http://informahealthcare.com/phd ISSN: 1083-7450 (print), 1097-9867 (electronic) Pharm Dev Technol, Early Online: 1–8 ! 2014 Informa UK Ltd. DOI: 10.3109/10837450.2014.910807
RESEARCH ARTICLE
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Thammasat University on 10/07/14 For personal use only.
Development and evaluation of a novel in situ gel of sparfloxacin for sustained ocular drug delivery: in vitro and ex vivo characterization Nazia Khan, Mohammed Aqil, Ameeduzzafar, Syed Sarim Imam, and Asgar Ali Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India
Abstract
Keywords
Conventional eye drops are the most popular delivery systems in the treatment of various eye infections. However, the major problem encountered in these dosage forms is precorneal elimination of the drug, resulting in poor bioavailability and therapeutic response. To overcome the side effects of pulsed dosing, an attempt has been made to formulate and evaluate a novel in situ gelling system of Sparfloxacin for sustained ocular drug delivery (ion and pH triggered gelling system). These gelling systems involve the use of sodium alginate (ion sensitive polymer) used as gelling agent and methylcellulose as viscosity-enhancing agent. The developed formulations were evaluated for clarity, pH, gelling capacity, rheological study, in vitro release study, ex vivo corneal permeation study, ocular irritation studies (HET-CAM test) and histopathological study using isolated goat corneas. The formulations were found to be stable, non-irritant and showed sustained release of the drug for a period up to 24 h with no ocular damage. In situ gel of sparfloxacin could be prepared successfully promising their use in ophthalmic delivery.
HET-CAM, in situ gel, methylcellulose, sodium alginate, sparfloxacin
Introduction The human eye is a unique organ, both anatomically and physiologically, comprising of several widely varied structures with each having independent physiological functions that make this organ highly impervious to foreign substances. Due to various complexities of the eye, pharmaceutical scientists are facing a number of challenges in ophthalmic drug delivery1. Also, human cornea which comprises of epithelium, stroma and endothelium restricts the entry of drug molecules in to the eye2. Therefore, the main aim of the formulator is to overcome the protective barriers of the eye without causing any type of permanent tissue damage and overcoming the restrictions. In ophthalmic drug delivery systems, the aim of pharmacotherapeutics is the attainment of drug concentration effectively at the intended site of action for a sufficient period of time to produce a response3. The efficient delivery of a drug while minimizing the side effects of the concerned drug is the key to treatment of ocular diseases. The major problem encountered with conventional ophthalmic delivery systems like eye drops is poor ocular drug bioavailability which is mainly due to ocular anatomical and physiological constraints as well as relative impermeability of the corneal epithelium membrane, nasolacrymal drainage and tears dynamics. It has been observed that only 1–10% of topically applied drug is absorbed whereas the major amount of drug is absorbed systemically resulting in systemic side effects4. The side effects of pulsed dosing, i.e. poor bioavailability and therapeutic response due to precorneal elimination of the drug and Address for correspondence: Asgar Ali, Department of Pharmaceutics, Jamia Hamdard, New Delhi 110062, India. Tel: +91 9899571726. E-mail: [email protected]
History Received 8 May 2013 Revised 16 February 2014 Accepted 23 March 2014 Published online 23 April 2014
rapid tear turnover, may be overcome by using delivery system based on the concept of in situ gel formation4. In situ forming gels are liquids when instilled into the eye which undergo rapid gelation in the cul de sac of the eye to form viscoelastic gels and this provides response to environmental changes5. Zhidong et al.6 formulated and evaluated an ophthalmic delivery system of an antibacterial agent, Gatifloxacin based on in situ gelling system and the results showed improved rheological behaviour, enhanced ocular bioavailability and better patient compliance when compared to conventional ophthalmic solutions. In situ gelling systems can be broadly classified in to three types depending upon the method employed to cause sol to gel transition systems. These are pH-triggered systems (including cellulose acetate hydrogen phthalate latex, carbopoletc), temperature-dependant systems (polaxamer, pluronics and tetronics) and ion-activated systems (sodium alginate and gelrite). Sparfloxacin is a fluroquinolone, newer generation antibacterial agent with a wide activity against a broad range of Gramnegative and Gram-positive organisms used to treat various dry eye syndromes like keratoconjunctivitis, bacterial conjunctivitis and for the treatment of corneal ulcer. As compared to ciprofloxacin, it has been reportedly found to be more active against Gram-positive bacteria and mycobacteria7. Keratoconjunctivitis sicca or dry eye syndrome is an ocular disease which is basically characterized by dryness of both conjunctiva and cornea. It is caused by either increased tear film evaporation or decreased tear production. The other causes of this disease include abnormal tear composition resulting in rapid evaporation or premature destruction of the tears8; whereas bacterial conjunctivitis is infection of the membrane lining the eyelids (conjunctivitis) and is caused due to infection with bacteria. The present research work deals with the development and evaluation of an ion and temperature triggered in situ gelling
2
N. Khan et al.
system of sparfloxacin, a newer generation fluroquinolone derivative used for the treatment of dry eye syndromes and corneal ulcer. A combination of sodium alginate (ion sensitive polymer) used as gelling agent and methylcellulose used as viscosity enhancing agent were investigated for the formulation of in situ gels of sparfloxacin (0.3%) to provide sustained release of the drug for prolonged periods of time with better patient compliance.
Material and methods
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Thammasat University on 10/07/14 For personal use only.
Materials Sparfloxacin was obtained as a gift sample from Radicura Pharmaceuticals Pvt. Ltd, New Delhi, India. Sodium alginate KeltonÕ , which is composed of (60% mannuronic acid and 40% gluronic + acid), was kindly gifted by ISP (Alginates, Ltd., Waterfield, UK). Methylcellulose (Methocel A4M) grade was kindly gifted by Pharmax Private Ltd, Mumbai, India. Dialysis membrane (mol. wt. cut-off: 12 000 Da, flat with 25 mm, diameter of 16 mm, capacity 60 ml ft) was purchased from Sigma Aldrich Chemicals, Saint Louis, MO. Preparation of formulations Selection of vehicle Solubility of sparfloxacin was tested mainly in four different buffer solution namely, phosphate buffer USP (pH 6.0, 6.5 and 7.2), acetate buffer I.P. (pH 4.6 and 4.8), boric acid buffer (pH 4.7) and citrophosphate buffer B.P. (pH 6.0 and 6.2). Buffer in which Sparfloxacin (0.3%) was found to be soluble was selected as vehicle and formulation were developed. Selection of polymers The polymers used in the formulations were selected on the basis of gelling capacity, gelling time, gelling consistency and viscosity. The various polymers used in formulation are Polaxamer, Gelrite, Hydroxypropyl cellulose, Methyl cellulose, Sodium alginate, Hydroxypropyl methyl cellulose and results shown in Table 1. Preparation of in situ gelling systems After suitable selection of polymers, solutions of the selected polymers of varying concentrations were prepared in the selected buffer system to check there solubility and evaluate gelling capacity in order to identify suitable compositions for the preparation of in situ gelling systems of sparfloxacin as shown in Table 2. The gelling capacity was determined by placing a drop of the placebo formulation in a vial containing 2 ml of simulated tear fluid (STF pH 7.4) prepared artificially and equilibrated at 37 C. The formation of the gel was assessed and the time for gelation and time taken for gel to dissolve was noted down9. The composition of STF (pH 7.4) used was sodium chloride 0.67 g, sodium bicarbonate 0.2 g, calcium chloride dehydrate 0.008 g and purified water (q.s.) 100 ml. The selected placebo formulation on the basis of gelling capacity was used for medicated sol to gel development. For the preparation of in situ gel of sparfloxacin, selected polymers of different concentrations were separately dissolved and mixed with each other on a magnetic stirrer slowly. Drug solution was prepared in the selected buffer (boric acid buffer pH 4.7) system and then incorporated into the polymeric solution along with a suitable preservative till a uniform solution was obtained. The volume was adjusted up to required volume by the buffer and pH (4.7) was adjusted with sodium hydroxide solution (0.1 ml) and hydrochloric solution (0.1 ml)10. The developed formulations were filled in amber coloured vials since, the drug
Pharm Dev Technol, Early Online: 1–8
Table 1. Screening of various polymers for in situ gel. S. No. 1 2 3 4 5 6
Polymer
Gelling capacity Time required for gelation
HPMC Sodium alginate Methylcellulose HPC Gelrite Polaxamer
+++ +++
+++ +++
++++ ++
++ +
, No gelation; + , Gels after a few minutes, dissolves rapidly; + +, Gelation after a few seconds, remains for a few hours; + ++, Gelation immediate and of good consistency, remains for extended periods of time; + +++, Very thick gel formed. Table 2. Composition of sodium alginate and methylcellulose studied. Concentration used (%w/v) Formulation codes SM1 SM2 SM3 SM4 SM5 SM6 SM7 SM8 SM9 SM10 SM11 SM12
Sodium alginate
Methylcellulose
Gelling capacity
0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1.0 2.0
0.2 0.25 0.3 0.4 0.5 0.5 0.5 0.5 0.25 0.3 0.25 0.35
+ ++ ++ +++ +++ +++ +++ ++ ++++ ++++ ++++
, No gelation; + , Gels after a few minutes, dissolves rapidly; + +, Gelation after a few seconds, remains for a few hours; + ++, Gelation immediate and of good consistency, remains for extended periods of time; + +++, Very thick gel formed.
showed photosensitive reactions, under aseptic conditions, sterilized in the autoclave (121 C and 15 psi)11 for 20 min and further evaluations were carried out. Evaluation of developed formulations Determination of visual appearance, pH, clarity and drug content Appearance and clarity were determined visually against black and white background in a well lit room. The pH of the formulations was determined using pH meter (CL 54, Toshniwal Instrument Pvt. Ltd., Ajmer, India). The drug content was determined by taking 1 ml of each formulation and diluting it with suitable buffer. The concentration was determined using a double beam UV spectrophotometer at 290 nm12 (UV-1700, Shimadzu, Kyoto, Japan). Rheological evaluation Rheological study of the formulations were determined at 25 C and 37 C for both sol and gel phase at different angular velocities or speed conditions (30, 50, 75 and 100 rpm), respectively, using a Brookfield viscometer (RV model) using spindle C50-1 (small sample adaptor)13. Differential scanning calorimetry analysis Differential scanning calorimetry (DSC) analysis of pure drug, physical mixture (Drug + polymers) and, freeze dried formulation were carried out using Perkin Elmer 7 DSC (Waltham, MA) and calibrated with indium. The sample (5 mg) was placed into a standard aluminium pan, crimped and scanned from 40 C to
Noval in situ gel for ocular drug delivery
DOI: 10.3109/10837450.2014.910807
400 C at a heating rate of 5 C/min with continuous purging of nitrogen (20 ml/min). An empty sealed pan was used as reference.
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Thammasat University on 10/07/14 For personal use only.
In vitro release studies The in vitro release profile of sparfloxacin from in situ gel and drug solution (marketed preparation, Sparfloxacin eye dropsSPAT eye drops, Cadila Pharmaceuticals, Ahmedabad, India) was performed using dialysis bag. The formulation (containing 6 mg of drug) was placed in pre-treated dialysis bag which was immersed into 100 ml of STF (pH 7.4), at 37 C with continuous stirring at 50 rpm. At predetermined time intervals, aliquots were withdrawn from the release medium and replaced with the same amount. The samples were analyzed in triplicate by U.V. spectrophotometer at 290 nm14. The data obtained from in vitro drug release studies were fitted to various release kinetic models like zero order, first order, Higuchi model, Korsmeyer–Peppas and Hixon–Crowell model to understand the mechanism of drug release from in situ gel.
3
through the window and left in contact for 5 min. The membranes were examined for vascular damage, and time taken for injury was observed and scores were given. Normal saline and 0.1 M NaOH were used as negative and positive controls. Ocular irritation potential of the substances was assessed for three basic parameters, namely, hyperaemia, haemorrhage and coagulation17. Histopathological studies Histopathological studies in ophthalmology are another way of assessing the irritations of the formulation. The presence or absence of irritancy depends upon the changes in corneal structure using excised goat cornea. The formulations were kept in contact with cornea for 12 h. Cornea was removed, immediately fixed with 8% (w/w) formalin solution. The tissue was dehydrated with an alcohol gradient, put in melted paraffin and solidified in block form. Cross-sections were cut, stained with haematoxylin and eosin, and microscopically examined by MOTIC electronic microscope for modifications18 (MOTIC, Japan). Results obtained were compared with control.
Ex vivo corneal permeation studies An ex vivo corneal permeation study was performed using freshly excised goat cornea. Fresh whole eyeballs were procured from the slaughter house at under 4 C in normal saline solution (0.9% NaCl solution). Corneas were carefully removed with the help of forceps and scissors along with a 5–6 mm of surrounding sclera tissue and washed with saline. Cornea was mounted by fixing surrounding scleral tissue clamped between upper and lower chamber of the Franz diffusion cell such that lower chamber which served as the receiver compartment was infused continuously with STF (pH 7.4) at the rate 20 ml/min at 37 ± 0.5 C. The perfusate was collected at periodic time intervals for up to 12 h in eppendorf tubes and were subjected to quantification of sparfloxacin by using ultraviolet spectroscopy at 290 nm15.
Confocal laser scanning microscopy study The penetration of optimized in situ gel and market formulation on excise goat cornea were determined by confocal laser scanning microscopy (CLSM). For this ex vivo goat permeation study was carried out as described above. After 8 h, the cornea was removed and washed with distilled water. The treated area was cut out and positioned on the microscopic slide and cover with the cover slip. CLSM was carried out with the Laser Confocal Microscope with Fluorescence Correlation Spectroscope-Olympus FluoView FV1000 (Olympus, Melville, NY) with an argon laser beam with excitation at 295 nm and emission at 525 nm. The permeated depths of formulation were detected at z-axis with Fluoview software19.
Antimicrobial efficacy studies
Sterility testing
The microbiological studies were carried out for ascertaining the biological activity of the formulated in situ gel systems against specified microorganisms. The organisms used in this study were Staphylococcus aureus (Gram-positive bacteria) and Pseudomonas aeruginosa (Gram-negative bacteria) using agar diffusion medium employing ‘‘cup plate technique’’. The developed formulation and marketed sparfloxacin eye drops (used as standard) were added, respectively, in agar plates. The plates were left to allow diffusion of solutions for 2 h and then incubated at 37 C in an incubator for 24 h. Zone of inhibition were measured after 12 and 24 h by an antibiotic zone finder16.
Test of sterility was carried out according to I.P. 1996 by membrane filtration method. Membrane filter of pore size 0.22 mm was used. The test was carried out by incubating the developed formulations in soybean casein digest medium (incubated at 25 C for 14 days) to analyze the growth of fungi and fluid thioglycolate medium (incubated at 35 C for 14 days) to analyze the growth of bacteria in the formulations20.
Ocular irritation studies Ocular irritation studies were carried out by HET-CAM assay (Hen’s egg chorioallantoic membrane test). It is a non-animalbased ocular toxicity test which has been used for irritation study. HET-CAM assay has been nominated by the EPA for evaluation as an alternative to the Draize test for ocular irritancy in rabbits. Three fertile eggs weighing 45–65 g were taken and observed under light and found non-defective. The eggs were placed in a tray filled with cotton in such a way that they were in an upside position. These eggs were then incubated in an incubator for a period of 10 days at a temperature of 37 ± 0.5 C and relative humidity 55 ± 7% for a period of 9 days. Eggs were rotated manually in a gentle manner after every 12 h. At the end of 10th day eggs were very carefully taken out with the help of a blunt forceps, egg shell was scratched off and then pared off. The inner membrane was carefully removed to expose the chorioallantoic membrane (CAM). Formulations (0.5 ml) were instilled directly
Stability studies Stability studies were carried out as per ICH guidelines21. The selected formulations were subjected to 25 ± 0.5 C, 30 ± 0.5 C and 40 ± 0.5 C/75% R.H. at 0, 30, 60, 90 and 180 days. The formulations were periodically evaluated for drug content, pH, clarity, viscosity. Using Arrhenius plot the shelf life of the formulations was calculated at room temperature, i.e. 25 C as per ICH guidelines.
Results and discussion Selection of vehicle In ophthalmic drug delivery systems, buffer systems must be nontoxic, non-irritating and having low buffer capacity and should be isotonic in nature. Isotonic boric acid buffer of pH 4.7 was selected in the formulation of sparfloxacin in situ gel. One main advantage of using boric acid buffer was that it has antimicrobial effects, which will enhance the anti microbial potency of the formulation. The boric acid buffer of pH 4.7 (Martin, 1993) was prepared by adding 1.75 g of boric acid crystals in 100 ml of double-distilled water.
4
N. Khan et al.
Pharm Dev Technol, Early Online: 1–8
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Thammasat University on 10/07/14 For personal use only.
Selection of polymers Various polymers were used for screening of polymers in formulation of in situ gel system. The selection of polymer was done on the basis of gelling capacity, gelling time, gelling consistency and viscosity of gel by using Rheometer (Table 1). Sodium alginate and Methylcellulose were the polymers selected for the preparation of in situ gels of sparfloxacin. Sodium alginate was used as gelling agent because it has high gluronic acid content, which forms a low viscosity, free flowing liquid. It improves the gelling properties hence reduction of polymer concentration to be introduced into the eye. Therefore, to improve viscosity methylcellulose was incorporated as viscosity enhancing agent. Both the polymers were found to exhibit favourable biological properties such as non-toxicity and biodegradability. Preparation of in situ gelling systems The polymers, namely sodium alginate and methylcellulose were found to be soluble in isotonic boric acid buffer (pH 4.7). Isotonicity calculations were done individually for drug and the polymers by sodium chloride equivalent method. Sparfloxacin (0.3 g) was first dissolved in isotonic boric acid buffer (pH 4.7) Table 3. Formulation codes of medicated ophthalmic sol to gel formulations. Formulation codes Ingredients
SM3
SM4
SM5
SM6
SM7
SM8
Sparfloxacin (g) Sodium alginate (g) Methylcellulose A4M (g) Benzalkonium chloride (ml) Modified boric acid buffer (pH 4.7) q.s. (ml) Tween-80 (ml)
0.3 0.6 0.3 0.01 100
0.3 0.65 0.4 0.01 100
0.3 0.7 0.5 0.01 100
0.3 0.75 0.5 0.01 100
0.3 0.8 0.5 0.01 100
0.3 0.85 0.5 0.01 100
0.1
0.1
0.1
0.1
0.1
0.1
Table 4. Physiochemical evaluation of selected formulations. Formulation codes
Appearances
pH
Clarity
Drug content (%w/v) ± SDa
SM3 SM4 SM5 SM6 SM7 SM8
Light Light Light Light Light Light
4.8 4.9 4.9 5.1 5.1 5.2
Clear Clear Clear Clear Clear Clear
98.3 ± 0.553 99.0 ± 0.614 99.2 ± 0.581 99.8 ± 0.742 98.7 ± 0.680 99.4 ± 0.598
yellow yellow yellow yellow yellow yellow
a
Standard deviation.
Figure 1. Viscosity of medicated formulation code SM6 in Sol and Gel state at 25 C and 37 C.
along with a cosolvent Tween-80 (0.1 ml). Tween-80 was used to enhance the solubility of the drug in isotonic boric acid buffer (pH 4.7). The mixture was then incorporated into the selected placebo formulation and the volume was adjusted up to the required volume as shown in Table 3. The pH was adjusted with 0.1 ml sodium hydroxide solution and 0.1 ml hydrochloric solution. The developed formulations were filled in amber coloured vials since, the drug showed photosensitive reactions. Evaluation of developed formulations Determination of visual appearance, pH, clarity and drug content Developed in situ gel of sparfloxacin was light yellow in colour and all formulations were found to be clear. Terminal sterilization by autoclaving had no effect on the appearance, clarity, pH and drug content of the formulations. The formulations were liquid at room temperature and at pH of the formulations which is within the range of 4.7–5.2 and a rapid transition to the gel phase was observed at pH of the tear fluid, i.e. pH 7.4. Drug content of all formulations was within the range of 98.31–100.03%, which inclined towards the uniform distribution of drug in developed ophthalmic formulations and results are shown in Table 4. Rheological evaluation The sol and gel phase of all the formulations showed shear thinning, and pseudoplastic behaviour of the formulation was observed which is a desirable phenomenon in case of ophthalmic formulations. All the formulations were found to be in liquid phase at pH 4.7 and an increase in the viscosity was observed when the pH of the formulations was raised to 7.4 which transformed into gel phase. Decrease in viscosity was observed on increasing the angular velocity in all formulations and in both sol as well as gel phases. Result for viscosity of medicated formulation code SM6 in Sol and Gel state at 25 C and 37 C is as shown in Figure 1. At higher angular velocities, viscosities were nearly constant. Pseudo plastic character of the precorneal tear film should be least affected by the administration of ophthalmic products DSC analysis DSC analysis of optimized formulation was done to determined interaction of drug with polymers. The drug and physical mixture (Drug + polymers) show an intense characteristic peak (Figure 2). The optimized in situ gel formulation does not show any peak because of drug was dispersed or converted into the amorphous form from the crystalline form, which implies that the drug has been fully entrapped in to the polymer matrix as shown in Figure 2.
Noval in situ gel for ocular drug delivery
DOI: 10.3109/10837450.2014.910807
5
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Thammasat University on 10/07/14 For personal use only.
In vitro release studies The cumulative percentage release of sparfloxacin from in situ gel was 94.3% over a period of 24 h and 11.8% in 1 h whereas, the percentage drug release of sparfloxacin from eye drop was 95.62% in 4 h and 42.34% in 1 h (Figure 3). The co-efficient of correlation (R2) of zero order, first order, Higuchi, Hixon–Crowell model and Korsmeyer–Peppas model for in situ gel was found to be 0.931, 0.989, 0.992, 0.967 and 0.987, respectively. Since, the co-efficient of correlation (R2) for Higuchi model was nearer to unity, i.e. (0.992) therefore, it was selected as the best fit model (Table 5). The linear relationships in conjunction with the slow dissolution rate suggest that the in vitro drug release from in situ gel formulation under physiological conditions occurs primarily by diffusion22. Ex vivo corneal permeation studies
Figure 2. Differential scanning calorimetry thermogram of physical mixture (A), Sparfloxacin (B) and optimized in situ gel formulation SM6 (C).
Ex vivo transcorneal permeation studies were conducted on optimized in situ gel formulation SM6, because it selected as optimized formulation on the basis of gelation capacity, gelation time and rheological properties. The extent of permeation of that formulation was compared to market Sparfloxacin eye dropsSPAT eye drop. A cumulative percent permeation of 62.56% done for 6 h was obtained for formulation SM6 and 36.87% from eye drop, respectively (Figure 4). The higher permeation of in situ gel formulation was due to bioadhesive and permeation enhancing properties of the polymers. It also enhanced the permeation and miscibility with corneal tissues compared to marketed eye drops of sparfloxacin due increased corneal contact time. Antimicrobial efficacy studies The zones of inhibition were measured after 12 and 24 h by an antibiotic zone finder. It was observed that diameter of zones of inhibitions from 44 to 48 mm for marketed formulation at 12 and Table 5. Kinetic release models data of formulation SM6. Zero order R2
First order K0
R2
K1
Higuchi model R2
KH
0.93 3.49 0.98 0.05 0.99 21.58 Figure 3. In vitro release of in situ gelling system and marketed formulation (mean ± SD).
Figure 4. In vitro transcorneal permeation study (mean ± SD).
Korsmeyer–Peppas model
Hixon–Crowell model
R2
KKP
R2
KHC
0.98
0.65
0.96
0.01
R2, regressed values; K, rate constant for different models.
6
N. Khan et al.
Pharm Dev Technol, Early Online: 1–8
24 h compared to 46 and 50 mm at 12 and 24 h for the optimized in situ gel formulation (Table 6). Results revealed that optimized formulation SM6 showed a comparable antimicrobial efficacy with marketed formulation against S. aureus (Gram-positive organism) and P. aeruginosa (Gram-negative bacteria). Table 6. Zone of inhibition produced by ophthalmic sol to gel formulation SM6. Zone diameter (mm) against S. aureus
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Thammasat University on 10/07/14 For personal use only.
Incubation time (h)
Zone diameter (mm) against P. aeruginosa
Formulation (SM6)
Sparfloxacin eye drops
Formulation (SM6)
Sparfloxacin eye drops
46 50
44 48
43 45
44 46
12 24
Table 7. Cumulative scores for assessment of ocular irritation potential. Cumulative scores
Irritation assessment
0–0.9 1–4.9 5–8.9 9–21
Practically none Slight Moderate Strong
Ocular irritation studies Formulation SM6 was tested in sol as well as gel form for their irritation potential if any. Normal saline (0.9% NaCl) and mild shampoo were used as negative and positive controls, respectively, as these substances have shown practically non-irritating and slightly irritating effects as reported in some literature. A cumulative score of 0 and 3.99 were obtained for normal saline solution and mild shampoo, respectively. Cumulative scores for the optimized formulation SM6 in solution as well as in gel form were 0.33 and 0.33, respectively, and these were50.9 (standard for being practically non-irritant) (Table 7). Therefore the optimized formulation (SM6) was found to be safe and nonirritant. Histopathological studies Histopathological studies revealed no change in histopathology of the tissues for the formulation SM6. Hence, it was safe and free from any ocular irritation and did not produce any changes in cell structure of corneal membrane. There was no rupture was observed in the corneal section when compared to eye drop (Figure 5). CLSM study The extent of penetration was measured by CLSM after application of formulations (i.e. in situ gel and marketed eye drop).
Figure 5. Histopathological study of cornea section of formulation SM6 (a) and marketed eye drop of sparfloxacin (b).
Noval in situ gel for ocular drug delivery
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Thammasat University on 10/07/14 For personal use only.
DOI: 10.3109/10837450.2014.910807
7
Figure 6. CLSM image: (A) optimized in situ gel formulation SM6 and (B) marketed eye drop. Table 8. Degradation rate constant of sparfloxacin in ophthalmic sol to gel preparation SM6. Temperature ( C) 25 30 40
Degradation rate constant (K) 1.2647 10 1.3818 10 1.5353 10
to conventional eye drops by virtue of its ability to sustained drug release and longer pre-corneal residence time. The formulation was stable for longer period of time with shelf life of 2.28 years.
4 4 4
Acknowledgements The authors are thankful to Radicura Pharmaceuticals Ltd. (India) for providing gift sample of Sparfloxacin. The authors are also thankful to Jamia Hamdard for carrying out the relevant studies.
The in situ gel system penetrates the cornea 110.64 mm in depth with fluorescence intensity as compared to eye drop (58.67 mm) depth during 8 h of study (Figure 6). The higher penetration of in situ gel of sparfloxacin is could be due to increased corneal contact time and penetration property of polymer used in formulation. Sterility testing Formulation SM6 passed the test for sterility as there was no turbidity was observed. The formulation did not showed any fungal and bacterial growth up to 14 days of incubation at 25 C in soyabean case in digest medium and 35 C in fluid thioglycolate medium. Stability studies The optimizedformulation was found to be clear; no change was observed in viscosity and pH after storage of 6 months at different temperature as per ICH guidelines. The degradation rate constant was found to be low (1.2647 10 4at 25 C, Table 8), i.e. negligible degradation (55%) and followed the first-order rate kinetics. The shelf life of was found to be 2.28 years.
Conclusion Sparfloxacin is fluroquinolone antibiotic antibacterial agent used in the treatment of ocular infections was successfully prepared by using polymers sodium alginate as gelling agent and methylcellulose A4M as viscosity enhancing agent. The formulation was in sol form at pH (4.7) and underwent rapid gelation upon raising pH to 7.4. This in situ gel formulation showed in vitro sustained drug release (p50.05) over period of 24 h as compared to eye drop. The ex vivo goat corneal permeation study showed significant enhanced permeation (p50.05) as compared to eye drop. Ocular tolerance tests and histopathology study showed that developed formulation was non-irritant, good ocular tolerance and significant antimicrobial study. This formulation is a viable alternative
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.
References 1. Kumar M, Kulkarni GT. Recent advances in ophthalmic drug delivery. Int J Pharm Sci 2012;4:387–394. 2. Gaudana R, Jwala J, Boddu SHS, Mitra KA. Recent perspectives in ocular drug delivery. Pharm Res 2009;26:1197–1216. 3. Rajas JN, Kavitha K, Gounder T, Mani T. In situ ophthalmic gels: a developing trend. Int J Pharm Sci Rev Res 2011;7:8–14. 4. Mohanambal E, Arun K, Abdul HSA. Formulation and evaluation of pH-triggered in situ gelling system of levofloxacin. Ind J Pharm Edu Res 2011;45:58–64. 5. Kashikar VS, Ophthalmic mucoadhesive polymers – a literature review of levofloxacin. Int J Pharm Edu Res 2011;7:68–73. 6. Zhidong LA, Jianier LB, Shufang NA, et al. Study of an alginate/ HPMC based in situ gelling ophthalmic delivery system for gatifloxacin. Int J Pharm 2006;315:12–17. 7. Gupta H, Aqil M, Khar KR, et al. Sparfloxacin-loaded PLGA nanoparticles for sustained ocular drug delivery. Nanomed Nanotechnol Biol Med 2010;6:324–333. 8. Thomas K, Patrichia B, Friedemann K. Treatment of patients with keratoconjunctivitis sicca with optiveÔ: results of a multicenter, open-label observational study in Germany. Clin Ophthalmol 2009; 3:33–39. 9. Johan C, Katarina E, Roger P, Katarina J. Rheological evaluation of gelrite in situ gels for ophthalmic use. Eur J Pharm Sci 1998;6: 113–119. 10. Sultana Y, Aqil M, Ali A. Ion activated Gelrite-based in situ ophthalmic gels of pefloxacinmesylate: comparison with conventional eye drops. Drug Deliv 2006;13:215–219. 11. Gupta D, Tator, HC, Shoichet SM. Fast gelling injectable blend of hyaluronan and methylcellulose for intrathecal, localized delivery to the spinal cord. Biomaterials 2006;27:2370–2379. 12. Eaga CM, Jagan MK, Venkatesh A. Preparation and evaluation of in situ gels for ocular drug delivery. J Pharm Res 2009;2: 1089–1094.
8
N. Khan et al.
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Thammasat University on 10/07/14 For personal use only.
13. Srividya B, Cardoza RM, Amin PD. Sustained ophthalmic delivery of ofloxacin from a pH-triggered in situ gelling system. J Control Rel 2001;73:205–211. 14. Gupta H, Jain S, Mathur R, et al. Sustained ocular drug delivery from a temperature and pH triggered novel in situ gel system. Drug Deliv 2007;14:507–515. 15. Rathore SM, Majumdar KD. Effect of formulation factors on in vitro transcorneal permeation of gatifloxacin from aqueous eye drops. AAPS PharmSciTech 2006;7:E1–E6. 16. Charoo NA, Kohli K, Ali A. Preparation of in situ forming ophthalmic gels of ciprofloxacin hydrochloride for the treatment of bacterial conjunctivitis: in vitro and in vivo studies. J Pharm Sci 2003;92:407–415. 17. Gilleron L, Coecke S, Sysmans S, et al. Evaluation of a modified HET-CAM assay as a screening test for eye irritancy. Toxicol In Vitro 1996;10:431–446.
Pharm Dev Technol, Early Online: 1–8
18. Sindhu A, Sharon F, Bharath S, et al. Sustained ophthalmic delivery of ofloxacin from an ion-activated in situ gelling system. Pak J Pharm Sci 2009;22:175–179. 19. Ahad A, Aqil M, Kohli K, et al. Formulation and optimization of nanotransfersomes using experimental design technique for accentuated transdermal delivery of valsartan. Nanomed Nanotechnol Biol Med 2012;8:237–249. 20. Stahlmann R, Zippel U, Forster C, et al. Chondrotoxicity and toxicokinetics of sparfloxacin in juvenile rats. Antimicrob Agents Chemother 1998;42:1470–1475. 21. ICH Topic Q 1 A (R2), Stability Testing of new Drug Substances and Products CPMP/ICH/2736/99, August 2003. 22. Liu Z, Li J, Nie S, et al. Study of an alginate/HPMC-based in situ gelling ophthalmic delivery system for gatifloxacin. Int J Pharm 2006;315:12–17.
RESEARCH ARTICLE
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Thammasat University on 10/07/14 For personal use only.
Development and evaluation of a novel in situ gel of sparfloxacin for sustained ocular drug delivery: in vitro and ex vivo characterization Nazia Khan, Mohammed Aqil, Ameeduzzafar, Syed Sarim Imam, and Asgar Ali Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India
Abstract
Keywords
Conventional eye drops are the most popular delivery systems in the treatment of various eye infections. However, the major problem encountered in these dosage forms is precorneal elimination of the drug, resulting in poor bioavailability and therapeutic response. To overcome the side effects of pulsed dosing, an attempt has been made to formulate and evaluate a novel in situ gelling system of Sparfloxacin for sustained ocular drug delivery (ion and pH triggered gelling system). These gelling systems involve the use of sodium alginate (ion sensitive polymer) used as gelling agent and methylcellulose as viscosity-enhancing agent. The developed formulations were evaluated for clarity, pH, gelling capacity, rheological study, in vitro release study, ex vivo corneal permeation study, ocular irritation studies (HET-CAM test) and histopathological study using isolated goat corneas. The formulations were found to be stable, non-irritant and showed sustained release of the drug for a period up to 24 h with no ocular damage. In situ gel of sparfloxacin could be prepared successfully promising their use in ophthalmic delivery.
HET-CAM, in situ gel, methylcellulose, sodium alginate, sparfloxacin
Introduction The human eye is a unique organ, both anatomically and physiologically, comprising of several widely varied structures with each having independent physiological functions that make this organ highly impervious to foreign substances. Due to various complexities of the eye, pharmaceutical scientists are facing a number of challenges in ophthalmic drug delivery1. Also, human cornea which comprises of epithelium, stroma and endothelium restricts the entry of drug molecules in to the eye2. Therefore, the main aim of the formulator is to overcome the protective barriers of the eye without causing any type of permanent tissue damage and overcoming the restrictions. In ophthalmic drug delivery systems, the aim of pharmacotherapeutics is the attainment of drug concentration effectively at the intended site of action for a sufficient period of time to produce a response3. The efficient delivery of a drug while minimizing the side effects of the concerned drug is the key to treatment of ocular diseases. The major problem encountered with conventional ophthalmic delivery systems like eye drops is poor ocular drug bioavailability which is mainly due to ocular anatomical and physiological constraints as well as relative impermeability of the corneal epithelium membrane, nasolacrymal drainage and tears dynamics. It has been observed that only 1–10% of topically applied drug is absorbed whereas the major amount of drug is absorbed systemically resulting in systemic side effects4. The side effects of pulsed dosing, i.e. poor bioavailability and therapeutic response due to precorneal elimination of the drug and Address for correspondence: Asgar Ali, Department of Pharmaceutics, Jamia Hamdard, New Delhi 110062, India. Tel: +91 9899571726. E-mail: [email protected]
History Received 8 May 2013 Revised 16 February 2014 Accepted 23 March 2014 Published online 23 April 2014
rapid tear turnover, may be overcome by using delivery system based on the concept of in situ gel formation4. In situ forming gels are liquids when instilled into the eye which undergo rapid gelation in the cul de sac of the eye to form viscoelastic gels and this provides response to environmental changes5. Zhidong et al.6 formulated and evaluated an ophthalmic delivery system of an antibacterial agent, Gatifloxacin based on in situ gelling system and the results showed improved rheological behaviour, enhanced ocular bioavailability and better patient compliance when compared to conventional ophthalmic solutions. In situ gelling systems can be broadly classified in to three types depending upon the method employed to cause sol to gel transition systems. These are pH-triggered systems (including cellulose acetate hydrogen phthalate latex, carbopoletc), temperature-dependant systems (polaxamer, pluronics and tetronics) and ion-activated systems (sodium alginate and gelrite). Sparfloxacin is a fluroquinolone, newer generation antibacterial agent with a wide activity against a broad range of Gramnegative and Gram-positive organisms used to treat various dry eye syndromes like keratoconjunctivitis, bacterial conjunctivitis and for the treatment of corneal ulcer. As compared to ciprofloxacin, it has been reportedly found to be more active against Gram-positive bacteria and mycobacteria7. Keratoconjunctivitis sicca or dry eye syndrome is an ocular disease which is basically characterized by dryness of both conjunctiva and cornea. It is caused by either increased tear film evaporation or decreased tear production. The other causes of this disease include abnormal tear composition resulting in rapid evaporation or premature destruction of the tears8; whereas bacterial conjunctivitis is infection of the membrane lining the eyelids (conjunctivitis) and is caused due to infection with bacteria. The present research work deals with the development and evaluation of an ion and temperature triggered in situ gelling
2
N. Khan et al.
system of sparfloxacin, a newer generation fluroquinolone derivative used for the treatment of dry eye syndromes and corneal ulcer. A combination of sodium alginate (ion sensitive polymer) used as gelling agent and methylcellulose used as viscosity enhancing agent were investigated for the formulation of in situ gels of sparfloxacin (0.3%) to provide sustained release of the drug for prolonged periods of time with better patient compliance.
Material and methods
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Thammasat University on 10/07/14 For personal use only.
Materials Sparfloxacin was obtained as a gift sample from Radicura Pharmaceuticals Pvt. Ltd, New Delhi, India. Sodium alginate KeltonÕ , which is composed of (60% mannuronic acid and 40% gluronic + acid), was kindly gifted by ISP (Alginates, Ltd., Waterfield, UK). Methylcellulose (Methocel A4M) grade was kindly gifted by Pharmax Private Ltd, Mumbai, India. Dialysis membrane (mol. wt. cut-off: 12 000 Da, flat with 25 mm, diameter of 16 mm, capacity 60 ml ft) was purchased from Sigma Aldrich Chemicals, Saint Louis, MO. Preparation of formulations Selection of vehicle Solubility of sparfloxacin was tested mainly in four different buffer solution namely, phosphate buffer USP (pH 6.0, 6.5 and 7.2), acetate buffer I.P. (pH 4.6 and 4.8), boric acid buffer (pH 4.7) and citrophosphate buffer B.P. (pH 6.0 and 6.2). Buffer in which Sparfloxacin (0.3%) was found to be soluble was selected as vehicle and formulation were developed. Selection of polymers The polymers used in the formulations were selected on the basis of gelling capacity, gelling time, gelling consistency and viscosity. The various polymers used in formulation are Polaxamer, Gelrite, Hydroxypropyl cellulose, Methyl cellulose, Sodium alginate, Hydroxypropyl methyl cellulose and results shown in Table 1. Preparation of in situ gelling systems After suitable selection of polymers, solutions of the selected polymers of varying concentrations were prepared in the selected buffer system to check there solubility and evaluate gelling capacity in order to identify suitable compositions for the preparation of in situ gelling systems of sparfloxacin as shown in Table 2. The gelling capacity was determined by placing a drop of the placebo formulation in a vial containing 2 ml of simulated tear fluid (STF pH 7.4) prepared artificially and equilibrated at 37 C. The formation of the gel was assessed and the time for gelation and time taken for gel to dissolve was noted down9. The composition of STF (pH 7.4) used was sodium chloride 0.67 g, sodium bicarbonate 0.2 g, calcium chloride dehydrate 0.008 g and purified water (q.s.) 100 ml. The selected placebo formulation on the basis of gelling capacity was used for medicated sol to gel development. For the preparation of in situ gel of sparfloxacin, selected polymers of different concentrations were separately dissolved and mixed with each other on a magnetic stirrer slowly. Drug solution was prepared in the selected buffer (boric acid buffer pH 4.7) system and then incorporated into the polymeric solution along with a suitable preservative till a uniform solution was obtained. The volume was adjusted up to required volume by the buffer and pH (4.7) was adjusted with sodium hydroxide solution (0.1 ml) and hydrochloric solution (0.1 ml)10. The developed formulations were filled in amber coloured vials since, the drug
Pharm Dev Technol, Early Online: 1–8
Table 1. Screening of various polymers for in situ gel. S. No. 1 2 3 4 5 6
Polymer
Gelling capacity Time required for gelation
HPMC Sodium alginate Methylcellulose HPC Gelrite Polaxamer
+++ +++
+++ +++
++++ ++
++ +
, No gelation; + , Gels after a few minutes, dissolves rapidly; + +, Gelation after a few seconds, remains for a few hours; + ++, Gelation immediate and of good consistency, remains for extended periods of time; + +++, Very thick gel formed. Table 2. Composition of sodium alginate and methylcellulose studied. Concentration used (%w/v) Formulation codes SM1 SM2 SM3 SM4 SM5 SM6 SM7 SM8 SM9 SM10 SM11 SM12
Sodium alginate
Methylcellulose
Gelling capacity
0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1.0 2.0
0.2 0.25 0.3 0.4 0.5 0.5 0.5 0.5 0.25 0.3 0.25 0.35
+ ++ ++ +++ +++ +++ +++ ++ ++++ ++++ ++++
, No gelation; + , Gels after a few minutes, dissolves rapidly; + +, Gelation after a few seconds, remains for a few hours; + ++, Gelation immediate and of good consistency, remains for extended periods of time; + +++, Very thick gel formed.
showed photosensitive reactions, under aseptic conditions, sterilized in the autoclave (121 C and 15 psi)11 for 20 min and further evaluations were carried out. Evaluation of developed formulations Determination of visual appearance, pH, clarity and drug content Appearance and clarity were determined visually against black and white background in a well lit room. The pH of the formulations was determined using pH meter (CL 54, Toshniwal Instrument Pvt. Ltd., Ajmer, India). The drug content was determined by taking 1 ml of each formulation and diluting it with suitable buffer. The concentration was determined using a double beam UV spectrophotometer at 290 nm12 (UV-1700, Shimadzu, Kyoto, Japan). Rheological evaluation Rheological study of the formulations were determined at 25 C and 37 C for both sol and gel phase at different angular velocities or speed conditions (30, 50, 75 and 100 rpm), respectively, using a Brookfield viscometer (RV model) using spindle C50-1 (small sample adaptor)13. Differential scanning calorimetry analysis Differential scanning calorimetry (DSC) analysis of pure drug, physical mixture (Drug + polymers) and, freeze dried formulation were carried out using Perkin Elmer 7 DSC (Waltham, MA) and calibrated with indium. The sample (5 mg) was placed into a standard aluminium pan, crimped and scanned from 40 C to
Noval in situ gel for ocular drug delivery
DOI: 10.3109/10837450.2014.910807
400 C at a heating rate of 5 C/min with continuous purging of nitrogen (20 ml/min). An empty sealed pan was used as reference.
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Thammasat University on 10/07/14 For personal use only.
In vitro release studies The in vitro release profile of sparfloxacin from in situ gel and drug solution (marketed preparation, Sparfloxacin eye dropsSPAT eye drops, Cadila Pharmaceuticals, Ahmedabad, India) was performed using dialysis bag. The formulation (containing 6 mg of drug) was placed in pre-treated dialysis bag which was immersed into 100 ml of STF (pH 7.4), at 37 C with continuous stirring at 50 rpm. At predetermined time intervals, aliquots were withdrawn from the release medium and replaced with the same amount. The samples were analyzed in triplicate by U.V. spectrophotometer at 290 nm14. The data obtained from in vitro drug release studies were fitted to various release kinetic models like zero order, first order, Higuchi model, Korsmeyer–Peppas and Hixon–Crowell model to understand the mechanism of drug release from in situ gel.
3
through the window and left in contact for 5 min. The membranes were examined for vascular damage, and time taken for injury was observed and scores were given. Normal saline and 0.1 M NaOH were used as negative and positive controls. Ocular irritation potential of the substances was assessed for three basic parameters, namely, hyperaemia, haemorrhage and coagulation17. Histopathological studies Histopathological studies in ophthalmology are another way of assessing the irritations of the formulation. The presence or absence of irritancy depends upon the changes in corneal structure using excised goat cornea. The formulations were kept in contact with cornea for 12 h. Cornea was removed, immediately fixed with 8% (w/w) formalin solution. The tissue was dehydrated with an alcohol gradient, put in melted paraffin and solidified in block form. Cross-sections were cut, stained with haematoxylin and eosin, and microscopically examined by MOTIC electronic microscope for modifications18 (MOTIC, Japan). Results obtained were compared with control.
Ex vivo corneal permeation studies An ex vivo corneal permeation study was performed using freshly excised goat cornea. Fresh whole eyeballs were procured from the slaughter house at under 4 C in normal saline solution (0.9% NaCl solution). Corneas were carefully removed with the help of forceps and scissors along with a 5–6 mm of surrounding sclera tissue and washed with saline. Cornea was mounted by fixing surrounding scleral tissue clamped between upper and lower chamber of the Franz diffusion cell such that lower chamber which served as the receiver compartment was infused continuously with STF (pH 7.4) at the rate 20 ml/min at 37 ± 0.5 C. The perfusate was collected at periodic time intervals for up to 12 h in eppendorf tubes and were subjected to quantification of sparfloxacin by using ultraviolet spectroscopy at 290 nm15.
Confocal laser scanning microscopy study The penetration of optimized in situ gel and market formulation on excise goat cornea were determined by confocal laser scanning microscopy (CLSM). For this ex vivo goat permeation study was carried out as described above. After 8 h, the cornea was removed and washed with distilled water. The treated area was cut out and positioned on the microscopic slide and cover with the cover slip. CLSM was carried out with the Laser Confocal Microscope with Fluorescence Correlation Spectroscope-Olympus FluoView FV1000 (Olympus, Melville, NY) with an argon laser beam with excitation at 295 nm and emission at 525 nm. The permeated depths of formulation were detected at z-axis with Fluoview software19.
Antimicrobial efficacy studies
Sterility testing
The microbiological studies were carried out for ascertaining the biological activity of the formulated in situ gel systems against specified microorganisms. The organisms used in this study were Staphylococcus aureus (Gram-positive bacteria) and Pseudomonas aeruginosa (Gram-negative bacteria) using agar diffusion medium employing ‘‘cup plate technique’’. The developed formulation and marketed sparfloxacin eye drops (used as standard) were added, respectively, in agar plates. The plates were left to allow diffusion of solutions for 2 h and then incubated at 37 C in an incubator for 24 h. Zone of inhibition were measured after 12 and 24 h by an antibiotic zone finder16.
Test of sterility was carried out according to I.P. 1996 by membrane filtration method. Membrane filter of pore size 0.22 mm was used. The test was carried out by incubating the developed formulations in soybean casein digest medium (incubated at 25 C for 14 days) to analyze the growth of fungi and fluid thioglycolate medium (incubated at 35 C for 14 days) to analyze the growth of bacteria in the formulations20.
Ocular irritation studies Ocular irritation studies were carried out by HET-CAM assay (Hen’s egg chorioallantoic membrane test). It is a non-animalbased ocular toxicity test which has been used for irritation study. HET-CAM assay has been nominated by the EPA for evaluation as an alternative to the Draize test for ocular irritancy in rabbits. Three fertile eggs weighing 45–65 g were taken and observed under light and found non-defective. The eggs were placed in a tray filled with cotton in such a way that they were in an upside position. These eggs were then incubated in an incubator for a period of 10 days at a temperature of 37 ± 0.5 C and relative humidity 55 ± 7% for a period of 9 days. Eggs were rotated manually in a gentle manner after every 12 h. At the end of 10th day eggs were very carefully taken out with the help of a blunt forceps, egg shell was scratched off and then pared off. The inner membrane was carefully removed to expose the chorioallantoic membrane (CAM). Formulations (0.5 ml) were instilled directly
Stability studies Stability studies were carried out as per ICH guidelines21. The selected formulations were subjected to 25 ± 0.5 C, 30 ± 0.5 C and 40 ± 0.5 C/75% R.H. at 0, 30, 60, 90 and 180 days. The formulations were periodically evaluated for drug content, pH, clarity, viscosity. Using Arrhenius plot the shelf life of the formulations was calculated at room temperature, i.e. 25 C as per ICH guidelines.
Results and discussion Selection of vehicle In ophthalmic drug delivery systems, buffer systems must be nontoxic, non-irritating and having low buffer capacity and should be isotonic in nature. Isotonic boric acid buffer of pH 4.7 was selected in the formulation of sparfloxacin in situ gel. One main advantage of using boric acid buffer was that it has antimicrobial effects, which will enhance the anti microbial potency of the formulation. The boric acid buffer of pH 4.7 (Martin, 1993) was prepared by adding 1.75 g of boric acid crystals in 100 ml of double-distilled water.
4
N. Khan et al.
Pharm Dev Technol, Early Online: 1–8
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Thammasat University on 10/07/14 For personal use only.
Selection of polymers Various polymers were used for screening of polymers in formulation of in situ gel system. The selection of polymer was done on the basis of gelling capacity, gelling time, gelling consistency and viscosity of gel by using Rheometer (Table 1). Sodium alginate and Methylcellulose were the polymers selected for the preparation of in situ gels of sparfloxacin. Sodium alginate was used as gelling agent because it has high gluronic acid content, which forms a low viscosity, free flowing liquid. It improves the gelling properties hence reduction of polymer concentration to be introduced into the eye. Therefore, to improve viscosity methylcellulose was incorporated as viscosity enhancing agent. Both the polymers were found to exhibit favourable biological properties such as non-toxicity and biodegradability. Preparation of in situ gelling systems The polymers, namely sodium alginate and methylcellulose were found to be soluble in isotonic boric acid buffer (pH 4.7). Isotonicity calculations were done individually for drug and the polymers by sodium chloride equivalent method. Sparfloxacin (0.3 g) was first dissolved in isotonic boric acid buffer (pH 4.7) Table 3. Formulation codes of medicated ophthalmic sol to gel formulations. Formulation codes Ingredients
SM3
SM4
SM5
SM6
SM7
SM8
Sparfloxacin (g) Sodium alginate (g) Methylcellulose A4M (g) Benzalkonium chloride (ml) Modified boric acid buffer (pH 4.7) q.s. (ml) Tween-80 (ml)
0.3 0.6 0.3 0.01 100
0.3 0.65 0.4 0.01 100
0.3 0.7 0.5 0.01 100
0.3 0.75 0.5 0.01 100
0.3 0.8 0.5 0.01 100
0.3 0.85 0.5 0.01 100
0.1
0.1
0.1
0.1
0.1
0.1
Table 4. Physiochemical evaluation of selected formulations. Formulation codes
Appearances
pH
Clarity
Drug content (%w/v) ± SDa
SM3 SM4 SM5 SM6 SM7 SM8
Light Light Light Light Light Light
4.8 4.9 4.9 5.1 5.1 5.2
Clear Clear Clear Clear Clear Clear
98.3 ± 0.553 99.0 ± 0.614 99.2 ± 0.581 99.8 ± 0.742 98.7 ± 0.680 99.4 ± 0.598
yellow yellow yellow yellow yellow yellow
a
Standard deviation.
Figure 1. Viscosity of medicated formulation code SM6 in Sol and Gel state at 25 C and 37 C.
along with a cosolvent Tween-80 (0.1 ml). Tween-80 was used to enhance the solubility of the drug in isotonic boric acid buffer (pH 4.7). The mixture was then incorporated into the selected placebo formulation and the volume was adjusted up to the required volume as shown in Table 3. The pH was adjusted with 0.1 ml sodium hydroxide solution and 0.1 ml hydrochloric solution. The developed formulations were filled in amber coloured vials since, the drug showed photosensitive reactions. Evaluation of developed formulations Determination of visual appearance, pH, clarity and drug content Developed in situ gel of sparfloxacin was light yellow in colour and all formulations were found to be clear. Terminal sterilization by autoclaving had no effect on the appearance, clarity, pH and drug content of the formulations. The formulations were liquid at room temperature and at pH of the formulations which is within the range of 4.7–5.2 and a rapid transition to the gel phase was observed at pH of the tear fluid, i.e. pH 7.4. Drug content of all formulations was within the range of 98.31–100.03%, which inclined towards the uniform distribution of drug in developed ophthalmic formulations and results are shown in Table 4. Rheological evaluation The sol and gel phase of all the formulations showed shear thinning, and pseudoplastic behaviour of the formulation was observed which is a desirable phenomenon in case of ophthalmic formulations. All the formulations were found to be in liquid phase at pH 4.7 and an increase in the viscosity was observed when the pH of the formulations was raised to 7.4 which transformed into gel phase. Decrease in viscosity was observed on increasing the angular velocity in all formulations and in both sol as well as gel phases. Result for viscosity of medicated formulation code SM6 in Sol and Gel state at 25 C and 37 C is as shown in Figure 1. At higher angular velocities, viscosities were nearly constant. Pseudo plastic character of the precorneal tear film should be least affected by the administration of ophthalmic products DSC analysis DSC analysis of optimized formulation was done to determined interaction of drug with polymers. The drug and physical mixture (Drug + polymers) show an intense characteristic peak (Figure 2). The optimized in situ gel formulation does not show any peak because of drug was dispersed or converted into the amorphous form from the crystalline form, which implies that the drug has been fully entrapped in to the polymer matrix as shown in Figure 2.
Noval in situ gel for ocular drug delivery
DOI: 10.3109/10837450.2014.910807
5
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Thammasat University on 10/07/14 For personal use only.
In vitro release studies The cumulative percentage release of sparfloxacin from in situ gel was 94.3% over a period of 24 h and 11.8% in 1 h whereas, the percentage drug release of sparfloxacin from eye drop was 95.62% in 4 h and 42.34% in 1 h (Figure 3). The co-efficient of correlation (R2) of zero order, first order, Higuchi, Hixon–Crowell model and Korsmeyer–Peppas model for in situ gel was found to be 0.931, 0.989, 0.992, 0.967 and 0.987, respectively. Since, the co-efficient of correlation (R2) for Higuchi model was nearer to unity, i.e. (0.992) therefore, it was selected as the best fit model (Table 5). The linear relationships in conjunction with the slow dissolution rate suggest that the in vitro drug release from in situ gel formulation under physiological conditions occurs primarily by diffusion22. Ex vivo corneal permeation studies
Figure 2. Differential scanning calorimetry thermogram of physical mixture (A), Sparfloxacin (B) and optimized in situ gel formulation SM6 (C).
Ex vivo transcorneal permeation studies were conducted on optimized in situ gel formulation SM6, because it selected as optimized formulation on the basis of gelation capacity, gelation time and rheological properties. The extent of permeation of that formulation was compared to market Sparfloxacin eye dropsSPAT eye drop. A cumulative percent permeation of 62.56% done for 6 h was obtained for formulation SM6 and 36.87% from eye drop, respectively (Figure 4). The higher permeation of in situ gel formulation was due to bioadhesive and permeation enhancing properties of the polymers. It also enhanced the permeation and miscibility with corneal tissues compared to marketed eye drops of sparfloxacin due increased corneal contact time. Antimicrobial efficacy studies The zones of inhibition were measured after 12 and 24 h by an antibiotic zone finder. It was observed that diameter of zones of inhibitions from 44 to 48 mm for marketed formulation at 12 and Table 5. Kinetic release models data of formulation SM6. Zero order R2
First order K0
R2
K1
Higuchi model R2
KH
0.93 3.49 0.98 0.05 0.99 21.58 Figure 3. In vitro release of in situ gelling system and marketed formulation (mean ± SD).
Figure 4. In vitro transcorneal permeation study (mean ± SD).
Korsmeyer–Peppas model
Hixon–Crowell model
R2
KKP
R2
KHC
0.98
0.65
0.96
0.01
R2, regressed values; K, rate constant for different models.
6
N. Khan et al.
Pharm Dev Technol, Early Online: 1–8
24 h compared to 46 and 50 mm at 12 and 24 h for the optimized in situ gel formulation (Table 6). Results revealed that optimized formulation SM6 showed a comparable antimicrobial efficacy with marketed formulation against S. aureus (Gram-positive organism) and P. aeruginosa (Gram-negative bacteria). Table 6. Zone of inhibition produced by ophthalmic sol to gel formulation SM6. Zone diameter (mm) against S. aureus
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Thammasat University on 10/07/14 For personal use only.
Incubation time (h)
Zone diameter (mm) against P. aeruginosa
Formulation (SM6)
Sparfloxacin eye drops
Formulation (SM6)
Sparfloxacin eye drops
46 50
44 48
43 45
44 46
12 24
Table 7. Cumulative scores for assessment of ocular irritation potential. Cumulative scores
Irritation assessment
0–0.9 1–4.9 5–8.9 9–21
Practically none Slight Moderate Strong
Ocular irritation studies Formulation SM6 was tested in sol as well as gel form for their irritation potential if any. Normal saline (0.9% NaCl) and mild shampoo were used as negative and positive controls, respectively, as these substances have shown practically non-irritating and slightly irritating effects as reported in some literature. A cumulative score of 0 and 3.99 were obtained for normal saline solution and mild shampoo, respectively. Cumulative scores for the optimized formulation SM6 in solution as well as in gel form were 0.33 and 0.33, respectively, and these were50.9 (standard for being practically non-irritant) (Table 7). Therefore the optimized formulation (SM6) was found to be safe and nonirritant. Histopathological studies Histopathological studies revealed no change in histopathology of the tissues for the formulation SM6. Hence, it was safe and free from any ocular irritation and did not produce any changes in cell structure of corneal membrane. There was no rupture was observed in the corneal section when compared to eye drop (Figure 5). CLSM study The extent of penetration was measured by CLSM after application of formulations (i.e. in situ gel and marketed eye drop).
Figure 5. Histopathological study of cornea section of formulation SM6 (a) and marketed eye drop of sparfloxacin (b).
Noval in situ gel for ocular drug delivery
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Thammasat University on 10/07/14 For personal use only.
DOI: 10.3109/10837450.2014.910807
7
Figure 6. CLSM image: (A) optimized in situ gel formulation SM6 and (B) marketed eye drop. Table 8. Degradation rate constant of sparfloxacin in ophthalmic sol to gel preparation SM6. Temperature ( C) 25 30 40
Degradation rate constant (K) 1.2647 10 1.3818 10 1.5353 10
to conventional eye drops by virtue of its ability to sustained drug release and longer pre-corneal residence time. The formulation was stable for longer period of time with shelf life of 2.28 years.
4 4 4
Acknowledgements The authors are thankful to Radicura Pharmaceuticals Ltd. (India) for providing gift sample of Sparfloxacin. The authors are also thankful to Jamia Hamdard for carrying out the relevant studies.
The in situ gel system penetrates the cornea 110.64 mm in depth with fluorescence intensity as compared to eye drop (58.67 mm) depth during 8 h of study (Figure 6). The higher penetration of in situ gel of sparfloxacin is could be due to increased corneal contact time and penetration property of polymer used in formulation. Sterility testing Formulation SM6 passed the test for sterility as there was no turbidity was observed. The formulation did not showed any fungal and bacterial growth up to 14 days of incubation at 25 C in soyabean case in digest medium and 35 C in fluid thioglycolate medium. Stability studies The optimizedformulation was found to be clear; no change was observed in viscosity and pH after storage of 6 months at different temperature as per ICH guidelines. The degradation rate constant was found to be low (1.2647 10 4at 25 C, Table 8), i.e. negligible degradation (55%) and followed the first-order rate kinetics. The shelf life of was found to be 2.28 years.
Conclusion Sparfloxacin is fluroquinolone antibiotic antibacterial agent used in the treatment of ocular infections was successfully prepared by using polymers sodium alginate as gelling agent and methylcellulose A4M as viscosity enhancing agent. The formulation was in sol form at pH (4.7) and underwent rapid gelation upon raising pH to 7.4. This in situ gel formulation showed in vitro sustained drug release (p50.05) over period of 24 h as compared to eye drop. The ex vivo goat corneal permeation study showed significant enhanced permeation (p50.05) as compared to eye drop. Ocular tolerance tests and histopathology study showed that developed formulation was non-irritant, good ocular tolerance and significant antimicrobial study. This formulation is a viable alternative
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.
References 1. Kumar M, Kulkarni GT. Recent advances in ophthalmic drug delivery. Int J Pharm Sci 2012;4:387–394. 2. Gaudana R, Jwala J, Boddu SHS, Mitra KA. Recent perspectives in ocular drug delivery. Pharm Res 2009;26:1197–1216. 3. Rajas JN, Kavitha K, Gounder T, Mani T. In situ ophthalmic gels: a developing trend. Int J Pharm Sci Rev Res 2011;7:8–14. 4. Mohanambal E, Arun K, Abdul HSA. Formulation and evaluation of pH-triggered in situ gelling system of levofloxacin. Ind J Pharm Edu Res 2011;45:58–64. 5. Kashikar VS, Ophthalmic mucoadhesive polymers – a literature review of levofloxacin. Int J Pharm Edu Res 2011;7:68–73. 6. Zhidong LA, Jianier LB, Shufang NA, et al. Study of an alginate/ HPMC based in situ gelling ophthalmic delivery system for gatifloxacin. Int J Pharm 2006;315:12–17. 7. Gupta H, Aqil M, Khar KR, et al. Sparfloxacin-loaded PLGA nanoparticles for sustained ocular drug delivery. Nanomed Nanotechnol Biol Med 2010;6:324–333. 8. Thomas K, Patrichia B, Friedemann K. Treatment of patients with keratoconjunctivitis sicca with optiveÔ: results of a multicenter, open-label observational study in Germany. Clin Ophthalmol 2009; 3:33–39. 9. Johan C, Katarina E, Roger P, Katarina J. Rheological evaluation of gelrite in situ gels for ophthalmic use. Eur J Pharm Sci 1998;6: 113–119. 10. Sultana Y, Aqil M, Ali A. Ion activated Gelrite-based in situ ophthalmic gels of pefloxacinmesylate: comparison with conventional eye drops. Drug Deliv 2006;13:215–219. 11. Gupta D, Tator, HC, Shoichet SM. Fast gelling injectable blend of hyaluronan and methylcellulose for intrathecal, localized delivery to the spinal cord. Biomaterials 2006;27:2370–2379. 12. Eaga CM, Jagan MK, Venkatesh A. Preparation and evaluation of in situ gels for ocular drug delivery. J Pharm Res 2009;2: 1089–1094.
8
N. Khan et al.
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Thammasat University on 10/07/14 For personal use only.
13. Srividya B, Cardoza RM, Amin PD. Sustained ophthalmic delivery of ofloxacin from a pH-triggered in situ gelling system. J Control Rel 2001;73:205–211. 14. Gupta H, Jain S, Mathur R, et al. Sustained ocular drug delivery from a temperature and pH triggered novel in situ gel system. Drug Deliv 2007;14:507–515. 15. Rathore SM, Majumdar KD. Effect of formulation factors on in vitro transcorneal permeation of gatifloxacin from aqueous eye drops. AAPS PharmSciTech 2006;7:E1–E6. 16. Charoo NA, Kohli K, Ali A. Preparation of in situ forming ophthalmic gels of ciprofloxacin hydrochloride for the treatment of bacterial conjunctivitis: in vitro and in vivo studies. J Pharm Sci 2003;92:407–415. 17. Gilleron L, Coecke S, Sysmans S, et al. Evaluation of a modified HET-CAM assay as a screening test for eye irritancy. Toxicol In Vitro 1996;10:431–446.
Pharm Dev Technol, Early Online: 1–8
18. Sindhu A, Sharon F, Bharath S, et al. Sustained ophthalmic delivery of ofloxacin from an ion-activated in situ gelling system. Pak J Pharm Sci 2009;22:175–179. 19. Ahad A, Aqil M, Kohli K, et al. Formulation and optimization of nanotransfersomes using experimental design technique for accentuated transdermal delivery of valsartan. Nanomed Nanotechnol Biol Med 2012;8:237–249. 20. Stahlmann R, Zippel U, Forster C, et al. Chondrotoxicity and toxicokinetics of sparfloxacin in juvenile rats. Antimicrob Agents Chemother 1998;42:1470–1475. 21. ICH Topic Q 1 A (R2), Stability Testing of new Drug Substances and Products CPMP/ICH/2736/99, August 2003. 22. Liu Z, Li J, Nie S, et al. Study of an alginate/HPMC-based in situ gelling ophthalmic delivery system for gatifloxacin. Int J Pharm 2006;315:12–17.
