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Preparation, in vitro and in vivo evaluation of stomach-specific metronidazole-loaded alginate beads as local anti-Helicobacter pylori therapy
Journal of Controlled Release 119 (2007) 207 – 214 www.elsevier.com/locate/jconrel
Preparation, in vitro and in vivo evaluation of stomach-specific metronidazole-loaded alginate beads as local anti-Helicobacter pylori therapy Rania A.H. Ishak a,⁎, Gehanne A.S. Awad a , Nahed D. Mortada a , Samia A.K. Nour b a
Department of Pharmaceutics, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt b Department of Pharmaceutics, Faculty of Pharmacy, Cairo University, Cairo, Egypt Received 10 May 2006; accepted 16 February 2007 Available online 1 March 2007
Abstract Metronidazole (MZ), a common antibacterial drug used in treatment of H. pylori, was prepared in chitosan-treated alginate beads by the ionotropic gelation method. A (3 × 2 × 2) factorially designed experiment was used in which 3 viscosity-imparting polymers namely, methyl cellulose, carbopol 934P and κ-carrageenan, 2 concentrations (0.2 and 0.4% w/v) of chitosan as encapsulating polymer and 2 concentrations (2.5 and 5% w/w) of the low density magnesium stearate as a floating aid were tested. The drug entrapment efficiency (%), the percent of floating beads and the time for 80% of the drug to be released (T80%) were the responses evaluated. The bead formula containing 0.5% κ-carrageenan, 0.4% chitosan and 5% magnesium stearate showed immediate buoyancy, optimum drug entrapment efficiency and extended drug release. The histopathological examination of mice stomachs and in vivo H. pylori clearance tests were carried out by orally administering MZ floating alginate beads or MZ suspension, to H. pylori infected mice under fed conditions as a single daily dose for 3 successive days in different doses 5, 10, 15 and 20 mg/kg. The histopathological examination showed that groups receiving MZ in the form of floating alginate beads at doses 10, 15 and 20 mg/kg were better than the corresponding suspension form, regarding eradication of H. pylori infection. The in vivo H. pylori clearance tests showed that MZ floating beads with a dose of 15 mg/kg provided 100% clearance rate whereas the MZ suspension with a dose of 20 mg/kg gave only 33.33%. © 2007 Elsevier B.V. All rights reserved. Keywords: Alginate beads; Floating drug delivery systems (FDDS); Metronidazole; Sustained release; H. pylori clearance; Local delivery
1. Introduction The local treatment of H. pylori with conventional tablets or capsules may fail, since these may fall to the base of the stomach from where they are readily emptied [1]. Little, if any, drug is delivered to the body or fundus of the stomach and the main drug action is through systemic effect. Various approaches have been pursued to increase the retention of an oral dosage form in the stomach, including swelling and expanding systems [2,3], bioadhesive systems [4,5], modifiedshape systems [6,7], high-density systems [8] and floating systems [9]. Floating drug delivery systems (FDDS) with low bulk density will remain buoyant in the stomach without being affected by the ⁎ Corresponding author. E-mail address: [email protected] (R.A.H. Ishak). 0168-3659/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2007.02.012
gastric emptying rate for a prolonged period of time. While the system is floating on the gastric contents, the drug is slowly released at a desired rate [10]. Multiple-unit FDDS show several advantages over monolithic ones, among which: avoiding all-or-nothing emptying, absence or impairing of performance due to failure of a few units, more predictable drug release kinetics, less chance of localized mucosal damage and administration of units with different release profiles or those containing incompatible substances [11]. However various floating alginate beads suffered from rapid drug release for drugs [12,13], a problem which was overcome by addition of further additive [14]. Recently, gastroretentive systems for treating H. pylori have shown special interest. The prolongation of the local availability of the antibacterial agents has been reported to be an important factor to increase the effectiveness of H. pylori treatment [15]. This will ensure a high drug concentration in the gastric mucosa
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for better microbial eradication [16]. Amoxicillin was prepared as mucoadhesive floating microspheres (Amo-ad-ms) and proved its superiority over powder drug in H. pylori eradication [17]. Metronidazole (MZ) is an active adjunct in treatment of H. pylori [18] with the commonly reported side effects including anorexia, nausea, vomiting, and epigastric pain. Metallic taste, mouth dryness, probably caused by the presence of high concentrations of the drug in the saliva, and furring of the tongue are also reported [19]. The work described here is concerned with the formulation of MZ floating alginate beads. Such dosage form for MZ would be beneficial in delivering higher concentrations of the antibacterial agent in the gastric mucosa where H. pylori resides ensuring better microorganism eradication. Furthermore, such treatment may lead to drug dose reduction which will be an additional valuable advantage. Mg st. is universally and commonly used as tablet and capsule excipient up to 5% concentration. It is known to be of low bulk density and hydrophobic in nature. As a new approach in this work, Mg st. was used in the preparation of MZ floating beads to impart buoyancy and to retard MZ release. A factorially designed experiment was used to prepare MZ floating alginate beads. This enabled the study of the effect of different variables either separately or combined on different parameters including drug entrapment, percentage of floating beads and drug release. MZ floating beads with maximum drug entrapment, higher percent of floating beads and with the most extended drug release over 4 h were evaluated in vivo. 2. Materials and methods 2.1. Materials Micronized Metronidazole (MZ), hydroxypropylmethylcellulose E5 (HPMC E5) and hydroxypropylcellulose Mf50 (HPC) were kindly supplied by E.I.P.I. Co. (Cairo, Egypt). Magnesium stearate (Mg st.) was from Winlab Laboratory Chemicals (Leicestershire, U.K.). Carbopol 934P (CBP) was kindly supplied from Memphis Co. (Cairo, Egypt). Methylcellulose (MC) was gifted from Amoun Co. (Cairo, Egypt). Sodium alginate (Na alg.) (low viscosity), chitosan (medium viscosity) and κ-carrageenan were obtained from Sigma Co. (USA). All other chemicals and reagents were of the highest purity available from local sources. 2.2. Methods 2.2.1. Preparation of conventional MZ calcium alginate beads Conventional calcium (Ca) alginate beads were prepared by the ionotropic gelation method [20]. In this method a dispersion of MZ in different concentrations of Na alg. (1, 2, 3 and 4% w/v) was extruded, using a 21G syringe needle into a solution of calcium chloride (CaCl2) of different concentrations (1, 2.5, 5 and 7.5% w/v) with gentle stirring for 20 min at room temperature. The formed beads were then separated, washed and dried at room temperature for 24 h. Different drug to sodium alginate ratios (D/Na alg.) were tried (1:1, 2:1, 3:1 and 4:1 w/w).
Table 1 The formulae of different metronidazole floating beads used in the factorial design Polymer type Chitosan concentration
Magnesium stearate concentration
Methyl cellulose
Carbopol κ-carrageenan 934P
0.2%
2.5% 5% 2.5% 5%
1 2 3 4
5 6 7 8
0.4%
9 10 11 12
2.2.1.1. Improvement of drug entrapment efficiency and modulation of MZ release. The MZ entrapment efficiency and control of drug release from the beads prepared with 3% w/ w Na alg. and 1% w/v CaCl2 solution at 3:1 D/Na alg. ratio were improved either by coating the beads with different concentrations of chitosan (0.2, 0.3 and 0.4% w/v) or by increasing the viscosity of Na alg. solution using different concentrations (0.5 and 1.5% w/v) of each of: κ-carrageenan, CBP, MC, HPMC E5 or HPC. 2.2.2. Preparation of MZ floating alginate beads using factorial design experiment A factorial design experiment was conducted to study the effect of three factors, namely: the polymer type in 0.5% (w/v) concentration at three levels (MC, CBP, and κ-carrageenan), the chitosan and Mg st. concentration, each at two levels (0.2 and 0.4%), and (2.5 and 5%), respectively. This gave a 3 × 2 × 2 = 12 formulae. The responses were the drug entrapment efficiency (DEE), the percent of floating beads, and the time required for 80% MZ to be released (T80%). The method used under Section 2.2.1 was adopted for the preparation of the floating beads, with the inclusion of Mg st. in the drug–alginate mixtures. The assigned number for each formula and the typical design of the factorial experiment are shown in Table 1. All experimental data were analyzed statistically according to the established factorial design using MINITAB for Windows (release 12.2, 1998) computer program by which the analysis of variance (ANOVA) was performed and the main effects and interactions were calculated. 2.2.3. In vitro evaluation of the prepared MZ alginate beads 2.2.3.1. Determination of DEE of beads. The beads were dispersed in Sorensen's phosphate buffer (pH 7.4) and the content of MZ was assayed spectrophotometrically (Shimadzu UV visible 1601 PC, Kyoto, Japan) at the predetermined λmax (319 nm) after filtration. The determinations were made in triplicate and DEE was calculated according to the following equation: DEEð%Þ ¼ ðActual drug content=Theoretical drug contentÞ 100 2.2.3.2. In vitro MZ release studies. The in vitro dissolution studies were carried out using USP rotating basket apparatus– Pharma test, type PTW-2, Germany (apparatus I). The baskets,
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each containing an amount of beads equivalent to 250 mg MZ, were rotated at 50 rpm in 500 ml 0.1 N HCl pH 1.2, maintained at 37 °C ± 0.5 °C. An aliquot of 5 ml of the solution was withdrawn at predetermined time intervals and replaced by fresh dissolution medium. The withdrawn samples were analyzed for MZ content spectrophotometrically at λmax 278 nm. None of the ingredients used in the bead formulations interfered with the assay. T80% was determined for the different formulae. The results expressed were the mean of three experiments. 2.2.3.3. Determination of the beads buoyancy. The floating ability was determined using USP dissolution tester apparatus II (paddle method). Fifty beads were introduced in the vessels and the paddles were rotated at 50 rpm in 500 ml 0.1 N HCl pH 1.2, maintained at 37 ± 0.5 °C. The floating ability of the beads was measured by visual observation and the percent of floating beads was the average of three determinations. The preparation was considered to have buoyancy only when all beads floated on the test solution [21] immediately or within a lag time which did not exceed 2 min. 2.2.4. In vivo evaluation of MZ floating beads 2.2.4.1. Preparation of pathogenic H. pylori culture. Gastric biopsy specimens were taken from selected patients (n = 10) with gastritis, gastric and duodenal ulcer through the gastroduodenoscope. The procedure was done in GIT endoscope unit at El Demerdash Hospital, Ain Shams University, after receiving board approval and consent from the patient. Each taken biopsy was homogenized with sterile saline (0.5 ml/ stomach) and plated on the selective H. pylori medium (Skirrow's medium). The plates were incubated for 5 days at 37 °C under microaerophilic conditions in GasPak jars. The detection of H. pylori was assessed microscopically and by biochemical tests including catalase and oxidase tests [22]. 2.2.4.2. Inoculation of H. pylori and administration of MZ floating beads to mice. Four-week-old specific-pathogen-free male albino mice were used following the approval of the experimental protocol by the Ethics Committee of EAPRU (Experimental and Advanced Pharmaceutical Research Unit, Faculty of Pharmacy — Ain Shams University). All mice were fasted overnight before inoculation and prior to euthanasia. One ml of broth containing about 1010 colony-forming units (CFUs) of H. pylori per ml was inoculated into the stomach of each mouse via an orogastric tube. Bacterial colonization was assessed four weeks post inoculation in a given mouse by bacterial culture and histology of stomach tissue. After then, MZ was orally administered for 4 mice groups (n = 6) for 3 consecutive days with a once daily dose of 5, 10, 15 and 20 mg/kg for each group in the form of floating alginate beads and suspension. Placebo-alginate beads and a 0.5% aqueous methylcellulose solution, used as controls, were also administered in the same manner. One day after administration of the final dose, the mice were sacrificed, their stomachs removed and subjected to the following tests.
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2.2.4.3. Histopathological examination of mice stomach. Histopathological examination was done for stomach specimens reserved in 10% formalin which were ranked according to the intensity of H. pylori colonization as follows: severe infection, moderate infection, mild infection and free from infection. 2.2.4.4. Clearance of H. pylori from mice stomach. Each stomach was homogenized with sterile normal saline (3 ml/ stomach) from which serial dilutions were plated on the selective H. pylori medium (Skirrow's medium). The plates were incubated for 5 days at 37 °C under microaerophilic conditions in GasPak jars. The viable cell count for each gastric wall was calculated by counting the number of colonies on the agar plates. The logarithm of CFU per gastric wall was expressed as % bacterial recovery. The clearance rate (%) was also calculated by the number of mice cleared from infection per total number of mice used in each group. 3. Results 3.1. Effect of Na alg., D/Na alg. ratio and CaCl2 concentration on DEE DEE increased by increasing Na alg. concentration from 1 to 3% w/v (results not shown). Increasing Na alg. concentration above 3% w/v increased the viscosity of the alginate solution to the extent that the formation of drops was strongly hindered. Thus the 3% w/v Na alg. was maintained in all next formulae. DEE increased from 22.76 to 79.08% by increasing the MZ: Na alg. ratio from 1:1 to 4:1, respectively. Due to the statistically non-significant difference (t-test, P N 0.05) in DEE observed between the MZ:Na alg. ratio 3:1 and 4:1 respectively, the smaller ratio (3:1) was chosen in all the next formulae. It was found unexpectedly that increasing CaCl2 concentration (1, 2.5, 5 and 7.5% w/v) decreased the drug loading in the beads (77.03, 74.6, 70.62 and 65.8%, respectively). Thus CaCl2 concentration was maintained at 1% w/v in all next formulae. Formula that showed 77.03% drug loading and consisting of 3% w/v Na alg. and 1% w/v CaCl2 at 3:1 D/Na alg. ratio was selected for further investigations. 3.2. Effect of chitosan and increasing Na alg. solution viscosity on DEE and T80% of MZ release Coating the alginate beads with chitosan had a significant effect (t-test, P b 0.05) on the MZ DEE, where increasing chitosan concentration from 0.2 to 0.4%, increased the DEE from 82.98 to 88.75% respectively compared to that of uncoated beads (77.03%). This was also reflected on MZ release where the T80% of the drug increased from 30 min for uncoated beads to 60 min for beads coated with 0.2% chitosan. Further increase in chitosan concentration up to 0.4% did not affect MZ release. Increasing viscosifier concentration decreased DEE. Based on DEE obtained with 0.5% of different polymers, they could be
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Table 2 shows the values of the dependent variables (responses) for the evaluation of MZ floating alginate beads according to the factorial design experiment. It is to be noted that Mg st. gave buoyancy for all beads, decreased DEE and delayed drug release.
Fig. 1. Release of MZ from floating alginate beads using 0.2% (w/v) of chitosan solution in 0.1 N HCl (pH 1.2).
arranged in a descending manner as follows: κ-carrageenan N MCN CBP N HPC N HPMC E5 with respective values of 73.98, 71.45, 69.43, 68.51 and 66.35%. The used polymers decreased drug release in comparison with the untreated beads. Increasing all polymers concentration from 0.5 to 1.5% decreased MZ T80% except with HPMC E5 and HPC. The highest T80% of MZ release was 66.5 min obtained with 0.5% κ-carrageenan. It is to be noted that all prepared beads showed 0% buoyancy. 3.3. Evaluation of MZ floating alginate beads prepared according to factorial design experiment Figs. 1 and 2 show the release profile of MZ from floating alginate beads using 0.2 and 0.4% of chitosan solution, respectively, in 0.1 N HCl (pH 1.2). By fitting the in vitro drug release data (0–80%) into zeroorder, first-order and Higuchi model, it was concluded that the release followed zero-order kinetics as the correlation coefficient R2 value was higher than those of the other 2 release models, ranging between 0.9571 and 0.9835 in comparison to (0.8321–0.8623) according to Higuchi model and (0.6553– 0.716) according to first-order model.
3.3.1. Drug entrapment efficiency The ANOVA test shows that all the main factors under study, namely: polymer type, chitosan and Mg st. concentrations, had a significant effects (P b 0.0001) on DEE with the highest one being that of the chitosan concentration and the lowest one the polymer type. According to the magnitude of their main effects, the different polymers can be arranged as follows: κ-carrageenan N MC N CBP, where their mean DEE were: 88.676 N 86.415 N 83.971% respectively. Increasing chitosan concentration from 0.2 to 0.4% w/v was found to increase significantly DEE, with the mean respective values of their main effects being 82.430 and 90.279%. It was also found that the lower the Mg st. concentration the higher was the loading efficiency. The ANOVA test showed that all the interactions between the studied variables were non-significant, thus, confirming the results of the main effects. So, it can be Table 2 Evaluation of metronidazole floating alginate beads prepared according to the factorial design Formula Composition code
Responses (mean⁎ ± S.D.)
1
84.65 ± 1.18 79.71 ± 1.11 92.70 ± 1.45 88.60 ± 1.39 83.96 ± 1.49 79.31 ± 3.10 88.50 ± 1.54 84.12 ± 1.38 85.87 ± 1.89 81.08 ± 1.32 95.79 ± 1.10 92.09 ± 1.47 97.87 ± 0.85
2 3 4 5 6 7 8 9 10 11 12 0
Fig. 2. Release of MZ from floating alginate beads using 0.4% of chitosan solution in 0.1 N HCl (pH 1.2).
Methyl cellulose/ 0.2% chitosan/2.5% Mg st. Methyl cellulose/ 0.2% chitosan/5% Mg st. Methyl cellulose/ 0.4% chitosan/2.5% Mg st. Methyl cellulose/ 0.4% chitosan/5% Mg st. Carbopol 934P/ 0.2% chitosan/2.5% Mg st. Carbopol 934P/ 0.2% chitosan/5% Mg st. Carbopol 934P/ 0.4% chitosan/2.5% Mg st. Carbopol 934P/0.4% chitosan/5% Mg st. κ-carrageenan/0.2% chitosan/2.5% Mg st. κ-carrageenan/0.2% chitosan/5% Mg st. κ-carrageenan/0.4% chitosan/2.5% Mg st. κ-carrageenan/0.4% chitosan/5% Mg st. κ-carrageenan/0.4% chitosan/0% Mg st.
Drug % of entrapment floating efficiency beads (%)
T80% (min)
71.00 ± 3.61 40.00 ± 0.56 100.00 ± 0
60.00 ± 1.90
73.67 ± 3.79 42.00 ± 0.27 100.00 ± 0
62.00 ± 0.73
4.00 ± 2.65 44.00 ± 1.07 100.00 ± 0
60.00 ± 0.60
4.67 ± 1.53 46.50 ± 0.81 100.00 ± 0
76.50 ± 0.72
49.00 ± 6.08 75.50 ± 0.23 100.00 ± 0
198.00 ± 0.18
54.33 ± 4.04 80.00 ± 0.39 100.00 ± 0
215.00 ± 0.50
0.00 ± 0
70.00 ± 1.05
S.D.: standard deviation ⁎ Average of 3 readings. N.B.: The beads are prepared with 3% w/w Na alg. and 1% w/v CaCl2 solution at 3:1 D/Na alg. ratio. Polymers used in the factorial design are maintained at concentration 0.5% (w/v).
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Fig. 3. Histopathological examination showing the gastric mucosa of mice suffering from severe infection. Arrows are pointed to H. pylori rod forms.
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Fig. 5. Histopathological examination showing the gastric mucosa of mice suffering from mild infection. Arrows are pointed to H. pylori rod forms.
3.3.2. Percent of floating beads According to ANOVA test, both the polymer type and Mg st. concentration have significant effects on percent of floating beads (P b 0.0001) with the effect of chitosan concentration being non-significant; i.e. varying chitosan concentration did not affect the percent of floating beads. The main effect on % of floating beads caused by different polymers can be arranged in a descending order as follows: MC N carrageenanN CBP, where the reported values were 86.167N 75.833 N 52.167% respectively. The use of 5% Mg st. had significantly higher effect on the % of floating beads when compared to 2.5%, where the reported values were 100 and 42.778% respectively. It is to be remembered that beads with 0% Mg st. had 0% buoyancy. A significant interaction (ANOVA) was only present between the polymer type and Mg st. concentration (p b 0.0001), while all other interactions were non-significant. Accordingly all polymers used gave higher % of floating beads with 5% Mg st. than with 2.5%.
3.3.3. T80% of MZ release The ANOVA test showed that both polymer type and Mg st. concentration have significant effects on T80% of the drug (p b 0.0001) with the effect of chitosan concentration being nonsignificant. According to the magnitude of the effect of the different polymers, they can be arranged in a descending order as follows: κ-carrageenan N CBP N MC, with their respective T80% 142.13, 56.75 and 51.00 min. Mg st. yielded beads with average T80% values of 54.67 and 111.92 min with 2.5 and 5% concentration, respectively. The only significant interaction was between the polymer type and the Mg st. concentration (p b 0.0001). So replacing 2.5% Mg st. by 5% in the beads containing carrageenan, CBP and MC led to a significant extension in T80%, from 77.75 to 206.50 min, from 45.25 to 68.25 min and from 41.00 to 61.00 min, respectively. Finally, according to ANOVA test, the 3-way interactions for the 3 responses were found to be non-significant, this means that the 2-way interactions described earlier will not differ depending on the level of a third variable. From the previously presented factorial design results, it could be concluded that the formula containing 0.5% κ-carrageenan,
Fig. 4. Histopathological examination showing the gastric mucosa of mice suffering from moderate infection. Arrows are pointed to H. pylori rod forms.
Fig. 6. Histopathological examination showing the gastric mucosa of mice free from H. pylori infection.
concluded that better MZ loading in alginate beads was possible by using κ-carrageenan in combination with 0.4% chitosan and 2.5% Mg st.
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Table 3 Effect of different doses of oral metronidazole suspension and floating alginate beads on H. pylori induced gastric infection in mice
4. Discussion
charged drug. This latter might be due to a competition between the Ca+ 2 and the drug on the same binding sites of alginate chains. MZ being also cationic in nature was subjected to the same effect. It was also assumed that gelation proceeds radially from the surface of the bead to its center. As gelation proceeds, water is expelled due to cross-links formed by the cations and the contraction of the gel volume. Therefore, more cross-linked structure, due to increased CaCl2 concentration, lead to larger water loss containing dissolved drug [26]. The polycationic nature of chitosan acted as an encapsulating coat onto the beads' surface, through crosslinking with Na alg. This allowed for an increased DEE, as reported [23,24]. The failure of increasing chitosan concentration to retard drug release might be due to its known solubility in the acidic dissolution medium [27]. The relatively high viscosity of the extruded alginate solutions might have hindered the interaction between CaCl2 and Na alg. leading to a decrease in DEE. In case of carrageenan, CBP and MC, increasing polymer concentration from 0.5 to 1.5% decreased MZ T80%. This might be due to the remarkable high viscosity of the extruded viscosified alginate solutions leading to a rapid bead formation only cross-linked with Ca+ 2 at the surface. The HPC and HPMC E5, with their relatively low viscosity, showed a reversed behavior where raising polymer concentration raised MZ T80%. Relying on factorial design experiment, better DEE was achieved by the anionic water soluble carrageenan. Being negatively charged, it interacted with both the positively charged chitosan and Ca+ 2 [28], engulfing more drug in the beads. In addition the presence of Ca+ 2 increased carrageenan gelling properties [29]. This was also reflected on the drug T80% which was the longest with this polymer as a complex network with higher binding capacity for drug molecules was formed. Despite its anionic nature CBP showed the least DEE due to its water insolubility [30] and consequently the T80% of MZ decreased with this viscosifier. Being non-ionic, MC did not show any chemical interaction but by its viscosity inducing properties it increased MZ DEE. On the other hand, the acid-catalyzed hydrolysis of the glucose– glucose linkages and reduction in the viscosity of MC solutions at pH b 3 [31] led to relatively lower MZ T80% in comparison to the other 2 polymers. The main effect on % of floating beads, caused by different polymers was arranged in a descending order: MC N κ-carrageenan N CBP and was inversely related to their bulk densities with the respective values 0.276, 0.6751 and 1.92 g/cm3, respectively [28]. MZ encapsulation increased with increased chitosan concentration. Similar results were reported with dextran [23]. The insignificant effect of varying chitosan concentrations on T80% is due to the acidic nature of the dissolution medium solubilizing it by protonation of its amine groups [26]. Chitosan swelling is favoured by both protonation and repulsion between chitosan free
In previous works [20,23–25], increasing alginate concentration and D/Na alg. on one hand and decreasing CaCl2 concentration on the other hand, increased DEE of positively
1 The bulk density (BD) of κ-carrageenan was measured by measuring cylinder method using the following equation: BD = weight / bulk volume.
Preparation
Dose Clearance (mg/kg) rate (%)
% Bacterial recovery (log of CFU per gastric wall)
Placebo–suspension Metronidazole suspension
0 5 10 15 20 0
0 (0/6) 0 (0/6) 0 (0/6) 16.67 (1/6) 33.33 (2/6) 0 (0/6)
9.23 ± 0.95 9.17 ± 0.32 8.72 ± 0.45 6.33 ± 0.36⁎ 5.11 ± 0.43⁎ 9.02 ± 0.54
16.67 (1/6) 66.67 (4/6) 100 (6/6) 100 (6/6)
6.03 ± 0.68⁎ 3.18 ± 0.36⁎ ND ND
Placebo–floating alginate beads Metronidazole floating 5 alginate beads 10 15 20
N.B.: ND: not detected. Values are means ± standard deviation. ⁎P b 0.05 (versus respective controls by t-test).
0.4% chitosan and 5% magnesium stearate had immediate buoyancy, optimum DEE and extended drug release. Thus the latter formula was chosen for the in vivo studies. 3.4. In vivo evaluation 3.4.1. Histopathological examination of mice stomach Groups of mice receiving placebo preparation either suspension or beads in addition to that receiving MZ suspension at the 2 lowest doses (5 and 10 mg/kg), showed severe infection with a large population of H. pylori, as presented in Fig. 3. From Fig. 4 it is obvious that the stomach tissue of the mice group receiving MZ suspension at doses of 15 and 20 mg/kg and MZ floating beads at dose of 5 mg/kg, showed similar moderate infection with few characteristic H. pylori forms colonizing the gland lumens. Fig. 5 presenting the mice stomach tissue receiving MZ floating beads at a dose of 10 mg/kg, showed mild H. pylori infection. On the other hand, Fig. 6 illustrating the gastric tissue of the mice stomach receiving MZ floating beads (15 and 20 mg/kg) showed total absence of H. pylori characteristic forms. The gastric mucosa revealed focal mild superficial infiltration by lymphocytes and plasma cells, indicating focal mild superficial gastritis due to previous infection with H. pylori. 3.4.2. In vivo clearance of H. pylori As shown in Table 3, the % bacterial recovery, after 3 days of treatment with MZ suspension with a dose of 5 mg/kg and placebo-suspension did not differ significantly (t-test, P b 0.05). Complete clearance of H. pylori (clearance rate, 100%) was obtained after the administration of MZ floating beads with either MZ doses of 15 and 20 mg/kg.
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ammonium groups. If the decrease in pH is too large, the charge density decreases due to dissociation of ionic linkages leading to network dissolution, instead of swelling [32,33]. Higher Mg st. concentration decreased the loading efficiency due to competition for encapsulation between the amount of drug powder and Mg st. According to the main effect, 2.5% Mg st. was not a suitable floating aid for MZ floating alginate beads with any of the used polymers as 100% floating was not achieved with any combination, as shown in Table 2. Due to its hydrophobic nature [34], Mg st. yielded beads with higher T80% with the 5% than with the 2.5% concentration. The significantly retarding effect in drug release noticed when combining 5% Mg st. with carrageenan than with the other polymers was mainly due to the hydrophobicity of Mg st. and the rigid character of carrageenan in comparison to the other two polymers. From the histopathological examination, it is obvious that groups receiving MZ in the form of floating alginate beads at doses 10, 15 and 20 mg/kg were better than the corresponding suspension form, in point of view eradication of H. pylori infection. The mean bacterial count after oral administration of both MZ suspension and floating beads decreased as the dose of MZ increased from 5 to 20 mg/kg. Complete clearance of H. pylori was obvious with MZ floating beads in a dose of 15 mg/kg. However, MZ suspension failed to give such results even with the 20 mg/kg dose. The MZ beads with a dose of 5 mg/kg achieved the same clearance rate (16.67%) as MZ suspension with a dose of 15 mg/kg. This means that MZ beads provided 3 times greater anti-H. pylori activity than MZ suspension due to the increased residence time in the stomach. In a study involving human volunteers, Whitehead et al. [35] have demonstrated prolonged gastric residence times (5.5 versus 1 h) for floating calcium alginate beads against non-floating beads. Formulating MZ as floating alginate beads with the use of 0.5% κ-carrageenan, 0.4% chitosan and 5% magnesium stearate, had a 92.09% DEE, immediate buoyancy for all beads with 100% drug release after 4 h. Complete clearance of H. pylori was observed when used the latter formula at a lower dose than the drug suspension. Although the work was a short-term treatment for H. pylori in mice, it proved its success. However, the authors suggest that clinical studies should be conducted for longer duration to ensure complete H. pylori eradication. Acknowledgements The authors thank Dr. Ibrahim Abdel Naby, the director of GIT endoscopy unit at El Demerdash Hospital – Ain Shams University, Cairo, Egypt – for permission to take gastric biopsies from patients suffering from gastritis, gastric and duodenal ulcer history. Thanks go to El-Borg Laboratory, Cairo, Egypt for preparing the histopathological slides for mice stomach specimens. The authors gratefully acknowledge Dr. Hanaa Morkos, head of the Microbiology Department of
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Tropical Countries Medicine Center – El Kasr El Eny, Cairo, Egypt – for her help in H. pylori culture. References [1] C.G. Wilson, N. Washington, The stomach: its role in oral drug delivery, in: M.H. Rubinstein (Ed.), Physiological Pharmaceutics: Biological Barriers to Drug Absorption, Ellis Horwood, Chichester, 1989, pp. 47–70. [2] R.C. Mamajek, E.S. Moyer, Drug-dispensing device and method, US Patent (1980) 4,207,890. [3] J. Urquhart, F. Theeuwes, Drug delivery system comprising a reservoir containing a plurality of tiny pills. US Patent (1984) 4,434,153. [4] C.M. Lehr, Bioadhesion technologies for the delivery of peptide and protein drugs to the gastrointestinal tract, Crit. Rev. Ther. Drug Carr. Syst. 11 (1994) 119–160. [5] G. Ponchel, J.M. Irache, Specific and non-specific bioadhesive particulate systems for oral delivery to the gastrointestinal tract, Adv. Drug Deliv. Rev. 34 (1998) 191–219. [6] L.J. Caldwell, C.R. Gardner, R.C. Cargill, Drug delivery device which can be retained in the stomach for a controlled period of time. US Patent (1988) 4,767,627. [7] R.I. Cargill, J. Caldwell, K.J. Engle, A. Fix, P.A. Porter, C.R. Gardner, Controlled gastric emptying. I. Effects of physical properties on gastric residence times of non-disintegrating geometric shapes in beagle dogs, Pharm. Res. 5 (8) (1988) 533–536. [8] S.S. Davis, A.F. Stockwell, M.J. Taylor, J.G. Hardy, D.R. Whalley, C.G. Wilson, H. Bechgaard, F.N. Christensen, The effect of density on the gastric emptying of single- and multiple-unit dosage forms, Pharm. Res. 3 (1986) 208–213. [9] A.A. Deshpande, N.H. Shah, C.T. Rhodes, W. Malick, Development of a novel controlled-release system for gastric retention, Pharm. Res. 14 (1997) 815–819. [10] B.N. Singh, K.H. Kim, Floating drug delivery systems: an approach to oral controlled drug delivery via gastric retention, J. Control. Release 63 (2000) 235–259. [11] V. Iannucelli, G. Coppi, M.T. Bernabei, R. Cameroui, Air compartment multiple-unit system for prolonged gastric residence, I: Formulation study, Int. J. Pharm. 174 (1998) 47–54. [12] L. Whitehead, J.T. Fell, J.H. Collett, Preparation of a gastroretentive dosage form, Eur. J. Pharm. Sci. 4 (1996) S182. [13] B.Y. Choi, H.J. Park, S.J. Hwang, J.B. Park, Preparation of alginate beads for floating drug delivery system: effects of CO2 gas-forming agents, Int. J. Pharm. 239 (2002) 81–91. [14] L. Whitehead, J.H. Collett, J.T. Fell, Amoxicillin release from a floating dosage form based on alginates, Int. J. Pharm. 210 (2000) 45–49. [15] S. Burton, N. Washington, R.J.C. Steele, R. Masson, L. Feely, Intragastric distribution of ion-exchange resins: a drug delivery system for the topical treatment of the gastric mucosa, J. Pharm. Pharmacol. 47 (1995) 901–906. [16] M.J. Blaser, Hypotheses on the pathogenesis and natural history of Helicobacter pylori-induced inflammation, Gastroenterology 102 (1992) 720–727. [17] Z. Liu, W. Lu, L. Qian, X. Zhang, P. Zeng, J. Pan, In vitro and in vivo studies on mucoadhesive microspheres of amoxicillin, J. Control. Release 102 (2005) 135–144. [18] J.J. Sung, S.C. Chung, T.K. Ling, Antibacterial treatment of gastric ulcers associated with Helicobacter pylori, N. Engl. J. Med. 332 (3) (1995) 139–142. [19] C. Dollery, 2nd edition, Therapeutic Drugs, vol. 2, Churchill Livingstone, Edinburgh, 1999. [20] S. Takka, O.H. Ocak, F. Acartürk, Formulation and investigation of nicardipine HCl-alginate gel beads with factorial design-based studies, Eur. J. Pharm. Sci. 6 (3) (1998) 241–246. [21] M.P. Cooreman, P. Krausgrill, K.J. Hengels, Local gastric and serum amoxicillin concentrations after different oral application forms, Antimicrob. Agents Chemother. 37 (7) (1993) 1506–1509. [22] C.S. Goodwin, B.W. Worsely, Helicobacter pylori, Biology and Clinical Practice, CRC Press, Boca Raton, NY, 1993, pp. 209–222.
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[23] A.D. Sezer, J. Akbuga, Release characteristics of chitosan-treated alginate beads, 1: Sustained release of a macromolecular drug from chitosan treated alginate beads, J. Microencapsul. 193 (1999) 197–203. [24] P.R. Hari, T. Chandy, C.P. Sharma, Chitosan/calcium alginate microcapsules for intestinal delivery of nitrofurantoin, J. Microencapsul. 13 (3) (1996) 319–329. [25] L.Y. Lim, L.S.C. Wan, Propranolol hydrochloride binding in calcium alginate beads, Drug Dev. Ind. Pharm. 23 (10) (1997) 973–980. [26] P. Aslani, R.A. Kennedy, Effect of gelation conditions and dissolution media on the release of paracetamol from alginate gel beads, J. Microencapsul. 13 (5) (1996) 601–614. [27] H.H. Tønnesen, J. Karisen, Alginate in drug delivery systems, Drug Dev. Ind. Pharm. 28 (6) (2002) 621–630. [28] www. medicinescomplete.com. [29] J.L. Zatz, J.J. Berry, D.A. Alderman, Viscosity-imparting agents in disperse systems, in: H.A. Lieberman (Ed.), 2nd edition, Pharmaceutical Dosage Forms: Disperse Systems, vol. 1, Marcel Dekker Inc., New York, 1996, p. 300.
[30] Carbopol Water Soluble Resins, B.F. Goodrich Technical Brochure. [31] A. Huikari, A. Karlsson, Viscosity stability of methyl cellulose solutions at different pH and temperature, Acta Pharm. Fenn. 984 (1989) 231–238. [32] X.Z. Shu, K.J. Zhu, W. Song, Novel pH-sensitive citrate cross-linked chitosan film for drug controlled release, Int. J. Pharm. 212 (1) (2001) 19–28. [33] F.L. Mi, S.S. Shyu, C.T. Chen, J.Y. Schoung, Porous chitosan microsphere for controlling the antigen release of Newcastle disease vaccine: preparation of antigen-adsorbed microsphere and in vitro release, Biomaterials 20 (17) (1999) 1603–1612. [34] T. Durig, R. Fassihi, Mechanistic evaluation of binary effects of magnesium stearate and talc as dissolution retardants at 85% drug loading in an experimental extended-release formulation, J. Pharm. Sci. 86 (10) (1997) 1092–1098. [35] L. Whitehead, J.T. Fell, J.H. Collett, H.L. Sharma, A.-M. Smith, Floating dosage forms: an in vivo study demonstrating prolonged gastric retention, J. Control. Release 55 (1998) 3–12.
Preparation, in vitro and in vivo evaluation of stomach-specific metronidazole-loaded alginate beads as local anti-Helicobacter pylori therapy Rania A.H. Ishak a,⁎, Gehanne A.S. Awad a , Nahed D. Mortada a , Samia A.K. Nour b a
Department of Pharmaceutics, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt b Department of Pharmaceutics, Faculty of Pharmacy, Cairo University, Cairo, Egypt Received 10 May 2006; accepted 16 February 2007 Available online 1 March 2007
Abstract Metronidazole (MZ), a common antibacterial drug used in treatment of H. pylori, was prepared in chitosan-treated alginate beads by the ionotropic gelation method. A (3 × 2 × 2) factorially designed experiment was used in which 3 viscosity-imparting polymers namely, methyl cellulose, carbopol 934P and κ-carrageenan, 2 concentrations (0.2 and 0.4% w/v) of chitosan as encapsulating polymer and 2 concentrations (2.5 and 5% w/w) of the low density magnesium stearate as a floating aid were tested. The drug entrapment efficiency (%), the percent of floating beads and the time for 80% of the drug to be released (T80%) were the responses evaluated. The bead formula containing 0.5% κ-carrageenan, 0.4% chitosan and 5% magnesium stearate showed immediate buoyancy, optimum drug entrapment efficiency and extended drug release. The histopathological examination of mice stomachs and in vivo H. pylori clearance tests were carried out by orally administering MZ floating alginate beads or MZ suspension, to H. pylori infected mice under fed conditions as a single daily dose for 3 successive days in different doses 5, 10, 15 and 20 mg/kg. The histopathological examination showed that groups receiving MZ in the form of floating alginate beads at doses 10, 15 and 20 mg/kg were better than the corresponding suspension form, regarding eradication of H. pylori infection. The in vivo H. pylori clearance tests showed that MZ floating beads with a dose of 15 mg/kg provided 100% clearance rate whereas the MZ suspension with a dose of 20 mg/kg gave only 33.33%. © 2007 Elsevier B.V. All rights reserved. Keywords: Alginate beads; Floating drug delivery systems (FDDS); Metronidazole; Sustained release; H. pylori clearance; Local delivery
1. Introduction The local treatment of H. pylori with conventional tablets or capsules may fail, since these may fall to the base of the stomach from where they are readily emptied [1]. Little, if any, drug is delivered to the body or fundus of the stomach and the main drug action is through systemic effect. Various approaches have been pursued to increase the retention of an oral dosage form in the stomach, including swelling and expanding systems [2,3], bioadhesive systems [4,5], modifiedshape systems [6,7], high-density systems [8] and floating systems [9]. Floating drug delivery systems (FDDS) with low bulk density will remain buoyant in the stomach without being affected by the ⁎ Corresponding author. E-mail address: [email protected] (R.A.H. Ishak). 0168-3659/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2007.02.012
gastric emptying rate for a prolonged period of time. While the system is floating on the gastric contents, the drug is slowly released at a desired rate [10]. Multiple-unit FDDS show several advantages over monolithic ones, among which: avoiding all-or-nothing emptying, absence or impairing of performance due to failure of a few units, more predictable drug release kinetics, less chance of localized mucosal damage and administration of units with different release profiles or those containing incompatible substances [11]. However various floating alginate beads suffered from rapid drug release for drugs [12,13], a problem which was overcome by addition of further additive [14]. Recently, gastroretentive systems for treating H. pylori have shown special interest. The prolongation of the local availability of the antibacterial agents has been reported to be an important factor to increase the effectiveness of H. pylori treatment [15]. This will ensure a high drug concentration in the gastric mucosa
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for better microbial eradication [16]. Amoxicillin was prepared as mucoadhesive floating microspheres (Amo-ad-ms) and proved its superiority over powder drug in H. pylori eradication [17]. Metronidazole (MZ) is an active adjunct in treatment of H. pylori [18] with the commonly reported side effects including anorexia, nausea, vomiting, and epigastric pain. Metallic taste, mouth dryness, probably caused by the presence of high concentrations of the drug in the saliva, and furring of the tongue are also reported [19]. The work described here is concerned with the formulation of MZ floating alginate beads. Such dosage form for MZ would be beneficial in delivering higher concentrations of the antibacterial agent in the gastric mucosa where H. pylori resides ensuring better microorganism eradication. Furthermore, such treatment may lead to drug dose reduction which will be an additional valuable advantage. Mg st. is universally and commonly used as tablet and capsule excipient up to 5% concentration. It is known to be of low bulk density and hydrophobic in nature. As a new approach in this work, Mg st. was used in the preparation of MZ floating beads to impart buoyancy and to retard MZ release. A factorially designed experiment was used to prepare MZ floating alginate beads. This enabled the study of the effect of different variables either separately or combined on different parameters including drug entrapment, percentage of floating beads and drug release. MZ floating beads with maximum drug entrapment, higher percent of floating beads and with the most extended drug release over 4 h were evaluated in vivo. 2. Materials and methods 2.1. Materials Micronized Metronidazole (MZ), hydroxypropylmethylcellulose E5 (HPMC E5) and hydroxypropylcellulose Mf50 (HPC) were kindly supplied by E.I.P.I. Co. (Cairo, Egypt). Magnesium stearate (Mg st.) was from Winlab Laboratory Chemicals (Leicestershire, U.K.). Carbopol 934P (CBP) was kindly supplied from Memphis Co. (Cairo, Egypt). Methylcellulose (MC) was gifted from Amoun Co. (Cairo, Egypt). Sodium alginate (Na alg.) (low viscosity), chitosan (medium viscosity) and κ-carrageenan were obtained from Sigma Co. (USA). All other chemicals and reagents were of the highest purity available from local sources. 2.2. Methods 2.2.1. Preparation of conventional MZ calcium alginate beads Conventional calcium (Ca) alginate beads were prepared by the ionotropic gelation method [20]. In this method a dispersion of MZ in different concentrations of Na alg. (1, 2, 3 and 4% w/v) was extruded, using a 21G syringe needle into a solution of calcium chloride (CaCl2) of different concentrations (1, 2.5, 5 and 7.5% w/v) with gentle stirring for 20 min at room temperature. The formed beads were then separated, washed and dried at room temperature for 24 h. Different drug to sodium alginate ratios (D/Na alg.) were tried (1:1, 2:1, 3:1 and 4:1 w/w).
Table 1 The formulae of different metronidazole floating beads used in the factorial design Polymer type Chitosan concentration
Magnesium stearate concentration
Methyl cellulose
Carbopol κ-carrageenan 934P
0.2%
2.5% 5% 2.5% 5%
1 2 3 4
5 6 7 8
0.4%
9 10 11 12
2.2.1.1. Improvement of drug entrapment efficiency and modulation of MZ release. The MZ entrapment efficiency and control of drug release from the beads prepared with 3% w/ w Na alg. and 1% w/v CaCl2 solution at 3:1 D/Na alg. ratio were improved either by coating the beads with different concentrations of chitosan (0.2, 0.3 and 0.4% w/v) or by increasing the viscosity of Na alg. solution using different concentrations (0.5 and 1.5% w/v) of each of: κ-carrageenan, CBP, MC, HPMC E5 or HPC. 2.2.2. Preparation of MZ floating alginate beads using factorial design experiment A factorial design experiment was conducted to study the effect of three factors, namely: the polymer type in 0.5% (w/v) concentration at three levels (MC, CBP, and κ-carrageenan), the chitosan and Mg st. concentration, each at two levels (0.2 and 0.4%), and (2.5 and 5%), respectively. This gave a 3 × 2 × 2 = 12 formulae. The responses were the drug entrapment efficiency (DEE), the percent of floating beads, and the time required for 80% MZ to be released (T80%). The method used under Section 2.2.1 was adopted for the preparation of the floating beads, with the inclusion of Mg st. in the drug–alginate mixtures. The assigned number for each formula and the typical design of the factorial experiment are shown in Table 1. All experimental data were analyzed statistically according to the established factorial design using MINITAB for Windows (release 12.2, 1998) computer program by which the analysis of variance (ANOVA) was performed and the main effects and interactions were calculated. 2.2.3. In vitro evaluation of the prepared MZ alginate beads 2.2.3.1. Determination of DEE of beads. The beads were dispersed in Sorensen's phosphate buffer (pH 7.4) and the content of MZ was assayed spectrophotometrically (Shimadzu UV visible 1601 PC, Kyoto, Japan) at the predetermined λmax (319 nm) after filtration. The determinations were made in triplicate and DEE was calculated according to the following equation: DEEð%Þ ¼ ðActual drug content=Theoretical drug contentÞ 100 2.2.3.2. In vitro MZ release studies. The in vitro dissolution studies were carried out using USP rotating basket apparatus– Pharma test, type PTW-2, Germany (apparatus I). The baskets,
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each containing an amount of beads equivalent to 250 mg MZ, were rotated at 50 rpm in 500 ml 0.1 N HCl pH 1.2, maintained at 37 °C ± 0.5 °C. An aliquot of 5 ml of the solution was withdrawn at predetermined time intervals and replaced by fresh dissolution medium. The withdrawn samples were analyzed for MZ content spectrophotometrically at λmax 278 nm. None of the ingredients used in the bead formulations interfered with the assay. T80% was determined for the different formulae. The results expressed were the mean of three experiments. 2.2.3.3. Determination of the beads buoyancy. The floating ability was determined using USP dissolution tester apparatus II (paddle method). Fifty beads were introduced in the vessels and the paddles were rotated at 50 rpm in 500 ml 0.1 N HCl pH 1.2, maintained at 37 ± 0.5 °C. The floating ability of the beads was measured by visual observation and the percent of floating beads was the average of three determinations. The preparation was considered to have buoyancy only when all beads floated on the test solution [21] immediately or within a lag time which did not exceed 2 min. 2.2.4. In vivo evaluation of MZ floating beads 2.2.4.1. Preparation of pathogenic H. pylori culture. Gastric biopsy specimens were taken from selected patients (n = 10) with gastritis, gastric and duodenal ulcer through the gastroduodenoscope. The procedure was done in GIT endoscope unit at El Demerdash Hospital, Ain Shams University, after receiving board approval and consent from the patient. Each taken biopsy was homogenized with sterile saline (0.5 ml/ stomach) and plated on the selective H. pylori medium (Skirrow's medium). The plates were incubated for 5 days at 37 °C under microaerophilic conditions in GasPak jars. The detection of H. pylori was assessed microscopically and by biochemical tests including catalase and oxidase tests [22]. 2.2.4.2. Inoculation of H. pylori and administration of MZ floating beads to mice. Four-week-old specific-pathogen-free male albino mice were used following the approval of the experimental protocol by the Ethics Committee of EAPRU (Experimental and Advanced Pharmaceutical Research Unit, Faculty of Pharmacy — Ain Shams University). All mice were fasted overnight before inoculation and prior to euthanasia. One ml of broth containing about 1010 colony-forming units (CFUs) of H. pylori per ml was inoculated into the stomach of each mouse via an orogastric tube. Bacterial colonization was assessed four weeks post inoculation in a given mouse by bacterial culture and histology of stomach tissue. After then, MZ was orally administered for 4 mice groups (n = 6) for 3 consecutive days with a once daily dose of 5, 10, 15 and 20 mg/kg for each group in the form of floating alginate beads and suspension. Placebo-alginate beads and a 0.5% aqueous methylcellulose solution, used as controls, were also administered in the same manner. One day after administration of the final dose, the mice were sacrificed, their stomachs removed and subjected to the following tests.
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2.2.4.3. Histopathological examination of mice stomach. Histopathological examination was done for stomach specimens reserved in 10% formalin which were ranked according to the intensity of H. pylori colonization as follows: severe infection, moderate infection, mild infection and free from infection. 2.2.4.4. Clearance of H. pylori from mice stomach. Each stomach was homogenized with sterile normal saline (3 ml/ stomach) from which serial dilutions were plated on the selective H. pylori medium (Skirrow's medium). The plates were incubated for 5 days at 37 °C under microaerophilic conditions in GasPak jars. The viable cell count for each gastric wall was calculated by counting the number of colonies on the agar plates. The logarithm of CFU per gastric wall was expressed as % bacterial recovery. The clearance rate (%) was also calculated by the number of mice cleared from infection per total number of mice used in each group. 3. Results 3.1. Effect of Na alg., D/Na alg. ratio and CaCl2 concentration on DEE DEE increased by increasing Na alg. concentration from 1 to 3% w/v (results not shown). Increasing Na alg. concentration above 3% w/v increased the viscosity of the alginate solution to the extent that the formation of drops was strongly hindered. Thus the 3% w/v Na alg. was maintained in all next formulae. DEE increased from 22.76 to 79.08% by increasing the MZ: Na alg. ratio from 1:1 to 4:1, respectively. Due to the statistically non-significant difference (t-test, P N 0.05) in DEE observed between the MZ:Na alg. ratio 3:1 and 4:1 respectively, the smaller ratio (3:1) was chosen in all the next formulae. It was found unexpectedly that increasing CaCl2 concentration (1, 2.5, 5 and 7.5% w/v) decreased the drug loading in the beads (77.03, 74.6, 70.62 and 65.8%, respectively). Thus CaCl2 concentration was maintained at 1% w/v in all next formulae. Formula that showed 77.03% drug loading and consisting of 3% w/v Na alg. and 1% w/v CaCl2 at 3:1 D/Na alg. ratio was selected for further investigations. 3.2. Effect of chitosan and increasing Na alg. solution viscosity on DEE and T80% of MZ release Coating the alginate beads with chitosan had a significant effect (t-test, P b 0.05) on the MZ DEE, where increasing chitosan concentration from 0.2 to 0.4%, increased the DEE from 82.98 to 88.75% respectively compared to that of uncoated beads (77.03%). This was also reflected on MZ release where the T80% of the drug increased from 30 min for uncoated beads to 60 min for beads coated with 0.2% chitosan. Further increase in chitosan concentration up to 0.4% did not affect MZ release. Increasing viscosifier concentration decreased DEE. Based on DEE obtained with 0.5% of different polymers, they could be
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Table 2 shows the values of the dependent variables (responses) for the evaluation of MZ floating alginate beads according to the factorial design experiment. It is to be noted that Mg st. gave buoyancy for all beads, decreased DEE and delayed drug release.
Fig. 1. Release of MZ from floating alginate beads using 0.2% (w/v) of chitosan solution in 0.1 N HCl (pH 1.2).
arranged in a descending manner as follows: κ-carrageenan N MCN CBP N HPC N HPMC E5 with respective values of 73.98, 71.45, 69.43, 68.51 and 66.35%. The used polymers decreased drug release in comparison with the untreated beads. Increasing all polymers concentration from 0.5 to 1.5% decreased MZ T80% except with HPMC E5 and HPC. The highest T80% of MZ release was 66.5 min obtained with 0.5% κ-carrageenan. It is to be noted that all prepared beads showed 0% buoyancy. 3.3. Evaluation of MZ floating alginate beads prepared according to factorial design experiment Figs. 1 and 2 show the release profile of MZ from floating alginate beads using 0.2 and 0.4% of chitosan solution, respectively, in 0.1 N HCl (pH 1.2). By fitting the in vitro drug release data (0–80%) into zeroorder, first-order and Higuchi model, it was concluded that the release followed zero-order kinetics as the correlation coefficient R2 value was higher than those of the other 2 release models, ranging between 0.9571 and 0.9835 in comparison to (0.8321–0.8623) according to Higuchi model and (0.6553– 0.716) according to first-order model.
3.3.1. Drug entrapment efficiency The ANOVA test shows that all the main factors under study, namely: polymer type, chitosan and Mg st. concentrations, had a significant effects (P b 0.0001) on DEE with the highest one being that of the chitosan concentration and the lowest one the polymer type. According to the magnitude of their main effects, the different polymers can be arranged as follows: κ-carrageenan N MC N CBP, where their mean DEE were: 88.676 N 86.415 N 83.971% respectively. Increasing chitosan concentration from 0.2 to 0.4% w/v was found to increase significantly DEE, with the mean respective values of their main effects being 82.430 and 90.279%. It was also found that the lower the Mg st. concentration the higher was the loading efficiency. The ANOVA test showed that all the interactions between the studied variables were non-significant, thus, confirming the results of the main effects. So, it can be Table 2 Evaluation of metronidazole floating alginate beads prepared according to the factorial design Formula Composition code
Responses (mean⁎ ± S.D.)
1
84.65 ± 1.18 79.71 ± 1.11 92.70 ± 1.45 88.60 ± 1.39 83.96 ± 1.49 79.31 ± 3.10 88.50 ± 1.54 84.12 ± 1.38 85.87 ± 1.89 81.08 ± 1.32 95.79 ± 1.10 92.09 ± 1.47 97.87 ± 0.85
2 3 4 5 6 7 8 9 10 11 12 0
Fig. 2. Release of MZ from floating alginate beads using 0.4% of chitosan solution in 0.1 N HCl (pH 1.2).
Methyl cellulose/ 0.2% chitosan/2.5% Mg st. Methyl cellulose/ 0.2% chitosan/5% Mg st. Methyl cellulose/ 0.4% chitosan/2.5% Mg st. Methyl cellulose/ 0.4% chitosan/5% Mg st. Carbopol 934P/ 0.2% chitosan/2.5% Mg st. Carbopol 934P/ 0.2% chitosan/5% Mg st. Carbopol 934P/ 0.4% chitosan/2.5% Mg st. Carbopol 934P/0.4% chitosan/5% Mg st. κ-carrageenan/0.2% chitosan/2.5% Mg st. κ-carrageenan/0.2% chitosan/5% Mg st. κ-carrageenan/0.4% chitosan/2.5% Mg st. κ-carrageenan/0.4% chitosan/5% Mg st. κ-carrageenan/0.4% chitosan/0% Mg st.
Drug % of entrapment floating efficiency beads (%)
T80% (min)
71.00 ± 3.61 40.00 ± 0.56 100.00 ± 0
60.00 ± 1.90
73.67 ± 3.79 42.00 ± 0.27 100.00 ± 0
62.00 ± 0.73
4.00 ± 2.65 44.00 ± 1.07 100.00 ± 0
60.00 ± 0.60
4.67 ± 1.53 46.50 ± 0.81 100.00 ± 0
76.50 ± 0.72
49.00 ± 6.08 75.50 ± 0.23 100.00 ± 0
198.00 ± 0.18
54.33 ± 4.04 80.00 ± 0.39 100.00 ± 0
215.00 ± 0.50
0.00 ± 0
70.00 ± 1.05
S.D.: standard deviation ⁎ Average of 3 readings. N.B.: The beads are prepared with 3% w/w Na alg. and 1% w/v CaCl2 solution at 3:1 D/Na alg. ratio. Polymers used in the factorial design are maintained at concentration 0.5% (w/v).
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Fig. 3. Histopathological examination showing the gastric mucosa of mice suffering from severe infection. Arrows are pointed to H. pylori rod forms.
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Fig. 5. Histopathological examination showing the gastric mucosa of mice suffering from mild infection. Arrows are pointed to H. pylori rod forms.
3.3.2. Percent of floating beads According to ANOVA test, both the polymer type and Mg st. concentration have significant effects on percent of floating beads (P b 0.0001) with the effect of chitosan concentration being non-significant; i.e. varying chitosan concentration did not affect the percent of floating beads. The main effect on % of floating beads caused by different polymers can be arranged in a descending order as follows: MC N carrageenanN CBP, where the reported values were 86.167N 75.833 N 52.167% respectively. The use of 5% Mg st. had significantly higher effect on the % of floating beads when compared to 2.5%, where the reported values were 100 and 42.778% respectively. It is to be remembered that beads with 0% Mg st. had 0% buoyancy. A significant interaction (ANOVA) was only present between the polymer type and Mg st. concentration (p b 0.0001), while all other interactions were non-significant. Accordingly all polymers used gave higher % of floating beads with 5% Mg st. than with 2.5%.
3.3.3. T80% of MZ release The ANOVA test showed that both polymer type and Mg st. concentration have significant effects on T80% of the drug (p b 0.0001) with the effect of chitosan concentration being nonsignificant. According to the magnitude of the effect of the different polymers, they can be arranged in a descending order as follows: κ-carrageenan N CBP N MC, with their respective T80% 142.13, 56.75 and 51.00 min. Mg st. yielded beads with average T80% values of 54.67 and 111.92 min with 2.5 and 5% concentration, respectively. The only significant interaction was between the polymer type and the Mg st. concentration (p b 0.0001). So replacing 2.5% Mg st. by 5% in the beads containing carrageenan, CBP and MC led to a significant extension in T80%, from 77.75 to 206.50 min, from 45.25 to 68.25 min and from 41.00 to 61.00 min, respectively. Finally, according to ANOVA test, the 3-way interactions for the 3 responses were found to be non-significant, this means that the 2-way interactions described earlier will not differ depending on the level of a third variable. From the previously presented factorial design results, it could be concluded that the formula containing 0.5% κ-carrageenan,
Fig. 4. Histopathological examination showing the gastric mucosa of mice suffering from moderate infection. Arrows are pointed to H. pylori rod forms.
Fig. 6. Histopathological examination showing the gastric mucosa of mice free from H. pylori infection.
concluded that better MZ loading in alginate beads was possible by using κ-carrageenan in combination with 0.4% chitosan and 2.5% Mg st.
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Table 3 Effect of different doses of oral metronidazole suspension and floating alginate beads on H. pylori induced gastric infection in mice
4. Discussion
charged drug. This latter might be due to a competition between the Ca+ 2 and the drug on the same binding sites of alginate chains. MZ being also cationic in nature was subjected to the same effect. It was also assumed that gelation proceeds radially from the surface of the bead to its center. As gelation proceeds, water is expelled due to cross-links formed by the cations and the contraction of the gel volume. Therefore, more cross-linked structure, due to increased CaCl2 concentration, lead to larger water loss containing dissolved drug [26]. The polycationic nature of chitosan acted as an encapsulating coat onto the beads' surface, through crosslinking with Na alg. This allowed for an increased DEE, as reported [23,24]. The failure of increasing chitosan concentration to retard drug release might be due to its known solubility in the acidic dissolution medium [27]. The relatively high viscosity of the extruded alginate solutions might have hindered the interaction between CaCl2 and Na alg. leading to a decrease in DEE. In case of carrageenan, CBP and MC, increasing polymer concentration from 0.5 to 1.5% decreased MZ T80%. This might be due to the remarkable high viscosity of the extruded viscosified alginate solutions leading to a rapid bead formation only cross-linked with Ca+ 2 at the surface. The HPC and HPMC E5, with their relatively low viscosity, showed a reversed behavior where raising polymer concentration raised MZ T80%. Relying on factorial design experiment, better DEE was achieved by the anionic water soluble carrageenan. Being negatively charged, it interacted with both the positively charged chitosan and Ca+ 2 [28], engulfing more drug in the beads. In addition the presence of Ca+ 2 increased carrageenan gelling properties [29]. This was also reflected on the drug T80% which was the longest with this polymer as a complex network with higher binding capacity for drug molecules was formed. Despite its anionic nature CBP showed the least DEE due to its water insolubility [30] and consequently the T80% of MZ decreased with this viscosifier. Being non-ionic, MC did not show any chemical interaction but by its viscosity inducing properties it increased MZ DEE. On the other hand, the acid-catalyzed hydrolysis of the glucose– glucose linkages and reduction in the viscosity of MC solutions at pH b 3 [31] led to relatively lower MZ T80% in comparison to the other 2 polymers. The main effect on % of floating beads, caused by different polymers was arranged in a descending order: MC N κ-carrageenan N CBP and was inversely related to their bulk densities with the respective values 0.276, 0.6751 and 1.92 g/cm3, respectively [28]. MZ encapsulation increased with increased chitosan concentration. Similar results were reported with dextran [23]. The insignificant effect of varying chitosan concentrations on T80% is due to the acidic nature of the dissolution medium solubilizing it by protonation of its amine groups [26]. Chitosan swelling is favoured by both protonation and repulsion between chitosan free
In previous works [20,23–25], increasing alginate concentration and D/Na alg. on one hand and decreasing CaCl2 concentration on the other hand, increased DEE of positively
1 The bulk density (BD) of κ-carrageenan was measured by measuring cylinder method using the following equation: BD = weight / bulk volume.
Preparation
Dose Clearance (mg/kg) rate (%)
% Bacterial recovery (log of CFU per gastric wall)
Placebo–suspension Metronidazole suspension
0 5 10 15 20 0
0 (0/6) 0 (0/6) 0 (0/6) 16.67 (1/6) 33.33 (2/6) 0 (0/6)
9.23 ± 0.95 9.17 ± 0.32 8.72 ± 0.45 6.33 ± 0.36⁎ 5.11 ± 0.43⁎ 9.02 ± 0.54
16.67 (1/6) 66.67 (4/6) 100 (6/6) 100 (6/6)
6.03 ± 0.68⁎ 3.18 ± 0.36⁎ ND ND
Placebo–floating alginate beads Metronidazole floating 5 alginate beads 10 15 20
N.B.: ND: not detected. Values are means ± standard deviation. ⁎P b 0.05 (versus respective controls by t-test).
0.4% chitosan and 5% magnesium stearate had immediate buoyancy, optimum DEE and extended drug release. Thus the latter formula was chosen for the in vivo studies. 3.4. In vivo evaluation 3.4.1. Histopathological examination of mice stomach Groups of mice receiving placebo preparation either suspension or beads in addition to that receiving MZ suspension at the 2 lowest doses (5 and 10 mg/kg), showed severe infection with a large population of H. pylori, as presented in Fig. 3. From Fig. 4 it is obvious that the stomach tissue of the mice group receiving MZ suspension at doses of 15 and 20 mg/kg and MZ floating beads at dose of 5 mg/kg, showed similar moderate infection with few characteristic H. pylori forms colonizing the gland lumens. Fig. 5 presenting the mice stomach tissue receiving MZ floating beads at a dose of 10 mg/kg, showed mild H. pylori infection. On the other hand, Fig. 6 illustrating the gastric tissue of the mice stomach receiving MZ floating beads (15 and 20 mg/kg) showed total absence of H. pylori characteristic forms. The gastric mucosa revealed focal mild superficial infiltration by lymphocytes and plasma cells, indicating focal mild superficial gastritis due to previous infection with H. pylori. 3.4.2. In vivo clearance of H. pylori As shown in Table 3, the % bacterial recovery, after 3 days of treatment with MZ suspension with a dose of 5 mg/kg and placebo-suspension did not differ significantly (t-test, P b 0.05). Complete clearance of H. pylori (clearance rate, 100%) was obtained after the administration of MZ floating beads with either MZ doses of 15 and 20 mg/kg.
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ammonium groups. If the decrease in pH is too large, the charge density decreases due to dissociation of ionic linkages leading to network dissolution, instead of swelling [32,33]. Higher Mg st. concentration decreased the loading efficiency due to competition for encapsulation between the amount of drug powder and Mg st. According to the main effect, 2.5% Mg st. was not a suitable floating aid for MZ floating alginate beads with any of the used polymers as 100% floating was not achieved with any combination, as shown in Table 2. Due to its hydrophobic nature [34], Mg st. yielded beads with higher T80% with the 5% than with the 2.5% concentration. The significantly retarding effect in drug release noticed when combining 5% Mg st. with carrageenan than with the other polymers was mainly due to the hydrophobicity of Mg st. and the rigid character of carrageenan in comparison to the other two polymers. From the histopathological examination, it is obvious that groups receiving MZ in the form of floating alginate beads at doses 10, 15 and 20 mg/kg were better than the corresponding suspension form, in point of view eradication of H. pylori infection. The mean bacterial count after oral administration of both MZ suspension and floating beads decreased as the dose of MZ increased from 5 to 20 mg/kg. Complete clearance of H. pylori was obvious with MZ floating beads in a dose of 15 mg/kg. However, MZ suspension failed to give such results even with the 20 mg/kg dose. The MZ beads with a dose of 5 mg/kg achieved the same clearance rate (16.67%) as MZ suspension with a dose of 15 mg/kg. This means that MZ beads provided 3 times greater anti-H. pylori activity than MZ suspension due to the increased residence time in the stomach. In a study involving human volunteers, Whitehead et al. [35] have demonstrated prolonged gastric residence times (5.5 versus 1 h) for floating calcium alginate beads against non-floating beads. Formulating MZ as floating alginate beads with the use of 0.5% κ-carrageenan, 0.4% chitosan and 5% magnesium stearate, had a 92.09% DEE, immediate buoyancy for all beads with 100% drug release after 4 h. Complete clearance of H. pylori was observed when used the latter formula at a lower dose than the drug suspension. Although the work was a short-term treatment for H. pylori in mice, it proved its success. However, the authors suggest that clinical studies should be conducted for longer duration to ensure complete H. pylori eradication. Acknowledgements The authors thank Dr. Ibrahim Abdel Naby, the director of GIT endoscopy unit at El Demerdash Hospital – Ain Shams University, Cairo, Egypt – for permission to take gastric biopsies from patients suffering from gastritis, gastric and duodenal ulcer history. Thanks go to El-Borg Laboratory, Cairo, Egypt for preparing the histopathological slides for mice stomach specimens. The authors gratefully acknowledge Dr. Hanaa Morkos, head of the Microbiology Department of
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Tropical Countries Medicine Center – El Kasr El Eny, Cairo, Egypt – for her help in H. pylori culture. References [1] C.G. Wilson, N. Washington, The stomach: its role in oral drug delivery, in: M.H. Rubinstein (Ed.), Physiological Pharmaceutics: Biological Barriers to Drug Absorption, Ellis Horwood, Chichester, 1989, pp. 47–70. [2] R.C. Mamajek, E.S. Moyer, Drug-dispensing device and method, US Patent (1980) 4,207,890. [3] J. Urquhart, F. Theeuwes, Drug delivery system comprising a reservoir containing a plurality of tiny pills. US Patent (1984) 4,434,153. [4] C.M. Lehr, Bioadhesion technologies for the delivery of peptide and protein drugs to the gastrointestinal tract, Crit. Rev. Ther. Drug Carr. Syst. 11 (1994) 119–160. [5] G. Ponchel, J.M. Irache, Specific and non-specific bioadhesive particulate systems for oral delivery to the gastrointestinal tract, Adv. Drug Deliv. Rev. 34 (1998) 191–219. [6] L.J. Caldwell, C.R. Gardner, R.C. Cargill, Drug delivery device which can be retained in the stomach for a controlled period of time. US Patent (1988) 4,767,627. [7] R.I. Cargill, J. Caldwell, K.J. Engle, A. Fix, P.A. Porter, C.R. Gardner, Controlled gastric emptying. I. Effects of physical properties on gastric residence times of non-disintegrating geometric shapes in beagle dogs, Pharm. Res. 5 (8) (1988) 533–536. [8] S.S. Davis, A.F. Stockwell, M.J. Taylor, J.G. Hardy, D.R. Whalley, C.G. Wilson, H. Bechgaard, F.N. Christensen, The effect of density on the gastric emptying of single- and multiple-unit dosage forms, Pharm. Res. 3 (1986) 208–213. [9] A.A. Deshpande, N.H. Shah, C.T. Rhodes, W. Malick, Development of a novel controlled-release system for gastric retention, Pharm. Res. 14 (1997) 815–819. [10] B.N. Singh, K.H. Kim, Floating drug delivery systems: an approach to oral controlled drug delivery via gastric retention, J. Control. Release 63 (2000) 235–259. [11] V. Iannucelli, G. Coppi, M.T. Bernabei, R. Cameroui, Air compartment multiple-unit system for prolonged gastric residence, I: Formulation study, Int. J. Pharm. 174 (1998) 47–54. [12] L. Whitehead, J.T. Fell, J.H. Collett, Preparation of a gastroretentive dosage form, Eur. J. Pharm. Sci. 4 (1996) S182. [13] B.Y. Choi, H.J. Park, S.J. Hwang, J.B. Park, Preparation of alginate beads for floating drug delivery system: effects of CO2 gas-forming agents, Int. J. Pharm. 239 (2002) 81–91. [14] L. Whitehead, J.H. Collett, J.T. Fell, Amoxicillin release from a floating dosage form based on alginates, Int. J. Pharm. 210 (2000) 45–49. [15] S. Burton, N. Washington, R.J.C. Steele, R. Masson, L. Feely, Intragastric distribution of ion-exchange resins: a drug delivery system for the topical treatment of the gastric mucosa, J. Pharm. Pharmacol. 47 (1995) 901–906. [16] M.J. Blaser, Hypotheses on the pathogenesis and natural history of Helicobacter pylori-induced inflammation, Gastroenterology 102 (1992) 720–727. [17] Z. Liu, W. Lu, L. Qian, X. Zhang, P. Zeng, J. Pan, In vitro and in vivo studies on mucoadhesive microspheres of amoxicillin, J. Control. Release 102 (2005) 135–144. [18] J.J. Sung, S.C. Chung, T.K. Ling, Antibacterial treatment of gastric ulcers associated with Helicobacter pylori, N. Engl. J. Med. 332 (3) (1995) 139–142. [19] C. Dollery, 2nd edition, Therapeutic Drugs, vol. 2, Churchill Livingstone, Edinburgh, 1999. [20] S. Takka, O.H. Ocak, F. Acartürk, Formulation and investigation of nicardipine HCl-alginate gel beads with factorial design-based studies, Eur. J. Pharm. Sci. 6 (3) (1998) 241–246. [21] M.P. Cooreman, P. Krausgrill, K.J. Hengels, Local gastric and serum amoxicillin concentrations after different oral application forms, Antimicrob. Agents Chemother. 37 (7) (1993) 1506–1509. [22] C.S. Goodwin, B.W. Worsely, Helicobacter pylori, Biology and Clinical Practice, CRC Press, Boca Raton, NY, 1993, pp. 209–222.
214
R.A.H. Ishak et al. / Journal of Controlled Release 119 (2007) 207–214
[23] A.D. Sezer, J. Akbuga, Release characteristics of chitosan-treated alginate beads, 1: Sustained release of a macromolecular drug from chitosan treated alginate beads, J. Microencapsul. 193 (1999) 197–203. [24] P.R. Hari, T. Chandy, C.P. Sharma, Chitosan/calcium alginate microcapsules for intestinal delivery of nitrofurantoin, J. Microencapsul. 13 (3) (1996) 319–329. [25] L.Y. Lim, L.S.C. Wan, Propranolol hydrochloride binding in calcium alginate beads, Drug Dev. Ind. Pharm. 23 (10) (1997) 973–980. [26] P. Aslani, R.A. Kennedy, Effect of gelation conditions and dissolution media on the release of paracetamol from alginate gel beads, J. Microencapsul. 13 (5) (1996) 601–614. [27] H.H. Tønnesen, J. Karisen, Alginate in drug delivery systems, Drug Dev. Ind. Pharm. 28 (6) (2002) 621–630. [28] www. medicinescomplete.com. [29] J.L. Zatz, J.J. Berry, D.A. Alderman, Viscosity-imparting agents in disperse systems, in: H.A. Lieberman (Ed.), 2nd edition, Pharmaceutical Dosage Forms: Disperse Systems, vol. 1, Marcel Dekker Inc., New York, 1996, p. 300.
[30] Carbopol Water Soluble Resins, B.F. Goodrich Technical Brochure. [31] A. Huikari, A. Karlsson, Viscosity stability of methyl cellulose solutions at different pH and temperature, Acta Pharm. Fenn. 984 (1989) 231–238. [32] X.Z. Shu, K.J. Zhu, W. Song, Novel pH-sensitive citrate cross-linked chitosan film for drug controlled release, Int. J. Pharm. 212 (1) (2001) 19–28. [33] F.L. Mi, S.S. Shyu, C.T. Chen, J.Y. Schoung, Porous chitosan microsphere for controlling the antigen release of Newcastle disease vaccine: preparation of antigen-adsorbed microsphere and in vitro release, Biomaterials 20 (17) (1999) 1603–1612. [34] T. Durig, R. Fassihi, Mechanistic evaluation of binary effects of magnesium stearate and talc as dissolution retardants at 85% drug loading in an experimental extended-release formulation, J. Pharm. Sci. 86 (10) (1997) 1092–1098. [35] L. Whitehead, J.T. Fell, J.H. Collett, H.L. Sharma, A.-M. Smith, Floating dosage forms: an in vivo study demonstrating prolonged gastric retention, J. Control. Release 55 (1998) 3–12.