in-vivo characterization

Journal of Drug Delivery Science and Technology 31 (2016) 22e34

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Research paper

Levofloxacin hemihydrate ocular semi-sponges for topical treatment of bacterial conjunctivitis: Formulation and in-vitro/in-vivo characterization Osama Saher*, Dalia M. Ghorab, Nadia M. Mursi Department Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Egypt

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 October 2015 Received in revised form 29 October 2015 Accepted 13 November 2015 Available online xxx

This study aimed to formulate and evaluate lyophilized, long acting, levofloxacin hemihydrate ocular semi-sponges that would fit cul-de-sac shape for bacterial conjunctivitis treatment. Formulae were prepared using casting/freeze drying technique employing a (41
31) full factorial design to examine the effects of polymer type (Gelrite, chitosan (low and high molecular weight), and sodium carboxymethylcellulose), and concentration (1%, 1.5%, 2%) on viscosity of the formed solutions, quantity of drug released after 12 h (Q12h) and time for 50% of the drug to be released (T50%). Formulae were evaluated for weight and content uniformity, surface pH, water uptake, and in vitro drug release with its kinetic analysis. The optimal formula was chosen using Design-Expert® software and subjected to scanning electron microscope imaging, g-sterilization and in-vivo evaluation. Results showed that formula G 2 (2% w/w Gelrite) had the highest desirability (0.894), a zero order drug release profile, and stability after gsterilization. Formula G 2 showed longer residence time (12 h) in rabbits' eye fluids compared to the commercial Levoxin® eye drops (4 h) with good correlations between in vitro and in vivo results. Conclusively, Gelrite ocular levofloxacin hemihydrate semi-sponges are promising drug delivery systems that would improve both patient compliance and treatment efficacy. © 2015 Elsevier B.V. All rights reserved.

Keywords: Levofloxacin Casting/freeze drying technique Bacterial conjunctivitis Sustained release sponge In vivo study

1. Introduction Over one third of eye-related conditions reported by the health service organizations worldwide are either infectious conjunctivitis, or conditions associated with infectious conjunctivitis [1]. It may be caused by bacteria, viruses or even fungi, but bacterial conjunctivitis is more prevalent in children [2]. The main threat of infectious conjunctivitis lies in eye complications that may cause blindness, if left untreated. Fortunately, most ocular, bacterial infections can be treated using topical fluoroquinolones. They inhibit bacterial DNA synthesis by inactivation of topoisomerases enzymes causing rapid bacterial cell death [3,4]. Levofloxacin is a third generation fluoroquinolone antibacterial agent and it has higher solubility in water at neutral pH than its parent ofloxacin. This allows the use of higher concentration of levofloxacin and achievement of higher ocular tissue

* Corresponding author. Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Kasr El Aini Street, 11562, Cairo, Egypt. E-mail address: [email protected] (O. Saher). http://dx.doi.org/10.1016/j.jddst.2015.11.004 1773-2247/© 2015 Elsevier B.V. All rights reserved.

concentrations, higher clinical efficacy and lower risk of fluoroquinolones resistance [5e8]. Levofloxacin has a broad spectrum against Gram-positive and Gram-negative bacteria with a higher activity against Streptococci compared to previous generations [9]. The main dosage form used to deliver most of eye medications (including levofloxacin) is eye drops. They may offer many advantages as simplicity of formulation, favorable cost advantage and ease of administration [10]. However, eye drops major drawback is the need for frequent instillations because topically applied drugs are rapidly washed off from the eye. Typically, not more than 5% of the topically applied drugs reach their target in the eye [11]. Levofloxacin eye drops for example, need to be administered up to 8 times in the first two days of bacterial eye infection treatment [12]. This dosage regimen usually leads to patient incompliance, unsuccessful treatment and emergence of bacterial resistance. Sponges are an example of ocular soluble inserts that showed promising results as controlled drug delivery matrices [13]. Simply, sponges are a dispersion of air in a solid matrix prepared by lyophilization. The end product consists of a sponge like hydrophilic polymer matrix, in which the drug is embedded [14]. Sponges as ocular solid dosage forms are a newly emerging candidate that

O. Saher et al. / Journal of Drug Delivery Science and Technology 31 (2016) 22e34

offers more flexibility, easiness of preparation, and less foreign body sensation when compared with ocular minitables. However, few studies were concerned to use sponges to deliver eye medications. The term semi-sponge is derived from the semi-circular shape of the formulae produced. This semi-circular shape was intended to ensure the resemblance and fitting of the formulae in the lower cul-de-sac, and to minimize the foreign body sensation. In view of the previously mentioned information, our study aimed to prepare sustained release ocular semi-sponges of levofloxacin hemihydrate. The polymers used are known to be biodegradable polymers that are approved by FDA for their safety and they are commonly used in many ocular drug delivery systems. The prepared formulae would eliminate the need of repetitive administration aiming to better patient compliance. 2. Materials and methods 2.1. Materials Levofloxacin hemihydrate was kindly supplied by El-Gomhouria Company, Cairo, Egypt. Chitosan [low molecular weight (30e200 cps) and high molecular weight (800e2000 cps)], Gelrite (phytagel) and sodium carboxy methyl cellulose (1500e4500 cps) were obtained from Sigma-aldrich Co., St. Louis, USA. Sodium chloride powder, sodium bicarbonate powder, and calcium chloride dihydrate were purchased from El Nasr pharmaceutical company (Cairo, Egypt). Glacial acetic acid was purchased from El Nasr chemical company (Cairo, Egypt). Spectra/Pore® dialysis membrane (12,000e14,000 molecular weight cut off) was purchased from Spectrum Laboratories Inc. (CA, USA). 2.2. DSC study Differential scanning calorimetry (DSC) studies were executed for the used excipients namely; low MW Chitosan (LCh), high MW Chitosan (HCh), sodium carboxy methyl cellulose (NaCMC) and Gelrite (G). Physical mixtures of equal amounts of levofloxacin hemihydrate with the excipients were prepared. Samples (3e4 mg) were placed in the aluminum pan of Shimadzu differential scanning calorimeter (DSC-60, Shimadzu, Kyoto, Japan). They were heated in the range 10e400  C at a rate of 10  C/min, with indium in the reference pan, in a nitrogen atmosphere. DSC and studies were done for the drug, excipients and drug-excipients mixtures. 2.3. FTIR study Fourier transform infrared spectroscopy (FTIR) spectra determination was done for 1:1 physical mixtures of the drug and the excipients according to potassium bromide disc technique. FTIR spectrophotometer (Model 22, Bruker, UK) was used and the wavenumber range was between 4000 and 500 cm1. FTIR spectra were established for the drug, excipients and drug-excipients mixtures. 2.4. Preparation of levofloxacin hemihydrate semi-sponges using casting/freeze drying technique The semi-sponges were prepared using casting/freeze drying technique. Levofloxacin hemihydrate was dissolved in 1% v/v acetic acid (in case of using chitosan) or in water for injection (in case of using other polymers) to give a dose of 0.5 mg levofloxacin hemihydrate per 100 mg solution. Accurately weighed quantities of the polymers were gradually added to the required amount of the proper solvent (containing the dissolved drug) with constant stirring (Magnetic stirrer, WiseStir, Wisd Lab. Instruments, USA) to

23

prepare the required concentrations (1, 1.5, 2% w/w). 200 mg of each polymer solution was poured in each pocket of a specially designed cylindrical plastic mold (Poly vinyl chloride (PVC blister)) with an internal diameter of 9 mm and a thickness of 3 mm. The pockets of the mold were divided into two identical halves by the use of plastic barriers to produce the desired semi-sponges. Plastic molds were then stored in a freezer at 22  C for 24 h. The frozen solutions were then placed in a lyophilizer (Novalyphe-NL 500; Savant Instruments Corp., Holbrook, NY, USA) for 24 h and the condenser temperature was - 45  C and under vacuum of 7
102 mBAR. To eliminate the possibility of moisture absorbance and to obtain soft, flexible sponges [15], the produced semi-sponges were stored in a desiccator containing anhydrous calcium chloride as a desiccant and stored at room temperature to be used within one week from their preparation and storage. g-sterilization was performed for the optimum formula in the presence of dry ice to avoid the problems that might occur due to temperature elevation associated with g-irradiation [16]. Irradiation was performed using a 60Co irradiator (National Center for Radiation Research & Technology, subordinate to Atomic Energy Authority, Nasr City, Egypt). Irradiation was done at a dose of 25 kGy at a dose rate of 1.88 kGy/hr [17].

2.5. In-vitro evaluation of the prepared levofloxacin hemihydrate ocular semi-sponges 2.5.1. Physical characterization Weight variation testing was done using ten randomly selected semi-sponges from each formula, which were separately weighed (Electric balance, AND Co, Ltd, Japan) and mean weight was estimated. Content uniformity was evaluated for the tested semi-sponge. To assure complete dissolution, formulae were stirred overnight in 100 mL simulated tear fluid (STF-pH 7.4). STF was freshly prepared using sodium chloride 0.67 g, sodium bicarbonate 0.2 g, calcium chloride.2H2O 0.008 g, and purified water added to 100 g [18]. The solution was then filtered through a 0.45 millipore filter and analyzed spectrophotometrically at 288 nm after adequate dilution with STF. The test was done in triplicate and the average drug content was calculated. The surface pH of the semi-sponges was also determined. Each semi-sponge was allowed to swell by keeping it in contact with 2 mL of STF for 2 h at room temperature [19]. The pH was estimated by bringing a pH strip (Merck Millipore®, Merck KGaA, Darmstadt, Germany) in contact with the surface of the semi-sponge and allowing it to equilibrate for 1 min.

2.5.2. Water uptake Water uptake of the prepared formulae was assessed gravimetrically at room temperature [20]. A small filter paper (d ¼ 15 mm, Whatman® Inc., Piscataway, NJ, USA) was cut into small circular pieces sufficient to hold tested semi-sponges over them. Filter paper pieces were then placed over an agar gel plate (1% w/v). This set-up was equilibrated for one hr and filter paper pieces were weighed (mfilter). The accurately weighed semisponges (md) were then placed on the upper side of the filter paper pieces in the covered plates. At certain time intervals up to 3 h, weights of the swollen semi-sponges (mw) were recorded where (mw ¼ mtotalmfilter). The study was done in triplicate and the mean was calculated. Percentage water uptake of semi-sponges (W) was calculated using the following equation:

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W¼

O. Saher et al. / Journal of Drug Delivery Science and Technology 31 (2016) 22e34

mw  md
100 md

(1)

2.5.3. Rheological characterization Viscosities of different polymer solutions were determined before casting using cone and plate viscometer (Brookfield Co., Model HBDV-I þ CP, Middleboro, MA, USA). The study temperature was (35  C þ 0.5) to simulate eye temperature and the angular velocity was increased gradually from 0.5 to 100 rpm using CPE-41 spindle. Accurate weight of each tested solution was applied to the plate and viscosities were determined at minimum and maximum rate of shear over the aforementioned range of speeds with 10 s between each two successive speeds. The torque was confirmed to be within the acceptable range (10e100%) before taking any results [21]. Rheological data obtained were shear stress and viscosity at different rates of shear. These data were fitted to power law model to study the type of flow:

t ¼ Kgn

(2)

Where, t is the shear stress, g is the rate of shear, K is the consistency index (sec.) and n is the flow index (dimensionless). For shear thinning fluids, n lies between zero and one while it approximates one in case of Newtonian systems and exceeds one in dilatant systems [22]. 2.5.4. In-vitro release studies In vitro release of levofloxacin hemihydrate from the prepared semi-sponges and lyophilized drug solution was determined using membrane diffusion technique [23e25]. A glass cylinder having the length of 10 cm and diameter of 2.5 cm fitted at its lower end with cellulose dialysis membrane presoaked in STF. Semi-sponges were transferred inside the glass cylinder to rest over the inner surface of the dialysis membrane. The cylinder was attached from the other end to the shaft of the dissolution apparatus, instead of the baskets, and then lowered to the vessels of a USP dissolution apparatus (Hanson Research Corporation, California, USA) containing 50 mL STF. The shafts were rotated at a constant speed (20 rpm) and the release medium was kept at a temperature of 35 ± 0.5  C [26e28]. Samples (1 mL) were withdrawn at specific time intervals and replaced by equivalent volume of fresh STF kept at the same temperature. Spectrophotometric assay for drug content was done at the predetermined lmax (288 nm) after sufficient dilution with STF. The test was performed in triplicate and the mean drug released was estimated. 2.5.5. Kinetic analysis of the release data The release data was analyzed to determine the mechanism and the order of drug release from different formulae. Models used for analysis of the release kinetics were zero order, first order, and diffusion controlled mechanism according to simplified Higuchi model [29]. KorsemeyerePeppas model was also chosen to analyze the drug release kinetics using the following equation (Korsmeyer et al., 1983):

Mt ¼ Kt n M∞

(3)

The diffusional exponent (n) depends on mechanism of release and drug delivery device shape [30]. For our case, we used parameters of cylindrical tablets. So, n  0.45 corresponds to a Fickian (case I) diffusion, 0.45 < n < 0.89 means an anomalous (non-Fickian) transport (where a combination of diffusion and polymer relaxation controls the release), n ¼ 0.89 indicates a zero order

(case II) transport (where the release rate is independent of time and involves polymer relaxation), and n > 0.89 suggests a super case II transport [31]. 2.5.6. Analysis of the factorial design In order to evaluate the effects of polymer type and concentration on water uptake results, viscosity results, and in vitro release results, a 41
31 full factorial experimental design was employed using Social Package for Statistical Study software (SPSS 19.0®). In this design (Table 1), polymer type (Lch, Hch, NaCMC, Gelrite) and concentration (1, 1.5 and 2%) were selected as independent variables, whereas percentage water uptake after 3 h, viscosity at minimum rate of shear, (Q12h), and (T50%) were chosen as dependent variable. The analysis was done using one way ANOVA test followed by Post Hoc's least significant difference (LSD) test was used in order to compare between individual variable levels and to confirm where the differences occurred. Difference at P < 0.05 was considered to be significant. 2.5.7. Selection of optimal formula through the determination of the desirability factor Desirability factor for the prepared formulae was determined by Design Expert® software. The criteria for the optimization were set at the highest polymer solution viscosity, the lowest Q12 and the highest T50%. 2.5.8. Scanning electron microscope imaging (SEM) Scanning electron microscope (SEM) was used to examine surface morphology and cross-sections of the optimum formula. A thin piece of the selected semi-sponge was fixed on the SEM sample holder with double-sided adhesive tape and coated with gold using Edwards Sputter coater under an argon atmosphere to achieve a  film of 150 A thickness. The sample was then examined using SEM (Jeol, JXA-840A, Tokyo, Japan) [15,32]. 2.5.9. Effect of g-sterilization Physical characteristics and content uniformity were evaluated for the optimum formula before and after sterilization. Similarity factor (f2) was calculated to detect any change in the release profile that might occur by sterilization using this equation:

" f2 ¼ 50
log10

1þ

W n

1 2

#
100

(4)

Where W is the sum of squares of differences in the cumulative percent dissolved between reference (release profile before sterilization) and test (release profile after sterilization) and n is the number of sampling times with a percent 85% [33]. Table 1 41
31 full factorial design independent variables and optimization criteria of the prepared formulae. Factors (independent variables)

Levels

X1: Polymer type X2: Polymer concentration

LCh 1%

Responses selected

Desirability constraints (optimization criteria)

Y1: Polymer solution viscosity Y2: Q12 Y3: T50%

maximize minimize maximize

HCh

NaCMC 1.5%

G 2%

Low MW Chitosan (LCh), high MW Chitosan (HCh), sodium carboxy methyl cellulose (NaCMC) and Gelrite (G).

O. Saher et al. / Journal of Drug Delivery Science and Technology 31 (2016) 22e34

2.6. In vivo characterization of prepared levofloxacin hemihydrate semi-sponges 2.6.1. Eye irritation test The in vivo characterization of safety of the selected formula was done by performing eye irritation test. Rabbits were selected as animal models for the test as their eyes showed to be more vulnerable to the exposure to irritating substances than the eyes of humans [34]. A group of 3 animals was used for the selected semisponge. The formula was g-sterilized (dose: 25 kGy) prior to application, and then was inserted into the lower conjunctival sac of one eye, whilst the other eye served as control. Both eyes of the rabbits under test were checked for any irritation signs (redness, inflammation, or tear production increase) based on direct visual observation using a slit lamp, before treatment, and 1, 4, 8, 12 and 24 h after instillation [28,35,36].

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adopted between fraction released and fraction diameter of inhibition zone. In both relations, the linear regression coefficient (R2) was calculated. 3. Results 3.1. DSC In the present study, DSC thermograms of levofloxacin hemihydrate showed peaks that were in agreement with the documented DSC chart under the same heating rate 10  C/min [38]. Pure levofloxacin hemihydrate exhibited four endothermic peaks the first at ~91  C due to the dehydration of levofloxacin hemihydrate, the second at ~227  C due to the melting of the g form, the third at ~231  C due to the melting of the b form and the last one at ~234  C due to the melting of the a form. The observed exothermic peaks might be due to the crystallization of an amorphous form that resulted partially from levofloxacin dehydration [38]. Endothermic values of levofloxacin hemihydrate showed no considerable changes when mixed with other excipients compared to that of pure levofloxacin hemihydrate.

2.6.2. Microbiological susceptibility testing Assessment of levofloxacin hemihydrate level in the external eye tissue of rabbits is expected to indicate residence time of the selected semi-sponge. This assessment was achieved by comparing the level of levofloxacin hemihydrate from the selected semisponge and commercially available Levoxin® eye drops (0.5% levofloxacin hemihydrate, Amoun pharmaceutical Company, Egypt), via employing a microbiological susceptibility testing after topical application. The study was performed using a parallel design, in which six male rabbits (weighing 1.5e2 Kg and with no eye diseases) were chosen for the study. All the experimental procedures with animals were carried out after being approved by the institutional review board of the Research Ethics Committee of Faculty of Pharmacy, Cairo University, Egypt and were in agreement with the ethical principles of EU Directive 2010/63/EU for the use of animals in scientific ocular researches. The rabbits were randomly divided into two groups each consisted of 3 rabbits. The optimized formula was applied to the first group, while the second group took the same semi sponge dose as 100 mL of the commercially available Levoxin® eye drops (0.5% levofloxacin hemihydrate, Amoun pharmaceutical Company, Egypt). Sterile 6 mm diameter filter paper discs were placed under the lower eyelid of the rabbit's eye for only 1 min at specific time intervals (0.5,1,2,4,6,8,10 and 12 h) after application. Care was taken during sampling to avoid irritation of either the eyelid or the eye. Each disc was stored at 20  C in a plastic Eppendorf for 24 h. Discs were then placed using sterile forceps under aseptic laminar flow on the surface of Muller Hinton Agar (MHA, Difco) that was previously inoculated using non-sporulated Staphylococcus aureus bacteria, being one of the common causative pathogens of bacterial conjunctivitis. Incubation of the plates was done at 37 ± 0.5  C for 24 h. Average diameters of the inhibition zones around the discs were recorded and used to compare the antibiotic levels in the external eye tissue according to the following proposed score guide: zone diameter 25 mm ¼ “þþþþþ”, 20 mm ¼ “þþþþ”, 15 mm ¼ “þþþ”, 10 mm ¼ “þþ”, 6e10 ¼ “þ”, no inhibition zone ¼ “” [28].

3.3.1. Physical characteristics, weight variation and content uniformity Freeze-dried semi-sponges were white in colour with sponge like structure. Semi-sponges were hard enough to withstand handling and at the same time not too hard to avoid hurting the eye. The average thickness 2.7 ± 0.21 mm and average radius was 4.4 ± 0.13. Average weights of semi-sponges ranged from 1.63 ± 0.17 to 3.2 ± 0.3 mg. The weight of the prepared semi-sponges increased with increment in the used polymer concentration. The average percent drug content of all semi-sponges lied within 92.6 ± 0.03 to 111.8 ± 0.04% of the labeled claim. This complied with the pharmacopoeial limits (85%e115%) and proved there was homogenous drug distribution in the formulae. The pH of all formulae ranged from 7 to 8. The safe pH range for eye preparations is from 6.5 to 8.5. Within this range, there is no chance for eye irritation or damage. These results revealed that all formulae had an acceptable pH and would not produce any local irritation or corneal damage upon application.

2.6.3. In vitro e in vivo correlation (IVIVC) In vitro e in vivo correlation (IVIVC) is setting up a rational relationship between a dosage form produced biological property, or even a parameter derived from a biological property, and the same dosage form characteristic or physicochemical property [37]. Trials done to establish relations between the average diameters of inhibition zones at different time intervals with in vitro release results at the same intervals. In case of the optimum formula, first relation constructed was between log percentage drug released and square of average diameter of inhibition zone. Second relation was

3.3.2. Water uptake Percentage water uptake values of the prepared formulae after 3 h of the study varied from 1553 ± 92% to 3612 ± 45.29%. Results of the 41
31 full factorial design showed that the type of the polymer had significant effect (p < 0.05) on water uptake (Fig. 1a). LSD test revealed that the water uptake results of the sponges prepared from NaCMC was significantly higher from that of the Lch, Hch, or Gelrite sponges. Oppositely, water uptake values of the sponges prepared using Lch, Hch, or Gelrite were not significantly different from each other.

3.2. FTIR The FTIR spectra confirmed the absence of any chemical interactions with the used excipients as the DSC studies. Pure levofloxacin hemihydrate showed characteristic peaks for the eOH group of the eCOOH moiety at 3265 cm1 and eC e O peak at 1724 cm1. The aromatic CeH peaks were also observed at 2935 cm1 [39]. There were no significant changes in these peaks in the prepared mixtures. 3.3. In-vitro evaluation of the prepared levofloxacin hemihydrate ocular semi-sponges

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O. Saher et al. / Journal of Drug Delivery Science and Technology 31 (2016) 22e34

Fig. 1. Effect of (a) polymer type and (b) polymer concentration on water uptake values. “Lch: Low chitosan, Hch: High chitosan, NaCMC: Sodium carboxymethyl cellulose, G: Gelrite”.

Regarding the polymer concentration, results reveal that it had a significant effect (p < 0.05) over values of water uptake (Fig. 1b). LSD test showed that the water uptake result of the sponges containing 2% of the polymer was significantly lower than the values of Lch, Hch, or Gelrite sponges. However, water uptake values of the sponges prepared using 1% or 1.5% showed no significant difference from each other.

3.3.3. Rheological characterization All tested solutions exhibited pseudoplastic behavior associated with thixotropy, which is vital to decrease interference with blinking. Viscosities of polymer solutions at minimum and maximum rate of shear are recorded in Table 2. Results of the 41
31 full factorial design showed that the type of the polymer had significant effect (p < 0.05) on viscosity results (Fig. 2a). LSD test revealed that all polymer types were significantly different from each other. The order of viscosity of polymer solutions according to polymer type was NaCMC > Gelrite > Hch > Lch. Regarding polymer concentration, It worth noting that increasing the concentration caused a significant increase (p < 0.05) in viscosity values (Fig. 2b). LSD test revealed that all concentrations were significantly different from each other.

3.3.4. In-vitro release studies The results of the in-vitro release of levofloxacin hemihydrate from the different ocular semi-sponges were graphically represented in Fig. 3. In order to understand the barrier presented by the dialysis membrane, the in vitro release study for plain lyophilized drug solution of the same concentration was carried out in the same manner. It was clear from the figure that the release of levofloxacin hemihydrate from the lyophilized drug solution was markedly faster than that from the prepared formulae. Results of the 41
31 full factorial design were analyzed (Fig. 4), and revealed that both polymer type and concentration had significant effect (p < 0.05) on (Q12h) and (T50%). Subsequent LSD test showed that both Hch and NaCMC semi-sponges were not significantly different from each other. (Fig. 5) shows the combined effects of the type and concentration of the polymer on both (Q12h) and (T50%). From the figure, there was significant interaction (p < 0.05) between the two factors. Formula containing 2% Gelrite was different from the other formulae, having the highest (T50%) and the lowest (Q12h) among all formulae. In addition, the figure reveals the minor significance of polymer type on the values of (Q12h) at low polymer concentration (1%).

Table 2 Viscosities of prepared polymer solutions at minimum and maximum rates of shear. Formula codea

Viscosity at minimum shear rate (Cp) ± S.D

LCh 1 LCh 1.5 LCh 2 HCh 1 HCh 1.5 HCh 2 NaCMC 1 NaCMC 1.5 NaCMC 2 G1 G 1.5 G2

1636 6250 12,857 4060 8186 14,190 13,016 21,333 25,620 3964 13,000 17,444

± ± ± ± ± ± ± ± ± ± ± ±

449 2173 1904 1126 1656 2086 4200 577 1579 3342 1520 1953

Low MW Chitosan (LCh), high MW Chitosan (HCh), sodium carboxy methyl cellulose (NaCMC) and Gelrite (G). a Mean of three experiments.

Viscosity at maximum shear rate (Cp) ± S.D 19 77 93 207 262 328 332 1481 18,356 40 88 212

± ± ± ± ± ± ± ± ± ± ± ±

3 9 40 8 31 6 10 46 134 11 11 70

O. Saher et al. / Journal of Drug Delivery Science and Technology 31 (2016) 22e34

27

Fig. 2. Effect of (a) polymer type and (b) polymer concentration on viscosity of polymer solution. “Lch: Low chitosan, Hch: High chitosan, NaCMC: Sodium carboxymethyl cellulose, G: Gelrite”.

Fig. 3. In vitro release profile of levofloxacin hemihydrate from semi sponges formulated using (a) Low chitosan; (b) High chitosan; (c) NaCMC and (d) Gelrite in STF (pH 7.4) at 35  C in comparison to lyophilized drug solution.

3.3.5. Kinetic analysis of the release data The preference of a certain mechanism was based on the

coefficient of determination (R2) deduced for the parameters studied. The highest coefficient of determination was preferred for

28

O. Saher et al. / Journal of Drug Delivery Science and Technology 31 (2016) 22e34

Fig. 4. Effect of (i) polymer type and (ii) polymer concentration on (a) Q12 h, (b) T50%. “Lch: Low chitosan, Hch: High chitosan, NaCMC: Sodium carboxymethyl cellulose, G: Gelrite”.

the selection of the order of release. The data is presented in Table 3 showed that the release of levofloxacin hemihydrate from all formulae followed diffusion controlled mechanism according to Higuchi model except formula G 2 that followed zero order of release. According to KorsemeyerePeppas model, all formulations had n values between 0.45 and 0.89 indicating an anomalous (non Fickian) transport. 3.3.6. Selection of optimal formula through the determination of the desirability factor The selection criteria of the best semi-sponge formulae were the highest polymer solution viscosity, the lowest Q12h and the highest T50%. Formula containing 2% w/w Gelrite (G 2) showed the highest desirability value (0.894) as shown in Fig. 6. This formula was selected for further testing. 3.3.7. Scanning electron microscope imaging (SEM) Scanning electron micrographs of formula G 2 was conducted. On top view (Fig. 7a); this semi-sponge had a uniform non-porous surface. Side views (Fig. 7bed) showed that the investigated formula possessed a porous nature. 3.3.8. Effect of g-sterilization Both physical characteristics and drug content showed no

extraordinary changes after g-sterilization at (25 kGy). Average drug content was 107.8 ± 0.021% and complied with pharmacopoeial limits (85%e115%). The release of levofloxacin hemihydrate from formula G 2 before and after g-sterilization showed no significant change in the release pattern, as similarity factor was found to be 67, which was within the acceptable range (50e100) [33,40]. 3.4. In vivo characterization of prepared levofloxacin hemihydrate semi-sponges 3.4.1. Eye irritation test Eye irritation test showed that the formula G 2 exhibited no signs of irritation (Fig. 8) over our study period (24 h). Thus, it could be concluded that the formula was safe and non-irritant to the eye. 3.4.2. Microbiological susceptibility testing Table 4 shows the score values of inhibition zone at different time intervals of either commercially available Levoxin® eye drops or formula G 2. Results revealed that formula G 2 had longer residence time (12 h) in the fluids of the outer tissues of the eye after application in comparison to Levoxin® eye drops (4 h only). Fig. 9 shows the pattern of inhibition zone diameter against time following the application of the market product Levoxin® and G 2 semi-sponge. Statistical analysis of inhibition zone diameters at

O. Saher et al. / Journal of Drug Delivery Science and Technology 31 (2016) 22e34

29

Fig. 5. Combined effects of polymer type and concentration on (a) T50% and (b) Q12h.

Table 3 Kinetic analysis of the release data of all levofloxacin hemihydrate semi-sponges. Formula

NaCMC 1 NaCMC 1.5 NaCMC 2 G1 G 1.5 G2 HCh 1 HCh 1.5 HCh 2 LCh 1 LCh 1.5 LCh 2

R2a

Mechanism

Zero

First

Diffusion

0.921 0.905 0.968 0.877 0.913 0.994 0.891 0.845 0.895 0.895 0.896 0.964

0.821 0.752 0.814 0.729 0.796 0.845 0.804 0.415 0.734 0.734 0.717 0.862

0.975 0.974 0.999 0.967 0.981 0.986 0.955 0.939 0.979 0.979 0.978 0.997

Diffusion Diffusion Diffusion Diffusion Diffusion Zero Diffusion Diffusion Diffusion Diffusion Diffusion Diffusion

Korsmeyer e Peppas model

T50% (minutes)

Diffusion exponent (n)

R2

Mechanism

0.718 0.799 0.573 0.734 0.743 0.681 0.569 0.829 0.761 0.718 0.799 0.573

0.999 0.995 0.992 0.990 0.996 0.999 0.992 0.998 0.998 0.996 0.999 0.998

Anomalous Anomalous Anomalous Anomalous Anomalous Anomalous Anomalous Anomalous Anomalous Anomalous Anomalous Anomalous

37.73 55.73 55.03 122.24 184.14 458.74 18.72 31.31 36.71 42.13 62.92 87.68

Low MW Chitosan (LCh), high MW Chitosan (HCh), sodium carboxy methyl cellulose (NaCMC) and Gelrite (G). a Underlined values indicate the highest R2 values in each formula.

different time intervals using (SPSS ver. 19®), showed that all diameters were not significantly different from each other except at 12 h (P < 0.05).

3.4.3. In vitro e in vivo correlation (IVIVC) Two in vitro e in vivo correlations were established for the optimized formula G 2. The first relation constructed was between log percentage drug released and square of average diameter of inhibition zone using third order polynomial regression (Fig. 10a) (R2 ¼ 0.951) [41]. Polynomial regression is a form of linear regression that had been generally used to describe nonlinear phenomena as the rate of growth of tissues [42] and epidemic diseases progression [43]. Second relation (Fig. 10b) was adopted between fraction released and fraction diameter of inhibition zone using third order polynomial regression (R2 ¼ 0.947).

4. Discussion Water absorption ability is associated with the presence of hydrophilic groups such aseOH, eCOOH, and eOSO3H. Water Entrance into polymer network results in the hydration of these functional groups, which leads to expansion and ordering of the polymer chains. The maximum water uptake is achieved, when balance occurs between osmotic forces of the functional groups and restrictive forces of the ordered polymer chains [44]. NaCMC results were similar to the results obtained by Refai & Tag [45] who found that NaCMC being a charged polymer had a higher extent of water uptake compared to neutral polymers. Bertram & Bodmeier [23] also found a good correlation between water uptake of NaCMC and its charge density. Chitosan and Gelrite are also charged polymers. Yet, their water uptake is lower than NaCMC. In a study made by Chu et al. [46] on chitosan, it was demonstrated that chitosan has neither free negative nor positive charges in neutral medium. This uncharged nature of chitosan in neutral media was

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Fig. 6. Desirability results of the prepared semi-sponges.

effect of polymer concentration on water uptake may be explained that upon increasing the polymer concentration, a highly viscous gel layer was formed at the contact area between the semi-sponge and the medium, hence decreasing water uptake. Another explanation stated by Chang et al. [47] that upon increasing polymer concentration, the porosity of the prepared sponges decreases, which will consequently decrease the degree of water uptake. Regarding rheological characterization, Ocular semi-sponges were supposed to take up tear fluids and transform to a gel or a viscous solution. The viscosity of polymer solution used for preparation and accordingly the viscosity of the formed gel is of great importance for the performance of semi-sponges with respect to water uptake and drug release. The resulted order of viscosity can be explained as there are many factors that may affect the solution viscosities and their resistance for the shear applied. In our case, secondary bonding (namely hydrogen bonds and Van der Waals forces) was believed to play the major role [48,49]. NaCMC structure contains free C]O and OH groups that can form strong intermolecular and intramolecular hydrogen bonds. Moreover, Van der Waals forces are highly dependent on the substance molecular

Fig. 7. Scanning electron micro-graphs of (G 2) formula (a) Top view (magnification 100), (b, c and d) Side views (magnifications 100,160 and 300 respectively).

responsible for the decrease in both the uptake of water and in its swelling. Regarding Gelrite, Chang et al. [47] suggested that water uptake behavior of Gelrite showed to be closely related to the porosity. Certain studies showed that scaffolds having higher porosity increased water storage space and hence increased the water uptake [38]. Other studies have suggested that small pore sizes may potentiate the capillary phenomenon leading to an increase in water absorption. The water uptake profile in our case showed an initial rapid water uptake that might be caused by capillary forces. The following reduction in water uptake rate may be resulted from the formation of a dense gel layer that restricted water entrance and produced slower hydration [45]. The significant

size and mass [50]. Thus, the considerable high molecular weight (reaching 700 KDa) of NaCMC may be responsible for significant higher Van der Waals forces and higher viscosity compared with other used polymers. Gelrite and high chitosan may both have similar molecular mass (around 200 KDa) and so, they have relatively the same Van der Waals forces. However, the number of OH and C]O groups of the Gelrite molecule is greater and more available for hydrogen bonding when compared to high chitosan. This will allow a higher degree of cross linking and hence higher viscosity. In our case, Gelrite showed higher viscosity than HCh at minimum shear rate, while it was more easily destroyed than HCh upon an increase in the shear rate as shown in the results of

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Fig. 8. Rabbit's right eye after (a) 1 h and (b) 8 h of formula G 2 application.

Table 4 Score values of inhibition zone of levoxin and formula G2 according to the aforementioned score guide. Time (hrs.)

0.5 1 2 4 6 8 10 12

Score of inhibition zones Levoxin®

Semi-sponge (G2)

þþþþþ þþþ þþþ þþ    

þþþþ þþþþ þþþ þþ þþ þþ þþ þþþ

Score guide: Inhibition zone diameter  25 mm ¼ “þþþþþ”, 20 mm ¼ “þþþþ”, 15 mm ¼ “þþþ”, 10 mm ¼ “þþ”, 6e10 ¼ “þ”, no inhibition zone ¼ ““.

Fig. 9. Average diameters of inhibition zones around discs indicating drug levels in the external eye fluids of albino rabbits versus time in hours following the instillation of formula (G 2) and levoxin® eye drops.

viscosity at maximum shear rate (Table 1). This may be explained by Rupenthal et al. [49] when they demonstrated that there was a high degree of elasticity (due to secondary bonding) in polymer samples at rest. However, these secondary bonds were easily destroyed upon an increase in the shear stress. Hence, it is expected that upon blinking there will be a decrease in viscosity, subsequent reduction in reflex tearing, and consequently prolonged residence. According to Srividya et al. [51], this would be beneficial. They

stated that viscoelastic substances with high viscosity at low shear rates and low viscosity at high shear rates are preferred, since the ocular shear rate is very high, ranging from 0.03 s1 during interblinking periods to 4250e28,500 s1 during blinking. Low chitosan has the least viscosity, because it has the lowest molecular weight, and hence the lowest Van der Waals forces among all the used polymers. The significant effect of concentration over viscosity may be attributed to the increase in the degree of cross linking with the increase of polymer concentration. The slower drug release from semi-sponges than the drug solution in all polymers may be a result of the influence of viscosity on the diffusion of the drug. The results were in accordance with what described by the StokeseEinstein equation [52], which demonstrates that an increased viscosity of the formulation causes slower diffusion of the drug across the gel matrix. The pattern of release seems to pass through three different mechanisms: (1) drug release from the surface of sponges, (2) diffusion through the swollen rubbery matrix or (3) drug release due to polymer erosion [53]. The release profiles of all formulae showed rapid drug release during the first 30 min. This result might be attributed to the release of the drug adhering to the surface and not entrapped in the inner matrix of the sponge. These results were in agreement with results of Foda et al. [15] and Kassem et al. [53]. They reported that a considerable percentage of their drugs were released within the first hour for both uncross-linked and crosslinked lyophilized matrices, and they attributed this to the presence of surface drug. After that, the drug release was due to the diffusion process, which was much slower when compared to the initial release. The results of NaCMC and Gelrite can be augmented by the study done by Michailova et al. [54] which correlated the water uptake, rheological properties of the used polymers with drug release rate. Moreover, Gelrite release results was confirmed by the results obtained by Rupenthal et al. [49] who found that release rates of carrageenan, xanthan gum and gellan gum were significantly lower than those of chitosan, alginate, and HPMC. They explained these result by the fact of slow diffusion of the drug through the gel matrix. They also assumed the presence of ionic interactions between the anionic polymer chains of gellan gum, xanthan gum and carrageenan and the positively charged model drug used, causing additional slowing down of the release rate. They added that there might be a repulsion caused between chitosan's positively charged backbone amino groups and the used drug, facilitating the drug diffusion through the chitosan gel matrix. Gorle and Gattani [55] provided a similar explanation for the zero order release from gellan gum films. They stated that the programmed release of Gatifloxacin from the ocular film prepared may

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Fig. 10. IVIVC correlations between (a) log % released and square of average diameter of inhibition zone using 3rd order polynomial regression, and (b) fraction released and fraction of average diameter of inhibition zone using 3rd order polynomial regression.

be due to the formation of hydrogen bonds between the drug and the polymers used. Gatifloxacin is a fourth-generation fluoroquinolone and similar in structure to Levofloxacin. Hence, formation of hydrogen bonds between the drug and the polymer is a possible scenario in our case and might has contributed to the controlled release rate of drug. On the other hand, HCh and LCh results in our study did not comply with the results obtained by Ko et al. [56] and Polk et al. [57] who both stated that the molecular weight of chitosan was a fundamental variable in the release of their drugs from chitosan. They reported that the release rate of their drugs increased with the decrease in chitosan molecular weight. However, in our study LCh showed more hindrance for the drug release than HCh. This result may be attributed to the difference in the pore size between LCh and HCh sponges. LCh had smaller pore size than HCh. This might cause a slower entry of the dissolution medium inside the sponge and hence a slower diffusion of the drug, resulting in more release retardation by LCh sponges. This results in the present study are consistent with the previous observations of Foda et al. [15] who demonstrated that release of tramadol HCl from LCh sponges was slower than from HCh sponges because of the smaller pore sizes in LCh sponges compared to sponges prepared by higher chitosan grades. The significant effect of polymer concentration over release

results might be explained by the assumption stated by Sankalia et al. [58] assuming that increasing in the amount of the polymer in the matrix would increase degree of hydration with simultaneous swelling that would result in lengthening of the drug diffusion pathway and reduction in drug release rate. Formula G 2 had the highest (T50%) and the lowest (Q12h) as a result to the low water uptake and relatively high viscosity resulted from the combined effects of polymer type and this high concentration (2 w/w %) used that consequently retarded the drug release from this formula. The minor significance of polymer type on the values of (Q12h) at low polymer concentration (1%) may be explained by the fact that, at this low polymer concentration, there were rapid release rates of the drug from the formulae regardless to the polymer type, due to the low viscosity and high water uptake values at this concentration. The zero order kinetics of release followed by formula G 2 might also have the same previous explanation as the high viscosity and the low water uptake, would make the passage of the drug through the gel layer a rate-limiting step for the release. The porous nature presented in the side views of the SEM were probably the cause of water penetration, drug dissolution, and release. The existence of only one site (straight side) for drug release might also explain the in-vitro zero ordered drug release from formula G 2.

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In vivo results showed to be in coherence with the in vitro drug release results from formula G 2. This in vivo behavior might be a result of a combination of the three release mechanisms that appeared in vitro: drug release from the surface of the sponge, diffusion through the swollen rubbery matrix and drug release due to polymer erosion [59]. The large inhibition zones at the first 2 h might indicate the rapid drug release from the semi-sponge owing to the dissolution of the drug adhering to the surface and not entrapped in the inner matrix of the sponge. After the first 2 h, the diameters of the zones decreased, but still insignificant (P < 0.05) and remained steady for 10 h. Drug release during this stage might be due to a combination of both the diffusion process, which was slower compared to initial rapid release, and the polymer erosion. The significant increase (P < 0.05) of the inhibition zone diameter after 10 h might be an indication for the beginning of polymer erosion predominance over the diffusion process. These close correlations appeared between in vitro dissolution results and in vivo data suggested that the stated dissolution method could be beneficial to predict the in vivo data of the sustained release semi-sponge formula G 2. 5. Conclusion Ocular semi-sponges containing levofloxacin hemihydrate can be considered as a promising new drug delivery systems to treat bacterial conjunctivitis with minimum patient discomfort. Ocular semi-sponges resemble the shape of the lower cul-de-sac, and provide sustainment of drug release in the tear film for a long period (up to 12 h). As a result, the need for frequent instillation of levofloxacin eye drops could be substituted by the insertion of semi-sponge in the lower cul-de-sac of the infected eye two times daily. This dosage regimen is expected to highly increase the compliance of patients, leading to better treatment in a shorter duration. Declaration of interest The author reports no conflicts of interest. The author alone is responsible for the content and writing of the paper. Acknowledgment The authors would like to thank Dr Heba attia, Microbiology and immunology department, Faculty of Pharmacy, Cairo University, for her help in the microbiological susceptibility testing. References [1] U. Ubani, Clinical features of infectious conjunctivitis, in: Z. Pelikan (Ed.), Conjunctivitis e a Complex and Multifaceted Disorder, 2011. InTech. [2] P.M. Hughes, et al., Topical and systemic drug delivery to the posterior segments, Adv. Drug Deliv. Rev. 57 (14) (2005) 2010e2032. [3] J.M. Blondeau, Fluoroquinolones: mechanism of action, classification, and development of resistance, Surv. Ophthalmol. 49 (Suppl. 2) (2004) S73eS78. [4] J.A. Garcia-Rodriguez, A.C. Gomez Garcia, The microbiology of moxifloxacin, Drugs Today (Barc) 36 (4) (2000) 215e227. [5] S.V. Scoper, Review of third-and fourth-generation fluoroquinolones in ophthalmology: in-vitro and in-vivo efficacy, Adv. Ther. 25 (10) (2008) 979e994. [6] D.P. Healy, et al., Concentrations of levofloxacin, ofloxacin, and ciprofloxacin in human corneal stromal tissue and aqueous humor after topical administration, Cornea 23 (3) (2004) 255e263. [7] H.R. Koch, et al., Corneal penetration of fluoroquinolones: aqueous humor concentrations after topical application of levofloxacin 0.5% and ofloxacin 0.3% eyedrops, J. Cataract Refract. Surg. 31 (7) (2005) 1377e1385. [8] T. Puustjarvi, et al., Penetration of topically applied levofloxacin 0.5% and ofloxacin 0.3% into the vitreous of the non-inflamed human eye, Graefes Arch. Clin. Exp. Ophthalmol. 244 (12) (2006) 1633e1637. [9] D. Miller, E.C. Alfonso, Comparative in vitro activity of levofloxacin, ofloxacin, and ciprofloxacin against ocular streptococcal isolates, Cornea 23 (3) (2004)

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