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In situ-gelling gellan formulations as vehicles for oral drug delivery
Journal of Controlled Release 60 (1999) 287–295
In situ-gelling gellan formulations as vehicles for oral drug delivery Shozo Miyazaki a , Hirotatsu Aoyama a , Naoko Kawasaki a , Wataru Kubo a , b, David Attwood * a
Faculty of Pharmaceutical Sciences, Health Science University of Hokkaido, Ishikari-Tohbetsu, Hokkaido 061 -0293, Japan b School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9 PL, UK Received 1 November 1998; received in revised form 11 March 1999; accepted 12 March 1999
Abstract Gels formed in situ following oral administration of 1% (w / v) aqueous solutions of gellan to rats and rabbits were evaluated as sustained-release vehicles. The formulation contained calcium ions in complexed form, the release of which in the acidic environment of the stomach caused gelation of the gellan gum. The in vitro release of theophylline from the rigid gellan gels followed root-time kinetics over a period of 6 h. Plasma levels of theophylline after oral administration of gellan solutions and a commercial oral sustained-release liquid dosage form containing an identical drug concentration were compared in both rats and rabbits. Bioavailability of theophylline from gellan gels formed by in situ gelation in the animal stomach was increased by four–fivefold in rats and threefold in rabbits compared with that from the commercial oral formulation. There was no significant difference in the mean residence times of theophylline when administered by these two vehicles. 1999 Elsevier Science B.V. All rights reserved. Keywords: Gellan gels; In situ gelation; Sustained release; Theophylline; Oral drug delivery
1. Introduction The use of natural polymers, such as chitosan [1–3], sodium alginate [4,5], hyaluronic acid esters [6] and xyloglucan [7], as vehicles for the sustained / controlled release of bioactive materials has received much attention recently. In this study, we report an assessment of the potential of the naturally occurring polysaccharide gellan as a vehicle for oral sustained delivery. Gellan gum (commercially available as *Corresponding author. Tel.: 144-161-275-2328; fax: 144161-275-2396. E-mail address: [email protected] (D. Attwood)
GelriteE or KelcogelE) is an anionic deacetylated exocellular polysaccharide secreted by Pseudomonas elodea, with a tetrasaccharide repeating unit of one a-L-rhamnose, one b-D-glucuronic acid and two b-Dglucose residues. It has the characteristic property of temperature-dependent and cation-induced gelation [8]. This gelation involves the formation of double helical junction zones followed by aggregation of the double-helical segments to form a three-dimensional network by complexation with cations and hydrogen bonding with water [9–11]. Much of the interest in the pharmaceutical application of this material has concentrated on its application for ophthalmic drug delivery [12–14]; aqueous solutions of gellan
0168-3659 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 99 )00084-X
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dropped into the eye undergo transition to the gel state due to the temperature and ionic conditions in the tear fluid. Drug release from these in-situ gels is prolonged due to longer precorneal contact times of the viscous gels compared with conventional eye drops. The use of gellan in the formulation of sustained delivery beads has recently been reported [15]. The present study is an examination of the possibility of exploiting the in-situ gelling characteristics of gellan for oral drug delivery. The formulation adopted was a gellan solution containing calcium chloride (as a source of Ca 11 ions), and sodium citrate, which complexes the free Ca 11 ions and releases them only in the highly acidic environment of the stomach. In this way, the formulation remains in liquid form until it reaches the stomach, when gelation is instantaneous. This paper reports in vitro and in vivo measurements of the release of a model drug, theophylline, and a comparison of the in vivo release characteristics with those from a commercial oral sustained release formulation.
2. Materials and methods
2.1. Materials Deacetylated gellan gum, KelcogelE, was supplied by Dainippon Pharmaceutical Co., Osaka, Japan, and was used as received. Theophylline was obtained from Wako Pure Chemical Ind. Ltd., Osaka, Japan. A commercially available product, TheoDurE syrup (20 mg ml 21 ), from Mitsubishi Kasei Co., Tokyo, Japan, was also studied. All other reagents were of analytical grade.
2.3. Measurement of gel strength Measurements were carried out using a rheometer (CR-200D, Sun Scientific Co.,Tokyo, Japan) by the method described previously [7,16]. Gels of gellan concentrations 0.25, 0.50 and 1.00% (w / v) were prepared by dissolving the weighed quantity of powder in 50 ml of water, heating to 908C, adding 0.016% (w / v) calcium chloride, and allowing the solution to cool to form a gel in a sample tube thermostatted at 208C. This tube was raised at a rate of 60 mm min 21 , pushing a probe slowly through the gel. The changes in the load on the probe were measured as a function of the depth of immersion of the probe below the gel surface.
2.4. Measurement of in vitro drug release The release rates of theophylline were measured by using plastic dialysis cells similar to those described previously [17]. The capacity of each halfcell was 4 ml and the surface area of the membranes was 2.67 cm 2 . The gellan gum solution, prepared in ultrapure water and loaded with a known weight of drug, was placed in the donor compartment and an equal volume of simulated gastric (pH 1.2) or intestinal (pH 6.8) fluid (as specified for the JP XIII disintegration test) was placed in the receptor compartment. The donor gel phase and the aqueous receptor phase were separated by a cellulose membrane (Viskase Sales Co., size 36 / 32). The assembled cell was shaken horizontally at a rate of 60 strokes min 21 in an incubator. The total volume of the receptor solution was removed at intervals and replaced by fresh release medium. The drug concentration of the sample was determined spectrophotometrically at a wavelength of 274 nm. All experiments were carried out in triplicate.
2.5. Animal experiments 2.2. Preparation of sols Gellan gum solutions were prepared by adding the gum to ultrapure water containing 0.05–0.17% (w / v) sodium citrate and heating to 908C while stirring. After cooling to below 408C, appropriate amounts of calcium chloride and theophylline were then dissolved in the resulting solution.
2.5.1. Rats Male Wistar rats, weighing ca. 250 g, were fasted for 24 h but with free access to water. The rats were anaesthetised with an i.p. injection of urethane and divided into three groups of four rats. One group received theophylline gel preparation given orally as 1 ml of gellan solution containing the drug (10 mg)
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through a stomach sonde needle for rats (Natume Seisakusho, KN-348). A second group was dosed in a similar manner with a commercial sustained release liquid dosage form, Theo-DurE syrup (10 mg in 0.5 ml). Theophylline solution (10 mg in 1 ml of saline) was administered intravenously to the third group of rats. At predetermined intervals, a blood sample was taken from the jugular vein of rats in each group.
2.5.2. Rabbits White male rabbits weighing 3.4–3.8 kg were fasted for two days prior to the experiments but were allowed free access to water. Gels containing theophylline were produced in situ by oral administration of 4 ml of the gellan solution containing 40 mg of drug using a stomach sonde for rabbits (Natume Seisakusho, KN-342). At given intervals, 1 ml blood samples were taken from the ear vein and analysed as described below. For intravenous administration, 40 mg doses of the drug in 4 ml of saline solution were injected through the ear vein. For oral administration of commercial Theo-DurE syrup, a dose of 40 mg in 2 ml was administered by the stomach tube. 2.6. Determination of theophylline The plasma samples were separated by centrifugation and assayed for theophylline by HPLC (Shimazu LC-10A with a Shimazu SPD-10A detector at a wavelength of 273 nm) using the method described by Schreiber-Deturmeny and Bruguerolle [18], with minor modifications. To 0.05 ml of plasma was
289
added 50 ml of caffeine solution (15 mg ml 21 ) as an internal standard and 20 ml of 20% perchloric acid, and the sample was vortex-mixed and centrifuged. The supernatant was passed through a Millipore filter (0.45 mm) and directly injected onto a 250346 mm I.D. column, packed with Inertsil-ODS. Elution was carried out with acetonitrile–tetrahydrofuran–concentrated acetic acid–distilled water (100:20:5:875, v / v) at a rate of 1.0 ml min 21 at 408C.
3. Results and discussion
3.1. Gelling properties Fig. 1 shows the rheological properties of gels of gellan concentrations 0.25, 0.50 and 1.00% (w / v), formed by dissolving the weighed quantity of powder in water, heating to 908C, adding 0.016% (w / v) calcium chloride, and allowing the solution to cool to 208C in the rheometer vessel. It was not possible to measure rheological properties of gels at pH 1.2 because of the low solubility of gellan at this pH. Values of gel strength (in kN m 22 ), calculated from Fig. 1, were 6.1860.20, 8.1360.47 and 17.6660.59 for gel concentrations of 0.25, 0.50 and 1.00% (w / v), respectively (each value representing the mean of four determinations). The values determined by this method are relative rather than absolute values but, nevertheless, they serve to show the influence of concentration on gel strength. The observed increase in gel strength with concentration has been noted previously for gellan [19,20] and is a consequence of
Fig. 1. Rheological properties of gellan gels of concentrations (a) 1.0, (b) 0.5 and (c) 0.25% (w / v) in water at 208C.
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increasing chain interaction with increasing polymer concentration. It is interesting to compare the rheological properties of the gellan gels with those of another natural product, enzyme-degraded xyloglucan, which were measured using the same apparatus and under similar conditions. The gel strength of the 0.5% (w / v) gellan gel was very similar to that of a 2% (w / w) xyloglucan gel (8.21 kN m 22 ) [7], indicating superior gelation characteristics of the gellan gum. The rheological behaviour shown in Fig. 1 is typical of that of elastic gels, with the abrupt decrease in stress after the maximum indicating a brittle system. In a comparison of the gelation properties of deacetylated gellan with those of other gums, Moorhouse et al. [19] noted similar characteristics (minimum gelling concentration, melting and setting temperatures) of the calcium gellan gels and those of agar, but a higher gel strength of the gellan gels.
3.2. In vitro release of theophylline In vitro release characteristics were determined at pH 1.2, pH 6.8 and at a combination of these two pH values in which the release medium was changed from a solution at pH 1.2 to one at pH 6.8 after 2.5 h, to simulate passage through the gastrointestinal (GI) tract. In each case, gellan sols were prepared in water at concentrations of 0.25, 0.50 and 1.00% (w / v) and separated from the release medium at the selected pH by a cellulose membrane, as described in Section 2.4. The initial theophylline concentration in all systems was 1% (w / v). Gelation of the sol in the donor compartment is a result of hydrogen bonding of the chains [9] by the H 1 ions that diffuse through the membrane separating the two compartments. Although the pH of the sol is reduced as a result of the influx of H 1 ions, there is clearly sufficient ionisation of the carboxylic groups of gellan for gelation to occur by this process. This process is rapid, with complete gelation of the donor solution occurring before the time of the first measurement (30 min after initial contact of the two solutions). Diffusion may thus be considered to be effectively from a gel block into the donor solution. It was not feasible to conduct a comparison of the release characteristics of the gellan preparations with those of the equivalent commercial product, Theo-DurE
syrup, because of osmotic effects in the apparatus used. A comparison of the in vivo release characteristics of the two preparations is presented below. Fig. 2 shows no significant influence of the gellan concentration on the release profile at pH 1.2, compared with the progressive decrease in the amount released with an increase of the gellan concentration seen at pH 6.8. The reason for this difference in release behaviour is attributable to the large difference in the H 1 ion concentrations of the two receptor solutions. The H 1 ion concentration at pH 6.8 is insufficiently high to cause the formation of rigid gels and the decreasing release rates observed in this system as the gellan concentration is increased reflect increasing viscosity of the soft gels formed. In contrast, rigid gels are formed when the donor solutions of all systems are placed in contact with a receptor solution at pH 1.2 and, as a consequence, the amount released is lower than that at pH 6.8 and independent of the gellan concentration. Fig. 3 shows no significant influence of gellan concentration on release when this was initially (2.5 h) into a receptor solution of pH 1.2 followed by a period of release into a receptor solution of higher pH. This is an expected consequence of rigid gel formation
Fig. 2. Cumulative release of theophylline as a function of time from gellan gels with an initial loading of 1% (w / v) drug and gellan concentrations of s 0.25, ^ 0.50 and h 1.0% (w / v) at pH 1.2 (filled symbols) and pH 6.8 (open symbols). Each value represents the mean6S.E. of three determinations.
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Fig. 3. Cumulative release of theophylline as a function of time from gellan gels with an initial loading of 1% (w / v) drug and gellan concentrations of s 0.25, ^ 0.50 and h 1.0% (w / v) at pH 1.2 for a period of 2.5 h and subsequently at pH 6.8. Each value represents the mean6S.E. of three determinations.
Fig. 4. Cumulative release of theophylline as a function of square root of time from gellan gels with an initial loading of 1% (w / v) drug and gellan concentrations of s 0.25, ^ 0.50 and h 1.0% (w / v) at pH 1.2 (filled symbols) and pH 6.8 (open symbols). Each value represents the mean6S.E. of three determinations.
during the initial release period at high H 1 ion concentration. Release data were analysed according to the treatment proposed by Higuchi [21] for drug release from semisolid vehicles containing dissolved drug. For the initial 50–60% release, the cumulative amount, Q, of drug released per unit surface area is proportional to the square root of time, t:
MMC released from a 1% (w / w) xyloglucan gel loaded with 0.025% drug, determined using the same equipment [22]. These two drugs have similar water solubilities and will partition predominantly in the water channels between the aggregated chains of the gel network. Comparison with the release characteristics of gels of the oxyethylene–oxypropylene– oxyethylene triblock copolymer Pluronic F127 [D of MMC53.61310 26 cm 2 s 21 from a 25% (w / w) Pluronic F127 gel [22]] shows the very much lower concentration of both xyloglucan and gellan gels required to achieve similar release characteristics of gels of the more widely studied polymer Pluronic F127. For the oral formulation, it was considered necessary to include a source of Ca 11 ions [0.016% (w / v) CaCl 2 ], the release of which in the stomach would ensure gelation of the gellan solution. The method by which gelation of the formulation before administration was avoided was through the inclusion of sodium citrate, which complexes the free Ca 11 ions and releases them only in the acidic environment of the stomach. The concentration of added sodium citrate was critical for the correct functioning of the formulation. Sodium citrate concentrations of
Q 5 2 C0 (Dt / p)1 / 2
(1)
Plots of Q vs. t 1 / 2 for the systems of Fig. 2 were linear after a short lag period (Fig. 4), indicating that drug release was controlled by diffusion of drug through the gel matrix. Diffusion coefficients calculated from the gradients of these plots were 8.48, 8.67 and 6.44310 26 cm 2 s 21 for gels with gellan concentrations of 0.25, 0.5 and 1.0% (w / v), respectively, at pH 1.2, and 3.07, 2.32 and 1.56310 25 cm 2 s 21 for gels with gellan concentrations of 0.25, 0.5 and 1.0% (w / v), respectively, at pH 6.8. The diffusion coefficients of theophylline from gellan gels at pH 1.2 are of a similar order of magnitude as those for the release of mitomycin c (MMC) from xyloglucan gels. For example, D57.28310 26 cm 2 s 21 for
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0.175% (w / v) and higher caused gelation of the formulation at room temperature, presumably due to the high Na 1 ion concentration (although not as effective as the divalent Ca 11 ions, Na 1 ions are able to cause gelation of this material at sufficiently high concentration [19,20]). The influence of sodium citrate concentration on the release properties was investigated at concentrations of 0.05, 0.10 and 0.17% (w / v). Fig. 5 shows no significant differences in release profiles over this concentration range, and the highest concentration [0.17% (w / v)] was selected for inclusion in the gellan formulation. The lack of influence of Ca 11 ions on the in vitro release characteristics shown in Fig. 5 is explicable by considering the gelation process. In formulations containing CaCl 2 , the sodium citrate complexes free Ca 11 ions, the release of which in acidic conditions causes gelation. However, in sols not containing Ca 11 ions, the sodium citrate will be in a predominantly ionised form and gelation is a consequence of the free Na 1 ions present in this solution, the relatively high concentration of which [0.17% (w / v) compared to 0.016% (w / v) Ca 11 ] is clearly
Fig. 5. Cumulative release of theophylline as a function of time from 1% (w / v) gellan gels with an initial loading of 1% (w / v) drug and containing sodium citrate at concentrations of s 0.05, ^ 0.10 and h 0.17% (w / v) but with no added calcium chloride, and j 0.17% (w / v) sodium citrate with 0.016% (w / v) added calcium chloride. Each value represents the mean6S.E. of three determinations.
sufficient to produce gels of comparable release characteristics to those produced by Ca 11 ions.
3.3. In vivo release of theophylline Plasma drug levels following oral administration to rats of 10 mg of theophylline from solutions of gellan are compared, in Fig. 6, with those following administration of an identical quantity of theophylline by i.v. injection of an aqueous solution, and from a commercial sustained release liquid dosage form (Theo-DurE syrup). It is clear from Fig. 6 that a sustained release of drug is achieved from the gellan preparations over a period of at least 6 h and this is at a significantly higher level (approx. four– fivefold) than that from the commercial product. Analysis of the release data of Fig. 6 showed no significant dependency on gellan concentration. Visual observation of the stomach contents of rats at time intervals after oral administration of 1% (w / v) gellan solutions containing 0.17% (w / v) citrate and 0.016% (w / v) Ca 11 (but no drug) indicated the formation of a gel block within 15 min (the first time interval at which observations were made), and its
Fig. 6. Plasma concentrations of theophylline in rats after d i.v. injection of aqueous solution; oral administration of s 0.25, ^ 0.50 and h 1.0% (w / v) gellan sols (containing 0.17% sodium citrate); and m oral administration of Theo-DurE syrup, containing identical initial amounts of theophylline (10 mg). Each value represents the mean6S.E. of four determinations.
S. Miyazaki et al. / Journal of Controlled Release 60 (1999) 287 – 295
Fig. 7. Plasma concentrations of theophylline in rats after oral administration of 1% (w / v) gellan sols containing 10 mg theophylline and sodium citrate at concentrations of s 0.05, ^ 0.10 and h 0.17% (w / v) but with no added calcium chloride and j 0.17% (w / v) sodium citrate with 0.016% (w / v) added calcium chloride. Each value represents the mean6S.E. of four determinations.
persistence for at least 6 h (the duration of observations). These observations are in agreement with the sustained release of theophylline from the gel formulations over the same time interval noted in Fig. 6. Fig. 7 shows a lack of any significant influence of the concentration of sodium citrate or the presence of Ca 11 ions, in agreement with the in vitro results. The area under the plasma concentration–time
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curve (AUC) and the mean residence time (MRT) were obtained from the theophylline plasma concentration–time data of each animal using a personal computer programme for model-independent analysis [23] and the results are summarised in Table 1. A more limited study of the release characteristics of gellan formulations after administration of 40 mg of theophylline to rabbits was conducted. Comparison was made of plasma levels following administration of a gellan formulation prepared from 1% (w / v) gellan, 0.17% (w / v) sodium citrate and 0.016% (w / v) CaCl 2 , with those from the commercial product (Theo-DurE syrup) and from i.v. injection of an aqueous solution. The results (see Fig. 8) were similar to those obtained using a rat model, and showed an approximately three-fold increase of the maximum plasma concentration, Cmax , and area under the curve, AUC, compared to Theo-DurE syrup. It is interesting to note that despite the large differences in these parameters, the two formulations had similar MRTs. Theo-DurE syrup is a colloidal suspension of microparticles of a cellulose derivative in concentrated sorbitol solution, which is formulated to produce sustained release of theophylline (see Fig. 8). The sustained release effect of the gellan formulation is a consequence of the gel structure. There is, however, a more prolonged residence of the gel within the stomach compared with the syrup and it is the balance of these two effects that is probably responsible for the similarity of the MRT values of the preparations. Plasma concentrations versus time following i.v.
Table 1 Comparison of bioavailability parameters in rats of theophylline from gellan gels and Theo-DurE syrup a Dosage form
Citrate % (w / v)
Ca 11 % (w / v)
Cmax mg / ml
t max h
AUC (0–6 h) mg h / ml
AUC oral ]] AUC i.v.
MRT h
Gellan gel 0.25% 0.50% 1.00% 1.00% 1.00% 1.00% Theo-DurE syrup i.v. injection
0.17 0.17 0.05 0.10 0.17 0.17 2 2
2 2 2 2 2 0.016 2 2
30.6661.33* 29.8760.12* 30.9262.43* 29.5261.12* 23.5863.20** 28.5461.19* 6.4960.97 2
3.5060.87 2.3860.38 3.0060.89 2.8860.77 3.3860.75 4.2560.48 3.3861.03 2
143.5368.28* 132.0163.01* 143.99611.44* 133.3863.83* 114.71616.43** 131.6066.42* 30.3364.74 206.5661.86
0.69560.04 0.63960.01 0.69760.06 0.64660.02 0.55660.08 0.63760.03 0.14760.02 2
3.3260.11 3.3560.09 3.3460.11 3.1560.13 3.1160.27 3.5160.04 3.3060.13 2.7860.02
a
Each value represents the mean6S.E. (n54). *P,0.001, **P,0.005, compared with Theo-DurE syrup.
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294
4. Conclusion This study has demonstrated the feasibility of forming gels in the stomach (of animal models) by the oral administration of aqueous solutions of gellan gum. Furthermore, a sustained release of the model drug, theophylline, is achievable from the gel vehicles over a period of at least 6 h. The bioavailability of theophylline from the gellan gels was increased four–fivefold in rats and threefold in rabbits compared with that from a commercial sustained release liquid dosage form.
Acknowledgements
Fig. 8. Plasma concentrations of theophylline in rabbits after d i.v. injection of aqueous solution, oral administration in either j 1.0% (w / v) gellan sols [containing 0.17% sodium citrate and 0.016% (w / v) calcium chloride] or m Theo-DurE syrup, containing identical initial amounts of theophylline (40 mg). Each value represents the mean6S.E. of four determinations.
injection were analysed using a two-compartment open model system. The curves were described by the sum of two exponentials according to Cp 5 A e 2 a t 1 B e 2 b t
(2)
where a and b are the distribution and elimination rate constants, respectively. The mean area under curve (AUC 0–24 h5154.77614.27 mg h ml 21 ) and the t 1 / 2 value (6.6360.93 h) are in reasonable 21 agreement with values of 113.73868.250 mg h ml and 5.21660.507 h determined for i.v. injection of theophylline (12 mg kg 21 ) into rabbits by Bouraoui et al. [24]. The pharmacokinetic parameters are summarised in Table 2.
This study was supported by the Royal Society of Great Britain, the Uehara Memorial Foundation and the Japan Society for the Promotion of Science (JSPS).
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Table 2 Comparison of bioavailability parameters in rabbits of theophylline from 1% gellan gels [containing 0.17% (w / v) citrate and 0.016% (w / v) Ca 11 ] and Theo-DurE syrup a Dosage form
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In situ-gelling gellan formulations as vehicles for oral drug delivery Shozo Miyazaki a , Hirotatsu Aoyama a , Naoko Kawasaki a , Wataru Kubo a , b, David Attwood * a
Faculty of Pharmaceutical Sciences, Health Science University of Hokkaido, Ishikari-Tohbetsu, Hokkaido 061 -0293, Japan b School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9 PL, UK Received 1 November 1998; received in revised form 11 March 1999; accepted 12 March 1999
Abstract Gels formed in situ following oral administration of 1% (w / v) aqueous solutions of gellan to rats and rabbits were evaluated as sustained-release vehicles. The formulation contained calcium ions in complexed form, the release of which in the acidic environment of the stomach caused gelation of the gellan gum. The in vitro release of theophylline from the rigid gellan gels followed root-time kinetics over a period of 6 h. Plasma levels of theophylline after oral administration of gellan solutions and a commercial oral sustained-release liquid dosage form containing an identical drug concentration were compared in both rats and rabbits. Bioavailability of theophylline from gellan gels formed by in situ gelation in the animal stomach was increased by four–fivefold in rats and threefold in rabbits compared with that from the commercial oral formulation. There was no significant difference in the mean residence times of theophylline when administered by these two vehicles. 1999 Elsevier Science B.V. All rights reserved. Keywords: Gellan gels; In situ gelation; Sustained release; Theophylline; Oral drug delivery
1. Introduction The use of natural polymers, such as chitosan [1–3], sodium alginate [4,5], hyaluronic acid esters [6] and xyloglucan [7], as vehicles for the sustained / controlled release of bioactive materials has received much attention recently. In this study, we report an assessment of the potential of the naturally occurring polysaccharide gellan as a vehicle for oral sustained delivery. Gellan gum (commercially available as *Corresponding author. Tel.: 144-161-275-2328; fax: 144161-275-2396. E-mail address: [email protected] (D. Attwood)
GelriteE or KelcogelE) is an anionic deacetylated exocellular polysaccharide secreted by Pseudomonas elodea, with a tetrasaccharide repeating unit of one a-L-rhamnose, one b-D-glucuronic acid and two b-Dglucose residues. It has the characteristic property of temperature-dependent and cation-induced gelation [8]. This gelation involves the formation of double helical junction zones followed by aggregation of the double-helical segments to form a three-dimensional network by complexation with cations and hydrogen bonding with water [9–11]. Much of the interest in the pharmaceutical application of this material has concentrated on its application for ophthalmic drug delivery [12–14]; aqueous solutions of gellan
0168-3659 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 99 )00084-X
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dropped into the eye undergo transition to the gel state due to the temperature and ionic conditions in the tear fluid. Drug release from these in-situ gels is prolonged due to longer precorneal contact times of the viscous gels compared with conventional eye drops. The use of gellan in the formulation of sustained delivery beads has recently been reported [15]. The present study is an examination of the possibility of exploiting the in-situ gelling characteristics of gellan for oral drug delivery. The formulation adopted was a gellan solution containing calcium chloride (as a source of Ca 11 ions), and sodium citrate, which complexes the free Ca 11 ions and releases them only in the highly acidic environment of the stomach. In this way, the formulation remains in liquid form until it reaches the stomach, when gelation is instantaneous. This paper reports in vitro and in vivo measurements of the release of a model drug, theophylline, and a comparison of the in vivo release characteristics with those from a commercial oral sustained release formulation.
2. Materials and methods
2.1. Materials Deacetylated gellan gum, KelcogelE, was supplied by Dainippon Pharmaceutical Co., Osaka, Japan, and was used as received. Theophylline was obtained from Wako Pure Chemical Ind. Ltd., Osaka, Japan. A commercially available product, TheoDurE syrup (20 mg ml 21 ), from Mitsubishi Kasei Co., Tokyo, Japan, was also studied. All other reagents were of analytical grade.
2.3. Measurement of gel strength Measurements were carried out using a rheometer (CR-200D, Sun Scientific Co.,Tokyo, Japan) by the method described previously [7,16]. Gels of gellan concentrations 0.25, 0.50 and 1.00% (w / v) were prepared by dissolving the weighed quantity of powder in 50 ml of water, heating to 908C, adding 0.016% (w / v) calcium chloride, and allowing the solution to cool to form a gel in a sample tube thermostatted at 208C. This tube was raised at a rate of 60 mm min 21 , pushing a probe slowly through the gel. The changes in the load on the probe were measured as a function of the depth of immersion of the probe below the gel surface.
2.4. Measurement of in vitro drug release The release rates of theophylline were measured by using plastic dialysis cells similar to those described previously [17]. The capacity of each halfcell was 4 ml and the surface area of the membranes was 2.67 cm 2 . The gellan gum solution, prepared in ultrapure water and loaded with a known weight of drug, was placed in the donor compartment and an equal volume of simulated gastric (pH 1.2) or intestinal (pH 6.8) fluid (as specified for the JP XIII disintegration test) was placed in the receptor compartment. The donor gel phase and the aqueous receptor phase were separated by a cellulose membrane (Viskase Sales Co., size 36 / 32). The assembled cell was shaken horizontally at a rate of 60 strokes min 21 in an incubator. The total volume of the receptor solution was removed at intervals and replaced by fresh release medium. The drug concentration of the sample was determined spectrophotometrically at a wavelength of 274 nm. All experiments were carried out in triplicate.
2.5. Animal experiments 2.2. Preparation of sols Gellan gum solutions were prepared by adding the gum to ultrapure water containing 0.05–0.17% (w / v) sodium citrate and heating to 908C while stirring. After cooling to below 408C, appropriate amounts of calcium chloride and theophylline were then dissolved in the resulting solution.
2.5.1. Rats Male Wistar rats, weighing ca. 250 g, were fasted for 24 h but with free access to water. The rats were anaesthetised with an i.p. injection of urethane and divided into three groups of four rats. One group received theophylline gel preparation given orally as 1 ml of gellan solution containing the drug (10 mg)
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through a stomach sonde needle for rats (Natume Seisakusho, KN-348). A second group was dosed in a similar manner with a commercial sustained release liquid dosage form, Theo-DurE syrup (10 mg in 0.5 ml). Theophylline solution (10 mg in 1 ml of saline) was administered intravenously to the third group of rats. At predetermined intervals, a blood sample was taken from the jugular vein of rats in each group.
2.5.2. Rabbits White male rabbits weighing 3.4–3.8 kg were fasted for two days prior to the experiments but were allowed free access to water. Gels containing theophylline were produced in situ by oral administration of 4 ml of the gellan solution containing 40 mg of drug using a stomach sonde for rabbits (Natume Seisakusho, KN-342). At given intervals, 1 ml blood samples were taken from the ear vein and analysed as described below. For intravenous administration, 40 mg doses of the drug in 4 ml of saline solution were injected through the ear vein. For oral administration of commercial Theo-DurE syrup, a dose of 40 mg in 2 ml was administered by the stomach tube. 2.6. Determination of theophylline The plasma samples were separated by centrifugation and assayed for theophylline by HPLC (Shimazu LC-10A with a Shimazu SPD-10A detector at a wavelength of 273 nm) using the method described by Schreiber-Deturmeny and Bruguerolle [18], with minor modifications. To 0.05 ml of plasma was
289
added 50 ml of caffeine solution (15 mg ml 21 ) as an internal standard and 20 ml of 20% perchloric acid, and the sample was vortex-mixed and centrifuged. The supernatant was passed through a Millipore filter (0.45 mm) and directly injected onto a 250346 mm I.D. column, packed with Inertsil-ODS. Elution was carried out with acetonitrile–tetrahydrofuran–concentrated acetic acid–distilled water (100:20:5:875, v / v) at a rate of 1.0 ml min 21 at 408C.
3. Results and discussion
3.1. Gelling properties Fig. 1 shows the rheological properties of gels of gellan concentrations 0.25, 0.50 and 1.00% (w / v), formed by dissolving the weighed quantity of powder in water, heating to 908C, adding 0.016% (w / v) calcium chloride, and allowing the solution to cool to 208C in the rheometer vessel. It was not possible to measure rheological properties of gels at pH 1.2 because of the low solubility of gellan at this pH. Values of gel strength (in kN m 22 ), calculated from Fig. 1, were 6.1860.20, 8.1360.47 and 17.6660.59 for gel concentrations of 0.25, 0.50 and 1.00% (w / v), respectively (each value representing the mean of four determinations). The values determined by this method are relative rather than absolute values but, nevertheless, they serve to show the influence of concentration on gel strength. The observed increase in gel strength with concentration has been noted previously for gellan [19,20] and is a consequence of
Fig. 1. Rheological properties of gellan gels of concentrations (a) 1.0, (b) 0.5 and (c) 0.25% (w / v) in water at 208C.
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increasing chain interaction with increasing polymer concentration. It is interesting to compare the rheological properties of the gellan gels with those of another natural product, enzyme-degraded xyloglucan, which were measured using the same apparatus and under similar conditions. The gel strength of the 0.5% (w / v) gellan gel was very similar to that of a 2% (w / w) xyloglucan gel (8.21 kN m 22 ) [7], indicating superior gelation characteristics of the gellan gum. The rheological behaviour shown in Fig. 1 is typical of that of elastic gels, with the abrupt decrease in stress after the maximum indicating a brittle system. In a comparison of the gelation properties of deacetylated gellan with those of other gums, Moorhouse et al. [19] noted similar characteristics (minimum gelling concentration, melting and setting temperatures) of the calcium gellan gels and those of agar, but a higher gel strength of the gellan gels.
3.2. In vitro release of theophylline In vitro release characteristics were determined at pH 1.2, pH 6.8 and at a combination of these two pH values in which the release medium was changed from a solution at pH 1.2 to one at pH 6.8 after 2.5 h, to simulate passage through the gastrointestinal (GI) tract. In each case, gellan sols were prepared in water at concentrations of 0.25, 0.50 and 1.00% (w / v) and separated from the release medium at the selected pH by a cellulose membrane, as described in Section 2.4. The initial theophylline concentration in all systems was 1% (w / v). Gelation of the sol in the donor compartment is a result of hydrogen bonding of the chains [9] by the H 1 ions that diffuse through the membrane separating the two compartments. Although the pH of the sol is reduced as a result of the influx of H 1 ions, there is clearly sufficient ionisation of the carboxylic groups of gellan for gelation to occur by this process. This process is rapid, with complete gelation of the donor solution occurring before the time of the first measurement (30 min after initial contact of the two solutions). Diffusion may thus be considered to be effectively from a gel block into the donor solution. It was not feasible to conduct a comparison of the release characteristics of the gellan preparations with those of the equivalent commercial product, Theo-DurE
syrup, because of osmotic effects in the apparatus used. A comparison of the in vivo release characteristics of the two preparations is presented below. Fig. 2 shows no significant influence of the gellan concentration on the release profile at pH 1.2, compared with the progressive decrease in the amount released with an increase of the gellan concentration seen at pH 6.8. The reason for this difference in release behaviour is attributable to the large difference in the H 1 ion concentrations of the two receptor solutions. The H 1 ion concentration at pH 6.8 is insufficiently high to cause the formation of rigid gels and the decreasing release rates observed in this system as the gellan concentration is increased reflect increasing viscosity of the soft gels formed. In contrast, rigid gels are formed when the donor solutions of all systems are placed in contact with a receptor solution at pH 1.2 and, as a consequence, the amount released is lower than that at pH 6.8 and independent of the gellan concentration. Fig. 3 shows no significant influence of gellan concentration on release when this was initially (2.5 h) into a receptor solution of pH 1.2 followed by a period of release into a receptor solution of higher pH. This is an expected consequence of rigid gel formation
Fig. 2. Cumulative release of theophylline as a function of time from gellan gels with an initial loading of 1% (w / v) drug and gellan concentrations of s 0.25, ^ 0.50 and h 1.0% (w / v) at pH 1.2 (filled symbols) and pH 6.8 (open symbols). Each value represents the mean6S.E. of three determinations.
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Fig. 3. Cumulative release of theophylline as a function of time from gellan gels with an initial loading of 1% (w / v) drug and gellan concentrations of s 0.25, ^ 0.50 and h 1.0% (w / v) at pH 1.2 for a period of 2.5 h and subsequently at pH 6.8. Each value represents the mean6S.E. of three determinations.
Fig. 4. Cumulative release of theophylline as a function of square root of time from gellan gels with an initial loading of 1% (w / v) drug and gellan concentrations of s 0.25, ^ 0.50 and h 1.0% (w / v) at pH 1.2 (filled symbols) and pH 6.8 (open symbols). Each value represents the mean6S.E. of three determinations.
during the initial release period at high H 1 ion concentration. Release data were analysed according to the treatment proposed by Higuchi [21] for drug release from semisolid vehicles containing dissolved drug. For the initial 50–60% release, the cumulative amount, Q, of drug released per unit surface area is proportional to the square root of time, t:
MMC released from a 1% (w / w) xyloglucan gel loaded with 0.025% drug, determined using the same equipment [22]. These two drugs have similar water solubilities and will partition predominantly in the water channels between the aggregated chains of the gel network. Comparison with the release characteristics of gels of the oxyethylene–oxypropylene– oxyethylene triblock copolymer Pluronic F127 [D of MMC53.61310 26 cm 2 s 21 from a 25% (w / w) Pluronic F127 gel [22]] shows the very much lower concentration of both xyloglucan and gellan gels required to achieve similar release characteristics of gels of the more widely studied polymer Pluronic F127. For the oral formulation, it was considered necessary to include a source of Ca 11 ions [0.016% (w / v) CaCl 2 ], the release of which in the stomach would ensure gelation of the gellan solution. The method by which gelation of the formulation before administration was avoided was through the inclusion of sodium citrate, which complexes the free Ca 11 ions and releases them only in the acidic environment of the stomach. The concentration of added sodium citrate was critical for the correct functioning of the formulation. Sodium citrate concentrations of
Q 5 2 C0 (Dt / p)1 / 2
(1)
Plots of Q vs. t 1 / 2 for the systems of Fig. 2 were linear after a short lag period (Fig. 4), indicating that drug release was controlled by diffusion of drug through the gel matrix. Diffusion coefficients calculated from the gradients of these plots were 8.48, 8.67 and 6.44310 26 cm 2 s 21 for gels with gellan concentrations of 0.25, 0.5 and 1.0% (w / v), respectively, at pH 1.2, and 3.07, 2.32 and 1.56310 25 cm 2 s 21 for gels with gellan concentrations of 0.25, 0.5 and 1.0% (w / v), respectively, at pH 6.8. The diffusion coefficients of theophylline from gellan gels at pH 1.2 are of a similar order of magnitude as those for the release of mitomycin c (MMC) from xyloglucan gels. For example, D57.28310 26 cm 2 s 21 for
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0.175% (w / v) and higher caused gelation of the formulation at room temperature, presumably due to the high Na 1 ion concentration (although not as effective as the divalent Ca 11 ions, Na 1 ions are able to cause gelation of this material at sufficiently high concentration [19,20]). The influence of sodium citrate concentration on the release properties was investigated at concentrations of 0.05, 0.10 and 0.17% (w / v). Fig. 5 shows no significant differences in release profiles over this concentration range, and the highest concentration [0.17% (w / v)] was selected for inclusion in the gellan formulation. The lack of influence of Ca 11 ions on the in vitro release characteristics shown in Fig. 5 is explicable by considering the gelation process. In formulations containing CaCl 2 , the sodium citrate complexes free Ca 11 ions, the release of which in acidic conditions causes gelation. However, in sols not containing Ca 11 ions, the sodium citrate will be in a predominantly ionised form and gelation is a consequence of the free Na 1 ions present in this solution, the relatively high concentration of which [0.17% (w / v) compared to 0.016% (w / v) Ca 11 ] is clearly
Fig. 5. Cumulative release of theophylline as a function of time from 1% (w / v) gellan gels with an initial loading of 1% (w / v) drug and containing sodium citrate at concentrations of s 0.05, ^ 0.10 and h 0.17% (w / v) but with no added calcium chloride, and j 0.17% (w / v) sodium citrate with 0.016% (w / v) added calcium chloride. Each value represents the mean6S.E. of three determinations.
sufficient to produce gels of comparable release characteristics to those produced by Ca 11 ions.
3.3. In vivo release of theophylline Plasma drug levels following oral administration to rats of 10 mg of theophylline from solutions of gellan are compared, in Fig. 6, with those following administration of an identical quantity of theophylline by i.v. injection of an aqueous solution, and from a commercial sustained release liquid dosage form (Theo-DurE syrup). It is clear from Fig. 6 that a sustained release of drug is achieved from the gellan preparations over a period of at least 6 h and this is at a significantly higher level (approx. four– fivefold) than that from the commercial product. Analysis of the release data of Fig. 6 showed no significant dependency on gellan concentration. Visual observation of the stomach contents of rats at time intervals after oral administration of 1% (w / v) gellan solutions containing 0.17% (w / v) citrate and 0.016% (w / v) Ca 11 (but no drug) indicated the formation of a gel block within 15 min (the first time interval at which observations were made), and its
Fig. 6. Plasma concentrations of theophylline in rats after d i.v. injection of aqueous solution; oral administration of s 0.25, ^ 0.50 and h 1.0% (w / v) gellan sols (containing 0.17% sodium citrate); and m oral administration of Theo-DurE syrup, containing identical initial amounts of theophylline (10 mg). Each value represents the mean6S.E. of four determinations.
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Fig. 7. Plasma concentrations of theophylline in rats after oral administration of 1% (w / v) gellan sols containing 10 mg theophylline and sodium citrate at concentrations of s 0.05, ^ 0.10 and h 0.17% (w / v) but with no added calcium chloride and j 0.17% (w / v) sodium citrate with 0.016% (w / v) added calcium chloride. Each value represents the mean6S.E. of four determinations.
persistence for at least 6 h (the duration of observations). These observations are in agreement with the sustained release of theophylline from the gel formulations over the same time interval noted in Fig. 6. Fig. 7 shows a lack of any significant influence of the concentration of sodium citrate or the presence of Ca 11 ions, in agreement with the in vitro results. The area under the plasma concentration–time
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curve (AUC) and the mean residence time (MRT) were obtained from the theophylline plasma concentration–time data of each animal using a personal computer programme for model-independent analysis [23] and the results are summarised in Table 1. A more limited study of the release characteristics of gellan formulations after administration of 40 mg of theophylline to rabbits was conducted. Comparison was made of plasma levels following administration of a gellan formulation prepared from 1% (w / v) gellan, 0.17% (w / v) sodium citrate and 0.016% (w / v) CaCl 2 , with those from the commercial product (Theo-DurE syrup) and from i.v. injection of an aqueous solution. The results (see Fig. 8) were similar to those obtained using a rat model, and showed an approximately three-fold increase of the maximum plasma concentration, Cmax , and area under the curve, AUC, compared to Theo-DurE syrup. It is interesting to note that despite the large differences in these parameters, the two formulations had similar MRTs. Theo-DurE syrup is a colloidal suspension of microparticles of a cellulose derivative in concentrated sorbitol solution, which is formulated to produce sustained release of theophylline (see Fig. 8). The sustained release effect of the gellan formulation is a consequence of the gel structure. There is, however, a more prolonged residence of the gel within the stomach compared with the syrup and it is the balance of these two effects that is probably responsible for the similarity of the MRT values of the preparations. Plasma concentrations versus time following i.v.
Table 1 Comparison of bioavailability parameters in rats of theophylline from gellan gels and Theo-DurE syrup a Dosage form
Citrate % (w / v)
Ca 11 % (w / v)
Cmax mg / ml
t max h
AUC (0–6 h) mg h / ml
AUC oral ]] AUC i.v.
MRT h
Gellan gel 0.25% 0.50% 1.00% 1.00% 1.00% 1.00% Theo-DurE syrup i.v. injection
0.17 0.17 0.05 0.10 0.17 0.17 2 2
2 2 2 2 2 0.016 2 2
30.6661.33* 29.8760.12* 30.9262.43* 29.5261.12* 23.5863.20** 28.5461.19* 6.4960.97 2
3.5060.87 2.3860.38 3.0060.89 2.8860.77 3.3860.75 4.2560.48 3.3861.03 2
143.5368.28* 132.0163.01* 143.99611.44* 133.3863.83* 114.71616.43** 131.6066.42* 30.3364.74 206.5661.86
0.69560.04 0.63960.01 0.69760.06 0.64660.02 0.55660.08 0.63760.03 0.14760.02 2
3.3260.11 3.3560.09 3.3460.11 3.1560.13 3.1160.27 3.5160.04 3.3060.13 2.7860.02
a
Each value represents the mean6S.E. (n54). *P,0.001, **P,0.005, compared with Theo-DurE syrup.
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4. Conclusion This study has demonstrated the feasibility of forming gels in the stomach (of animal models) by the oral administration of aqueous solutions of gellan gum. Furthermore, a sustained release of the model drug, theophylline, is achievable from the gel vehicles over a period of at least 6 h. The bioavailability of theophylline from the gellan gels was increased four–fivefold in rats and threefold in rabbits compared with that from a commercial sustained release liquid dosage form.
Acknowledgements
Fig. 8. Plasma concentrations of theophylline in rabbits after d i.v. injection of aqueous solution, oral administration in either j 1.0% (w / v) gellan sols [containing 0.17% sodium citrate and 0.016% (w / v) calcium chloride] or m Theo-DurE syrup, containing identical initial amounts of theophylline (40 mg). Each value represents the mean6S.E. of four determinations.
injection were analysed using a two-compartment open model system. The curves were described by the sum of two exponentials according to Cp 5 A e 2 a t 1 B e 2 b t
(2)
where a and b are the distribution and elimination rate constants, respectively. The mean area under curve (AUC 0–24 h5154.77614.27 mg h ml 21 ) and the t 1 / 2 value (6.6360.93 h) are in reasonable 21 agreement with values of 113.73868.250 mg h ml and 5.21660.507 h determined for i.v. injection of theophylline (12 mg kg 21 ) into rabbits by Bouraoui et al. [24]. The pharmacokinetic parameters are summarised in Table 2.
This study was supported by the Royal Society of Great Britain, the Uehara Memorial Foundation and the Japan Society for the Promotion of Science (JSPS).
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Table 2 Comparison of bioavailability parameters in rabbits of theophylline from 1% gellan gels [containing 0.17% (w / v) citrate and 0.016% (w / v) Ca 11 ] and Theo-DurE syrup a Dosage form
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