil phase, aqueous phase, primary surfactant, co-surfactant and sometimes an electrolyte. The surfactant and co-surfactants provide low interfacial tension, which is an essential requirement for the formation and stability of microemulsion. The hydrophilic–lipophilic balance (HLB) of the surfactant is a useful guide for the selection of surfactant. The low HLB value (3–6) surfactants are considered for the formation of water-in-oil microemulsion, whereas high HLB value (412) are preferred for oil-in-water (o/w) microemulsion. The oil component influences curvature by its ability to penetrate and hence swell the tail region of the surfactant monolayers. Short chain oils penetrate the tail region to a

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greater extent than long chain alkanes, and hence swell to a greater extent, resulting in an increased negative curvature and reduced effective HLB value (Kommuru et al., 2001; Kawakami et al., 2002). The role of temperature is important in the formation of microemulsion prepared with non-ionic surfactant. The microemulsions with non-ionic surfactants is affected by temperature as the hydrophobic end of surfactants is dehydrated with increased temperature, whereas ionic surfactants are not strongly influenced by temperature and increase in temperature does not cause an increase in positive curvature due to counter ion dissolution. Due to smaller molecular size of isopropyl myristate (IPM), as compared to medium chain triglycerides, it gives larger microemulsion region in the ternary phase diagram, hence used as oil component in this study (Trotta, 1999). TweenÕ 80 was used as surfactant in the formulation of gatifloxacin microemulsion as it helped in the corneal permeation of parabens and it was found non toxic and non-irritating to the eyes (Lee et al., 1991; Akhtar et al., 2011). Transcutol-P (diethylene glycol monoethyl ether), an oil water-soluble liquid, was used as co surfactant. It is a synthetic chemical penetration enhancers and widely used solvent, which also has a great penetration-enhancing ability of many hydrophilic and hydrophobic drug molecules (Liu et al., 2006; Shokri et al., 2012). Advantages of microemulsions are their spontaneous formation, thermodynamic stability and high solubilizing potential for both hydrophilic and hydrophobic drugs. Microemulsions undergo phase transition from microemulsions to liquid crystalline form and to coarse emulsion with a change in viscosity depending on water content and incorporation of drugs do not affect their phase transition behavior. So, microemulsions are good alternative for ophthalmic delivery as it offers the pseudoplastic rheology with increased viscosity after application and increased ocular retention and retaining the therapeutic efficacy (Gan et al., 2009). Ophthalmic microemulsions offer many advantages over conventional eye drops, like increased ocular retention, possibility of releasing drug in sustained and controlled way, accurate dosing, without preservatives and increased shelf life. Gatifloxacin, a broad-spectrum antimicrobial fluoroquinolone, is active against Gram-positive and Gramnegative bacteria and frequently used in infections like, conjunctivitis, keratitis, keratoconjunctivitis and endophthalmitis (Price et al., 2005); however daptomycin is also indicated in endophthalmitis (Silva et al., 2013), but less effective than gatifloxacin; gatifloxacin can effectively clear pathogens with fast and strong effect. Gatifloxacin has a low minimum inhibitory concentration value, hence negligible possibility of drug resistance development (Gong et al., 2010). The mechanism of antibacterial activity of gatifloxacin is the inhibition of DNA-gyrase (topoisomerase II) and topoisomerase IV, the enzymes that cause partitioning of the bacterial chromosomal DNA during their cell division (Blondeau, 2004). Commercially available gatifloxacin eye drops (0.3%, w/v) is administered 2–3 drops at a regular interval of 2 h in the affected eye (Patel et al., 2012) to treat bacterial conjunctivitis, but the developed formulations would reduce the dosing frequency and hence will enhance the patient compliance.

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Materials and methods Materials Gatifloxacin was obtained as kind gift from Dr. Reddy’s Lab. Ltd. (Hyderabad, India). IPM and TweenÕ 80 were purchased from MerckÕ (Mumbai, India). Diethylene glycol monoethyl ether (Transcutol-P) was generously supplied by Gattefossee (Nanterre, France). Dialysis membrane (MWCO ¼ 12 kDa) was obtained from Sigma Aldrich (St. Louis, MO). Reference eye drops solution, ZigatÕ Eyedrops, 0.3% w/v was obtained from FDC Ltd. (Mumbai, India). All other chemicals used in the study were of highest purity grade. Methods Development of pseudo-ternary phase diagram Construction of phase diagram is a useful approach to illustrate the various components and series of interactions that can occur when different components are mixed together. As the quaternary phase diagram is time consuming and difficult to interpret, pseudo-ternary phase diagram is often constructed to find the different zones including microemulsion zone; each corner of the diagram represents 100% of the particular components. Figure 1, represents schematically pseudo-ternary phase diagram at constant surfactant to co-surfactant ratio, showing o/w type microemulsion regions. Preparation of microemulsion o/w microemulsion of gatifloxacin was prepared by using appropriate amounts of deionized water (DW) as aqueous phase, IPM as oil phase, TweenÕ 80 and Transcutol-P were used as surfactant and co-surfactant phases, respectively. Appropriate amount of gatifloxacin (0.3%, w/w) was dissolved in oil phase. The oil phase was added to the mixture of surfactant and co-surfactant phase, and then the aqueous phase was added to the mixture and vigorously stirred and vortexed to get microemulsion. A total of seven formulations were selected from the microemulsion region for further study, formulae of which with different batches are listed in Table 1. Eye drops are generally dispensed in multidose containers, each of which is intended to be used within defined time duration, once it is opened. Thus, to maintain the sterility of the preparation during use and shelf life, benzalkonium chloride (0.005%, w/w) was added as a preservative to each formulation (Fialho & da Silva-Cunha, 2004). Characterization of o/w microemulsion Transmission electron microscopy. Structural morphology of microemulsion droplets were studied by using transmission electron microscopy (TEM), TOPCON 002B operating at 200 kV (Topcon, Paramus, NJ) and of a 0.18 nm capable of point-to-point resolution. The combinations of bright field imaging at increasing magnification and diffraction modes were used to reveal the morphology and structure of the microemulsions. To perform the TEM observations, a drop of the microemulsion was stained with 2%, w/v phosphotungstic acid solution and directly placed on the copper grids, dried at room temperature and were observed.

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Figure 1. Pseudo-ternary phase diagram of oil (IPM), water and Smix, presenting microemulsion zones (A) Tween-80:Transcutol-P (1:1), IPM and water (B) Tween80:Transcutol-P (1:2), IPM and water (C) Tween-80:Transcutol-P (2:1), IPM and water; (D) Tween-80:Transcutol-P (3:1), IPM and water.

Table 1. Few ternary phase compositions and some characterization parameters of o/w microemulsions. Surfactant mixtures (Smix) (%)a S:CoS ratio 1:0 1:1 2:1 3:1

IPM (%)a

Tween-80

Transcutol-P

Deionized water (%)a

Droplet size (nm) ± SD

PI ± SD

Zeta potential (mV) ± SD

Formulations

10.0 10.0 15.0 10.0 15.0 10.0 15.0

20.0 10.0 10.0 15.0 13.0 20.0 19.0

– 10.0 10.0 8.0 7.0 6.0 5.0

70.0 70.0 65.0 67.0 65.0 64.0 61.0

74.55 ± 5.45 51.42 ± 2.85 58.25 ± 3.55 70.21 ± 4.05 72.32 ± 6.23 63.46 ± 3.89 68.54 ± 4.56

0.232 ± 0.022 0.163 ± 0.015 0.189 ± 0.014 0.224 ± 0.023 0.281 ± 0.025 0.192 ± 0.018 0.215 ± 0.015

15.65 ± 3.15 24.35 ± 4.21 23.05 ± 4.65 18.42 ± 2.08 17.22 ± 2.01 21.85 ± 2.45 20.22 ± 1.98

M/E101 M/E110 M/E115 M/E210 M/E215 M/E310 M/E315

n ¼ 3, ± SD. a All are in terms of w/w.

Droplet size measurements. The average droplet size and size distributions of the microemulsions droplets were measured by dynamic light scattering (DLS) using Malvern Zetasizer Nanoseries-ZS (Malvern Instruments, Malvern, UK). Briefly, a few drops of each sample were added to 5 ml filtered distilled water (0.2 mm PVDF filter, Millipore, Billerica, MA) in a polystyrene disposable cuvette and placed the path of light that measures DLS. The scattered light signal is collected with a detector, at a 90  (right angle) scattering angle and at 25  C temperature. Measurements were done in triplicate; average droplet size, polydispersity index and zeta potential were determined (Driscoll et al., 2001). For the zeta potential measurements, each sample was diluted with double-distilled water and the electrophoretic mobility was determined at 25  C using dispersant dielectric constant of 78.5. The obtained electrophoretic mobility values were used to calculate the zeta potentials using the software DTS, version 4.1 (Malvern, England, UK) as illustrated by (Attama et al., 2007).

Physicochemical characterization of the optimized microemulsion (M/E110) Osmolarity, refractive index, clarity, pH and viscosity. Osmolarity of microemulsion (M/E110) was determined by Osmometer (Fiske Associate, Norwood, MA), refractive index by Abbe’s refractrometer (Scientific, Mumbai, India), clarity was observed by visual examination under light alternatively against white and black background and pH was determined by a calibrated pH meter (Mettler Toledo MP-220, Greifensee, Switzerland). Surface tension was measured by the method of Ferguson and Kennedy adopted by Tiffany et al. (1989), on small volume (0.3–0.4 mL) samples of microemulsions and ZigatÕ eyedrops. All the measurements were done in triplicate. Rheological study of microemulsion M/E110. Viscosity of ophthalmic preparations is an important parameter; hence, the viscosity of the microemulsion was determined by Cone and Plate Viscometer (Physica Rheolab, Graz, Austria) using

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Figure 2. Rheogram (A) and steady-flow viscosity versus shear rate of formulation M/E110 tested a 25  C (B) (n ¼ 3, ± SD).

MK-22 spindle. Approximately about 1 ml of formulation M/E110 was placed on the plate and spindle was touched with the sample, temperature was adjusted at a temperature of 25  C and system was started. The rheograms between shear rate (s1) and shear stress (Pa), as well as viscosity (PaS) versus shear rate of M/E110 were plotted (Figure 2). In vitro drug release study The developed microemulsions were subjected to in vitro drug release study by using flow-through cell. Briefly, onemilliliter sample was taken in the dialysis tubing (MWCO 10 kDa, cellulose membrane, Sigma Aldrich) and kept in the jacketed flow-through cell (simulating the artificial eye). The dissolution medium was simulated tear fluid (STF) pH 7.4. One-hundred twenty-five microliters of STF of pH 7.4 was placed in the flask and 1 ml sample was withdrawn at regular intervals and replaced with fresh buffer to maintain the sink condition. The buffer was allowed to flow through the artificial eye with the help of the peristaltic pump. The flow was regulated with flow regulator to 10 drops per minute. To compensate blinking of the eye, air bubbles were blown in the artificial eye through an aerator. The content of the flask was continuously stirred with the help of magnetic stirrer. The whole assembly was maintained at 37 ± 0.5  C by the circulation of warm water through the outer jacket. The water from the water bath maintained at 37 ± 0.5  C was circulated through the outer jacket of the flow-through cell and then through the outer jacket of the flask and finally to the sink. Withdrawn samples from the flow-through cell were analyzed to determine the drug concentration by UV-spectrophotometer at 293 nm; by using the standard calibration curve, cumulative amount of drug released was calculated in triplicate. Physicochemical stability testing Stability studies were conducted to determine the effect of the presence of other excipients on the stability of the drug in optimized o/w microemulsion (M/E110) and also to determine physicochemical properties on accelerated conditions (Srividya et al., 2001). The optimized M/E110 was packed in amber-colored 10 ml capacity amber-colored glass vials and sterilized by steam sterilization. The formulation was stored in a refrigerator at temperature 2–8  C for a period of six months that could be considered as long-term

stability (Ibrahim et al., 2009), and steam sterilization could be considered as the accelerated stability test (Benita & Levy, 1993). Antimicrobial effectiveness The antimicrobial property of the drug in to the formulation was determined by agar diffusion test employing well-plate technique. Marketed ZigatÕ eye drops (standard solution) of gatifloxacin and the optimized formulation M/E110 containing gatifloxacin (test solution) were poured into wells, bored into sterile nutrient agar previously seeded with test organisms (Staphylococcus aureus, Bacillus subtilis and Escherchia coli) (Kalam et al., 2008; Pokharkar et al., 2014) and left undisturbed for 2 h for complete diffusion of the poured solutions after that the agar plates were incubated at 37 ± 2  C for 24 h. Then zone of inhibition (ZOI) was measured around each well and then compared with the control one. The entire operation except the incubation was carried out in a laminar flow unit. Each solution was tested in triplicate. Transcorneal permeation study Transcorneal permeation was performed according to our previously used method (Kalam et al., 2013). Briefly, freshly excised goat cornea was fixed between the donor and receptor component of automated transdermal diffusion cells (sampling system-SFDC 6, LOGAN, Somerset, NJ) to determine the transcorneal permeation of the drug from the microemulsion up to 4-h study period. The sample was withdrawn from the receptor compartment and analyzed for gatifloxacin content by using UV-visible spectrophotometer at 293 nm max. The permeation parameters of gatifloxacin from the o/w microemulsion were calculated by plotting the amounts of drug permeated through cornea (mg cm2) versus time (h), and the slope of the linear portion of the graph was calculated. The steady-state flux (J) values across cornea were evaluated (Equation (1)) from the linear ascents of the permeation plots by following the expression:
dQ J mg cm2 s1 ¼ dt:A

ð1Þ

where, dQ/dt is the linear portion of the slope, A is the exposed area of the cornea (0.636 cm2), and t, is the

Delivery of gatifloxacin using microemulsion as vehicle

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exposure time. The coefficient of permeation (P) was calculated by using Equation (2):
J P cms1 ¼ C0

ð2Þ

where, C0 represents the initial drug concentration (mg mL1), i.e. 3000 mg mL1, in the donor compartment (Attama et al., 2008). Finally, corneal hydration level of each cornea used for this experiment was calculated at the end of the experiment (Liu et al., 2005).

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In vivo studies New Zealand white rabbits weighing 2–3 kg were used for irritation, corneal retention and pharmacokinetic studies. Animals were housed as one animal per cage and maintained at 20–30  C and 50–55% relative humidity in a natural light and dark cycle, with free access to food and water. Permission for the use of animals was obtained from animals’ ethics committee of institute of nuclear medicine and allied science, Delhi, India. Utmost care was taken to ensure that animals were treated in the most human and ethically acceptable manner.

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chromatography by calculating the percentage binding from a Gamma Counter (Electronics Corporation, Bangalore, India). Recording was started 5 s after instillation and continued for 10 min using 128  128 pixel matrix. Images were analyzed using dual head Gamma camera (Millenium VG). All the images were divided into three regions of interest (ROIs) (figure not shown), and the movement of the Gamma emitting material accurately followed within the zones were observed. The radioactivity in the anatomical region of interest in the first frame was assumed to be 100% of the instilled dose, and the remaining activity in the precorneal regions was plotted against time to evaluate the elimination parameters (Felt et al., 1999; Alany et al., 2006). A one-way analysis of variance (ANOVA) at 95% confidence intervals was used to test for significant difference between animals and formulations with regard to the % radioactivity remaining in the ocular ROIs at 10 min and the area under the radioactivity remaining (AUC) versus time (0–10 min) profile (Wei et al., 2002). After excluding any animal effect owing to insignificant differences between the six rabbits, a one-way ANOVA followed by t-test at a 95% confidence interval was used to test for significant difference between two formulations.

Ocular irritation studies For a drug delivery system intended for ophthalmic use, it is important to assay the ocular tolerability. In vivo ocular irritation was assessed in New Zealand white rabbits by the Draize’s eye test, which is the certified technique for the evaluation of cosmetic and pharmaceuticals for ocular instillation (Gonzalez-Mira et al., 2010). A single instillation of 50 mL of each preparation, containing 0.3%, w/v of gatifloxacin, was administered in the left eye of each rabbit, using untreated right eye as a control. Readings were executed 1 h after sample instillation, then after 1, 2, 3, 4 and 7 d. The Draize’s score was determined by visual assessment of changes in the ocular structures. Discomfort of the rabbit eyes were graded in such a way that the slight irritation was characterized by half closed and severe irritation by firm closure of the eyelid. The eyelid closure was expressed as the sum of full closure and half closure times of the eyes. Mucoidal discharge was scored from 0 to 2, where 0 is normal; any clear discharge different from normal is 1 and milky discharge moistening the lids scores 2, and then average total score was calculated (Araujo et al., 2010). Corneal retention by scintigraphy technique In vivo precorneal drainage of radionuclides was studied using dual head Gamma camera (Millenium VG, Milwaukee, WI), autotuned to detect the 140 KeV radiation of 99mTc. The optimized formulation M/E110 and ZigatÕ eye drops was assessed in six rabbits with a minimum washout period of three days. The rabbit was positioned 5 cm in front of the probe, and radiolabeled formulations, previously stored at 20  C for 30 min before use, were instilled onto the left corneal surface of the rabbits. Radiolabeled formulation was prepared by adding aqueous stannous chloride in to the formulation as a reducing agent, pH was adjusted to 7.2 and then activity (99mTc pertechnetate solution) was added and vortexed. The stability of binding was determined by instant thin-layer

Ocular pharmacokinetic study On the basis of physicochemical parameters, drug release in vitro, transcorneal permeation and irritation potential, as well as the constraints in animal use, only formulation M/E110 and ZigatÕ eye drops were subjected for ocular pharmacokinetic study (aqueous humor drug determination). The drug concentration in the aqueous humor after instillation in rabbit’s eyes was determined in order to evaluate the ocular availability of gatifloxacin from M/E110, which was compared with 0.3%, w/v aqueous solution of gatifloxacin (ZigatÕ eye drops). The study was performed on New Zealand albino rabbits of 3.0–4.0 kg body weight. Animals were divided into two groups of each containing seven rabbits. The availability of gatifloxacin in aqueous humor was estimated by administering in the cul-de-sac of both eyes of all rabbits of the two groups (Liu et al., 2007; Fu et al., 2008) a volume of 50 mL of the sterilized M/E110 and ZigatÕ eyedrops, respectively, each containing 0.3%, w/v gatifloxacin. Both the eyes of each rabbit of the one group received single topical instillation of M/E110, and both the eyes of each rabbit of the second group was given single topical instillation of ZigatÕ eye drops. After half-an-hour of the instillation, rabbits were anesthetized by i.v. administration of ketamine. After half-an-hour of the instillation, rabbits were anesthetized by i.v. administration of Ketamine hydrochloride injection to the ear vein in each rabbit, then a 29-gauge insulin syringe needle was used to aspirate 50 mL of aqueous humor through a partial thickness limbal incision at 0.5, 1, 2, 3, 4, 5 and 6 h time points (Levine et al., 2004). Aqueous humor was withdrawn from both eyes of one rabbit at each time point. The collected aqueous humor samples were analyzed for the drug concentration by HPLC method (Jain et al., 2010; Kalam et al., 2013). The obtained data of the pharmacokinetic parameters (calculated using PK Solver software, Nanjing, China) were

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compared by one-way ANOVA, by considering the value p50.05 as statistically significant (Zhang et al., 2010). The concentrations of drug were determined in the aspirated aqueous humor samples by HPLC technique, then the amount of drug was calculated and pharmacokinetic parameters were estimated by non-compartmental model approach. The elimination rate constant (kel) was calculated by log-linear decay of the drug in the elimination phase and the half-life (t1/2) was calculated with the relationship; t1/2 ¼ 0.693/kel. The peak aqueous humor drug concentration (Cmax) and time of peak (tmax) were obtained by visual examination of the concentration-time curve (Kozai et al., 2009). The area under the aqueous humor drug concentration versus time curve (AUC0– t) from zero to the time of last measured concentration (Clast) was calculated by the log-linear trapezoidal method. The AUC0–1 was obtained by the addition of AUC0–t and the extrapolated area estimated as Clast/kel. Statistical processing of results is performed with the help of MICROSOFT EXCEL 2007 software, Redmond, WA. Results are expressed as mean values ± SD.

Results and discussion Phase behavior Pseudo-ternary phase diagram of the systems (TweenÕ 80:Transcutol-P (1:1)), IPM and DW, TweenÕ Õ 80:Transcutol-P (1:2), IPM and DW, Tween 80:TranscutolP (2:1), IPM and DW, and TweenÕ 80:Transcutol-P (3:1), IPM and DW were constructed. The phase diagrams are presented in Figure 1, where DW was first component, TweenÕ 80 and Transcutol-P (Smix) was second and variable amount of IPM was used as third component. In all the phase diagrams, the surfactant phase was a mixture of TweenÕ 80 and Transcutol-P in different ratios (Table 1). The simple aqueous titration method was adopted to construct the pseudo-ternary phase diagrams. In the mixtures of surfactant and co-surfactant, appropriate amount of IPM was added and vortexed, then the mixture was titrated through drop-by-drop addition of DW by using micropipette; during the titration, samples were vortexed in order to reach the equilibrium quickly. The phase boundary was determined by observing the changes of sample appearance from turbid to transparent or from transparent to turbid. All samples that remained transparent and homogenous after vigorous stirring were considered to belong to the mono-phasic area in the phase diagram, and the dilution line was studied by adding phosphate buffer until solution became turbid (Kizilbash et al., 2011; Fouad et al., 2013). At the very low aqueous content, for example 510%, liquid crystal phase was often generated. In the pseudo ternary phase diagrams (Figure 1), V, X, Y and Z represents the region of o/w microemulsions. The factors that affect the phase behavior of the microemulsion system are the chain length of oil used and the quantity of surfactant and co-surfactant mixture. It can be seen clearly in Figure 1 that the microemulsion region varies as the surfactant-co-surfactant ratios varies. Chain length compatibility of surfactant and oil is very important factor regarding the formation of microemulsion. The maximum water solubilization by surfactants occurred when la + lo ¼ ls (where la, lo and ls are the lengths of

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hydrocarbon chains in alcohol, oil and surfactant, respectively). The equation of la + lo ¼ ls is also called BSO equation and reflects the requirements of chain length compatibility (Ezrahi et al., 2005). Formulation development After exploring a number of microemulsion systems, one microemulsion containing 10%, w/w of a non-ionic surfactant (TweenÕ 80), IPM as the oil phase (15% w/w), DW with a preservative benzalkonium chloride (0.005% w/w) as the aqueous phase and 10% w/w of Transcutol-P as the co-surfactant, was considered as the optimized one. Gatifloxacin was used at a concentration of 0.3% w/w. For a drug delivery system aimed for ocular instillation, it is important to assay the ocular tolerability. The selection of TweenÕ 80 as a surfactant has been reported as non-irritating to the rabbit eye up to a concentration of 10% and has been used in a number of marketed ophthalmic preparations (Jiao, 2008). However, the higher polydispersity index of colloidal dispersions is an important factor for ocular irritation, hence it should be low (Gonzalez-Mira et al., 2010). The phase behavior of the microemulsion was greatly influenced by IPM, as it produced a larger microemulsion region because of smaller molecular size of IPM as compared to medium chain triglycerides (Fialho & da Silva-Cunha, 2004). Depending on the size, chain length and volume of the oil molecule, surfactant penetration in the tails of hydrocarbon changes the chain volume of the hydrocarbon part of surfactant molecule and hence influences the effective geometric packing parameter, which can be achieve by the use of co-surfactant (Trotta, 1999). Similarly, it was observed in case of TweenÕ 80 and TweenÕ 20. TweenÕ 80 has longer hydrocarbon chain than TweenÕ 20 and increases the microemulsion region (Lv et al., 2005). This is the rationale of the use of IPM and TweenÕ 80 in the development of microemulsion in this work. Transcutol-P was used as co-surfactant in the formulation of microemulsion because it is considered as high purity solvent and solubilizer for poorly water soluble drugs and highly associated with improved drug penetration through the biological membranes (Liu et al., 2006; Ge et al., 2014). Morphology by TEM, droplet size and zeta potential measurements TEM results revealed that the microemulsion droplets in the developed system were circular in shape (Figure 3) with an

Figure 3. TEM microphotograph of microemulsion (M/E110).

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DOI: 10.3109/10717544.2014.920432

even distribution of contrast and can easily be distinguished (Kuntsche et al., 2009). Morphometrical properties (mean droplet/particle size and polydispersity index) and the physical stability of any colloidal systems (that is influenced by zeta potential) are considered as the important factors for the development of ophthalmic drug delivery systems to avoid ocular irritation and to enhance and control the particles or droplets retention time on the eye surfaces. For an optimized formulation intended for ocular use, the mean droplet size and polydispersity index should be as low as possible because with the larger sizes a grittiness feeling might occur. Therefore, a reduction in the droplet size improves the patient comfort during ocular instillation. The average droplet size of the developed microemulsions varied from 51.42 ± 2.85 (M/E110) to 74.55 ± 5.45 nm (M/E101), and the particle size that human eyes can tolerate is about 10 mm (Zimmer & Kreuter, 1995), so the developed formulation is considered as suitable for ocular use. The zeta potential values ranged between –15.65 ± 3.15 (M/E101) and –24.35 ± 4.21 mV (M/E110), and the value of polydispersity index ranged between 0.163 ± 0.015 (M/E110) and 0.281 ± 0.025 (M/E215) as mentioned in Table 1. The droplet size distribution plot and polydispersity index (measures the width of droplet size distribution) of the optimized M/E110 showed unimodal distribution of the droplets (Figure 4), that is indicative of the stable dispersion of the droplets. The zeta potential is an important factor that predicts the physical stability and the potential mucoadhesion of any colloidal systems (Araujo et al., 2009). Theoretically, high absolute values of zeta potentials have a propensity to stabilize colloidal systems, and droplet aggregation does not occur due to the electrostatic repulsion between the droplets with the same electrical charge (here the droplets are negatively charged). Physicochemical characterization The physicochemical characterization of the optimized o/w microemulsion (M/E110) and marketed eye drops was performed, and it was found that most of the parameters were satisfactory for ophthalmic use (Table 2). The pH of the

Figure 4. Droplet size distributions of the optimized M/E110.

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developed microemulsion was found to be 6.7 and that of the marketed eye drops was 7.0, which can be easily buffered by tear fluid (pH 7.2–7.4) hence did not cause any irritation, reflex tears and rapid blinking of the eye. The better solubility of gatifloxacin at lower pH would provide a good physical stability by preventing the crystallization at low ambient temperature, so the attained pH of the microemulsion is good for gatifloxacin stability in the formulation as well. The osmolarity of human eye fluid after prolonged eye closure is 293–288 mOs Kg1 and as eye is opened, it is progressively rises at a rate of 1.43 mOs Kg1 h1 up to 302–318 mOs Kg1 (Washington et al., 2001). The ophthalmic preparations intended for instillation into the cul-de-sac of eye should be isotonic to avoid irritation and here in the case of M/E110 the osmolarity was found to be 316 mOs Kg1, so the prepared microemulsion is good for ocular use in terms of isotonicity. The surface tension of tear fluid at the eye surface temperature (33  C) is in the range of 44–50 mN m1 and the administration of any solution containing actives and or excipients that may lower the surface tension, which in turn may disrupt the outermost lipid layer of the tear film (Tiffany et al., 1989). Therefore, surface tension measurement is important parameter for the ophthalmic preparations, and in the case of developed o/w microemulsion of gatifloxacin, it was found to be approximately 49 mN m1. This value was in agreement with the surface tension of tear fluid, hence any irritation and reflex blinking will not occurred due to the sensation of a foreign body (ophthalmic microemulsion) in the eye. Rheological evaluation of microemulsion M/E110 The administration of an ophthalmic formulation should not influence the pseudoplastic nature of the precorneal film, if they do so, it should be negligible. Continuous shear rheometry investigations displayed pseudoplastic flow characteristics for both the systems tested. In Figure 2(A), formulations exhibited pseudoplastic rheology as evidenced by shear thinning and an increase in the shear stress with increased shear rate, and Figure 2(B) supports the condition that the viscoelastic fluids with a viscosity that is high under the low shear rate, and low under the high shear rate conditions (enhance corneal retention) are appropriate for ophthalmic use due to the fact that the ocular shear rate is very high, particularly during the blinking period. Moreover, the viscosity of human tears ranges from 1.3 to 5.9 mPas with a mean value of 2.9 mPas (Washington et al., 2001); and in this case, the viscosity of the ophthalmic microemulsion was found to be about 5.5 mPas initially and 6.3 mPas even after a six-month storage period at the simulated shear rate condition (enhanced blinking) of the eye.

Table 2. Physicochemical characteristics of o/w microemulsion M/E110 of gatifloxacin.

Batch M/E110 ZigatÕ eyedrops n ¼ 3, ± SD

Clarity

pH

Refractive index ± SD

Surface tension (mN m1) ± SD

Osmolarity (mOs Kg1) ± SD

Viscosity (mPa s) ± SD

Clear transparent Clear solution

6.7 7.0

1.28 ± 0.14 1.32 ± 0.09

49.5 ± 4.2 45.5 ± 3.5

316.2 ± 15.5 309.5 ± 12.4

5.5 ± 1.12 –

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Figure 5. Cumulative drug released versus time (h) curve of formulations M/E101, M/E110, M/E115, M/E210, M/E215, M/E310 and M/E315.

Table 3. Zero-order (K0) and first-order (K1) release rate constants (n ¼ 3, ± SD) with percent coefficient of variation (% CV).

Table 4. Results of stability testing of M/E110 (physicochemical and physical parameters).

Formulations

Physicochemical characteristics

M/E101 M/E110 M/E115 M/E210 M/E215 M/E310 M/E315

K0 ± SD

% CV

K1 ± SD

% CV

0.3076 ± 0.4715 0.3405 ± 0.5965 0.3661 ± 0.6126 0.2097 ± 0.2940 0.1907 ± 0.2782 0.1986 ± 0.2869 0.2006 ± 0.2933

153.39 175.18 167.29 140.16 145.90 144.47 146.24

0.2360 ± 0.1436 0.2074 ± 0.0829 0.2088 ± 0.0807 0.1552 ± 0.0868 0.1570 ± 0.0933 0.1722 ± 0.0893 0.1713 ± 0.0838

60.84 39.95 38.66 55.91 59.42 51.87 48.92

Clarity pH Refractive index Osmolarity (mOs Kg1) Viscosity (mPa s) Droplet size (nm) Polydispersity index Zeta potential (mV) Drug content (%)

Results of batch M/E110 (n ¼ 3, ± SD) Clear transparent 6.8 1.31 ± 0.076 319 ± 16.4 6.3 ± 1.5 55.45 ± 3.65 0.145 ± 0.012 26.25 ± 45.25 98.99

In vitro drug release The developed o/w microemulsions were subjected to in vitro drug release studies. The graphs between cumulative % drug released versus time for o/w microemulsions (Figure 5) and cumulative % drug released versus square root of time was plotted, and release rate constants were calculated for zeroand first-order kinetics in order to establish the nature of release kinetics. The plot between cumulative drugs released versus square root of time represented almost a straight line for the formulation M/E110. Zero- and first-order release rate constants were determined. The coefficient of variation was less for first-order release rate constant and was found to be higher for zero-order release rate constant calculated for all the formulations; this indicated the release of drug from all the tested formulations followed first-order release rate kinetics (Table 3). Stability testing Following the stability testing, the M/E110 was found to be a clear transparent biphasic solution and exhibited no precipitation. There was no any phase separation and creaming on visual observation and was found stable after cooling centrifuge at 5000 rpm for 10 min. The results (Table 4) indicated that there are negligible changes (p50.05) in the parameters like pH, refractive index, osmolarity, viscosity, droplet size and polydispersity index of M/E110 after six months of storage at 2–8  C in amber colored glass vials. The droplet size and surface properties are the important parameters for the evaluation of the stability of any colloidal systems. Hence, the droplet size and the surface

electrical charge (zeta potential) were evaluated after six months of storage at 2–8  C. Stability studies revealed that there was a negligible effect on the drug content due to the presence of other excipients in the optimized microemulsion. Antimicrobial effectiveness An antibiotic may give assay approximately 100% by chemical or spectrophotometric methods of estimation, but may not give these values when tested microbiologically. This might be due to isomeric effect. Some isomers of the drugs are inactive against microorganisms, while they give good assay results by analytical methods other than microbiological type. In this study, gatifloxacin, a fluoroquinolone antibiotic, was used, so it was necessary to carry out microbiological studies to demonstrate the effect of drug in ophthalmic o/w microemulsion against the microorganisms (Bassetti et al., 2001). The ZOIs produced by the o/w microemulsion and marketed eye drops against the growth of microorganisms, i.e. S. aureus, E. coli and B. subtilis, can be represented graphically in Figure 6. The broad-spectrum antibacterial activity of gatifloxacin in microemulsion (against the growth of microorganisms: S. aureus, B. subtilis and E. coli) was observed to be almost similar to reference solution (ZigatÕ eyedrops, FDC Ltd.), suggesting that the preparation process used and the different conditions employed during preparation of formulation did not affect the intrinsic antimicrobial activity of the entrapped gatifloxacin.

Delivery of gatifloxacin using microemulsion as vehicle

Transcorneal permeation

of gatifloxacin and its ocular availability after 4 h. The pKa values of gatifloxacin are: 5.94 for the carboxyl group (pKa1) and 9.21 for the piperazinyl group (pKa2). The octanol/water partition coefficient of gatifloxacin at pH 5.1 was found to be 0.044, and at pH 7.0, the value increased to 0.145 (Lutsar et al., 1998). Thus, the shifting of pH of the formulation toward neutrality (pH of tear fluid) is a major cause for the existence of a larger fraction of the drug in non-ionized form, which has high lipid solubility, and higher lipid solubility at neutral pH encourage high permeation of gatifloxacin through the cornea. The permeation of drug from ZigatÕ eye drops was higher at very first hours of the study; this might be due to the fact that most of the drug would be in un-ionized form at the higher pH (7.2) of the ZigatÕ eyedrops. From the results of the permeation profiles, M/E110 delivered drug with a sustained rate as compared to conventional eye drops, which showed instant release of most of the drugs from its solution form. The addition of benzalkonium chloride as preservative in M/E110 is thought to be responsible for increased permeability coefficient (P) value of gatifloxacin, as benzalkonium chloride, a cationic surfactant has been reported to increase the corneal permeation of drugs by emulsification and disruption of the corneal epithelium (Ahuja et al., 2007; Mohanty et al., 2013).

The permeated amount per unit area through excised goat cornea was found to be higher but very fast in case of solution form (ZigatÕ eyedrops) when compared to its permeation from microemulsion (Figure 7). The amount permeating per unit area per unit time (steady-state fluxes, J) were calculated. The steady-state fluxes and maximum apparent corneal permeability coefficients (P), obtained for the two ophthalmic preparations and their corneal hydration potentials are listed in Table 5. Corneal hydration level remained in the normal range of 75%–80%, when the pH of the formulation was in the range of 6–7 (Rathore & Majumdar, 2006). Since the corneal hydration level was within the limit of normal range, then there was no corneal damage and if it happened, it would be reversible. Thus, the optimized microemulsion was considered safe and non-damaging to the eye. The pH of the formulation was found to be appropriate for permeation

9

Irritation study and statistical analysis The results of ocular irritation studies indicated that formulations were non-irritant and excellent ocular tolerance was observed (Table 6). Abnormal clinical signs to cornea, iris or conjunctiva were not seen. Thus, there was no ocular damage, indicating the relative safety of the developed microemulsion, which was evidenced by the statistical Figure 6. Zone of inhibition by the o/w microemulsion (M/E110) and marketed eye drops. Table 6. Ocular irritation studies and summary of paired t-test for mucoidal discharge and eyelid closure. 90 Cummulave amount of gafloxacin permeated (μg.cm-2)

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DOI: 10.3109/10717544.2014.920432

Mucoidal discharge

80

Õ

Zigat eye drops

70

Rabbits

60 50 40 30 20 Zigat® eyedrops M/E110

10 0

0

50

100

150 Time (min)

200

250

300

Figure 7. Transcorneal permeation of gatifloxacin from M/E110 and ZigatÕ eye drops (n ¼ 3, ± SD).

First 0 Second 0 Third 1 Fourth 0 Fifth 0 Sixth 1 Summary of paired t-test Average scores 0.3333 No. of points 6 Standard deviation 0.5164 a Lower 95% CI 0.2087 Upper 95% CIa 0.8753 a

M/E110 eye drops 1 0 1 1 0 0 0.5000 6 0.5477 0.0748 1.075

Eyelid closure ZigatÕ eye drops 0 1 0 1 0 1 0.5000 6 0.5477 0.0748 1.075

CI is confidence interval.

Table 5. Transcorneal permeation parameters of gatifloxacin-containing M/E110 and ZigatÕ eyedrops (n ¼ 3, ± SD).

Batch M/E110 ZigatÕ eyedrops

Amount permeated (mg cm2 at 4 h)

pH

Steady state flux, J (mg cm2 h1)

Permeability coefficient, p (cm h1)

Corneal hydration (%)

73.56 ± 4.98 79.63 ± 5.41

6.7 7.0

0.3855 0.2381

1.2851  104 0.7935  104

78.25 ± 0.238 79.34 ± 0.325

M/E110 eye drops 0 1 1 1 0 1 0.6667 6 0.5164 0.1247 1.209

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analysis. Statistical analysis of mucoidal discharge and eyelid closure by using the marketed and M/E110 microemulsion preparation was performed by the use of paired t-test. With the application of paired t-test, the two tailed p value was found to be 0.6109 for mucoidal discharge, which could be considered as not significant, t ¼ 0.5423 with five degree of freedom. Similarly, the two-tailed p value was found to be 0.3632 for eyelid closure, which could be considered as not significant, t ¼ 1.000 with five degree of freedom.

Gamma scintigraphy is a well-established method for in vivo evaluation of the ocular drug delivery. The M/E110 formulation showed at all times a significantly higher retention of the radioactive tracer in the corneal region of interest in comparison to commercial (ZigatÕ ) eye drops. ZigatÕ eye drops were rapidly cleared due to tear turnover and nasolachrymal drainage, giving decrease bioavailability. Presence of oily phase in the case of microemulsion prolonged the residence of the radioactive tracer in the precorneal region. An initial rapid clearance phase and then a slower basal drainage phase were noticed in both the formulations, which is in conformity with the reported biphasic nature of the clearance process of solutions (Alany et al., 2006). The parameters describing the precorneal drainage are summarized in Table 7; and Figure 8 shows the percentage of instilled radioactivity remaining in the ocular region as a function of time for the optimized microemulsion and ZigatÕ eye drops up to 10 min. One-way ANOVA followed by paired t-test at a 95% confidence interval was conducted to test for differences Table 7. 99mTc radioactivity remaining in the preocular regions after 10 min (a10) and area under the radioactivity remaining versus time curve (AUC0!10 min) for the two formulation (n ¼ 6, ± SD).

Formulations M/E110 ZigatÕ eye drops

% Binding efficiency

% Radioactivity remaining (a10) (Mean ± SD)

AUC0!10 min (Mean ± SD)

94.0 96.0

55.156 ± 7.85 39.755 ± 6.02

14675.5 ± 145.35 10282.4 ± 132.85

Ocular pharmacokinetic study The adopted HPLC method (Kalam et al., 2013) was used for the quantitative determination of gatifloxacin in aqueous humor samples collected following the topical administration of M/E110 and ZigatÕ eye drops to rabbit eyes. The estimated concentrations of drug measured in aqueous humor collected at 0.5, 1, 2, 3, 4, 5 and 6 h (Figure 9). The aqueous humor levels of gatifloxacin in the group of rabbits treated with ZigatÕ eye drops was detected only up to 4 h and remained undetectable after that, attributed to the rapid precorneal loss of eye drops in the solution form through lachrymal drainage and tear turn over. Quite the reverse, the drug was quantified in aqueous humor of rabbits for at least up to 6th h (99.96 ± 11.98 ng mL1) of the study period in the group treated with M/E110. The results of the study indicated the ocular bioavailability of gatifloxacin was found to be increased significantly (p50.05). A twofold increase in the relative bioavailability was found with the M/E110 when compared with ZigatÕ eye drops, the marketed formulation. A 2.32-fold increase in the AUC0!1 was calculated with the M/ E110 as compared to ZigatÕ eye drops. The half life (t1/2) of the formulation M/E110 was found to be a 3.17-fold longer than the marketed ZigatÕ eye drops, seem to be significantly increased (p50.05). The Cmax of gatifloxacin from M/E110 illustrated 1.12-fold elevated concentration as compared to ZigatÕ eye drops. The results indicate that the prolonged ocular residence time of M/E110 and hence maintained a higher fraction of 110M/E Zigat eyedrops

450 400 Aqueous humor gafloxacin concentraon (ngmL-1)

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Precorneal retention of radiolabeled M/E110 by gamma scintigraphy

between the two formulations at the final time point with regard to percentage activity remaining, and it was found to be significant (p50.005). M/E110 showed a significantly greater percentage radioactivity remaining in the preocular area after 10 min (a10) as compared to ZigatÕ eye drops and the AUC0!10 min was found significantly increased as compared to the marketed ZigatÕ eye drops reaching to 1.43fold increase approximately. This is due to the longer preocular retention of the microemulsion (Liu et al., 2006). But, no blinking of the eye, no conjunctival redness, no mucoidal discharge and no ocular swelling were observed with the microemulsion in the treated left eye as compared to the untreated right eye of the same rabbit.

350 300 250 200 150 100 50 0

Figure 8. Precorneal drainage (the percentage of instilled radioactivity remaining in the ocular region as a function of time) of 99mTc-labeled M/ E110 and ZigatÕ eye drops.

0

1

2

3 4 Time (h)

5

6

7

Figure 9. Aqueous humor concentration-time profile of gatifloxacin after topical administration of M/E110 and ZigatÕ eye drops in rabbit eyes (n ¼ 3, ± SD) up to 6 h.

Delivery of gatifloxacin using microemulsion as vehicle

DOI: 10.3109/10717544.2014.920432

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Table 8. Gatifloxacin in aqueous-humor: pharmacokinetic parameters following topical administration of M/E110 and ZigatÕ eye drops (n ¼ 3, ± SD). Batch

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M/E110 ZigatÕ eyedrops

AUC0!6 h (mg mL1 h)

AUC0!1 (mg mL1 h)

Cmax (ng mL1)

tmax (h)

t1/2 (h)

ke

1.3152 ± 0.124 0.6573 ± 0.087

1.5291 ± 0.265 0.6582 ± 0.016

410.54 ± 19.25 365.12 ± 13.74

3.0 2.0

1.483 ± 0.162 0.467 ± 0.101

0.467 ± 0.135 1.482 ± 0.178

transcorneal drug concentration gradient. It could be attributed to the presence of Transcutol-P that has enough ability to enhance the corneal permeation of drugs by loosening the intracellular tight junctions of corneal epithelium, stroma and endothelium and facilitate the influx of hydrophilic drugs. The mechanism of action of Transcutol-P on transcorneal permeation of gatifloxacin might be due to the changes in the structure of the epithelium of the cornea as Transcutol-P produces micelles in the lipid bilayer of epithelium. These micelles remove the phospholipids from the epithelial cell membranes, and hence lead to a higher transcorneal influx of drugs (Liu et al., 2006). Moreover, the presence of Tween-80 also contributes the higher corneal permeability of gatifloxacin as the rate of permeation is accelerated by Tween-80 (Jukanti et al., 2011). Thus o/w microemulsion increased the ocular bioavailability of gatifloxacin from M/E110 as compared to gatifloxacin solution (ZigatÕ eye drops). Aqueous humor concentration-time curves of gatifloxacin were good enough to perform the pharmacokinetic analysis of gatifloxacin, and their elimination phase was fitted well to the first-order kinetic model. Hence, the pharmacokinetic parameters (Table 8) obtained from less-variable data from a small population of subjects were trustworthy and would be clinically useful.

Conclusion The developed microemulsion was found in the limit of acceptable droplet size range for ocular use, and presented physical stability. Physicochemical parameters were found in the range that favors its suitability for ophthalmic use. In vitro release and transcorneal permeation of drug from microemulsion found good without any significant adverse effect on the corneal hydration level. The results of the study indicated that microemulsion could prolong the precorneal retention and drug corneal permeability. Hence, the bioavailability and relative bioavailability of gatifloxacin in the eyes were found to be significantly increased with the M/E110 as compared to ZigatÕ eye drops. The excellent ocular tolerance of the formulation and good pharmacokinetic profile of gatifloxacin indicated that the developed microemulsion is a promising alternative to the eye drop solutions in the delivery of gatifloxacin to the eye to offer a sustained pharmacological effect in the treatment of bacterial conjunctivitis, keratitis and other bacterial infections.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. The authors are thankful to the College of Pharmacy Research Center and the Deanship of Scientific Research at King Saud University, Riyadh, Saudi Arabia, for financial assistance.

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