Biomacromolecules 2004, 5, 2358-2365
In Situ Gelling of Alginate/Pluronic Solutions for Ophthalmic Delivery of Pilocarpine Hong-Ru Lin,† K. C. Sung,*,‡ and Wen-Jong Vong§ Department of Chemical Engineering, Southern Taiwan University of Technology, Tainan 710, Taiwan, and Department of Pharmacy and Department of Applied Chemistry, Chia-Nan University of Pharmacy and Science, Tainan 717, Taiwan Received May 24, 2004; Revised Manuscript Received July 19, 2004
We prepared a series of alginate and Pluronic-based solutions as the in situ gelling vehicles for ophthalmic delivery of pilocarpine. The rheological properties, in vitro release as well as in vivo pharmacological response of polymer solutions, including alginate, Pluronic solution, and alginate/Pluronic solution, were evaluated. The optimum concentration of alginate solution for the in situ gel-forming delivery systems was 2% (w/w) and that for Pluronic solution was 14% (w/w). The mixture of 0.1% alginate and 14% Pluronic solutions showed a significant increase in gel strength in the physiological condition; this gel mixture was also found to be free flowing at pH 4.0 and 25 °C. Both in vitro release and in vivo pharmacological studies indicated that the alginate/Pluronic solution retained pilocarpine better than the alginate or Pluronic solutions alone. The results demonstrated that the alginate/Pluronic mixture can be used as an in situ gelling vehicle to increase ocular bioavailability. Introduction The conventional liquid ophthalmic formulation is eliminated from the precorneal area immediately upon instillation because of lacrimal secretion and nasolacrimal drainage.1-5 As a result, frequent instillation of concentrated solutions is needed to achieve the desired therapeutic effects.6 Various ophthalmic vehicles, such as inserts, ointments, suspensions, and aqueous gels, have been developed to lengthen the residence times of the instilled dose and increase its ophthalmic bioavailability.7 These ocular drug delivery systems, however, have not been used extensively because of some drawbacks, such as blurred vision with ointments or low patient compliance with inserts.8 An ideal ophthalmic formulation should be administrated in eyedrop form, without causing blurred vision or irritation. It should also provide site-specific delivery, continuous drug release, and increased bioavailability.9 Recent research efforts have focused intensively on systems in which drugs can be administered as an eyedrop. Significant advancements have been made in in situ forming gels, oil-in-water emulsions, colloidal drug delivery systems, and microparticulates.9 Major progress has been made in in situ gel-forming systems to prolong the precorneal residence time of a drug and increase ocular bioavailability. Polymers are employed in such delivery systems to carry various drugs, and they may demonstrate a transition from sol (liquid)-to-gel state once * To whom correspondence should be addressed. Tel: 886-6-2664911. Fax: 886-6-3313309. E-mail: [email protected]
† Southern Taiwan University of Technology. ‡ Department of Pharmacy, Chia-Nan University of Pharmacy and Science. § Department of Applied Chemistry, Chia-Nan University of Pharmacy and Science.
instilled in the cul-de-sac of the eye.10 Various mechanisms are involved in the phase transition of these polymers. The viscosity of cellulose acetophthalate (CAP) latex11 and Carbopol solution increases when the pH is raised from its native value to that of the eye environment (pH ) 7.4). Gelrite12 gels in the presence of mono- or divalent cations. The Pluronics,13 a class of block copolymers of poly(oxyethylene) and poly(oxypropylene), and tetronics,14 as well as ethyl(hydroxyethyl) cellulose, 15 exhibit thermoreversible gelation. That is, their solutions show an increase in viscosity upon an increase in temperature. However, most of the systems require the use of high concentrations of polymers. To form a stiff gel upon instillation in the eye, for instance, requires 25% (w/v) Pluronics and 30% (w/v) CAP, respectively. As the concentration of Carbopol increases in the vehicle, its acidic nature may stimulate the eye tissue. To reduce total polymer content and improve gelling properties, Joshi et al.16 first used a combination of polymers in the delivery system. They demonstrated that aqueous compositions that reversibly gel in response to simultaneous variations in at least two physical parameters (e.g., pH, temperature, and ionic strength) can be formed by using a combination of polymers that exhibit reversible gelation properties.16 On the basis of this finding, Kumar et al.10 developed an ocular drug delivery system using a combination of Carbopol and methylcellulose. Carbopol is a poly(acrylic acid) (PAA) polymer, which shows a sol-to-gel transition in aqueous solution as the pH is raised above its pKa of about 5.5.17 Methylcellulose, a viscosity-enhancing polymer,18 exhibits a sol-to-gel transition in aqueous solution in the range of 50-55 °C. The rheological properties of this system were investigated, and the sol-to-gel transition
10.1021/bm0496965 CCC: $27.50 © 2004 American Chemical Society Published on Web 09/03/2004
Gelling of Alginate/Pluronic Solutions
occurred primarily because of an increase in pH due to the presence of Carbopol; the temperature-mediated effect occurred only at very low shear rates.10 Kumar et al.19 also developed a similar delivery system using a combination of Carbopol and hydroxypropylmethylcellulose. For both systems, they found that, by adding a suitable viscosityenhancing polymer, the Carbopol concentration could be reduced without compromising the in situ gelling properties or the overall rheological behaviors.10,19 We developed another system by combining Carbopol and Pluronic solutions as an in situ gelling vehicle.20 Our in vitro release and in vivo pharmacological studies indicated that the Carbopol/ Pluronic solution retained drugs better than Carbopol or Pluronic solutions alone. The use of combined in situ gelling polymers with various phase transition mechanisms in ophthalmic drug delivery vehicles has not been extensively explored, however. In the present study, aqueous solutions of different compositions containing either alginate or Pluronic were prepared to identify compositions suitable for use as in situ gel-forming systems. Alginate is a natural polysaccharide extracted from brown sea algae. It belongs to a family of linear block polyanionic copolymers, composed of (1-4)-linked β-Dmannuronic acid (M units) and (1-4)-linked R-L-guluronic acid (G unit) residues.21 Alginate forms stable hydrogels in the presence of certain divalent cations (e.g., Ca2+, Sr2+, and Ba2+) at low concentrations through the ionic interaction between the cation and the carboxyl functional group of G units located on the polymer chain.22,23 According to one model,24 divalent cations bridge the negatively charged G units on the alginate polymer chain and form an egg-box structure. Alginate is highly hydrophilic, biocompatible, and relatively economical, and it is widely utilized in drug delivery.25-28 The in situ gelling alginate system, based on polymers with high G contents, appears to be an excellent drug carrier for the prolonged delivery of pilocarpine.29 The alginic-acid vehicle is an excellent drug carrier, welltolerated, and usable for the development of a long-acting ophthalmic formulation of carteolol,25 and the release behavior of alginate-chitosan beads containing diclofenac salt depends on pH value and the alginate-chitosan ratio.26 In this paper, we describe an alternative in situ gelling system prepared by the combination of alginate and Pluronic. The rheological behaviors of various aqueous polymer solutions under controlled shear conditions of varying magnitude were evaluated. In addition, the in vitro pilocarpine release and in vivo pilocarpine pharmacological response of various drug-containing polymer solutions were characterized to evaluate the use of in situ gelling polymer solutions for ophthalmic drug delivery. Materials and Methods Materials. Alginic acid (sodium salt, 61% mannuronic acid, and 39% guluronic acid, low viscosity), Pluronic F-127, and pilocarpine hydrochloride were used as received from Sigma-Aldrich Corp (St Louis, MO). All the other chemicals, including sodium chloride, sodium hydrogen carbonate, calcium chloride dihydrate, and sodium hydroxide pellets,
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were purchased from Merck & Co, Inc. (Whitehouse Station, NJ) and were used as received. Sample Preparation. The alginate solutions (0.5-3.0 (w/ w)) were prepared by dispersing the required amount in distilled, deionized water and then mixing completely using a homogenizer (Poly Tron; Kinematica, AG, Switzerland) at 26 000 rpm. To prepare the Pluronic solutions (12-16% (w/w)), the required amount of polymer was dispersed in distilled deionized water with continuous stirring for 1 h. The partially dissolved Pluronic solutions were stored in the refrigerator until the entire polymer was completely dissolved (approximately 24 h). The alginate/Pluronic solutions were prepared by dispersing the required amount of Pluronic in the desired concentration of alginate solution; the resulting combination was mixed with a homogenizer at 26 000 rpm for 3 min. The partially dissolved solutions were then refrigerated until thoroughly mixed (approximately 24 h). The reported composition of alginate/Pluronic mixture was the final concentration of alginate and Pluronic content in the mixture. All the previous sample solutions were adjusted to pH 4.0 ( 0.1 by 0.5 M hydrochloric acid solution and then stored in the refrigerator before evaluation of their properties under nonphysiological conditions (pH 4.0 and 25 °C).10,19-20 The same procedure was used to prepare the solutions for characterization under physiological conditions (pH 7.4 and 37 °C); the simulated tear fluid (STF, composition: NaCl 0.67 g, NaHCO3 0.20 g, CaCl2‚2H2O 0.008 g, and distilled deionized water to 100 g), however, was used as a dispersion medium instead. The prepared solutions were adjusted to pH 7.4 ( 0.1 by 0.5 M sodium hydroxide solution and then stored in the refrigerator before evaluation of their properties under physiological conditions. To prepare the pilocarpine-containing polymer solutions, the desired amounts of pilocarpine were added to the alginate, Pluronic, and alginate/Pluronic solutions with continuous stirring until thoroughly mixed. Determination of Flow Behavior of Vehicles. The flow behavior of vehicles was determined by various signs obtained by observation as well as by using the Ubbelohde viscometer. The flow behavior with the + sign indicates the vehicle is in the liquid form and is very easy to flow, and the vehicle can flow from one mark to the other mark in the Ubbelohde viscometer (NO. 3C-C342, Cannon Instrument) for less than 20 s (0-20 s). The flow behavior with the ++ sign indicates the vehicle is in the liquid-gel like form and flows less readily, and the vehicle can flow from one mark to the other mark in the Ubbelohde viscometer within 2050 s. The flow behavior with the +++ sign indicates the sample is in the gel form and is very difficult to flow, and the vehicle can flow from one mark to the other mark in the Ubbelohde viscometer within 50-100 s. The flow behavior with the ++++ sign indicates the vehicle is a strong gel and cannot flow, and it will take more than 100 s for the vehicle flow from one mark to the other mark in the Ubbelohde viscometer. Rheological Studies. The rheological studies were carried out on a cone (4°) and plate geometry viscometer (RVCP DV-III; Brookfield, Middleboro, MA). The viscosity and shear stress of the sample solutions were measured at various
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Lin et al. Table 1. Flow Behavior of Alginate Solution at Different Concentrationsa alginate concentration (% w/w)
25 °C, pH 4.0
37 °C, pH 7.4
0.5 1.0 1.5 2.0 2.5 3.0
+ + ++ +++ +++ +++
+ + ++ +++ +++ +++
a +Liquid, very easy to flow; ++liquid-gel like, flow less readily; +++gel, difficult to flow.
Figure 1. Diagram of dissolution apparatus used in the in vitro drug release studies.
shear rates at 25 and 37 °C. The temperature was maintained within (0.1 °C by a recirculating bath (HIPOINT, LC-06, Taiwan) connected to the sample cup of the viscometer. The samples were equilibrated on the plate for 5 min to reach the running temperature before each measurement. A typical run consisted of changing the shear rate from 0 to 200 s-1 at a controlled ramp speed, a 0.1 min wait at 200 s-1, and finally a decrease in shear rate to 0 s-1 at the same controlled ramp speed. All measurements were performed in triplicate. In Vitro Release Studies.20 The in vitro drug release from various polymer solutions was first carried out by putting 3 g of 1% pilocarpine-containing polymer solution into open circular plastic containers (1.5 cm in diameter and 3.8 cm in depth) in triplicate and sticking each container in bottom of a 1000 mL beaker (Figure 1). Care was taken to make sure that no air bubbles were inside the polymer solutions. The beaker was then filled with 1000 mL of simulated tear fluid (STF) and placed in a circulating water bath equipped with stirrer to stir the release medium. The temperature and stirring rate were maintained at 37 °C and 75 rpm. Aliquots (1 mL) were withdrawn from the release medium at each sampling time. The samples were filtered through 0.45 µm syringe filters and subjected to HPLC analysis to determine the pilocarpine concentrations. The HPLC chromatographic system consisted of a pump (Hitachi L-7100; Tokyo, Japan), an autosampler (Hitachi L-7200), a UV detector (Hitachi L-7420), and an integrator (Hitachi D-2500). A reverse phase silica column (Inertsil 5µ ODS-2, 4.6 × 150 mm; Vercopak Co., Taipei, Taiwan) was used for drug separation, and a methanol pH 3.0 phosphate buffer system (3:97) was used as the mobile phase. The flow rate and UV wavelength were 1.4 mL min-1 and 220 nm, respectively. The injection volume was 100 µL. The drug concentrations were determined by measuring peak areas and by comparing them with peak areas of known standards. In Vivo Studies. New Zealand albino rabbits were used as the model animals in the in vivo experiments. Rabbits of either sex, free of gross ocular defects and weighing 2.5-3 kg, were positioned into restraining boxes and placed in an isolated room with 50 W controlled lighting. The pupillary diameters, which were used to evaluate the pharmacological response of pilocarpine, were measured using a micrometer
held at a fixed distance from each rabbit. All rabbits were acclimatized to laboratory testing conditions for 30 min before the study was initiated. After the 30 min period, the left and right pupil diameters were alternatively measured four times within 30 min to establish baseline values for both eyes. For each pair of readings, the differences in pupil diameter (control minus test eye) were determined. These predosing differences were averaged, and the mean was used to convert postadministration data to the baseline-corrected values. This process minimized both animal and day variation. Fifty microliters of polymer solutions or simulated tear fluid (STF), each with 1% pilocarpine hydrochloride, were dosed from a micropipet. Various drug-containing polymer solutions were administered at room temperature and were placed in the lower conjunctival sac, approximately midway between inner and outer canthus. To avoid experimental bias, the left eye of each rabbit was first administered with the control vehicle (formulation with no drug), followed by the application of drug-containing vehicle (formulation with drug) to the right eye. After administration of both the control vehicle and the drug-containing polymer solutions, the pupil diameters of both eyes were measured according to the following time schedule: 1, 15, 30, 45, 60, 90, 120, 150, 180, 240, 300, and 360 min. For each time point, the difference in pupil diameter (control minus test eye) was calculated; the data were then converted to baseline-corrected value (i.e., the pharmacological response of pilocarpine) by subtracting the average baseline difference in pupil diameter for each experiment on the basis of the readings obtained before dosing. To assess the extent of total pharmacological response of the various formulations, areas under the decrease in pupil diameter (∆pupil diameter, after baseline correction) versus time profiles in 360 min (AUC0-360) were calculated using the trapezoidal rule. Results and Discussion Optimum Concentration of Vehicles. We prepared aqueous solutions containing either alginate or Pluronic to evaluate the compositions suitable for in situ gel-forming systems. An ideal in situ gelling delivery system should be a free-flowing liquid with low viscosity under nonphysiological conditions to allow reproducible administration into the eye as drops; it should also undergo in situ phase transition to form a strong gel capable of withstanding shear forces in the cul-de-sac and to sustain drug release under physiological conditions.19 Table 1 lists the flow behavior
Gelling of Alginate/Pluronic Solutions
Figure 2. Effects of pH value on the shear stress of 2% (w/w) alginate solution at 25 and 37 °C. All measurements were performed in triplicate.
of alginate solutions at various concentrations. While preparing samples, we observed that alginate solutions with concentrations equal to or less than 1% (w/w) had freeflowing properties under both physiological and nonphysiological conditions. Alginate solution with a concentration of 1.5% (w/w) formed a gel under both conditions, but the gel was not strong enough under physiological conditions to withstand the shear forces in the cul-de-sac. On the other hand, alginate concentrations equal to or greater than 2% (w/w) formed a gel even at pH 4.0 and 25 °C because of its high viscosity. Accordingly, although the 2% (w/w) alginate solution was difficult to flow under nonphysiological conditions, it formed a relatively strong gel under physiological condition; thus, it may be able to sustain the release of pilocarpine at pH 7.4 and 37 °C. To further verify this formulation, we investigated the effects of pH value and temperature on the flow behavior of 2% (w/w) alginate solution. Figure 2 shows the effects of pH value on the shear stress of 2% (w/w) alginate solution at 25 and 37 °C. The shear stress of alginate solution clearly decreased with increasing pH value, and the corresponding values were very small. For instance, the shear stress of 2% (w/w) alginate solution was only about 3 Pa (Pascal) at pH 7.4 and 37 °C. Such low shear stress is not expected to withstand the shear forces in the cul-de-sac. Furthermore, at 37 °C, when the pH value of the alginate solutions was greater than 7.4, the solutions became free-flowing liquids with very low viscosity, and their rheological behavior could not be detected with a rheometer. Figure 3 shows the effects of temperature on the flow behavior of 2% (w/w) alginate solution prepared using either distilled deionized water or STF. The shear stress decreased with increasing temperature for both types of alginate solution preparations. The shear stresses of alginate solution with STF are even smaller than those with distilled deionized water. This suggests the concentrations of cations (Na+ (0.14 M) and Ca2+ (0.0005 M)) in the STF are not high enough for 2% (w/w) alginate solution to form a strong gel. In fact, the extent of alginate gelation depends on the
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Figure 3. Effects of temperature on the flow behavior of 2% (w/w) alginate solution prepared either by distilled deionized water or STF. All measurements were performed in triplicate. Table 2. Flow Behavior of Pluronic Solution at Different Concentrationsa pluronic concentration (% w/w)
25 °C, pH 4.0
37 °C, pH 7.4
12 13 14 15 16
+ + + +++ +++
+ + ++++ ++++ ++++
a +Liquid, very easy to flow; ++liquid-gel like, flow less readily; difficult to flow; ++++strong gel, cannot flow.
percentage of guluronic acid (G units) residues in the polymer backbone. Alginates with G contents of more than 65% formed gels instantaneously upon their addition to simulated lacrimal fluid, while those with low G contents formed weak gels at a relatively slow rate.29 The G content of sodium alginate used in this study was only 39%; therefore, the alginate solution could not form strong gels under physiological conditions. In the present study, to improve the gel strength of the polymer solution, we used Pluronic as a viscosity enhancer in alginate/Pluronic solutions. In the preparation of Pluronic solutions, it was found that for Pluronic concentrations equal to or less than 13% (w/ w), the solution was in the sol (liquid) state under physiological and nonphysiological conditions. For Pluronic concentrations equal to or greater than 15% (w/w), the solution was a stiff gel under nonphysiological conditions. The flow behavior of Pluronic solution at various concentrations is listed in Table 2. These observations suggest that the optimum concentration for Pluronic solution used as an in situ gel-forming system is 14% (w/w). The 14% (w/w) Pluronic solution was mixed with various concentrations of alginate solution. Table 3 lists the flow behavior of these combination solutions. We found that when the concentration of alginate solution was greater than 0.7%, the solution formed a gel under nonphysiological conditions. The gel strength increased directly with the concentration of alginate solution; therefore, alginate/Pluronic solutions with an alginate content greater than 0.7% are not suitable as eyedrops in an in situ gelling system. As indicated in Table
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Table 3. Flow Behavior of Alginate/14% Pluronic Solution at Different Alginate Concentrationsa alginate/ pluronic concentration (% w/w) 0.1% alginate/14% pluronic 0.3% alginate/14% pluronic 0.5% alginate/14% pluronic 0.7% alginate/14% pluronic 0.9% alginate/14% pluronic 1.1% alginate/14% pluronic
25 °C, pH 4.0
37 °C, pH 7.4
+ + + ++ +++ +++
++++ ++++ ++++ ++++ ++++ ++++
a +Liquid, very easy to flow; ++liquid-gel like, flow less readily; +++gel, difficult to flow; ++++strong gel, cannot flow.
Figure 4. Shear stress vs shear rate flow curves of different aqueous polymer solutions. (]): 2% alginate solution measured at pH 4.0 and 25 °C; ([): 2% alginate solution measured at pH 7.4 and 37 °C; (0): 14% Pluronic solution measured at pH 4.0 and 25 °C; (9): 14% Pluronic solution measured at pH 7.4 and 37 °C; (4): 0.1% alginate/ 14% Pluronic solution measured at pH 4.0 and 25 °C; and (2): 0.1% alginate/14% Pluronic solution measured at pH 7.4 and 37 °C. All the measurements were performed in triplicate, and the standard deviations were all within 3%.
3, when the concentration of alginate solution falls between 0.1 and 0.5%, the alginate/Pluronic solutions are free-flowing liquids under nonphysiological conditions and form a strong gel under physiological conditions. These solutions are all ideal ophthalmic formulations. To reduce dose concentration and improve patient acceptability, 0.1% alginate/14% Pluronic solution was chosen as an optimum formulation for the in situ gelling system. In the present study, aqueous solutions of alginate (2.0% (w/w)) and Pluronic (14% (w/w)) as well as a mixture of alginate (0.1% (w/w))/Pluronic (14% (w/w)) were prepared to evaluate the rheological behaviors and in vitro and in vivo performances of these vehicles. Rheological Behaviors of Vehicles without Pilocarpine. The rheological behaviors of various polymer solutions were investigated as a function of temperature and pH. All measurements were performed in triplicate with good reproducibility, and the standard deviations were all within 3%. Figure 4 shows the shear stress versus shear rate flow curves of alginate solution (2.0% (w/ w)), Pluronic solution (14% (w/w)), and the mixture of alginate (0.1% (w/w))/ Pluronic (14% (w/w)) solution under nonphysiological and
physiological conditions. The shear stress of alginate solution under both conditions increased linearly with an increase in shear rate, demonstrating a Newtonian flow behavior.18 The rheological behavior of Pluronic solution and the combined solutions under nonphysiological conditions also demonstrated a Newtonian flow behavior. Pluronic solution under physiological conditions, however, resisted the initial rotary motion, and a sudden increase in the shear stress was observed at higher shear rates. The solution began to flow after the shear stress reached its yield point. Accordingly, the flow curve for Pluronic solution under physiological conditions demonstrated pseudoplastic behavior with hysteresis.18,19,30 Although the flow curve of alginate/Pluronic solution at pH 4.0 and 25 °C shows Newtonian flow behavior (Figure 4), pseudoplastic flow behavior with hysteresis was observed for alginate/Pluronic solution at pH 7.4 and 37 °C. For all the polymer systems except the alginate solution, the shear stresses at pH 7.4 and 37 °C were higher than those at pH 4.0 and 25 °C. For instance, at the shear rate of 200 s-1, the shear stresses of Pluronic and alginate/Pluronic solutions under physiological conditions were both approximately 10 times greater than those under nonphysiological conditions, suggesting the occurrence of phase transition between these two conditions for both systems. The increase in shear stress for Pluronic solution from nonphysiological to physiological conditions was mediated by temperature and can be explained by the structural characteristics of Pluronic. Pluronic is a class of block copolymers, consisting of poly(oxyethylene) (PEO) and poly(oxypropylene) (PPO) units, with the general formula poly(oxyethylene)x-poly(oxypropylene)y-poly(oxyethylene)x.31 PEO is predominantly hydrophilic, whereas PPO is hydrophilic at low temperatures and becomes more hydrophobic at higher temperatures. Once blocks of PEO and PPO are combined, one can expect amphiphilic characteristics and aggregation phenomena at higher temperatures.32 That is, when the polymer concentration and the characteristic temperature are above a critical point, this tri-block copolymer forms micelles.33,34 The formation of micelles may increase the viscosity of vehicles and thus lead to the sol-gel transition at a higher temperature.20, 35, 36 The shear stresses of alginate solution under physiological conditions shown in Figure 4 are slightly lower than those under nonphysiological conditions, which is consistent with the result indicated in Figure 3. To improve this limitation, a stronger gel can be formed by combining Pluronic with alginate solutions. Figure 4 shows that, at pH 4.0 and 25 °C, the shear stress of alginate/Pluronic solution was higher than that of Pluronic solution and slightly lower than that of alginate solution at each shear rate. At pH 7.4 and 37 °C, however, the shear stress of Carbopol/ Pluronic solution was significantly greater than that of individual alginate and Pluronic solutions at each shear rate. For instance, at a shear rate of 200 s-1, the shear stress of the alginate/Pluronic solution under physiological conditions was about 2 and 10 times greater than that of Pluronic and alginate solutions, respectively. This observation can be explained by the formation of cross-links between the two polymers; that is,
Gelling of Alginate/Pluronic Solutions
the water molecules may act as a cross-linking agent to form hydrogen bonds between the carboxyl groups of alginate and ether groups of Pluronic, which may lead to the formation of a three-dimensional network and stronger gel.37 Accordingly, under physiological conditions, the ionic repulsion between the negatively charged carboxyl groups may produce a more stretched alginate structure and thus form increased hydrogen bonds with the exposed PEO structure, which may subsequently result in a significantly increased shear stress response and the phase transition phenomenon.20 The previous results and inferences clearly indicate that the gel strength of the polymer solution under physiological conditions can be significantly increased by combining the two individual solutions. We also observed, while preparing samples, that the 0.1% alginate/14% Pluronic solution flowed freely under nonphysiological conditions. Thus, without increasing the concentration of individual polymer solution, this mixed vehicle may be administered into the eye as drops and form a stronger gel following the phase transition. These results suggest that the combined polymer solution may have more strength to withstand the low shear forces likely to be encountered in the cul-de-sac of the eye as well as prolong the residence times of the drug in the eye. All the flow curves shown in Figure 4 were investigated by varying the shear rate from 0 to 200 s-1 at a controlled ramp speed, a 0.1 min wait at 200 s-1, and finally a decrease in shear rate to 0 s-1 at the same controlled ramp speed. Except for the Pluronic and alginate/Pluronic solutions, both under physiological conditions, most vehicles showed Newtonian flow behavior with no hysteresis. The hysteresis phenomenon observed in these two flow curves at pH 7.4 and 37 °C may have been due to the structural changes in the gel following the exposure to shear forces.19 The strong gels formed with both solutions can be characterized as viscoelastic materials; viscoelastic material normally shows hysteresis under cyclic deformation.10,38,39 Figure 5 shows the shear stress versus time of alginate solution (2.0% (w/w)), Pluronic solution (14% (w/w)) as well as the mixture of alginate (0.1% (w/w))/Pluronic (14% (w/ w)) solution at pH 4.0 and 25 °C and at pH 7.4 and 37 °C. Because all the measurements were performed at a fixed shear rate of 50 rpm, the results shown in Figure 5 indicated clearly that the flow curves of all formulations except Pluronic solution were time independent. A formulation with a time-independent flow curve is essential for topical administration into the eyes of drugs that may experience the same magnitude of shear stress caused by blinking during treatment period. However, the shear stress of alginate solution in the eyes under physiological conditions is only about 3 Pa. Such low shear stress is not expected to withstand the shear forces in the cul-de-sac. On the other hand, the high shear stress of alginate/Pluronic solution (about 150 Pa) is well-suited to sustained drug delivery. Rheological Behaviors of Pilocarpine-Containing Vehicles. To determine the rheological behavior of polymer solutions containing pilocarpine hydrochloride, studies were conducted under physiological conditions (Figure 6). For the 2% alginate solution, the incorporation of pilocarpine did
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Figure 5. Shear stress vs time of different aqueous polymer solutions. (]): 2% alginate solution measured at pH 4.0 and 25 °C; ([): 2% alginate solution measured at pH 7.4 and 37 °C; (0): 14% Pluronic solution measured at pH 4.0 and 25 °C; (9): 14% Pluronic solution measured at pH 7.4 and 37 °C; (4): 0.1% alginate/14% Pluronic solution measured at pH 4.0 and 25 °C; and (2): 0.1% alginate/ 14% Pluronic solution measured at pH 7.4 and 37 °C. The 2% alginate solution at pH 4.0 and 25 °C curve overlapped with the 2% alginate solution at pH 7.4 and 37 °C curve. All the measurements were performed at a fixed shear rate of 50 rpm, in triplicate, and the standard deviations were all within 3%.
Figure 6. Effect of drug on the shear stress vs shear rate flow curves of different aqueous polymer solutions at pH 7.4 and 37 °C. (]): 2% alginate solution; ([): pilocarpine-containing 2% alginate solution; (0): 14% Pluronic solution; (9): pilocarpine-containing 14% Pluronic solution; (4): 0.1% alginate/14% Pluronic solution; and (2): pilocarpine-containing 0.1% alginate/14% Pluronic solution. The insert shows that the curves of 2% alginate solution and pilocarpinecontaining 2% alginate solution overlapped. All the measurements were performed in triplicate, and the standard deviations were all within 3%.
not change the Newtonian flow behaviors. Pilocarpinecontaining 14% Pluronic solution produced a pseudoplastic flow behavior with hysteresis similar to that of Pluronic solution. The shear stresses of the pilocarpine-containing Pluronic solution, however, were lower than those of Pluronic solution without pilocarpine. When pilocarpine was added
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Figure 7. Cumulative amount of pilocarpine released as a function of time from various pilocarpine-containing solutions. All the measurements were performed in triplicate, and the standard deviations were all within 3%.
to Pluronic solution under physiological conditions, the positively charged amine group in pilocarpine, which is hydrophilic, may have reacted with the hydrophilic PEO in Pluronic; these hydrophilic interactions may have disrupted the micelle structure stabilized by hydrogen bonds.20 In addition, pilocarpine may act as a plasticizer to mask the hydrophobic interactions between backbone chains of PPO in Pluronic through selectively solvating the polymer chains.40 For the pilocarpine-containing 0.1% alginate/14% Pluronic solution, the flow behavior was similar to that of 0.1% alginate/14% Pluronic solution, suggesting that the incorporation of pilocarpine did not disrupt the strong threedimensional gel network formed under physiological conditions. Nevertheless, the shear stress of the pilocarpinecontaining solution at each shear rate was higher than that of the solution without pilocarpine. The reason for this positive effect is unclear and needs further study. In Vitro Release Studies. Figure 7 shows the cumulative amount of pilocarpine released versus time profiles for various polymer solutions and STF, all of which contained 1% (w/w) pilocarpine hydrochloride. Almost all the pilocarpine in the STF released immediately after the start of the experiment. The pilocarpine in 2% alginate solution released about 77% to the medium after 15 min, and after 90 min, almost all had been released. The release rate of pilocarpine in the 14% Pluronic solution was much slower than in the 2% alginate solution because of the difference in gel strength. Only about 21% released to the medium after 15 min, 53% after 60 min, and almost 100% after 4 h. The release rate in 0.1% alginate/14% Pluronic solution was significantly lower. Only about 12% released after 15 min, 34% after 60 min, 74% after 4 h, and about 90% after 6 h. The results indicate that the 0.1% alginate/14% Pluronic mixture was best able to retain pilocarpine, and they suggest that an alginate/Pluronic aqueous system can be used as an in situ gel-forming system for ophthalmic drug delivery. Furthermore, by plotting the logarithm of release percentage versus logarithm of time curve for the alginate/Pluronic formulation (up to 60% of total pilocarpine released), a linear
Lin et al.
Figure 8. Decrease in pupil diameter vs time profiles for various pilocarpine-containing solutions. All the measurements were performed in triplicate.
relationship with slope of 0.69 and correlation coefficients of 0.99 can be obtained. This observation was also reported in other systems;41 the linear relationship with slope of 0.69 indicates that the in vitro drug release from the polymer vehicle under physiological condition exhibited anomalous release kinetics.41 The results suggest that both drug diffusion as well as polymer swelling were important processes during drug release. In Vivo Studies. Figure 8 shows the pharmacological response (the decrease in pupil diameter, ∆pupil diameter) versus time profiles for the various polymer solutions and STF containing pilocarpine. Because some in vivo pharmacological response variations were observed, the following results and discussion focus on reporting the general trends of experimental data. In the first minute, the pharmacological responses of STF and alginate solutions were higher than those of Pluronic and alginate/Pluronic solutions due to their weak gel strength and fast release rate of pilocarpine. However, after 15 min, the pharmacological responses of Pluronic and the alginate/ Pluronic solutions were higher than those of STF and alginate solutions due to their sustained drug release. After 30 min, the decrease in pupil diameter of STF was the smallest, with Pluronic and alginate/Pluronic solutions showing almost the same pharmacological response. After 90 min, almost no pharmacological response was observed in STF or alginate solutions. The pharmacological response of the Pluronic solution was similar to that of alginate/Pluronic solution; the decrease in pupil diameter, however, was lower for the Pluronic solution, between 45 and 300 min. The overall miotic response of alginate/Pluronic solution was the greatest. Table 4 lists the area under the ∆pupil diameter versus time profiles in 360 min (AUC0-360) for various polymer as well as STF formulations. The results indicate that a 4.38fold increase (p < 0.05, t-test) in total miotic response was obtained for the alginate/Pluronic solution relative to the STF. Less pronounced increases in total pharmacological response were observed for the alginate (1.36-fold, p > 0.05, t-test) and the Pluronic (2.85-fold, p < 0.05, t-test) solutions as compared to the STF. These in vivo results, along with the
Biomacromolecules, Vol. 5, No. 6, 2004 2365
Gelling of Alginate/Pluronic Solutions Table 4. Area under the ∆Pupil Diameters versus Time Profiles in 360 min (AUC0-360) for Various Formulationsa vehicles
alginate pluronic alginate/ pluronic STF
160.8 (38.1) 337.2 (79.1)b 518.3 (93.8)b 118.4 (41.0)
1.36 2.85 4.38
a All measurements were performed in triplicate. The numbers in parentheses are standard errors of the mean. b Significant different comparing to STF-based vehicle (p < 0.05).
rheogram and in vitro pilocarpine-release studies, demonstrate that the alginate/Pluronic solution may significantly prolong drug contact time and thus increase its pharmacological response. The in vitro and in vivo results in the present study all support the hypothesis that the combined alginate/Pluronic solution is a promising in situ gelling vehicle for ophthalmic drug delivery. Conclusions In this study, we found that the optimum concentrations of alginate and Pluronic solutions for use as an in situ gelling vehicle for ophthalmic drug delivery were 2% (w/w) and 14% (w/w), respectively. When 0.1% alginate and 14% Pluronic solutions were combined, the gel strength under physiological conditions was significantly increased, and this combined solution could flow freely under nonphysiological conditions. Both in vitro and in vivo results indicated that the combined polymer systems performed better in retaining pilocarpine than the individual solutions. This combined solution may be reproducibly administered into the eye as drops and form a strong gel following the phase transition to withstand the shear force in the cul-de-sac. References and Notes (1) Patton, T. F.; Robinson, J. R. J. Pharm. Sci. 1976, 65, 1295-1301. (2) Makoid, M. C.; Sieg, J. W.; Robinson, J. R. J. Pharm. Sci. 1976, 65, 150-152. (3) Patton, T. F.; Robinson, J. R. J. Pharm. Sci. 1975, 64, 267-271. (4) Sieg, J. W.; Robinson, J. R. J. Pharm. Sci. 1975, 64, 931-936. (5) Sieg, J. W.; Robinson, J. R. J. Pharm. Sci. 1977, 66, 1223-1228. (6) Chein, Y. W.; Cabana, B. E.; Mares, S. E. Drugs and The Pharmaceutical Sciences; Chein, Y. W., Ed.; Marcel Dekker: New York, 1982; Vol. 14, pp 13-50. (7) Lee, V. H. L.; Robinson, J. R. J. Ocular Pharmacol. 1986, 2, 67108. (8) Lee, V. H. L. J. Ocular Pharmacol. 1990, 6, 157-164. (9) Ding, S. Pharm. Sci. Technol. Today 1998, 1, 328-335. (10) Kumar, S.; Haglund, B. O.; Himmelstein, K. J. J. Ocular Pharmacol. 1994, 10, 47-56.
(11) Gurny, R.; Boye, T.; Ibrahim, H. J. Contr. Release 1985, 2, 353361. (12) Rozier, A.; Mazuel, C.; Grove, J.; Plazonnet, B. Int. J. Pharm. 1989, 57, 163-168. (13) Miller, S. C.; Donovan, M. D. Int. J. Pharm. 1982, 12, 147-152. (14) Haslam, J. L.; Higuchi, T.; Mlodozeniec, A. R. U.S. Patent 4 474 752, 1984. (15) Lindell, K.; Engstro¨m, S. Int. J. Pharm. 1993, 95, 219-228. (16) Joshi, A.; Ding, S.; Himmelstein, K. J. U.S. Patent 5 252 318, 1993. (17) Davies, N. M.; Farr, S. J.; Hadgraft, J.; Kellaway, I. W. Pharm. Res. 1991, 8, 1039-1043. (18) Patton, T. F.; Robinson, J. R. J. Pharm. Sci. 1975, 64, 1312-1316. (19) Kumar, S.; Himmelstein, K. J. J. Pharm. Sci. 1995, 84, 344-348. (20) Lin, H. R.; Sung, K. C. J. Control. Release 2000, 69, 379-388. (21) Sutherland, I. W. Biomaterials: NoVel Materials from Biological Sources; Byron, D., Ed.; Stockton Press: New York, 1991; pp 309331. (22) Wang, Z.; Zhang, Q.; Konno, M.; Saito, S. Biopolymers 1993, 33, 703-711. (23) Honghe, Z. Carbohydr. Res. 1997, 302, 97-101. (24) Grant, G. T.; Morris, E. R.; Rees, D. A.; Smith, P. J.; Thom, D. FEBS Lett. 1973, 32, 195-198. (25) Sechoy, O.; Tissie, G.; Sebastian, C.; Maurin, F.; Driot, J. Y.; Trinquand, C. Int. J. Pharm. 2000, 207, 109-116. (26) Ferna´ndez-Herva´s, M. J.; Holgado, M. A.; Fini, A.; Fell, J. T. Int. J. Pharm. 1998, 163, 23-34. (27) Vandenberg, G. W.; Drolet, C.; Scott, S. L.; de la Noue, J. J. Control. Release 2001, 77, 297-307. (28) Mi, F. L.; Sung, H. W.; Shyu, S. S. Carbohydr. Polym. 2002, 48, 61-72. (29) Cohen, S.; Lobel, E.; Trevgoda, A.; Peled, Y. J. Control. Release 1997, 44, 379-388. (30) Schoenwald, R. D.; Ward, R. L.; DeSantis, L. M.; Roehrs, R. E. J. Pharm. Sci. 1978, 67, 1280-1283. (31) Vadnere, M.; Amidon, G.; Lindenbaum, S.; Haslam, J. L. Int. J. Pharm. 1984, 22, 207-218. (32) te Nijenhuis, K. ThermoreVersible Networks: Viscoelastic Properties and Structure of Gels; Advances in Polymer Science 130; Springer: New York, 1997. (33) Zhou, Z.; Chu, B. Macromolecules 1987, 20, 3089-3091. (34) Zhou, Z.; Chu, B. Macromolecules, 1988, 21, 2548-2554. (35) Glatter, O.; Scherf, G.; Schille´n, K.; Brown, W. Macromolecules 1994, 27, 6046-6054. (36) Hvidt, S.; Jørgensen, E. B.; Brown, W.; Schille´n, K. J. Phys. Chem. 1994, 98, 12320-12328. (37) Kulicke, W.-M.; Nottelmann, H. Polymers in Aqueous Media: Performance Through Association; Glass, J. E., Ed.; Advances in Chemistry Series 223; American Chemical Society: Washington, DC, 1989; pp 15-44. (38) Tschoegl, N. W. The Phenomenological Theory of Linear Viscoelastic BehaVior: An Introduction; Springer-Verlag: New York, 1989; pp 471-472. (39) Ferry, J. D. Viscoelastic Properties of Polymers; Wiley: New York, 1980. (40) Sears, J. K.; Darby, J. R. The Technology of Plasticizers; John Wiley & Sons: New York, 1982; pp 39-40. (41) Korsmeyer, R. W.; Gurny, R.; Doelker, E.; Buri, P.; Peppas, N. A. Int. J. Pharm. 1983, 15, 25-35.