Sustained Ophthalmic Delivery of Moxifloxacin Hydrochloride from an pH Triggered in Situ Gelling System

 

S.K. Gupta¹* and I.J. Singhvi²

 

ABSTRACT:

The poor bioavailability and therapeutic response exhibited by conventional ophthalmic solutions due to rapid pre-corneal elimination of the drug may be overcome by the use of in situ gel forming systems that are instilled as drops into the eye and then undergo a sol-gel transition in the cul-de-sac. The present work describes the formulation and evaluation of an ophthalmic delivery system of an antibacterial agent moxifloxacin hydrochloride, based on the concept of pH triggered in situ gelation. Carbopol 940 was used in different concentrations (0.3-0.5 w/v) as the gelling agent in combination with HPMC E50 LV (Hydroxy Propyl Methyl Cellulose) that acted as a viscosity-enhancing agent. The primary criteria for formulation optimization were gelling capacity and rheological behaviour. In addition, formulations were evaluated for pH, and antimicrobial efficacy and drug release. The clarity, pH, gelation in simulated tear fluid and rheological properties of the optimized formulations were satisfactory. In vitro release studies indicated that the Carbopol /HPMC solution retained the drug better than the Carbopol or HPMC solutions alone. The formulations were therapeutically efficacious, sterile, stable and provided sustained release of the drug over a period of time. These results demonstrate that the developed system is an alternative to conventional ophthalmic drops, patient compliance, industrially oriented and economical.

 

 

1. INTRODUCTION:

Moxifloxacin is a broad-spectrum antibiotic that is active against both Gram-positive and Gram-negative bacteria. It functions by inhibiting DNA gyrase, a type II topoisomerase, and topoisomerase IV¹ enzymes necessary to separate bacterial DNA, thereby inhibiting cell replication. It provides increased penetration into ocular tissues and fluids with improved activity against Streptococci and Staphylococci species and moderate to excellent activity against clinically relevant, gram-negative ocular pathogens.

 

The extent of absorption of an ophthalmic drug is severely limited by physiological constraints. The conventional liquid ocular formulation is eliminated from the precorneal area immediately upon instillation because of lachrymation and effective nasolacrimal drainage. Various preparations, such as ointments², suspensions³, inserts⁴, and hydrogels, have been developed for ophthalmic delivery system not only to slow down the drug elimination but also to lengthen the residence time of vehicle on ocular surface⁵.

 

However, they have not been used extensively because of some drawbacks, such as blurred vision with ointments or low patient compliance with inserts⁶.

 

An ideal ophthalmic formulation should be administrated in eye drop form, without causing blurred vision or irritation. This problem can be overcome using in situ gel-forming drug delivery systems prepared from polymers that exhibit sol-to-gel phase transitions due to a change in a specific physicochemical parameter in the cul-de-sac⁷. In situ gelation approach combines advantages of both solutions and gels, such as accuracy and ease of administration of the former and prolonged precorneal retention of the latter^{8, 9.}

 

In the past few years, an impressive number of pH (e.g., cellulose acetate phthalate and Carbopol) induced in situ forming systems have been reported to sustain ophthalmic drug delivery^10-13. These in situ gel-forming systems could prolong the precorneal residence time of a drug^14-18 and improve ocular bioavailability¹⁹. So the present work was aimed at the preparation of in situ gelling ophthalmic formulations of moxifloxacin Hcl. using carbopol 940 as the gelling agent.

 

 

Table 1: Composition of ophthalmic in situ gelling systems of moxifloxacin HCl

Ingredients	F1	F2	F3	F4	F5	F6	F7	F8	F9	F10	F11	F12
Moxifloxacin Hcl (%w/v)	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
Corbopol 940 (%w/v)	0.3	0.4	0.5	0.3	0.4	0.5	0.3	0.4	0.5	0.3	0.4	0.5
HPMC E 50 LV (%w/v)	---	---	---	0.5	0.5	0.5	1.0	1.0	1.0	1.5	1.5	1.5
Benzalkonium chloride (%v/v)	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02
Citric acid(%w/v)	0.407	0.407	0.407	0.407	0.407	0.407	0.407	0.407	0.407	0.407	0.407	0.407
Disodium hydrogen phosphate IP(%w/v)	1.125	1.125	1.125	1.125	1.125	1.125	1.125	1.125	1.125	1.125	1.125	1.125
Propylene gylcol (ml)	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0
Purified water (ml) q.s	100	100	100	100	100	100	100	100	100	100	100	100

 

The objective of the present study was to develop an pH triggered in situ gelling for moxifloxacin HCl, a fluoroquinolone derivative used to treat external infections of the eye such as acute and subacute bacterial conjunctivitis, conjuncttivitis, keratitis, keratoconjuctivitis and corneal ulcers which can prevent frequent drug administration and enhance patient compliance. Carbopol 940 was used as the gelling agent in combination with hydroxy propyl methyl cellulose E 50 LV (HPMC) as the viscosity enhancer for the formulation of moxifloxacin Hcl eye drops (0.5% w/v), which undergo gelation when instilled into the cul-de-sac of the eye and provide sustained release of the drug.

 

MATERIALS AND METHODS:

Materials

Moxifloxacin HCl. was obtained as a gift sample from Torrent Research, Village Bhat, Gandhinagar, Gujarat, India. Carbopol 940 and HPMC E50 were purchased from Colorcon Asia, Mumbai, India.  All other reagents were of analytical grade.

 

Method

Preparation of formulations

The solubility of moxifloxacin HCl was tested in various buffer such as (acetate buffer I.P. (pH 6.0, 6.5), citrophosphate buffer (pH 7.2 and 7.4) in order to select a suitable vehicle. Solution of moxifloxacin Hcl in the above buffer were prepared to test its solubility at the dosage lavel desired (0.5 % w/v). moxifloxacin Hcl was found to be completely dissolved in phosphate buffer (pH 7.4) which was selected as the vehicle for the preparation of in situ gelling systems.

 

Corbopol 940 in concentrations of 0.3-0.5% w/v and viscosity builders HPMC E50LV was used to prepare totally 09 formulations (Table 1). Buffer salt (citric acid, disodium hydrogen phosphate) were dissolve in 75 ml of purified water, HPMC E 50LV was added and allow to hydrate. Carbopol 940 was sprinkled over this solution and allows to hydrate overnight. The solution were stirred with an overhead stirrer, tween 20 was added while stirring. Moxifloxacin Hcl. (0.5% w/v) was dissolved in small quantity of purified water and the pH was adjusted to 6.2 using 0.5M NaOH. Benzalkonium chloride (0.02% v/v) solution was then added to the above solution. The drug solution was then added to the Carbopol /HPMC solutions under constant stirring to obtain a uniform solution. Purified water was then added to make the volume up to 100ml. The formulation were filled in vials under aseptic conditions, sterilized in the autoclave(121⁰C and 15 psi) for 20 minutes, and further evaluation were carried out.

 

EVALUATION STUDIES

Selection of formulations:

Gelling capacity and rheology are the main prerequisites of the in situ gelling systems. Therefore based on these two properties the formulations F6, F9 and F12 were selected and evaluated for further studies. The general appearance of the formulations was observed which included colour and clarity of solution. The pH of the prepared formulations was checked using a pH meter.

 

In situ gelling capacity:

The gelling capacity of the formulation was determined by placing the formulation in a vial containing artificial tear fluid in the proportion of 25:7. The composition of the simulated tear fluid (STF) was: NaCl 0.67 g, NaHCO₃ 0.₂ g, CaCl₂.2H₂O 0.008 g and water up to 100 g²⁰. The gel formation was assessed visually and the time was noted for the gelation and also noted the time taken for the gel formed to dissolve.

 

Rheological studies:

Viscosity of the instilled formulation is an important factor in determining residence time of drug in the eye. The prepared solutions were allowed to gel in the simulated tear fluid and then the viscosity determination were carried out by using Brookefield DV-II+ Rheometer with spindle LV-3 with angular velocity run from 6 to 60 rpm.

 

Determination of drug content:

The drug content was determined by taking 1ml of the formulation and diluting it to 100 ml with phosphate buffer pH 7.4. Aliquot of 5ml was withdrawn and further diluted to 25ml with phosphate buffer pH 7.4. Moxifloxacin HCl. concentration was determined at 287.0 nm by using UV-Visible spectrophotometer (UV-1700, Shimadzu Corporation, Japan).calibration curve were presented in Figure 1.

 

Antimicrobial efficacy studies: Antimicrobial efficacy was determined by agar diffusion employing 'cup plate techinique' on selected formulations²¹. Wells were made with borer in the solidified medium previously seeded with test organism (Pseudomonas aeruginosa and Staphylococcus aureus). After allowing diffusion of the solution for 2 hours, the agar plates were incubated at 37^oC for 24 hrs, and then observed for zone of inhibition.

Table 2: Evaluation of In Situ Gelling Formulations

Formulation

F1

F2

F3

F4

F5

F6

F7

F8

F9

F10

F11

F12

Appearance

light yellow

light yellow

light yellow

light yellow

light yellow

light yellow

light yellow

light yellow

light yellow

light yellow

light yellow

light yellow

clarity

clear

clear

clear

clear

clear

clear

clear

clear

clear

clear

clear

clear

pH

6.0

6.1

6.0

6.1

6.0

6.2

6.3

6.1

6.3

6.2

6.2

6.3

Viscosity(cps)

-

-

-

12.3

12.2

16.4

12.4

10.8

16.2

12.1

12.4

18.2

Gelling capacity

+

+

+

++

++

+++

++

++

+++

++

++

+++

 

 

 

In vitro drug release studies:

A number of approaches have been used by different workers to conduct in vitro drug release from controlled ocular drug delivery systems, including bottle method, diffusion method, modified rotating basket/paddle method and technique involving flow through apparatus²², drug release studies were performed using a diffusion technique ²³ with some modification.

 

In vitro release studies of Moxifloxacin HCl. from the formulations were studied through cellophane membrane using a modified USP XXIII dissolution testing apparatus²⁴. The dissolution medium used was simulated tear fluid freshly prepared of pH 7.4, cellophane membrane previously soaked overnight in the dissolution medium was tied to one end of a specifically designed glass cylinder (open at both ends of 5 cm diameter) a 1ml of the formulation was placed in to this assembly . The cylinder was attached to the metallic drive shaft and suspended in 50ml of simulated tear fluid (STF) maintained at 37±1^oC temperature so that the membrane just touched the receptor medium surface. The shaft was rotated at 50 rpm, at specified intervals of time (hourly), 1ml of the sample solution was withdrawn from the receptor compartment and replaced with the fresh STF. The samples were analyzed after necessary dilutions by UV-Visible spectrophotometer at 287.0 nm. The in vitro release studies were also carried out for the marketed conventional ophthalmic drops of Moxifloxacin HCl. (0.5% w/v) in order to compare the release profile of the drug with the prepared in situ gelling system.

 

Stability study

The selected formulations were stored at ambient humidity conditions between 2-8^oC, ambient temperature and at 40^oC for a period of one month. The samples were withdrawn at frequent intervals and evaluated for the parameters viz. pH, appearance, gelation studies and drug content.

 

RESULTS AND DISCUSSION:

The vehicle selected for the ocular formulations was phosphate buffer (pH 7.4) based on the solubility of moxifloxacin HCl. A buffered vehicle in ophthalmic preparations is appropriate as the pH plays a significant role with respect to stability of formulations, solubility of drugs, and also eye irritation. Viscosity enhancing polymers such as HPMC were included in the formulations in order to achieve the desired rheological behavior. Moreover cellulose polymers possess mucoadhesive property which may help in prolongation of the stay of drug in cul de sac. All the formulations prepared were clear, light yellow in colour without any turbidity or impurities and pH of the formulations were between 6.0-6.3 (Table 2) which is an acceptable range for ophthalmic preparations.

.

The stability of the formed gels with different formulations is given in (Table 2). The time taken for the gel to dissolve is denoted as -, +, ++, or +++ indicating no gelation, gel for few a minutes, dissolve rapidly, Gelation immediate, remain for few hours, Gelation immediate; remain for extended period of time respectively.

 

Figure 1: Calibration curve of moxifloxacin HCl in phosphate buffer pH 7.4

 

The gels formed with carbopol alone (F1, F2, F3) were stable only for 10 min but inclusion of viscosity builders was found to enhance the stability of the gel structure for a longer time. Among the rest of the formulations, the gels formed with two formulations F6, F9 were stable for one hour whereas, F12 gel remained undissolved for extended times.

 

The viscosities (cps) of all the formulations at angular velocities 60 rpm as shown in (Table 2), reveals the shear thinning nature of the solutions. The some formulations with greater gel stability (F6, F9, and F12) were found to possess higher viscosities at 6-60 rpm in (Figure 2).

 

Figure 2: Rheological profile of in situ gelling formulations (F6, F9, and F12)

 

Based on the results of rheological studies and gelling behaviour (Table 2) three formulations, F6, F9 and F12 were selected for further studies.

 

The drug contents of the formulations F6, F9 and F12 were found to be 98.75% w/v, 99.42% w/v, 99.86% w/v respectively.

The zones of inhibition observed in agar diffusion study on the formulations (Table 3 and 4) were found to be similar, which proves the sustained release of the drug from the in situ formed gels.

 

 

Table 3: Comparison of antimicrobial activity of the in situ gels with standard against Pseudomonas aeruginosa in cup and plate technique

Conc.(ug/ml)	Standard zone of inhibition (cm)	Tests zone of inhibition(cm)
		F6	F9	F12
20	3.5	3.3	3.5	3.5
40	4.0	3.7	3.0	3.7
60	4.2	3.0	3.1	3.1
80	4.4	3.3	3.3	3.3
100	4.8	3.7	3.8	3.5

 

Table 4: Comparison of antimicrobial activity of the in situ gels with standard against Staphylococcus aureus in cup and plate technique

Conc.(ug/ml)	Standard zone of inhibition (cm)	Tests zone of inhibition(cm)
		F6	F9	F12
20	3.4	3.2	3.4	3.4
40	3.9	3.7	3.7	3.8
60	4.3	4.2	4.2	4.2
80	4.6	4.3	4.5	4.5
100	4.9	4.8	4.8	4.7

 

Results of in vitro drug release studies by diffusion through cellophane membrane on the three formulations F6, F9 and F12 are given in the (Figure 3). The results clearly assure that a temporally controlled release of moxifloxacin HCl. in the cul de sac will occur for more than 8 h. A more prolonged release was observed with F12 which showed the maximum in situ formed gel stability, which may be due to the higher concentration of carbopol along with HPMC. The higher regression coefficient values (Table 4) for each formulation suggested that the formulations behaved matrix type of drug release, whereas formulation F-12 showed zero order drug release kinetics. This indicates that the gels have the ability to retain moxifloxacin resulting in sustained drug release. All the formulation the best fit model was higuchi matrix equation and suggesting diffusion controlled release may be due to the swelling nature of polymer. The n value obtained from peppas equation were less than 0.5, which indicated that all the formulation showed drug release by Fickian diffusion mechanism. The results were shown in table 4.

 

The comparative in vitro drug release profile (Figure 3) between the marketed conventional ophthalmic drops and the formulation F-12 showed 22% and 30 % after initial 1hrs. At the end of 4 hrs. The drug release was found to be 90.6% from the marketed product and 85% from the F-12 indicating that the drug release was significantly prolonged by using the in situ gelling systems.

 

Figure 3: comparative In vitro release profile of in situ gel formulations and marketed Preparation (moxifax 0.5% w/v).

 

Table 4: Release kinetics of formulations

Formulation	Zero order r2	First order r2	Higuchi r2	Kosermeyer peppas r2	n value
F6	0.9738	0.8845	0.9819	0.995	0.322
F9	0.984	0.936	0.982	0.900	0.1696
F12	0.992	0.987	0.963	0.949	0.4757

 

The results of stability study of selected formulations (F12) were indicated in Table 5. The samples were withdrawn at frequent intervals and evaluated for the parameters viz. pH, appearance, gelation studies and drug content.

 

Table 5: Stability studies of moxifloxacin HCl. In situ gel of F12 formulation at room temperature and 40^oC after 1 month

Formulation (F12)	Room temperature	40oC
pH	6.2	6.1
appearance	Clear light yellow	Clear light yellow
Gelation studies	+++	+++
Drug content	99.86% w/v	99.12 % w/v

 

CONCLUSION:

Moxifloxacin HCl, a broad spectrum antibacterial agent used in the treatment of ocular infections was successfully formulated as a pH triggered in situ gel forming ophthalmic solution using carbopol 940 in combination with HPMC E50 LV as a viscosity enhancer which sustained the drug release over a period of 8 hours.

 

In the present study, we found that the optimum concentrations of carbopol 940 and HPMC E 50LV solutions for ocular drug delivery system were 0.5% and 1.5% (w/v), respectively. When 0.4% carbopol and 1.0 % HPMC solutions were combined, the gel strength under physiological conditions was significantly increased and the combined solution was easy to administer during ocular instillation. The formulation also promises to reduce the frequency of drug administration, thus improving patient compliance. As the concept involved is novel and the methodology used for the preparation is simple as that of conventional ophthalmic liquid dosage form, it is industrially oriented and economical. The polymers used are inexpensive and easily available.

 

ACKNOWLEDGEMENT:

The authors wish to thank Pacific College of Pharmacy, Udaipur (Raj.) for providing necessary facilities to carry out the research work.

 

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Received on 06.10.2012          Modified on 20.10.2012

Accepted on 25.10.2012         © RJPT All right reserved