Beyond the Blink: Using In-Situ Gelling to Optimize Opthalmic Drug Delivery

Abstract

Conventional ophthalmic solutions frequently show poor bioavailability and a weak therapeutic response because they are often eliminated before they can reach the cornea, when patients blink or their eyes tear. Use of in-situ gel forming solutions may help improve performance and patient compliance. These solutions are delivered as eye drops, but undergo a sol-gel transition in the conjunctival sac (cul de sac). This article describes how an ion-activated in-situ gelling system was designed to deliver an ophthalmic formulation of the antibacterial agent, Levofloxacin. 

The delivery system uses gellan gum, a novel ophthalmic vehicle that gels in the presence of mono or divalent cations in the lacrimal fluid. This gum was used alone and combined with sodium alginate as a gelling agent and hydroxypropyl methylcellulose (HPMC) Methocel F4M as a viscosity enhancer. 

A 3² full factorial design approach was used, with two polymers: Gelrite and HPMC, as independent variables. Gelling strength, bioadhesion force, rheological behavior, and in-vitro drug release after 10 h were selected as dependent variables

Both in-vitro release studies and rheological profile studies indicated that the combined Gelrite-HPMC solution retained the drug better than the gellan gum alone or a combination of gellan gum-alginate-HPMC. The developed formulations were therapeutically efficacious and provide sustained release of the drug over a 12-h period  in vitro.  These results demonstrate that the Gelrite-HPMC Methocel F4M mixture can be used as an in-situ gelling vehicle to enhance ophthalmic bioavailability and patient compliance.

Opthalmic drug delivery systems, such as eye drops, ointments, and soft gel capsules, are typically used to treat diseases of the eye.  However, the eye’s protective mechanisms often reduce their therapeutic effect. When a drug solution is dropped into the eye, there is typically a 10-fold reduction in the drug concentration within 4-20 min, due to the effective tear drainage and blinking action (1). The cornea’s limited permeability contributes to the low absorption of ocular drugs. Due to tear drainage, most of the administered dose passes via the nasolacrimal duct into the gastrointestinal tract, leading to side effects (2). Rapid elimination of both the solutions and the suspended solid administered often results in blurred vision, poor patient acceptance, and short duration of the therapeutic effect, making more frequent dosing necessary (3)

New preparations have been developed to prolong the contact time on the ocular surface and slow down drug elimination (4, 5). Ocular inserts (5) and collagen shields (6)  can also be used, but they pose challenges.

These delivery challenges can be overcome by using in-situ gel-forming ophthalmic drug delivery systems prepared from polymers that exhibit reversible phase transitions (sol-gel-sol) and pseudoplastic behavior.  Such formulations minimize interference with blinking (7).

Changes to the gel phase (8) can increase pre-corneal residence time and enhance ocular bioavailability. Three types of systems have been used: pH-triggered systems including cellulose acetate hydrogen phthalate latex (9, 10) and carbopol (11-15); temperature-dependent systems including pluronics (7, 16-20), tetronics (21, 22), and polymethacrylates (23); and ion-activated systems including Gelrite (24-26), gellan (27-28), and sodium alginate (29).

The authors used an ion-activated in-situ gelling system to deliver Levofloxacin, a fourth-generation fluoroquinolone anti-infective agent, which can be used to treat conditions including acute and subacute conjunctivitis, bacterial keratitis, and keratoconjunctivitis. The goal was to demonstrate prolonged action and show antibacterial activity against gram-positive and gram-negative bacteria directly at the site of infection without loss of dosage. The combination of Gelrite (gellan gum) and hydroxypropyl methylcellulose (HPMC) (Methocel F4M) was used to prepare the gelling system, which was used with and without sodium alginate to prepare Levofloxacin eye drops (0.5% w/v). These drops would undergo gelation when instilled into the cul-de-sac of the eye, and provide controlled release of the drug in treatment of ocular infections.

Materials and methodsMaterials. Levofloxacin was obtained from Zydus Healthcare, Gelrite from CP Kelco, and HPMC (Methocel F4M) was provided by Colorcon Asia Pvt. Ltd. All other reagents, chemicals, and solvents were of analytical grade. 

Methods. Method of preparation. Gelrite-based in-situ gelling systems were prepared by dissolving gellan, alone and combined with sodium alginate and/or HPMC in hot phosphate buffer (pH 7.4, 70 °C), by continuous stirring at 40-45 °C for 24 h, as shown in Table I. Then the weighed quantities of levofloxacin (0.5% w/v), mannitol, and preservatives, such as methyl paraben and propyl paraben, were added to the solution and stirred until dissolved. The solutions were then transferred into previously sterilized amber-colored glass vials, capped, and sealed with aluminum caps. The formulations were sterilized by terminal autoclaving at 121 °C, 15 PSI for 20 min. The sterilized formulations were stored in a refrigerator at 4-8 °C until use.

Experimental design

A 3² full factorial approach was taken to design the gelling system. Two factors were selected, and a total of nine  experimental trials were performed using all possible combinations. The concentrations of Gelrite (cation-sensitive in-situ gelling polymer) as X₁ (0.3, 0.4, and 0.5%, m/V) and HPMC (viscosity imparting agent) as X₂ (0.3, 0.5, and 0.7%, m/V) were selected as independent variables. 

Gel strength (GS in s), bioadhesion force (BF in N), viscosity (VI) in Pa.s, and cumulative percent drug release after 10 h (CR10) were selected as dependent variables. The design is shown in Table II. Equation 1 summarizes the experimental design, using two independent variables and three levels (low, medium, and high) of each variable:

Y = b₀ + b₁X₁ + b₂X₂ + b₁₁X₁₁ + b₂₂X₂₂ + b₁₂X₁X₂ (Eq 1)

where Y is the dependent variable, b₀ is the mean response of the nine runs, and b_i is the estimated coefficient for factor X_i.

The main effects (X₁ and X₂) represent the average result of changing a factor at a time. The interaction term (X₁₂) shows how the response changes when the factors are simultaneously changed. Polynomial terms (X₁₁ and X₂₂) are included to investigate nonlinearity.

Statistical analysis and two-way analysis of variance (ANOVA) were used to evaluate the significance of each factor to the response at different levels. Three-dimensional response surface plots and two-dimensional contour plots of the data were generated using Design Expert software (Version 8).

Evaluation of formulation
The following were used to evaluate the formulation.

Gelation studies were carried out in a vial containing the gelation solution and simulated tear fluid (STF) solution, composed of 0.670 g of sodium chloride, 0.200 g of sodium bicarbonate, 0.008 g of calcium chloride dehydrate, and purified water, quantum satis to 100 g.

The preparation was carefully taken into the vial using a micropipette, and 2 mL of gelation solution (STF) was added slowly. Gelation was assessed by visual examination (26).

Rheological studies. Viscosities of sample solutions were measured in a Brookfield synchrolectric viscometer (LVDVI prime) at different angular velocities at a temperature of 37±1°C. The angular velocity was increased from 0.5 to 100 rpm with 6 s between two speeds. The sequence of the angular velocity was reversed. The average of two readings was used to calculate viscosity. Evaluations were conducted in triplicate (26).

Drug content uniformity. Vials containing the formulation were shaken for 2-3 min, and the preparation was transferred aseptically to sterile volumetric flasks. The final volume was made up with phosphate buffer pH 7.4. The concentration of Levofloxacin present was determined at 287 nm using UV spectrophotometry (26).

In-vitrodrug release studies. The studies were carried out using a Franz diffusion cell, with STF (pH 7.4) as dissolution medium. The cell consists of glass donor and receptor compartments, separated by a dialysis membrane. The optimized formulation was placed in the donor compartment, and freshly prepared STF was placed in the receptor compartment. The whole assembly was placed in a temperature-controlled shaker water bath maintained at 37 °C ± 0.5 °C. A sample (1 mL) was withdrawn at predetermined time intervals up to 24 h and the same volume of fresh medium was replaced. The withdrawn samples were analyzed by UV spectrophotometer at 287 nm. 

Bioadhesive strength measurement. Freshly excised goat conjuctival membrane was used to measure bioadhesive strength. The membrane was placed in an aerated saline solution at 4 °C until used. It was tied to the lower side of the hanging polytetrafluoroethylene (PTFE) cylinder using thread, and the cylinder was fixed beneath of left pan of a pan balance. The formulation was placed into a sterile petri plate that was kept on the platform beneath the left pan. The two sides of the pan balance were balanced by keeping a 2-g weight on the right pan

The 2-g weight was then removed, lowering the left pan and allowing the membrane to come in contact with the formulation. The membrane was kept in contact with the formulation for 5 min. Weight was slowly added to the right pan slowly, in increments of 0.5 g, until the formulation detached from the membrane surface. The excess weight on the right pan was taken as the measure of the bioadhesive strength. The force of adhesion was then calculated using the following formula (13).

Force of adhesion = Bioadhesive strength x 9.81 / 1000.

Infrared spectroscopy and DSC studies. Infrared (IR) spectroscopy and differential scanning calorimetry (DSC) were then used to analyze the pure drug, gellan, HPMC, physical mixture of drug-gellan-HPMC, and optimized formulation. Resulting spectra were then compared with reference spectra.

Antimicrobial efficacy studies. The solution’s antimicrobial efficacy was determined using agar diffusion and commercial Levofloxacin eyedrops as a control. The sterilized solutions were poured into cups bored into sterile agar nutrient seeded with test organisms (Pseudomonas aeruginosa and Staphylococcus aureus).

After allowing diffusion of the solutions for two hours, the plates were incubated at 37 ˚C for 24 h, and the zone of inhibition (ZOI) was measured around each cup and compared with control’s ZOI. The entire process, except for the incubation, was carried out under laminar flow units in an aseptic area (Class 10,000). Each solution was tested three times. Both positive and negative controls were maintained throughout the study (26).

In-vivo ocular irritation and stability studies. In-vivo ocular irritation studies were performed using the Draize technique (30) and guidelines set by the Organization for Economic Cooperation and Development (OECD) (31). Six albino rabbits, each weighing 2-3 kg, were used for this study. The sterile formulation was administered to the test rabbits twice a day for 21 days and the rabbits were observed periodically.

International Council for Harmonization (ICH) guidelines were used to determine the optimized formulation’s stability. The gel was stored in a stability chamber at ambient humidity between 2 °C to 8 °C, ambient temperature at 40 °C ±0.5 °C  for six months. The samples were withdrawn at regular intervals and analyzed. The logarithms of percent drug remaining were calculated and plotted against time in days. The degradation rate constant was calculated using the equation: slope = k/2.303, where k is a degradation rate constant. The shelf life of the developed formulation was calculated using the Arrhenius plot. 

Results and discussion
Composition of various batches of the prepared in-situ gelling formulations are shown in Table I. In the batch containing gellan alone, the concentration of gellan was kept at a maximum of 0.4% (w/v). Higher concentration beyond 0.4% caused gelation upon cooling to 40 °C.

In the combination batches, the concentration of gellan was varied and the concentration of sodium alginate was kept at approximately 0.25% (by compensating the concentration difference due to reduction in viscosity after autoclaving for sodium alginate) to give a maximum of 0.65% polymer concentration, because an increase beyond this concentration resulted in gelation during formulation.

To maintain the proper pseudoplastic behavior of formulation, HPMC was used with gellan alone and with combination of gellan and sodium alginate. The drug content and gelling capacity of the formulations were found to be satisfactory as mentioned in Table I, and the formulations were liquid at both room temperature and when refrigerated. Viscosity and gelling capacity (speed and extent of gelation) are the most important criteria for any in-situ gelling system.

All batches exhibited pseudoplastic behavior, as showed in Figure 1. All batches showed low viscosity at high shear rate and high viscosity at low shear rate. Autoclave process had not affected the viscosity of the formulations, except for those containing sodium alginate where viscosity was reduced around 8-15%. Therefore, the concentration of sodium alginate was adjusted to compensate (25). All measurements were taken three times and showed good reproducibility.

Figure 2 shows the cumulative percentage of Levofloxacin released versus time profiles for batches LV1 to LV8. These results suggested that Levofloxacin was sustainably released from formulation LV8, when the content of gellan gum was 0.4% and 0.5% of HPMC Methocel F4M (Table I). A similar release pattern is reported for pilocarpine (32) from alginate systems, wherein an inverse relationship between drug release and polymer concentration was observed. 

Experimental design 
Based on studies of response variables, the polynomial relationships are expressed in Equations 2 to 5.

GS (gel strength) = 115.33 + 7.33*X₁ + 7X₂ (Eq. 2)

BF (bioadhesion force) = 3069.44 + 989.83*X₁ + 314.33*X₂ + 116*X₁² (Eq. 3)

VI (viscosity) = 1771.11 + 220.66*X₁ + 390.66*X₂ + 57 X₁*X₂ (Eq. 4)

CR10 (cumulative percentage drug  release after 10 hs) = 90.51 - 9.03*X₁ - 6.1*X₂ (Eq. 5)

All the polynomial equations were found to be statistically significant (P < 0.05) and in good agreement with results. From Equation 2, it can be concluded that both gellan gum and HPMC significantly affect the gelling strength (25, 13).

Formulation batches LF1 and LF2 showed poor gelation strength, which might be due to the minimum amount of gellan and/or HPMC.  The results are shown in Table II.

The studies showed that, in the presence of HPMC, as the amount of gellan increased, gel strength increased as well; this effect must be due to the additional effect of concentration of polymer. Figure 3 (a) shows the response surface plot illustrating the effect of gellan gum and HPMC on the gelling strength. Studies confirmed that both polymers significantly affect the gelling strength.

From Equation 3, it can be concluded that both polymers have a predominant effect on bioadhesive force. The formulation contained gellan gum, which is a mucoadhesive agent. Studies show that polymers with charge can serve as good mucoadhesive agents. It has also been reported that polyanion polymers are more effective bioadhesives than polycations or nonionic polymers (33, 34). This polymer adheres to the mucin of the eye, which leads to prolonged retention of the formulation inside an eye (13).

Figure 3(b) depicts the response surface plot, showing the influence of both polymers on bioadhesion force. The studies confirmed that, as the concentration of gellan gum or HPMC is increased, bioadhesion also increases. 

Equation 4 shows that HPMC has a predominant effect on viscosity compared to gellan gum. Normally, water-soluble polymers such as HPMC produce two effects:

• Lowering surface tension and improving mixing with the precorneal tear film 

• Increasing viscosity and prolonging contact time, thereby resisting drainage of drug from eye (13).

Gellan gum can significantly increase viscosity of the formulation upon exposure to lachrymal fluid. So, by optimizing the concentration of HPMC viscosity-enhancing agent, one can decrease the amount of gellan gum in the preparation to improve patient compliance.

Figure 3(c) shows the response-surface plot of effect of gellan gum and HPMC on viscosity. From Equation 5, gellan gum and HPMC are inversely related to the amount of drug released.

The results of in-vitro release studies show that the formulations retain drug for the duration of the study (12 h). The movement of the eyelid and eyeball provide shearing action for faster dissolution of gels in the cul-de-sac. Figure 3(d) depicts the response-surface plot, showing the influence of both polymers on drug release after 10 h respectively. Checkpoint batches LF10 and LF11 were prepared (Table II) to validate the evolved model.  The actual values of GS, BF, VI, and CR10 of batches LF10 and LF11 are given in Table III.

Checkpoint batches were found in good agreement with the actual values. Results of ANOVA are shown in Table IV.

Release of an optimized batch fitted to a Higuchian matrix equation showed a high R-squared value (0.99), least SSR value, and F value {21} as compared to other batches. Thus, it can be concluded that release of drug was based on a Higuchian-matrix, diffusion-controlled mechanism. 

Bioadhesive strength and thermogram results
The bioadhesive strength measurement of designed batches is shown in Table II. Differential scanning calorimetry (DSC) thermograms showed characteristic peaks of Levofloxacin at 230.50 °C and 111.55 °C, gellan gum at 266.11°C, and HPMC at 288.55 °C and 79.79 °C (Figure 4).

The peak of Levofloxacin was found to be reduced in intensity in physical mixture of drug, gelling agent, and polymer (Figure 4e) and could not be seen in optimized formulations of DSC thermogram (Figure 4c), indicating the entrapment of drug in the in-situ matrix gel system of gellan gum and HPMC.

The optimized formulation (LF5) showed antimicrobial activity when tested microbiologically by the cup-plate technique. Clear ZOIs were obtained in the case of the optimized formulation and marketed eye drops.

The diameters of the ZOIs produced by the optimized formulation against both test organisms were either on par or higher than those produced by marketed eye drops as shown in Table V.

The antimicrobial effect of Levofloxacin gel formulation is probably due to its rapid initial release into the viscous solution and followed by formation of a drug reservoir that attributed to the slow and prolonged diffusion from the polymeric solution due to its higher viscosity (26).

Ocular irritation studies (35) indicated that the formulation is well tolerated by rabbit eyes (36). No ocular damage or abnormal clinical signs were observed (37).

The optimized formulation of Levofloxacin was kept for stability studies at refrigeration temperature (4 °C), ambient temperature (25 °C), and elevated temperature (40 °C) for a period of six months. Samples were withdrawn at regular time intervals and were evaluated for appearance, gelation studies, drug content, and in-vitro drug release. 

Stability studies
The formulation was found to be sterile at the end of six months. The drug degraded to a negligible extent and the degradation rate constant for optimized formulation was very low (1.12 x 10^-4). Because the overall degradation is <5%, a tentative shelf life of two years may be estimated the formulation (13). 

Conclusion 
An ion-activated in-situ gel formulation of Levofloxacin was successfully formulated using gellan gum in combination with HPMC. The formulation underwent gelation in the conjunctival sac (cul-de-sac), allowing for sustained drug release over a 12-h period without any adverse effect to the ocular tissues.

Stability data confirmed that the formulation is stable for a six-month period in given storage conditions. This new formulation can enhance bioavailability through its sustained drug release, higher viscosity, longer pre-corneal residence time, and better miscibility with the lacrimal fluid. These benefits promise to improve patient acceptance and compliance.

References
1. D.M. Maurice, “Kinetics of topically applied ophthalmic drugs,” in Ophthalmic Drug Delivery Biopharmaceutical, Technological and Clinical Aspects, M.F. Saettone, M. Bucci, and P. Speiser, Eds. (Fidia Research Series, Padova: Liviana Press, Springer, New York, 1987), pp. 19-26.
2. D.L. Middleton, S.S. Leung, and J.R. Robinson, “Ocular bioadhesive delivery systems,” in Bioadhesive Drug Delivery Systems, V. Lenaerts, and R. Gurny, Eds. (Boca Raton, FL: CRC Press, 1st ed., 1990), pp. 179-202.
3. O. Olejnik, “Conventional systems in ophthalmic drug delivery systems,” in Ophthalmic Drug Delivery Systems, A.K. Mitra, Ed. (Marcel Dekker, New York, 1993), pp. 179-193.
4. C.L. Boularis, et al., Prog. Retin. Eye Res. 17 (1), 33-58 (1998).
5. S. Ding, Pharm. Sci. Technol. Today, 8 (1), 328-335 (1998).
6. J.M. Hill et al, “Controlled collagen shields for ocular delivery,” in Ophthalmic Drug Delivery Systems, A.K. Mitra, Ed. (Marcel Dekker, New York 1993), pp. 261-275.
7. A.H. El-Kamel, Int. J. Pharm. 241 (1) 47-55 (2002).
8. O. Sechoy, et al., Int. J. Pharm. 207 (1-2) 109-116 (2000).
9. R. Gurny, Pharm. Acta. Helv. 56 (4-5) 130-132 (1981).
10. R. Gurny, T. Boye, and H. Ibrahim, J. Contr. Rel. (2), 353-361 (1985).
11. B. Srividya, et al, J. Contr. Rel. 73 (2-3), 205-211 (2001).
12. D. Aggarwal and I.P. Kaur, Int. J. Pharm. 290 (1-2) 155-159 (2005).
13. Y. Sultana et al., Pharm. Dev. Technol. 11 (3) 313-319 (2006).
14. C. Wu et al., Yakugaku Zasshi 127 (1) 183-191 (2007).
15. H.R. Lin and K.C. Sung, J. Contr. Rel. 69 (3) 379-388 (2000).
16. S.C. Miller and M.D. Donovan, Int. J. Pharm. (12), 147-152 (1982).
17. S.D. Desai and J. Blanchard, J. Pharm. Sci. 87 (2) 226-230 (1998).
18. K.Y. Cho et al.,  Int. J. Pharm., 260 (1), 83-91 (2003).
19. M.K. Yoo et al., Drug Dev. Ind. Pharm. 31 (4-5) 455-463 (2005).
20. H. Qi et al., Int. J. Pharm. 337 (1-2) 178-187 (2007).
21. M. Vadnere et al., Int. J. Pharm. 22 (2-3) 207-218 (1984).
22. C.W. Spancake et al, Int. J. Pharm. 75 (2-3) 231-239 (1989).
23. G.H. Hsiue et al., Biomaterials 24 (13), 2423-2430 (2003).
24. A. Rozier et al., Int. J. Pharm. 57 (2), 163-168 (1989).
25. J. Balasubramaniam et al, Acta Pharm. 53 (4) 251-261 (2003).
26. Balasubramaniam and J.K. Pandit, Drug Deliv. 10 (3) 185-191 (2003).
27.Y.D. Sanzgiri et al., J. Contr. Rel. 26 (3) 195-201 (1993).
28. J.N. Shah et al,  PharmaInfo.net, 5 (6) 2007 www.pharmainfo.net/reviews/gellan-gum-and-its-applications-%E2%80%93-review,  12 Sep. 2010.
29. Z. Liu et al., Int. J. Pharm. 315 (1-2) 12-17 (2006).
30. J.H. Draize et al,  J. Pharmacol. Exp. Ther. 82 (3) 377-390 (1944).
31. OECD Publication Office (Paris, Oct. 2012) Guidelines for testing the chemicals, Section 4, Test No. 405: Acute eye irritation/corrosion.
32. S. Cohen et al., J. Control. Rel. 44 (2-3) 201-208 (1997).
33. K. Park and J.R. Robinson, Int. J. Pharm. 19 (2) 107-127 (1984).
34. S. Wee and W.R. Gombotz, Adv. Drug Deliv. Rev. 31(3) 267-285 (1998).
35. J.F. Griffith et al., Toxic Appl. Pharmacol. 55 (3) 501-513 (1980).
36. H. Sasaki et al., J. Control. Rel. 27 (2) 127 -137 (1993).
37. Z. Liu et al., Drug Dev. Ind. Pharm. 31 (10) 969-975 (2005).