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Permeability and swelling studies on free films containing inulin in combination with different polymethacrylates aimed for colonic drug delivery
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 8 ( 2 0 0 6 ) 307–314
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Permeability and swelling studies on free films containing inulin in combination with different polymethacrylates aimed for colonic drug delivery A. Akhgari a,∗ , F. Farahmand a , H. Afrasiabi Garekani a , F. Sadeghi a , T.F. Vandamme b a
School of Pharmacy and Pharmaceutical Research Centre, Vakilabad blvd., PO Box 91775-1365, Mashhad University of Medical Science, Mashhad, Iran b D´epartement de Chimie Bioorganique, LC 01, UMR 7514, Facult´e de Pharmacie, Universit´e Louis Pasteur, Strasbourg, France
a r t i c l e
i n f o
a b s t r a c t
Article history:
The aim of this study was to assess some permeability and swelling characteristics of free
Received 30 January 2006
films prepared by combination of inulin as a bacterially degradable system and time- or pH-
Received in revised form 12 March
dependent polymers as a coating formulation for colonic drug delivery. Different free films
2006
were prepared by casting and solvent evaporation method. Formulations containing inulin
Accepted 19 March 2006
with Eudragit RS, Eudragit RL, Eudragit RS–Eudragit RL, Eudragit FS and Eudragit RS–Eudragit
Published on line 28 March 2006
S with different ratios of inulin were prepared. After preparation, free films were evaluated by water vapor transmission test, swelling experiment and permeability to indomethacin
Keywords:
and theophylline in different media. Formulations containing Eudragit FS had high resis-
Inulin
tance to water vapor permeation; but were unable to protect premature swelling and drug
Free film
release in simulated small intestine media. Also, combination of Eudragit RS and Eudragit
Permeability
S had no suitable characteristics for colon delivery. However, Eudragit RS and Eudragit RL in
Swelling
combination with inulin made free films which had more swelling and permeation of drug
Water vapor transmission
in the colonic medium rather than the other media. It was shown that formulations contain-
Colonic delivery
ing sustained release polymethacrylates in combination with inulin have more potential as a coating system for specific colon delivery compared with pH-dependent polymers.
Abbreviations:
© 2006 Elsevier B.V. All rights reserved.
ERS, Eudragit RS; ERL, Eudragit RL; EFS, Eudragit FS; ES, Eudragit S; In, inulin; WVT, water vapor transmission; SGF, simulated gastric fluid; SIF, simulated intestinal fluid; SCF, simulated colonic fluid
1.
Introduction
Targeting of drugs to the large intestine has a number of important implications in the field of pharmacotherapy. These are treatments of colonic disorders such as ulcerative col-
∗
Corresponding author. Tel.: +98 8823255/6; fax: +98 8823251. E-mail address: akhgari [email protected] (A. Akhgari).
0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2006.03.005
itis and Crohn’s disease and also the oral administration of protein and peptide drugs, which are normally degrade by the enzymes of the upper gastrointestinal tract (Zhou, 1994). Various approaches have been used for oral delivery of drugs to the colon, which include pH-dependent systems,
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time-dependent systems, and systems which are bacterially degraded in the colon. Among these strategies, systems relying on the metabolic activity of the colonic microflora can be more site-specific and a large number of polysaccharides have recently been proposed for development of colon-specific drug delivery devices (Sinha and Kumria, 2001). Inulin is a naturally occurring polysaccharide which is not significantly hydrolyzed by gastric or intestinal enzymes. Colonic bacteria however, and more specifically Bifidobacteria can degrade this polysaccharide (Van den Mooter et al., 2003). However, its fairly good water solubility makes it difficult to be used as a carrier for colonic drug delivery. Several methods such as synthesized different derivatives of inulin with reduced water solubility (Van den Mooter et al., 2003; Damian et al., 1999; Vervoort et al., 1998) or mixing it with Eudragit RS as a coating formulation (Vervoort and Kinget, 1996) have been used. The aim of this study was to evaluate the permeability and swelling characteristics of isolated films prepared by mixing of inulin with either sustained-release or pH-dependent polymethacrylates and also combination of these two kinds of polymers. To assess the resistance of a colon-specific coating to the gastric and intestinal fluids and also susceptibility to the colonic media, it is necessary to investigate the diffusion of drug molecules through the films and also the swelling of free films under conditions simulating these media. On the other hand, water vapor transmission study gives some valuable information on the protection of a coated dosage form against environment humidity (Van den Mooter et al., 1994). Permeability studies were investigated for two drugs having different solubilities; theophylline as a soluble and indomethacin as a low soluble drug model.
2.
Materials and methods
2.1.
Materials
Eudragit RS100 (ERS), Eudragit RL100 (ERL), Eudragit S100 (ES) and Eudragit FS30D (EFS) (Rohm Pharma, Germany), inulin (Raftiline HP, Orafti, France), inulinase from aspergillus niger (Sigma, USA), triethyl citrate (Morflex, USA), theophylline monohydrate (Cooper Rhone, France), indomethacin (Fluka, Italy), magnesium nitrate hexahydrate (Mg(NO3 )2 ·6H2 O) (Aldrich, Germany), phosphorus pentoxide (Acros organics, USA) were supplied from indicated sources. All chemicals were of analytical grade.
2.2.
Preparation of free films
2.2.1. Preparation of free films containing inulin–Eudragit RS–Eudragit RL A 10% (w/v) solution of Eudragit RS100, Eudragit RL100 or 1:1 ratio of two polymers was prepared by dissolving granules of these polymers in isopropyl alcohol:distilled water (9:1 ratio) solvent. Then, a fixed amount of triethyl citrate (1:6 ratio related to total polymer content) was added as a plasticizer. Specific amounts of inulin powder were dissolved in distilled water by keeping warming the solution. Inulin solution was gently added to Eudragit solution with continuous
stirring. The ratios of inulin to Eudragit content were 0, 10, 20 and 30% (w/w). The resulted suspension was transferred to Teflon plates. Volume of suspension was 25 ml in each plate. The plates were then placed in an oven at 50 ◦ C for 24 h for complete drying. After that, plates were transferred to a desiccator with 100% relative humidity (RH) for 24 h. Then, the films were cut with a scalpel to different special pieces for various tests. The thickness of the films was measured at five different ¨ places by using a micrometer (Kafer, Germany) and the average thickness of 150–170 m was selected. Free films were stored in a desiccator with 50% RH resulted by a saturated solution of magnesium nitrate hexahydrate at room temperature until use.
2.2.2. Preparation of free films containing inulin–Eudragit FS30D Firstly, specific amounts of inulin were dissolved in warmed distilled water. Then, a fixed amount of triethyl citrate (1:30 ratio related to total polymer content) was added to solution as a plasticizer. Then, predetermined amounts of Eudragit FS30D were added to this solution with stirring. Final dispersion was stirred by using a magnetic stirrer for 5 h and then poured into Teflon plates. The ratios of inulin to Eudragit content and also next steps of film preparation and storage were the same with Section 2.2.1.
2.2.3. Preparation of free films containing inulin–Eudragit RS–Eudragit S Various amounts of granules of Eudragit RS100 were dissolved in isopropyl alcohol:distilled water (9:1 ratio). The solution was plasticized with a constant amount of triethyl citrate (1:6 ratio related to total polymer content). Separately, specific amounts of inulin were dissolved in warmed distilled water and specific amounts of Eudragit S100 powder were added to inulin solution. The resulted suspension was dissolved by adding droplets of ammonia and partial neutralizing of Eudragit S. Then, Eudragit RS solution was gently added with continuous stirring to the solution containing inulin and Eudragit S. Neutralization of Eudragit S made it possible to mix this polymer with inulin and Eudragit RS and film preparation. The resulted suspension was poured into teflon plates. The ratio of Eudragit RS to Eudragit S was 1:1 in all of formulations. The ratios of inulin to Eudragit content and also next steps of film preparation and storage were the same with Section 2.2.1.
2.3.
Water vapor transmission test
Water vapor permeation of free films was determined gravimetrically in triplicate. The permeability cups were 3.5 cm in diameter. The inside of the cup was filled with 10 ml of distilled water (100% RH). A circular piece of free films was placed on the cup and a gasket placed on the film followed by a metal ring. The film was held in place with the help of three screw clamps. Another circular piece of the same free film fixed on another cup without water as a reference. Both sample and reference were accurately weighed (±0.0001 g) and then placed in a desiccator containing phosphorus pentoxide (P2 O5 ) as a desiccant and filled with silicagel (0% RH). At specific intervals (24, 48, 72, 96 and 120 h) the cups were weighed (±0.0001 g) and the profile of mass change was plotted versus time for
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 8 ( 2 0 0 6 ) 307–314
each free film. Water vapor transmission (WVT) was calculated using following equation: WVT =
wx tAP0 (RH1 − RH2 )
(1)
where w/t is the mass change (flux, g/h) resulted from slope of profile of the mass change versus time, x the film thickness (mm), A the area of the film surface exposed to the permeant (m2 ), P0 the vapor pressure of pure water (kPa), and (RH1 − RH2 ) is the relative humidity gradient. At 25 ◦ C, P0 is 3.159 kPa (Carvalho and Grosso, 2004).
2.4.
Swelling experiments
Firstly, a piece of 1 cm2 of each free film was dried in an oven at 50 ◦ C for 24 h. Then, dried film was accurately weighed (±0.0001 g) and immersed in a flask of dissolution test containing 250 ml of different media at 37 ◦ C. At specific intervals, the swollen sample was withdrawn from the medium and weighed (±0.0001 g) after removal of excess surface water ¨ by light blotting with a filter paper (Carl Schleicher & Schull, Germany). During the first 10 min, intervals of sampling were 1 min. Sampling time was gradually increased after this period until 3 h. To quantify the swelling process, the swelling index, Is (%), was calculated as follows (Blanchon et al., 1991): Is (%) =
W s − Wd × 100 Wd
(2)
where Wd is the weight of the dried polymer film and Ws denotes the weight after swelling. Swelling tests were separately carried out in simulated gastric fluid (SGF) without pepsin, simulated intestinal fluid (SIF) without pancreatin with pH 6.8 (USP XXVI, 2003) and also simulated colonic fluid (SCF) with adding of 1 ml/l of inulinase to the media with pH 6.4. In case of formulations containing In–EFS and In–ERS–ES, swelling characteristics were also investigated in media with pH 6.4 as SIF. All of the experiments were carried out in triplicate.
2.5.
Permeability test
Isolated films of the polymers were mounted between the donor and acceptor compartments of a side-by-side diffusion cell (diffusion area 3.46 cm2 ). Temperature of the cells was kept at 37 ◦ C throughout the experiments by continuous circulating of water and each compartment was stirred continuously with a magnetic stirrer. Different experimental conditions were set up to examine the permeability of drugs through polymer films; for theophylline, the initial concentration of drug in the donor compartment was 3 g/l. The donor and acceptor compartments were both composed of SGF, SIF and SCF with inulinase. SGF and SCF were the same for all of the formulations. SIF was the medium with pH 6.8 for all of the formulations except formulations containing In–EFS for which the medium with pH 6.4 was supposed as SIF. For indomethacin, the initial concentrations of drug solutions in the donor compartment were 300 mg/l for SIF with pH 6.4 and 6.8 and SCF, and 500 mg/l for SIF with pH 7.2. To achieve the sink conditions for permeability experiments, nearly saturated concentration
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of drug was used in the donor compartment. Regarding the low solubility of indomethacin in more acidic pH, the lower concentration of drug solution was used in the media with pH 6.4 and 6.8 rather than medium with pH 7.2. The donor and acceptor compartment were both filled with SIF or SCF. The medium with pH 7.2 was used as SIF for evaluating formulations containing In–ERS–ERL; but in case of formulations containing In–EFS and In–ERS–ES, regarding dissolution of pHdependent polymers in this pH, the media with pH 6.4 and 6.8 were used as SIF, respectively. SCF medium was the same for all of the formulations. All of the permeability experiments were carried out for 3 h. At predetermined time intervals, samples of 10 ml were taken from the receptor cells and replaced with fresh medium. The contents of the acceptor cells were assayed spectrophotometrically for theophylline and indomethacin at 272 and 318 nm, respectively. Each permeation experiment was repeated three times and the cumulative amount of drug permeated and corrected for the acceptor sample replacement was plotted against time. Applying Fick’s first law of diffusion in sink conditions, the permeation rate of drug was defined as dM = PSCd dt
(3)
where M was the amount of drug diffused (mg) at time t, S the effective diffusion area (cm2 ), Cd the concentration of drug in the donor compartment and P was the permeability (cm/s). Then, the permeability was obtained by the following equation: P=
slope SCd
(4)
where the slope was obtained from the plot of the amount of drug permeated versus time (Lin and Lu, 2002; Ingels et al., 2002).
2.6.
Statistical analysis of data
One-way analysis of variance was used to assess the significance of the differences among different groups. Tukey–Kramer post-test was used to compare the means of different treatment groups. Results with P < 0.05 were considered to be statistically significant.
3.
Results and discussion
3.1.
WVT experiments
According to Fig. 1 the rate of water vapor permeation was constant for free films containing Eudragit FS and inulin. This linear relation between amount of vapor permeated and time was also observed for the other formulations. Table 1 lists the results of WVT experiments for all the formulations. As shown in Table 1, free films containing Eudragit FS and Eudragit RS had the lowest permeability to water vapor in all the formulations (P < 0.01). It is well known that increasing the hydrophilic
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Fig. 1 – Profiles of water vapor transmission through free films containing Eudragit FS–inulin. Error bars indicate S.D. (n = 3).
nature of a polymer membrane induces water vapor tendency and as a result increases water vapor permeation (Wang et al., 2004; Parra et al., 2004). Eudragit RS and RL are acrylic and methacrylic acid esters with some hydrophilic properties due to the presence of quaternary ammonium groups where ERL possesses higher amount of such groups than ERS. Then, it is expected that formulations containing ERL exhibit higher water vapor permeability than ERS. The order of WVT for free films containing Eudragit RL and RS was as followed: ERL > ERL–ERS > ERS. Also, low permeability of formulations containing Eudragit FS to water vapor is due to low hydrophilic character of this polymer. Nevertheless, formulations with EFS and ERS had no significant difference in WVT (P > 0.05) except in the case of free films containing 30% inulin. This result can be explained by a different film preparation for formulations of EFS and ERS. Free films containing Eudragit FS were prepared from aqueous dispersion of polymer, whereas Eudragit RS films were made from an organic solution. Bodmeier and Paeratakul (1994) have shown that films cast from organic solutions have a tighter and
a more compact structure than those prepared from aqueous dispersions and this is due to the tighter bound plasticizer in their polymeric chains. Therefore, this phenomenon could lower water vapor permeability of polymeric films and compensate the effect of hydrophobic character of Eudragit FS. As the overall result, WVT of formulations containing EFS and ERS was almost the same. As shown in Table 1, formulations containing Eudragit S in combination with Eudragit RS had the high WVT. This is due to partial neutralization of carboxylic groups of Eudragit S by ammonia that causes less hydrophobic character and finally less resistance to water vapor. Also, the presence of ammonia in the film solution and its rapid evaporation could make some free volume which leads to more diffusion of penetrant in the polymeric chains (Pandey and Chauhan, 2001; George and Thomas, 2001). Addition of inulin to the film formulation significantly increased WVT (P < 0.05). This result is also in agreement with increasing the hydrophilic nature of membrane by inulin and as a result increasing vapor permeability of water. Only in formulations containing ERL in the ratio of 70–90%, addition of inulin had no significant effect (P > 0.05).
3.2.
Swelling test
The results of swelling experiments are listed in Table 2. Formulations containing only ERS had low swelling index in SGF and SIF. Swelling of free films in SCF significantly increased with addition of inulin up to the ratio of 30% (P < 0.05). This is due to the presence of inulinase enzyme in the media which can diffuse into the polymeric chains, hydrolyze the fructose backbone of inulin, reduce the network density and finally increase swelling (Vervoort et al., 1998). In the case of formulations containing ERS–ERL, swelling was higher in SIF than SGF. This is in contrast with pH-independency of these polymers and can be due to difference in the swelling media.
Table 1 – Water vapor transmission of free films Formulation ERS 100% ERS–In 90–10% ERS–In 80–20% ERS–In 70–30% ERS–ERL 50–50% ERS–ERL–In 45–45–10% ERS–ERL–In 40–40–20% ERS–ERL–In 35–35–30% ERL 100% ERL–In 90–10% ERL–In 80–20% ERL–In 70–30% EFS 100% EFS–In 90–10% EFS–In 80–20% EFS–In 70–30% ERS–ES 50–50% ERS–ES–In 45–45–10% ERS–ES–In 40–40–20% ERS–ES–In 35–35–30%
Film thickness (mean ± S.D.; n = 15) (m)
Mass change (mg/h)
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.54 5.47 7.73 15.82 10.80 12.33 20.32 20.72 22.86 25.58 27.26 27.32 3.95 6.32 9.32 10.06 14.32 21.15 24.83 25.16
158 160 167 160 159 159 162 170 163 160 160 160 152 160 152 161 160 162 159 160
8 8 5 6 5 8 8 5 6 10 8 8 6 11 9 7 5 4 7 8
WVT (mean ± S.D.; n = 3) (mg mm/m2 h kPa) 2.365 2.886 4.248 8.347 5.650 6.451 10.855 11.616 12.262 13.498 14.381 14.412 1.997 3.336 4.671 5.352 7.555 11.298 12.991 13.275
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.060 0.344 0.078 0.039 0.041 0.188 0.192 0.430 0.230 0.672 0.377 0.566 0.030 0.047 0.221 0.076 0.291 0.139 0.095 1.304
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Table 2 – Swelling of formulations in different media (data represent mean ± S.D.; n = 3) Formulation
Maximum swelling index, Is (%) SGF
ERS 100% ERS–In 90–10% ERS–In 80–20% ERS–In 70–30% ERS–ERL 50–50% ERS–ERL–In 45–45–10% ERS–ERL–In 40–40–20% ERS–ERL–In 35–35–30% ERL 100% ERL–In 90–10% ERL–In 80–20% ERL–In 70–30% EFS 100% EFS–In 90–10% EFS–In 80–20% EFS–In 70–30% ERS–ES 50–50% ERS–ES–In 45–45–10% ERS–ES–In 40–40–20% ERS–ES–In 35–35–30% a
10.3 17.3 15.8 19.3 30.6 36.7 31.0 36.5 44.8 50.1 54.0 58.2 18.6 18.1 21.1 31.9 87.8 63.8 18.9 24.5
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.4 8.0 2.1 3.9 1.0 3.1 8.1 4.9 8.6 11.3 2.8 4.3 8.3 3.7 3.7 3.4 6.2 13.2 3.2 2.0
SIF 12.4 13.6 20.2 19.9 41.9 47.1 62.7 50.8 62.3 106.5 103.9 108.1 52.0 46.8 76.1 82.5 151.5 156.9 147.5 117.9
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
SCF 0.3 1.0 5.1 3.3 2.0 2.7 4.9 2.4 3.2 21.3 13.4 11.2 7.0a 1.8a 7.5a 1.1a 15.0a 4.0a 0.1a 1.8a
13.6 15.4 23.8 37.1 47.5 71.2 81.4 77.3 80.1 222.5 183.4 150.4 66.6 78.4 70.1 58.4 185.7 162.5 165.2 141.7
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.6 0.3 1.9 7.1 4.4 6.9 8.4 10.9 6.6 35.4 33.3 7.9 11.9 4.9 5.0 1.1 4.1 6.8 3.0 1.6
Data are swelling in the media with pH 6.4.
Eudragit RL and RS have quaternary ammonium groups which are in the chloride salt form. The dissociation of these quaternary ammonium groups in aqueous media is responsible for the hydration and swelling of the polymer films. Indeed, the exchange of chloride ion with the buffer anions of the dissolution medium could govern the degree of hydration and swelling. The SGF media had chloride anion due to hydrochloric acid and sodium chloride which had less selectivity to ion exchange than phosphate and then reduce swelling of polymer in SGF compared with SIF media (Wagner and McGinity, 2002; Bodmeier et al., 1996). On the other hand, regarding more ammonium groups in ERL than ERS, swelling of formulations containing ERL is higher and also the difference in swelling in the SGF and SIF can be more pronounced. Also, in the case of formulations containing only ERL, swelling in SIF was higher than in SGF which confirms this fact. Swelling of formulations containing EFS was lower in SGF than in SIF and in SCF (P < 0.001) because this polymer is pHdependent and in the acidic pH, it is resistant to dissolution media with low pH whereas in SIF polymer tends to ionize and swell, so that in the media with pH 6.8, free films were dissolved and the swelling was not possible. Therefore, the media with pH 6.4 was used as SIF. Swelling index in SIF increased with the addition of inulin in the ratios of 20 and 30% (P < 0.01) which is due to hygroscopic characteristics of inulin and the higher water uptake of polymer. Addition of inulin up to 10% caused increasing swelling in SCF which is due to enzyme activity. However, swelling was surprisingly decreased in the higher ratios of inulin and in the mixture of EFS–In (70:30), swelling ratio in SCF was lower than in SIF (P < 0.01). This result can be explained by leaching out of inulin in the high ratios of this polysaccharide. Regarding the ionization of carboxylic groups of Eudragit FS and its swelling, this polymer cannot protect inulin anymore and the polysaccharide
311
which is partially degraded before by enzyme, excludes from the polymeric chains and as the overall result, the swelling index is decreased. This decrease in swelling by inulin ratio of 30% compared with the other ratios was also shown in the other formulations except for films containing only ERS. Totally, formulations containing EFS have not good potential as a coating system for colonic delivery because of dissolution of the polymer in the media simulating small intestine and EFS cannot protect inulin in this media. Formulations containing ERS–ES had high swelling ratios in SGF and in SIF except for formulations containing 20 and 30% of inulin which had low swelling index in SGF. The high swelling of the formulations having the higher ratios of Eudragit S can be attributed to the presence of some voids resulted by ammonia evaporation and therefore more easiness to water uptake. The decrease in the swelling by increasing inulin level in SCF was observed and could be the result of leaching it through free volumes of polymers in the higher ratio of polysaccharide. Formulations containing ES had so high swelling index in SIF with pH 6.8 that it was not possible to take and weight the films after about 1 h. Therefore, swelling in SIF with pH 6.4 was determined. This is due to the increase in dissolution of ES with the increase in media pH. Totally, formulations containing pH-dependent polymers could not protect free films from high swelling in the media simulating small intestine which is important for a colonic drug delivery system. Also, the ratio of swelling index in SCF to SIF as a key parameter to assess potential of polymeric system for colonic delivery was higher in the formulations containing time-dependent polymers with inulin than free films with pHdependent polymethacrylates.
3.3.
Permeability experiments
Permeability tests were carried out for two kinds of drugs: theophylline as a soluble model and indomethacin as a low soluble drug. The data of permeability of theophylline in different media are shown in Table 3. As shown, permeability in SGF was lower than in SIF for formulations containing EFS and ES which is due to the presence of pH-dependent polymers in the structure of free films and resistance to the acidic medium. Lower permeability in SGF than in SIF was also observed for formulations with ERL and it was in agreement with the swelling results and was due to the effect of ion exchange in different dissolution buffers. Formulations containing only ERS showed a higher permeability in SGF than in SIF that may be explained by the difference between the solubility of theophylline in acidic and in neutral pH which is a little lower in the latter pH (Knop, 1996). Formulations containing In–ERS and In–ERL had lower permeability in SGF and in SIF than in SCF (P < 0.05) which demonstrates the susceptibility of inulin in these formulations to enzyme and degradation in colonic media. However, this difference in the permeability was not significant for the other formulations (P > 0.05). According to these results, inulin in combination with sustained release polymers is more susceptible to the colonic media compared to combination with pH-dependent polymethacrylates. Permeability of formulations containing ERS–ES was not as high as WVT and swelling data of these formulations and the addition of ES to ERS did not increase the permeability
312
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Table 3 – Permeability of different free films to theophylline (data represent mean ± S.D.; n = 3) Ptheo (×10−5 cm/s)
Formulation SGF ERS 100% ERS–In 90–10% ERS–In 80–20% ERS–In 70–30% ERS–ERL 50–50% ERS–ERL–In 45–45–10% ERS–ERL–In 40–40–20% ERS–ERL–In 35–35–30% ERL 100% ERL–In 90–10% ERL–In 80–20% ERL–In 70–30% EFS 100% EFS–In 90–10% EFS–In 80–20% EFS–In 70–30% ERS–ES 50–50% ERS–ES–In 45–45–10% ERS–ES–In 40–40–20% ERS–ES–In 35–35–30%
0.016 0.059 0.209 0.230 1.959 2.296 3.152 3.548 3.495 6.257 6.985 8.146 0.018 0.012 0.032 0.043 0.014 0.198 0.166 0.193
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0 0.009 0.056 0.019 0.028 0.016 0.067 0.128 0.025 0.049 0.778 0.260 0.013 0.005 0 0.009 0.002 0.019 0.025 0.028
to drug as much as ERL. This difference could be explained by difference in the mechanism of water uptake by free films containing ERL and ES. The first polymer swells in the presence of buffer media based on ion exchange and mutual repulsion of the cationic groups which create some large pores and expand the polymeric chains which results in more hydration and also more permeability to drug molecules (Okor, 1982). On the other hand, in our study, free films containing ES could have some voids resulted by evaporation of ammonia molecules before the exposure of films to the media. When exposing free films, water molecules penetrate to these free volumes and cause high water uptake and also water vapor permeation. However, due to the smaller size of pores, it is hard for larger drug molecules to diffuse from these voids and as a result, permeability of polymeric film to drug is not as high as swelling. This phenomenon was confirmed with the observation of the apparent figures of two different kinds of free films. After swelling experiments, films containing ERL had been expanded in size; whereas formulations with ES did not show so much expansion. The results of permeability of indomethacin through different compositions of free films are listed in Table 4. As depicted in Table 4, addition of inulin is effective on high permeability in SCF for all of the formulations (P < 0.05) except for formulations containing ERS–ES–In in the ratio of 45% of each Eudragit. The permeation increasing effect of inulin was also seen in SIF and only formulations with ERL in the ratios of 80 and 70% and EFS in the ratio of 70% had the higher permeability to indomethacin in SCF compared with SIF (P < 0.05). Formulations containing EFS showed the highest permeability in two different media and can be attributed to ionization of functional groups of this polymer, as referred before. Permeability of indomethacin through films constituted of ERL was more than those made of ERS; but the difference in the permeability of indomethacin was not as high as theophylline. Also, permeability of indomethacin through free
SIF 0.012 0.016 0.112 0.198 2.821 2.847 3.891 4.598 5.143 8.981 13.129 13.038 6.861 9.741 11.443 13.343 0.198 0.359 0.385 0.439
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
SCF 0.002 0 0 0.019 0.025 0.114 0.046 0.061 0.088 0.179 0.249 0.367 1.770 3.192 0.252 0.530 0.009 0.040 0.056 0.019
0.007 0.225 0.498 0.594 2.772 3.436 5.074 6.037 8.665 17.427 20.220 30.432 6.920 10.640 11.834 13.648 0.246 0.450 0.476 0.648
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.002 0.016 0.167 0.016 0.033 0.096 0.028 0.070 0.828 0.887 0.931 0.715 0.153 0.567 0.612 1.099 0.009 0.058 0.040 0.074
films containing ERL was lower than the one observed with theophylline, whereas formulations with EFS showed more permeation to indomethacin than for theophylline. This result could be explained by two different causes. The first reason could be the difference in solubility of the two drugs. Studies of film permeation have demonstrated two types of mechanisms for the drug diffusion through a polymeric membrane: either a pore mechanism within water-filled pores present in the film or a solution–diffusion mechanism within the polymeric chains where solvent is not in a bulk state (Blanchon
Table 4 – Permeability of different free films to indomethacin (data represent mean ± S.D.; n = 3) Pindo (×10−5 cm/s)
Formulation SIF ERS 100% ERS–In 90–10% ERS–In 80–20% ERS–In 70–30% ERS–ERL 50–50% ERS–ERL–In 45–45–10% ERS–ERL–In 40–40–20% ERS–ERL–In 35–35–30% ERL 100% ERL–In 90–10% ERL–In 80–20% ERL–In 70–30% EFS 100% EFS–In 90–10% EFS–In 80–20% EFS–In 70–30% ERS–ES 50–50% ERS–ES–In 45–45–10% ERS–ES–In 40–40–20% ERS–ES–In 35–35–30%
1.028 2.087 2.087 2.152 2.280 2.730 2.826 4.399 2.280 3.147 3.789 4.432 30.721 38.964 51.809 61.925 2.087 1.873 2.676 3.854
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
SCF 0.056 0.310 0.310 0.294 0.056 0.729 0.895 0.890 0.874 0.556 1.350 1.015 7.304 5.933 7.688 8.605 0.579 0.093 0.884 1.766
0.963 1.713 1.873 2.194 1.659 3.586 3.104 4.603 1.927 5.245 8.403 7.547 30.454 45.012 54.217 94.466 1.820 2.034 3.532 3.639
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.278 0.093 0.093 0.463 0.093 0.884 1.298 0.093 0 1.298 0.245 1.400 6.748 4.876 5.705 4.657 0.185 0.608 0.278 0.245
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 8 ( 2 0 0 6 ) 307–314
313
Fig. 2 – Eudragit RL or RS molecules scheme.
et al., 1991; Zentner et al., 1979). Regarding the low solubility of indomethacin, when the drug molecules move through the polymeric chain, they have some limitation in diffusing by the second mechanism and then are entrapped in the polymeric film. The difference in the permeability of the two drugs resulted by this mechanism is not seen in the erodible and soluble films like for the formulations containing EFS and also in the films containing low permeable ERS. In the formulations with ERL which have high swelling characteristics, entrapment of indomethacin molecules could be more. The second reason for lower permeation of indomethacin trough free films containing ERL could be the interaction between indomethacin (anionic drug) and ERL (cationic polymer) (Fig. 2) which has been demonstrated in the other studies (Heun et al., 1998). The anionic carboxylic moieties in indomethacin can interact with the cationic ammonio groups of membrane and this ion-exchange reaction immobilizes drug molecules. Obviously, this phenomenon is not occurred by theophylline which is a non ionic drug. Fig. 3 shows the permeability of formulations containing ERL–In in the SIF. In the case of theophylline, permeation rate is linear. However, indomethacin permeability is biphasic which confirms the above mechanism and is in agreement with the other studies involved with ionic drug permeability (Sun et al., 2001). The first step is related to non-interactive solute diffusion through the polymer region and the second slow phase is due to interaction and also entrapment of drug molecules in the membrane. However, regarding the high level of hydration of ERL compared with ERS, inulin is more accessible to enzyme and as a result, formulations with ERL showed a significant higher permeability in SCF than in SIF. Also, as shown in Fig. 4, permeability of indomethacin in SCF for formulations containing higher ratios of inulin becomes a three-phase release system with a higher rate at the third step which is attributed to the more degradation of inulin and the formation of more pores that causes diffusion of drug from these channels. Therefore, free films containing ERL and inulin which have significant difference in the swelling and permeability of drugs in SCF compared to SGF and SIF especially for the low soluble drugs like indomethacin are more promising as a coating system for colonic delivery of these kinds of drugs. Meanwhile, in the case of soluble drugs
Fig. 3 – Permeability profiles of formulations containing Eudragit RL–inulin in SIF for two drugs: (a) theophylline and (b) indomethacin. Error bars indicate S.D. (n = 3).
Fig. 4 – Permeability profiles of free films containing Eudragit RL–inulin in SCF for indomethacin. Error bars indicate S.D. (n = 3).
such as theophylline, the use of ERS in the film structure could be suitable to protect drug from the higher regions of the GI tract and to assure the drug release in colon.
4.
Conclusion
The results of this study revealed that inulin is a suitable polysaccharide for colonic delivery systems because of the susceptibility to enzymes present in the colon. It was shown that formulations containing sustained release polymethacrylates in combination with inulin have more potential as coating systems for specific colon delivery rather than pHdependent polymers. Depending of the solubility and also the ionic properties of drugs, the use of time-dependent polymethacrylates with inulin would be suitable for film coating aimed for colonic drug delivery.
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Acknowledgements This work was done during the scholarship which was supported by I.R. Iran Ministry of Health and Medical Education. The authors also acknowledge Rohm Pharma for free supplying of Eudragit samples.
references
Blanchon, S., Couarraze, G., Rieg-Falson, F., Cohen, G., Puisieux, F., 1991. Permeability of progesterone and a synthetic progestin through methacrylic films. Int. J. Pharm. 72, 1–10. Bodmeier, R., Paeratakul, O., 1994. Mechanical properties of dry and wet cellulosic and acrylic films prepared from aqueous colloidal polymer dispersions used in the coating of solid dosage forms. Pharm. Res. 11, 882–888. Bodmeier, R., Guo, X., Sarabia, R.E., Skultety, P.F., 1996. The influence of buffer species and strength on diltiazem HCl release from beads coated with the aqueous cationic polymer dispersions, Eudragit RS, RL 30D. Pharm. Res. 13, 52–56. Carvalho, R.A., Grosso, C.R.F., 2004. Characterization of gelatin based films modified with transglutamine, glyoxal and formaldehyde. Food Hydrocolloids 18, 717–726. Damian, F., Van den Mooter, G., Samyn, C., Kinget, R., 1999. In vitro biodegradation study of acetyl and methyl inulins by bifidobacteria and inulinase. Eur. J. Pharm. Biopharm. 47, 275–282. George, S.C., Thomas, S., 2001. Transport phenomena through polymeric systems. Prog. Polym. Sci. 26, 985–1017. Heun, G., Lambov, N., Groning, R., 1998. Experimental and molecular modeling studies on interactions between drugs and Eudragit RL/RS resins in aqueous environment. Pharm. Acta Helv. 73, 57–62. Ingels, F., Deferme, S., Destexhe, E., Oth, M., Van den Mooter, G., Augustijns, P., 2002. Simulated intestinal fluid as transport medium in the Caco-2 cell culture model. Int. J. Pharm. 232, 183–192. Knop, K., 1996. Influence of buffer solution composition on drug release from pellets coated with neutral and quaternary acrylic polymers and on swelling of free polymer films. Eur. J. Pharm. Sci. 4, 293–300.
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Lin, W.J., Lu, C.H., 2002. Characterization and permeation of microporous poly(-caprolactone) films. J. Membr. Sci. 198, 109–118. Okor, R.S., 1982. Effect of polymer cation content on certain film properties. J. Pharm. Pharmacol. 34, 83–86. Pandey, P., Chauhan, R.S., 2001. Membranes for gas separation. Prog. Polym. Sci. 26, 853–893. Parra, D.F., Tadini, C.C., Ponce, P., Lugao, A.B., 2004. Mechanical properties and water vapor transmission in some blends of cassava starch edible films. Carbohydr. Polym. 58, 475–481. Sinha, V.R., Kumria, R., 2001. Polysaccharides in colon-specific drug delivery. Int. J. Pharm. 224, 19–38. Sun, Y.M., Hsu, S.C., Lai, J.Y., 2001. Transport properties of ionic drugs in the ammonia methacrylate copolymer membranes. Pharm. Res. 18, 304–310. 2003. US Pharmacopeia XXVI. US Pharmacopeial Convention, Rockville, MD, pp. 2524–2528. Van den Mooter, G., Vervoort, L., Kinget, R., 2003. Characterization of methacrylated inulin hydrogels designed for colon targeting: in vitro release of BSA. Pharm. Res. 20, 303–307. Van den Mooter, G., Samyn, C., Kinget, R., 1994. Characterization of colon-specific azo polymers: a study of the swelling properties and the permeability of isolated polymer films. Int. J. Pharm. 111, 127–136. Vervoort, L., Rombaut, P., Van den Mooter, G., Augustijns, P., Kinget, R., 1998. Inulin hydrogels. II. In vitro degradation study. Int. J. Pharm. 172, 137–145. Vervoort, L., Kinget, R., 1996. In vitro degradation by colonic bacteria of inulin HP incorporated in Eudragit RS films. Int. J. Pharm. 129, 185–190. Wagner, K.G., McGinity, J.W., 2002. Influence of chloride ion exchange on the permeability and drug release of Eudragit RS 30D films. J. Contr. Release 82, 385–397. Wang, Z.F., Wang, B., Ding, X.M., Zhang, M., Liu, L.M., Qi, N., Hu, J.L., 2004. Effect of temperature and structure on the free volume and water vapor permeability in hydrophilic polyurethanes. J. Membr. Sci. 241, 355–361. Zentner, G.M., Cardinal, J.R., Feijen, J., Song, S.S., 1979. Progestin permeation through polymer membranes. IV. Mechanism of steroid permeation and functional group contributions to diffusion through hydrogel films. J. Pharm. Sci. 68, 970–975. Zhou, H.W., 1994. Overcoming enzymatic and absorption barriers to non-parenterally administered protein and peptide drugs. J. Contr. Release 29, 239–252.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ejps
Permeability and swelling studies on free films containing inulin in combination with different polymethacrylates aimed for colonic drug delivery A. Akhgari a,∗ , F. Farahmand a , H. Afrasiabi Garekani a , F. Sadeghi a , T.F. Vandamme b a
School of Pharmacy and Pharmaceutical Research Centre, Vakilabad blvd., PO Box 91775-1365, Mashhad University of Medical Science, Mashhad, Iran b D´epartement de Chimie Bioorganique, LC 01, UMR 7514, Facult´e de Pharmacie, Universit´e Louis Pasteur, Strasbourg, France
a r t i c l e
i n f o
a b s t r a c t
Article history:
The aim of this study was to assess some permeability and swelling characteristics of free
Received 30 January 2006
films prepared by combination of inulin as a bacterially degradable system and time- or pH-
Received in revised form 12 March
dependent polymers as a coating formulation for colonic drug delivery. Different free films
2006
were prepared by casting and solvent evaporation method. Formulations containing inulin
Accepted 19 March 2006
with Eudragit RS, Eudragit RL, Eudragit RS–Eudragit RL, Eudragit FS and Eudragit RS–Eudragit
Published on line 28 March 2006
S with different ratios of inulin were prepared. After preparation, free films were evaluated by water vapor transmission test, swelling experiment and permeability to indomethacin
Keywords:
and theophylline in different media. Formulations containing Eudragit FS had high resis-
Inulin
tance to water vapor permeation; but were unable to protect premature swelling and drug
Free film
release in simulated small intestine media. Also, combination of Eudragit RS and Eudragit
Permeability
S had no suitable characteristics for colon delivery. However, Eudragit RS and Eudragit RL in
Swelling
combination with inulin made free films which had more swelling and permeation of drug
Water vapor transmission
in the colonic medium rather than the other media. It was shown that formulations contain-
Colonic delivery
ing sustained release polymethacrylates in combination with inulin have more potential as a coating system for specific colon delivery compared with pH-dependent polymers.
Abbreviations:
© 2006 Elsevier B.V. All rights reserved.
ERS, Eudragit RS; ERL, Eudragit RL; EFS, Eudragit FS; ES, Eudragit S; In, inulin; WVT, water vapor transmission; SGF, simulated gastric fluid; SIF, simulated intestinal fluid; SCF, simulated colonic fluid
1.
Introduction
Targeting of drugs to the large intestine has a number of important implications in the field of pharmacotherapy. These are treatments of colonic disorders such as ulcerative col-
∗
Corresponding author. Tel.: +98 8823255/6; fax: +98 8823251. E-mail address: akhgari [email protected] (A. Akhgari).
0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2006.03.005
itis and Crohn’s disease and also the oral administration of protein and peptide drugs, which are normally degrade by the enzymes of the upper gastrointestinal tract (Zhou, 1994). Various approaches have been used for oral delivery of drugs to the colon, which include pH-dependent systems,
308
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 8 ( 2 0 0 6 ) 307–314
time-dependent systems, and systems which are bacterially degraded in the colon. Among these strategies, systems relying on the metabolic activity of the colonic microflora can be more site-specific and a large number of polysaccharides have recently been proposed for development of colon-specific drug delivery devices (Sinha and Kumria, 2001). Inulin is a naturally occurring polysaccharide which is not significantly hydrolyzed by gastric or intestinal enzymes. Colonic bacteria however, and more specifically Bifidobacteria can degrade this polysaccharide (Van den Mooter et al., 2003). However, its fairly good water solubility makes it difficult to be used as a carrier for colonic drug delivery. Several methods such as synthesized different derivatives of inulin with reduced water solubility (Van den Mooter et al., 2003; Damian et al., 1999; Vervoort et al., 1998) or mixing it with Eudragit RS as a coating formulation (Vervoort and Kinget, 1996) have been used. The aim of this study was to evaluate the permeability and swelling characteristics of isolated films prepared by mixing of inulin with either sustained-release or pH-dependent polymethacrylates and also combination of these two kinds of polymers. To assess the resistance of a colon-specific coating to the gastric and intestinal fluids and also susceptibility to the colonic media, it is necessary to investigate the diffusion of drug molecules through the films and also the swelling of free films under conditions simulating these media. On the other hand, water vapor transmission study gives some valuable information on the protection of a coated dosage form against environment humidity (Van den Mooter et al., 1994). Permeability studies were investigated for two drugs having different solubilities; theophylline as a soluble and indomethacin as a low soluble drug model.
2.
Materials and methods
2.1.
Materials
Eudragit RS100 (ERS), Eudragit RL100 (ERL), Eudragit S100 (ES) and Eudragit FS30D (EFS) (Rohm Pharma, Germany), inulin (Raftiline HP, Orafti, France), inulinase from aspergillus niger (Sigma, USA), triethyl citrate (Morflex, USA), theophylline monohydrate (Cooper Rhone, France), indomethacin (Fluka, Italy), magnesium nitrate hexahydrate (Mg(NO3 )2 ·6H2 O) (Aldrich, Germany), phosphorus pentoxide (Acros organics, USA) were supplied from indicated sources. All chemicals were of analytical grade.
2.2.
Preparation of free films
2.2.1. Preparation of free films containing inulin–Eudragit RS–Eudragit RL A 10% (w/v) solution of Eudragit RS100, Eudragit RL100 or 1:1 ratio of two polymers was prepared by dissolving granules of these polymers in isopropyl alcohol:distilled water (9:1 ratio) solvent. Then, a fixed amount of triethyl citrate (1:6 ratio related to total polymer content) was added as a plasticizer. Specific amounts of inulin powder were dissolved in distilled water by keeping warming the solution. Inulin solution was gently added to Eudragit solution with continuous
stirring. The ratios of inulin to Eudragit content were 0, 10, 20 and 30% (w/w). The resulted suspension was transferred to Teflon plates. Volume of suspension was 25 ml in each plate. The plates were then placed in an oven at 50 ◦ C for 24 h for complete drying. After that, plates were transferred to a desiccator with 100% relative humidity (RH) for 24 h. Then, the films were cut with a scalpel to different special pieces for various tests. The thickness of the films was measured at five different ¨ places by using a micrometer (Kafer, Germany) and the average thickness of 150–170 m was selected. Free films were stored in a desiccator with 50% RH resulted by a saturated solution of magnesium nitrate hexahydrate at room temperature until use.
2.2.2. Preparation of free films containing inulin–Eudragit FS30D Firstly, specific amounts of inulin were dissolved in warmed distilled water. Then, a fixed amount of triethyl citrate (1:30 ratio related to total polymer content) was added to solution as a plasticizer. Then, predetermined amounts of Eudragit FS30D were added to this solution with stirring. Final dispersion was stirred by using a magnetic stirrer for 5 h and then poured into Teflon plates. The ratios of inulin to Eudragit content and also next steps of film preparation and storage were the same with Section 2.2.1.
2.2.3. Preparation of free films containing inulin–Eudragit RS–Eudragit S Various amounts of granules of Eudragit RS100 were dissolved in isopropyl alcohol:distilled water (9:1 ratio). The solution was plasticized with a constant amount of triethyl citrate (1:6 ratio related to total polymer content). Separately, specific amounts of inulin were dissolved in warmed distilled water and specific amounts of Eudragit S100 powder were added to inulin solution. The resulted suspension was dissolved by adding droplets of ammonia and partial neutralizing of Eudragit S. Then, Eudragit RS solution was gently added with continuous stirring to the solution containing inulin and Eudragit S. Neutralization of Eudragit S made it possible to mix this polymer with inulin and Eudragit RS and film preparation. The resulted suspension was poured into teflon plates. The ratio of Eudragit RS to Eudragit S was 1:1 in all of formulations. The ratios of inulin to Eudragit content and also next steps of film preparation and storage were the same with Section 2.2.1.
2.3.
Water vapor transmission test
Water vapor permeation of free films was determined gravimetrically in triplicate. The permeability cups were 3.5 cm in diameter. The inside of the cup was filled with 10 ml of distilled water (100% RH). A circular piece of free films was placed on the cup and a gasket placed on the film followed by a metal ring. The film was held in place with the help of three screw clamps. Another circular piece of the same free film fixed on another cup without water as a reference. Both sample and reference were accurately weighed (±0.0001 g) and then placed in a desiccator containing phosphorus pentoxide (P2 O5 ) as a desiccant and filled with silicagel (0% RH). At specific intervals (24, 48, 72, 96 and 120 h) the cups were weighed (±0.0001 g) and the profile of mass change was plotted versus time for
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 8 ( 2 0 0 6 ) 307–314
each free film. Water vapor transmission (WVT) was calculated using following equation: WVT =
wx tAP0 (RH1 − RH2 )
(1)
where w/t is the mass change (flux, g/h) resulted from slope of profile of the mass change versus time, x the film thickness (mm), A the area of the film surface exposed to the permeant (m2 ), P0 the vapor pressure of pure water (kPa), and (RH1 − RH2 ) is the relative humidity gradient. At 25 ◦ C, P0 is 3.159 kPa (Carvalho and Grosso, 2004).
2.4.
Swelling experiments
Firstly, a piece of 1 cm2 of each free film was dried in an oven at 50 ◦ C for 24 h. Then, dried film was accurately weighed (±0.0001 g) and immersed in a flask of dissolution test containing 250 ml of different media at 37 ◦ C. At specific intervals, the swollen sample was withdrawn from the medium and weighed (±0.0001 g) after removal of excess surface water ¨ by light blotting with a filter paper (Carl Schleicher & Schull, Germany). During the first 10 min, intervals of sampling were 1 min. Sampling time was gradually increased after this period until 3 h. To quantify the swelling process, the swelling index, Is (%), was calculated as follows (Blanchon et al., 1991): Is (%) =
W s − Wd × 100 Wd
(2)
where Wd is the weight of the dried polymer film and Ws denotes the weight after swelling. Swelling tests were separately carried out in simulated gastric fluid (SGF) without pepsin, simulated intestinal fluid (SIF) without pancreatin with pH 6.8 (USP XXVI, 2003) and also simulated colonic fluid (SCF) with adding of 1 ml/l of inulinase to the media with pH 6.4. In case of formulations containing In–EFS and In–ERS–ES, swelling characteristics were also investigated in media with pH 6.4 as SIF. All of the experiments were carried out in triplicate.
2.5.
Permeability test
Isolated films of the polymers were mounted between the donor and acceptor compartments of a side-by-side diffusion cell (diffusion area 3.46 cm2 ). Temperature of the cells was kept at 37 ◦ C throughout the experiments by continuous circulating of water and each compartment was stirred continuously with a magnetic stirrer. Different experimental conditions were set up to examine the permeability of drugs through polymer films; for theophylline, the initial concentration of drug in the donor compartment was 3 g/l. The donor and acceptor compartments were both composed of SGF, SIF and SCF with inulinase. SGF and SCF were the same for all of the formulations. SIF was the medium with pH 6.8 for all of the formulations except formulations containing In–EFS for which the medium with pH 6.4 was supposed as SIF. For indomethacin, the initial concentrations of drug solutions in the donor compartment were 300 mg/l for SIF with pH 6.4 and 6.8 and SCF, and 500 mg/l for SIF with pH 7.2. To achieve the sink conditions for permeability experiments, nearly saturated concentration
309
of drug was used in the donor compartment. Regarding the low solubility of indomethacin in more acidic pH, the lower concentration of drug solution was used in the media with pH 6.4 and 6.8 rather than medium with pH 7.2. The donor and acceptor compartment were both filled with SIF or SCF. The medium with pH 7.2 was used as SIF for evaluating formulations containing In–ERS–ERL; but in case of formulations containing In–EFS and In–ERS–ES, regarding dissolution of pHdependent polymers in this pH, the media with pH 6.4 and 6.8 were used as SIF, respectively. SCF medium was the same for all of the formulations. All of the permeability experiments were carried out for 3 h. At predetermined time intervals, samples of 10 ml were taken from the receptor cells and replaced with fresh medium. The contents of the acceptor cells were assayed spectrophotometrically for theophylline and indomethacin at 272 and 318 nm, respectively. Each permeation experiment was repeated three times and the cumulative amount of drug permeated and corrected for the acceptor sample replacement was plotted against time. Applying Fick’s first law of diffusion in sink conditions, the permeation rate of drug was defined as dM = PSCd dt
(3)
where M was the amount of drug diffused (mg) at time t, S the effective diffusion area (cm2 ), Cd the concentration of drug in the donor compartment and P was the permeability (cm/s). Then, the permeability was obtained by the following equation: P=
slope SCd
(4)
where the slope was obtained from the plot of the amount of drug permeated versus time (Lin and Lu, 2002; Ingels et al., 2002).
2.6.
Statistical analysis of data
One-way analysis of variance was used to assess the significance of the differences among different groups. Tukey–Kramer post-test was used to compare the means of different treatment groups. Results with P < 0.05 were considered to be statistically significant.
3.
Results and discussion
3.1.
WVT experiments
According to Fig. 1 the rate of water vapor permeation was constant for free films containing Eudragit FS and inulin. This linear relation between amount of vapor permeated and time was also observed for the other formulations. Table 1 lists the results of WVT experiments for all the formulations. As shown in Table 1, free films containing Eudragit FS and Eudragit RS had the lowest permeability to water vapor in all the formulations (P < 0.01). It is well known that increasing the hydrophilic
310
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 8 ( 2 0 0 6 ) 307–314
Fig. 1 – Profiles of water vapor transmission through free films containing Eudragit FS–inulin. Error bars indicate S.D. (n = 3).
nature of a polymer membrane induces water vapor tendency and as a result increases water vapor permeation (Wang et al., 2004; Parra et al., 2004). Eudragit RS and RL are acrylic and methacrylic acid esters with some hydrophilic properties due to the presence of quaternary ammonium groups where ERL possesses higher amount of such groups than ERS. Then, it is expected that formulations containing ERL exhibit higher water vapor permeability than ERS. The order of WVT for free films containing Eudragit RL and RS was as followed: ERL > ERL–ERS > ERS. Also, low permeability of formulations containing Eudragit FS to water vapor is due to low hydrophilic character of this polymer. Nevertheless, formulations with EFS and ERS had no significant difference in WVT (P > 0.05) except in the case of free films containing 30% inulin. This result can be explained by a different film preparation for formulations of EFS and ERS. Free films containing Eudragit FS were prepared from aqueous dispersion of polymer, whereas Eudragit RS films were made from an organic solution. Bodmeier and Paeratakul (1994) have shown that films cast from organic solutions have a tighter and
a more compact structure than those prepared from aqueous dispersions and this is due to the tighter bound plasticizer in their polymeric chains. Therefore, this phenomenon could lower water vapor permeability of polymeric films and compensate the effect of hydrophobic character of Eudragit FS. As the overall result, WVT of formulations containing EFS and ERS was almost the same. As shown in Table 1, formulations containing Eudragit S in combination with Eudragit RS had the high WVT. This is due to partial neutralization of carboxylic groups of Eudragit S by ammonia that causes less hydrophobic character and finally less resistance to water vapor. Also, the presence of ammonia in the film solution and its rapid evaporation could make some free volume which leads to more diffusion of penetrant in the polymeric chains (Pandey and Chauhan, 2001; George and Thomas, 2001). Addition of inulin to the film formulation significantly increased WVT (P < 0.05). This result is also in agreement with increasing the hydrophilic nature of membrane by inulin and as a result increasing vapor permeability of water. Only in formulations containing ERL in the ratio of 70–90%, addition of inulin had no significant effect (P > 0.05).
3.2.
Swelling test
The results of swelling experiments are listed in Table 2. Formulations containing only ERS had low swelling index in SGF and SIF. Swelling of free films in SCF significantly increased with addition of inulin up to the ratio of 30% (P < 0.05). This is due to the presence of inulinase enzyme in the media which can diffuse into the polymeric chains, hydrolyze the fructose backbone of inulin, reduce the network density and finally increase swelling (Vervoort et al., 1998). In the case of formulations containing ERS–ERL, swelling was higher in SIF than SGF. This is in contrast with pH-independency of these polymers and can be due to difference in the swelling media.
Table 1 – Water vapor transmission of free films Formulation ERS 100% ERS–In 90–10% ERS–In 80–20% ERS–In 70–30% ERS–ERL 50–50% ERS–ERL–In 45–45–10% ERS–ERL–In 40–40–20% ERS–ERL–In 35–35–30% ERL 100% ERL–In 90–10% ERL–In 80–20% ERL–In 70–30% EFS 100% EFS–In 90–10% EFS–In 80–20% EFS–In 70–30% ERS–ES 50–50% ERS–ES–In 45–45–10% ERS–ES–In 40–40–20% ERS–ES–In 35–35–30%
Film thickness (mean ± S.D.; n = 15) (m)
Mass change (mg/h)
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.54 5.47 7.73 15.82 10.80 12.33 20.32 20.72 22.86 25.58 27.26 27.32 3.95 6.32 9.32 10.06 14.32 21.15 24.83 25.16
158 160 167 160 159 159 162 170 163 160 160 160 152 160 152 161 160 162 159 160
8 8 5 6 5 8 8 5 6 10 8 8 6 11 9 7 5 4 7 8
WVT (mean ± S.D.; n = 3) (mg mm/m2 h kPa) 2.365 2.886 4.248 8.347 5.650 6.451 10.855 11.616 12.262 13.498 14.381 14.412 1.997 3.336 4.671 5.352 7.555 11.298 12.991 13.275
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.060 0.344 0.078 0.039 0.041 0.188 0.192 0.430 0.230 0.672 0.377 0.566 0.030 0.047 0.221 0.076 0.291 0.139 0.095 1.304
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 8 ( 2 0 0 6 ) 307–314
Table 2 – Swelling of formulations in different media (data represent mean ± S.D.; n = 3) Formulation
Maximum swelling index, Is (%) SGF
ERS 100% ERS–In 90–10% ERS–In 80–20% ERS–In 70–30% ERS–ERL 50–50% ERS–ERL–In 45–45–10% ERS–ERL–In 40–40–20% ERS–ERL–In 35–35–30% ERL 100% ERL–In 90–10% ERL–In 80–20% ERL–In 70–30% EFS 100% EFS–In 90–10% EFS–In 80–20% EFS–In 70–30% ERS–ES 50–50% ERS–ES–In 45–45–10% ERS–ES–In 40–40–20% ERS–ES–In 35–35–30% a
10.3 17.3 15.8 19.3 30.6 36.7 31.0 36.5 44.8 50.1 54.0 58.2 18.6 18.1 21.1 31.9 87.8 63.8 18.9 24.5
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.4 8.0 2.1 3.9 1.0 3.1 8.1 4.9 8.6 11.3 2.8 4.3 8.3 3.7 3.7 3.4 6.2 13.2 3.2 2.0
SIF 12.4 13.6 20.2 19.9 41.9 47.1 62.7 50.8 62.3 106.5 103.9 108.1 52.0 46.8 76.1 82.5 151.5 156.9 147.5 117.9
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
SCF 0.3 1.0 5.1 3.3 2.0 2.7 4.9 2.4 3.2 21.3 13.4 11.2 7.0a 1.8a 7.5a 1.1a 15.0a 4.0a 0.1a 1.8a
13.6 15.4 23.8 37.1 47.5 71.2 81.4 77.3 80.1 222.5 183.4 150.4 66.6 78.4 70.1 58.4 185.7 162.5 165.2 141.7
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.6 0.3 1.9 7.1 4.4 6.9 8.4 10.9 6.6 35.4 33.3 7.9 11.9 4.9 5.0 1.1 4.1 6.8 3.0 1.6
Data are swelling in the media with pH 6.4.
Eudragit RL and RS have quaternary ammonium groups which are in the chloride salt form. The dissociation of these quaternary ammonium groups in aqueous media is responsible for the hydration and swelling of the polymer films. Indeed, the exchange of chloride ion with the buffer anions of the dissolution medium could govern the degree of hydration and swelling. The SGF media had chloride anion due to hydrochloric acid and sodium chloride which had less selectivity to ion exchange than phosphate and then reduce swelling of polymer in SGF compared with SIF media (Wagner and McGinity, 2002; Bodmeier et al., 1996). On the other hand, regarding more ammonium groups in ERL than ERS, swelling of formulations containing ERL is higher and also the difference in swelling in the SGF and SIF can be more pronounced. Also, in the case of formulations containing only ERL, swelling in SIF was higher than in SGF which confirms this fact. Swelling of formulations containing EFS was lower in SGF than in SIF and in SCF (P < 0.001) because this polymer is pHdependent and in the acidic pH, it is resistant to dissolution media with low pH whereas in SIF polymer tends to ionize and swell, so that in the media with pH 6.8, free films were dissolved and the swelling was not possible. Therefore, the media with pH 6.4 was used as SIF. Swelling index in SIF increased with the addition of inulin in the ratios of 20 and 30% (P < 0.01) which is due to hygroscopic characteristics of inulin and the higher water uptake of polymer. Addition of inulin up to 10% caused increasing swelling in SCF which is due to enzyme activity. However, swelling was surprisingly decreased in the higher ratios of inulin and in the mixture of EFS–In (70:30), swelling ratio in SCF was lower than in SIF (P < 0.01). This result can be explained by leaching out of inulin in the high ratios of this polysaccharide. Regarding the ionization of carboxylic groups of Eudragit FS and its swelling, this polymer cannot protect inulin anymore and the polysaccharide
311
which is partially degraded before by enzyme, excludes from the polymeric chains and as the overall result, the swelling index is decreased. This decrease in swelling by inulin ratio of 30% compared with the other ratios was also shown in the other formulations except for films containing only ERS. Totally, formulations containing EFS have not good potential as a coating system for colonic delivery because of dissolution of the polymer in the media simulating small intestine and EFS cannot protect inulin in this media. Formulations containing ERS–ES had high swelling ratios in SGF and in SIF except for formulations containing 20 and 30% of inulin which had low swelling index in SGF. The high swelling of the formulations having the higher ratios of Eudragit S can be attributed to the presence of some voids resulted by ammonia evaporation and therefore more easiness to water uptake. The decrease in the swelling by increasing inulin level in SCF was observed and could be the result of leaching it through free volumes of polymers in the higher ratio of polysaccharide. Formulations containing ES had so high swelling index in SIF with pH 6.8 that it was not possible to take and weight the films after about 1 h. Therefore, swelling in SIF with pH 6.4 was determined. This is due to the increase in dissolution of ES with the increase in media pH. Totally, formulations containing pH-dependent polymers could not protect free films from high swelling in the media simulating small intestine which is important for a colonic drug delivery system. Also, the ratio of swelling index in SCF to SIF as a key parameter to assess potential of polymeric system for colonic delivery was higher in the formulations containing time-dependent polymers with inulin than free films with pHdependent polymethacrylates.
3.3.
Permeability experiments
Permeability tests were carried out for two kinds of drugs: theophylline as a soluble model and indomethacin as a low soluble drug. The data of permeability of theophylline in different media are shown in Table 3. As shown, permeability in SGF was lower than in SIF for formulations containing EFS and ES which is due to the presence of pH-dependent polymers in the structure of free films and resistance to the acidic medium. Lower permeability in SGF than in SIF was also observed for formulations with ERL and it was in agreement with the swelling results and was due to the effect of ion exchange in different dissolution buffers. Formulations containing only ERS showed a higher permeability in SGF than in SIF that may be explained by the difference between the solubility of theophylline in acidic and in neutral pH which is a little lower in the latter pH (Knop, 1996). Formulations containing In–ERS and In–ERL had lower permeability in SGF and in SIF than in SCF (P < 0.05) which demonstrates the susceptibility of inulin in these formulations to enzyme and degradation in colonic media. However, this difference in the permeability was not significant for the other formulations (P > 0.05). According to these results, inulin in combination with sustained release polymers is more susceptible to the colonic media compared to combination with pH-dependent polymethacrylates. Permeability of formulations containing ERS–ES was not as high as WVT and swelling data of these formulations and the addition of ES to ERS did not increase the permeability
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Table 3 – Permeability of different free films to theophylline (data represent mean ± S.D.; n = 3) Ptheo (×10−5 cm/s)
Formulation SGF ERS 100% ERS–In 90–10% ERS–In 80–20% ERS–In 70–30% ERS–ERL 50–50% ERS–ERL–In 45–45–10% ERS–ERL–In 40–40–20% ERS–ERL–In 35–35–30% ERL 100% ERL–In 90–10% ERL–In 80–20% ERL–In 70–30% EFS 100% EFS–In 90–10% EFS–In 80–20% EFS–In 70–30% ERS–ES 50–50% ERS–ES–In 45–45–10% ERS–ES–In 40–40–20% ERS–ES–In 35–35–30%
0.016 0.059 0.209 0.230 1.959 2.296 3.152 3.548 3.495 6.257 6.985 8.146 0.018 0.012 0.032 0.043 0.014 0.198 0.166 0.193
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0 0.009 0.056 0.019 0.028 0.016 0.067 0.128 0.025 0.049 0.778 0.260 0.013 0.005 0 0.009 0.002 0.019 0.025 0.028
to drug as much as ERL. This difference could be explained by difference in the mechanism of water uptake by free films containing ERL and ES. The first polymer swells in the presence of buffer media based on ion exchange and mutual repulsion of the cationic groups which create some large pores and expand the polymeric chains which results in more hydration and also more permeability to drug molecules (Okor, 1982). On the other hand, in our study, free films containing ES could have some voids resulted by evaporation of ammonia molecules before the exposure of films to the media. When exposing free films, water molecules penetrate to these free volumes and cause high water uptake and also water vapor permeation. However, due to the smaller size of pores, it is hard for larger drug molecules to diffuse from these voids and as a result, permeability of polymeric film to drug is not as high as swelling. This phenomenon was confirmed with the observation of the apparent figures of two different kinds of free films. After swelling experiments, films containing ERL had been expanded in size; whereas formulations with ES did not show so much expansion. The results of permeability of indomethacin through different compositions of free films are listed in Table 4. As depicted in Table 4, addition of inulin is effective on high permeability in SCF for all of the formulations (P < 0.05) except for formulations containing ERS–ES–In in the ratio of 45% of each Eudragit. The permeation increasing effect of inulin was also seen in SIF and only formulations with ERL in the ratios of 80 and 70% and EFS in the ratio of 70% had the higher permeability to indomethacin in SCF compared with SIF (P < 0.05). Formulations containing EFS showed the highest permeability in two different media and can be attributed to ionization of functional groups of this polymer, as referred before. Permeability of indomethacin through films constituted of ERL was more than those made of ERS; but the difference in the permeability of indomethacin was not as high as theophylline. Also, permeability of indomethacin through free
SIF 0.012 0.016 0.112 0.198 2.821 2.847 3.891 4.598 5.143 8.981 13.129 13.038 6.861 9.741 11.443 13.343 0.198 0.359 0.385 0.439
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
SCF 0.002 0 0 0.019 0.025 0.114 0.046 0.061 0.088 0.179 0.249 0.367 1.770 3.192 0.252 0.530 0.009 0.040 0.056 0.019
0.007 0.225 0.498 0.594 2.772 3.436 5.074 6.037 8.665 17.427 20.220 30.432 6.920 10.640 11.834 13.648 0.246 0.450 0.476 0.648
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.002 0.016 0.167 0.016 0.033 0.096 0.028 0.070 0.828 0.887 0.931 0.715 0.153 0.567 0.612 1.099 0.009 0.058 0.040 0.074
films containing ERL was lower than the one observed with theophylline, whereas formulations with EFS showed more permeation to indomethacin than for theophylline. This result could be explained by two different causes. The first reason could be the difference in solubility of the two drugs. Studies of film permeation have demonstrated two types of mechanisms for the drug diffusion through a polymeric membrane: either a pore mechanism within water-filled pores present in the film or a solution–diffusion mechanism within the polymeric chains where solvent is not in a bulk state (Blanchon
Table 4 – Permeability of different free films to indomethacin (data represent mean ± S.D.; n = 3) Pindo (×10−5 cm/s)
Formulation SIF ERS 100% ERS–In 90–10% ERS–In 80–20% ERS–In 70–30% ERS–ERL 50–50% ERS–ERL–In 45–45–10% ERS–ERL–In 40–40–20% ERS–ERL–In 35–35–30% ERL 100% ERL–In 90–10% ERL–In 80–20% ERL–In 70–30% EFS 100% EFS–In 90–10% EFS–In 80–20% EFS–In 70–30% ERS–ES 50–50% ERS–ES–In 45–45–10% ERS–ES–In 40–40–20% ERS–ES–In 35–35–30%
1.028 2.087 2.087 2.152 2.280 2.730 2.826 4.399 2.280 3.147 3.789 4.432 30.721 38.964 51.809 61.925 2.087 1.873 2.676 3.854
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
SCF 0.056 0.310 0.310 0.294 0.056 0.729 0.895 0.890 0.874 0.556 1.350 1.015 7.304 5.933 7.688 8.605 0.579 0.093 0.884 1.766
0.963 1.713 1.873 2.194 1.659 3.586 3.104 4.603 1.927 5.245 8.403 7.547 30.454 45.012 54.217 94.466 1.820 2.034 3.532 3.639
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.278 0.093 0.093 0.463 0.093 0.884 1.298 0.093 0 1.298 0.245 1.400 6.748 4.876 5.705 4.657 0.185 0.608 0.278 0.245
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 8 ( 2 0 0 6 ) 307–314
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Fig. 2 – Eudragit RL or RS molecules scheme.
et al., 1991; Zentner et al., 1979). Regarding the low solubility of indomethacin, when the drug molecules move through the polymeric chain, they have some limitation in diffusing by the second mechanism and then are entrapped in the polymeric film. The difference in the permeability of the two drugs resulted by this mechanism is not seen in the erodible and soluble films like for the formulations containing EFS and also in the films containing low permeable ERS. In the formulations with ERL which have high swelling characteristics, entrapment of indomethacin molecules could be more. The second reason for lower permeation of indomethacin trough free films containing ERL could be the interaction between indomethacin (anionic drug) and ERL (cationic polymer) (Fig. 2) which has been demonstrated in the other studies (Heun et al., 1998). The anionic carboxylic moieties in indomethacin can interact with the cationic ammonio groups of membrane and this ion-exchange reaction immobilizes drug molecules. Obviously, this phenomenon is not occurred by theophylline which is a non ionic drug. Fig. 3 shows the permeability of formulations containing ERL–In in the SIF. In the case of theophylline, permeation rate is linear. However, indomethacin permeability is biphasic which confirms the above mechanism and is in agreement with the other studies involved with ionic drug permeability (Sun et al., 2001). The first step is related to non-interactive solute diffusion through the polymer region and the second slow phase is due to interaction and also entrapment of drug molecules in the membrane. However, regarding the high level of hydration of ERL compared with ERS, inulin is more accessible to enzyme and as a result, formulations with ERL showed a significant higher permeability in SCF than in SIF. Also, as shown in Fig. 4, permeability of indomethacin in SCF for formulations containing higher ratios of inulin becomes a three-phase release system with a higher rate at the third step which is attributed to the more degradation of inulin and the formation of more pores that causes diffusion of drug from these channels. Therefore, free films containing ERL and inulin which have significant difference in the swelling and permeability of drugs in SCF compared to SGF and SIF especially for the low soluble drugs like indomethacin are more promising as a coating system for colonic delivery of these kinds of drugs. Meanwhile, in the case of soluble drugs
Fig. 3 – Permeability profiles of formulations containing Eudragit RL–inulin in SIF for two drugs: (a) theophylline and (b) indomethacin. Error bars indicate S.D. (n = 3).
Fig. 4 – Permeability profiles of free films containing Eudragit RL–inulin in SCF for indomethacin. Error bars indicate S.D. (n = 3).
such as theophylline, the use of ERS in the film structure could be suitable to protect drug from the higher regions of the GI tract and to assure the drug release in colon.
4.
Conclusion
The results of this study revealed that inulin is a suitable polysaccharide for colonic delivery systems because of the susceptibility to enzymes present in the colon. It was shown that formulations containing sustained release polymethacrylates in combination with inulin have more potential as coating systems for specific colon delivery rather than pHdependent polymers. Depending of the solubility and also the ionic properties of drugs, the use of time-dependent polymethacrylates with inulin would be suitable for film coating aimed for colonic drug delivery.
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Acknowledgements This work was done during the scholarship which was supported by I.R. Iran Ministry of Health and Medical Education. The authors also acknowledge Rohm Pharma for free supplying of Eudragit samples.
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