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Colon specific chitosan microspheres for chronotherapy of chronic stable angina
Colloids and Surfaces B: Biointerfaces 83 (2011) 277–283
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Colon specific chitosan microspheres for chronotherapy of chronic stable angina S. Jose a , M.T. Prema a , A.J. Chacko a , A. Cinu Thomas a , E.B. Souto b,c,∗ a
Department of Pharmaceutical Sciences, Mahatma Gandhi University, Cheruvandoor Campus, Ettumanoor-686 631, Kerala, India Institute of Biotechnology and Bioengineering, Centre of Genomics and Biotechnology University of Trás-os-Montes and Alto Douro (CGB-UTAD/IBB), P.O. Box 1013, P-5001-801, Vila Real, Portugal c Faculty of Health Sciences, Fernando Pessoa University, Rua Carlos da Maia, 296, P-4200-150 Porto, Portugal b
a r t i c l e
i n f o
Article history: Received 17 September 2010 Received in revised form 23 November 2010 Accepted 23 November 2010 Available online 1 December 2010 Keywords: Chitosan Chronotherapeutics Colon specific drug delivery Eudragit S-100 Microspheres
a b s t r a c t In the present work, chitosan microspheres with a mean diameter between 6.32 m and 9.44 m, were produced by emulsion cross-linking of chitosan, and tested for chronotherapy of chronic stable angina. Aiming at developing a suitable colon specific strategy, diltiazem hydrochloride (DTZ) was encapsulated in the microspheres, following Eudragit S-100 coating by solvent evaporation technique, exploiting the advantages of microbiological properties of chitosan and pH dependent solubility of Eudragit S-100. Different microsphere formulations were prepared varying the ratio DTZ:chitosan (1:2 to 1:10), stirring speed (1000–2000 rpm), and the concentration of emulsifier Span 80 (0.5–1.5% (w/v)). The effect of these variables on the particle size and encapsulation parameters (production yield (PY), loading capacity (LC), encapsulation efficiency (EE)) was evaluated to develop an optimized formulation. In vitro release study of non-coated chitosan microspheres in simulated gastrointestinal (GI) fluid exhibited a burst release pattern in the first hour, whereas Eudragit S-100 coating allowed producing systems of controlled release diffusion fitting to the Higuchi model, and thus suitable for colon-specific drug delivery. DSC analysis indicated that DTZ was dispersed within the microspheres matrix. Scanning electron microscopy revealed that the microspheres were spherical and had a smooth surface. Chitosan biodegradability was proven by the enhanced release rate of DTZ in presence of rat caecal contents. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Chronotherapy refers to a therapeutic scheme in which in vivo drug availability is time-dependent on the circadian rhythm to produce the maximum health benefit and minimum harm to the patient [1]. Among cardiovascular diseases, hypertension, angina, acute myocardial infarction, and ischemic stroke, have a circadian pattern. Large database analyses and epidemiological studies, have demonstrated that acute myocardial infarction, sudden cardiac death, thrombotic stroke, and angina occur several-fold more frequently in the initial early morning (i.e., 6–12 h), compared with any other time of the day. Colon specific drug delivery systems (CDDS) have been developed as one of the site-specific delivery systems for several therapeutic agents for both local and systemic effects [2,3]. A CDDS could be of additional value when a delay in systemic absorption is desirable from a therapeutic point of view, as for diseases that have peak symptoms in the early morning and exhibit circadian
∗ Corresponding author at: Faculty of Health Sciences, Fernando Pessoa University, Rua Carlos da Maia, 296, P-4200-150 Porto, Portugal. Tel.: +351 225 074630; fax: +351 225 074637. E-mail address: [email protected] (E.B. Souto). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.11.033
rhythm, e.g. angina [4]. Angina pectoris is a clinical syndrome due to myocardial ischemia as a result of an imbalance in the myocardial oxygen supply-demand relationship and may be caused by an increase in myocardial oxygen demand (determined by heart rate, ventricular wall tension and ventricular contractility) or by a decrease in myocardial oxygen supply (primarily determined by coronary blood flow) or both. Diltiazem hydrochloride (DTZ), a calcium channel blocker, inhibits calcium influx into cardiac and vascular smooth muscle tissue and hence, acts as a potent dilator of coronary arteries, thereby reducing myocardial oxygen demand. A therapeutic system that would synchronize the drug delivery with the circadian variation in periods of increased risk is highly desirable for an anti-anginal regimen. This can be achieved by a bed time administration of a drug delivery system which, with a delayed start of drug release, can provide adequate protection in the early morning. In this context, CDDS have been used for chronotherapeutic drug administration [5,6]. CDDS based in microspheres show several advantages in comparison to single unit forms, because the former can: (i) provide more uniform drug dispersion in the gastrointestinal (GI) tract and a more homogeneous drug absorption, (ii) decrease the inter- and intra-individual variability, (iii) allow predicting the gastric emptying, (iv) increase the colonic residence time, and (v) decrease the local irritation. Furthermore, when compared to conventional
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dosage forms, microspheres-based CDDS provide more consistent and reproducible transit through the GI tract [7]. Following bedtime administration, microspheres are expected to maintain a low drug plasma concentration overnight when the cardiovascular risks are reported to be the minimum and release the optimal concentrations in the morning between 6 and 12 h when the ischemic risk is found to be the maximum. DTZ undergoes high pre-systemic metabolism (oral bioavailability is 40%) due to the inhibitory effects of cytochrome P-450 (CYP3A4) and P-glycoprotein, both found in the small intestine. By formulating CDDS, it is expected that DTZ bioavailability will increase as it escapes the interaction with CYP3A4 and P-glycoprotein [8]. In this study, DTZ-loaded chitosan microspheres for chronotherapy of chronic stable angina were produced for colon specific delivery and for that purpose, the systems were coated with Eudragit S-100. Being a pH-sensitive polymer and soluble above pH 7, Eudragit S-100 was used to prevent drug release from microspheres in the small intestine, until they reach the terminal ileum where chitosan ensures a controlled release of DTZ, following degradation by the abundant colonic microflora. 2. Materials and methods
Table 1 Processing variable parameters of the developed chitosan microspheres (rpm, rotations per minute; % (w/v), percentage weight per volume). Parameters
Process variables
Formulation code
Drug–polymer ratio (constant: 1500 rpm; 1%)
1:2 1:4 1:6 1:8 1:10
P1 P2 P3 P4 P5
Rotational speed (constant: 1:6; 1%)
1000 rpm 1500 rpm 2000 rpm
R1 R2 R3
Emulsifier concentration (constant: 1:6; 1500 rpm)
0.5% 1.0% 1.5%
S1 S2 S3
Immediately, 2 ml of ethanol was added drop-wise to form a stable emulsion. The emulsification was maintained for 3 h at 1000 rpm with mechanical stirrer. The Eudragit S-100 coated microspheres were collected, rinsed with petroleum ether and dried in hot air oven at 50 ◦ C. The processing variable parameters of the coated samples are shown in Table 2.
2.1. Materials Diltiazem hydrochloride (Sangrose Laboratories Ltd., Kerala, India), chitosan (Central Institute of Fisheries Technology, Cochin, India) and Eudragit S-100 (poly(methacylic acid-co-methyl methacrylate) 1:2) from Matrix Laboratories (Hyderabad, India) were kindly supplied as gift samples. Glacial acetic acid and Span 80 (sorbitan monooleate) were purchased from Central Drug House (New Delhi, India). All other reagents of analytical grade were supplied by Nice Chemicals (Cochin, India) which include petroleum ether, acetone, methanol, light liquid paraffin and heavy liquid paraffin. 2.2. Methods 2.2.1. Preparation of DTZ-loaded chitosan microspheres Chitosan microspheres were prepared by emulsion crosslinking method [9,10]. Briefly, 2% (w/v) chitosan solution was prepared in 1% aqueous solution of glacial acetic acid following dispersion of DTZ. A volume of 3 ml of resulting solution was then injected with a syringe (No. 23) into 20 ml of oil phase containing Span 80 (1%, v/v). Stirring was performed with a mechanical stirrer (Remi Motors, Mumbai, India) to form a w/o emulsion. The external oil phase was composed of heavy and light liquid paraffins in 1:1 ratio. After 30 min of homogenization, 1.5 ml of toluene-saturated glutaraldehyde (8:1) was added to the emulsion. It was then left for stabilization and cross-linking for a period of 7 h. The formed microspheres were centrifuged at 4000 rpm and the sediment washed with petroleum ether and acetone and dried in hot air oven at 50 ◦ C. The processing variable parameters were adjusted to prepare different batches, as shown in Table 1. For P1–P5 rotational speed selected was 1500 rpm and the emulsifier concentration was 1%. In case of R1–R3, drug–polymer ratio was maintained constant at 1:6, with the emulsifier concentration of 1%. For S1–S3 drug–polymer ratio was maintained constant at 1:6, with a rotational speed of 1500 rpm. 2.2.2. Polymeric coating of DTZ-loaded chitosan microspheres DTZ-loaded chitosan microspheres were coated with Eudragit S-100 by emulsion-solvent evaporation technique [11,12]. The previously obtained chitosan microspheres were suspended in 2.5 ml of ethanolic solution of Eudragit S-100 (10%, w/v) and then emulsified with 40 ml of light liquid paraffin containing Span 80 (1%, v/v).
2.2.3. Particle size and surface morphology analysis The particle size of the uncoated and Eudragit S-100 coated chitosan microspheres were first evaluated using an optical microscope fitted with a calibrated eyepiece micrometer under a magnification of 40×. The particle diameters of about 100 microspheres were measured randomly and the average particle size was determined using the Edmondson’s equation: Dmean =
nd n
where n stands for the number of counted microspheres, and d for the mean size range. The shape and surface morphology of the microspheres were studied using scanning electron microscopy (SEM). Microspheres were fixed with carbon tape, mounted on metal stubs and then coated with platinum, keeping the acceleration voltage at 10 kV. Photographs were taken using scanning electron microscope (6390, Jeol JSM). 2.2.4. Determination of percentage yield, loading capacity and encapsulation efficiency The percentage yield (PY) was calculated based on the dry weight of DTZ and the polymers, applying the following equation: Percentage Yield (PY) =
Obtained mass of microspheres × 100 Initial mass of drug + Initial mass of polymer
10 mg of microspheres were taken and triturated with 20 ml of methanol and kept for overnight drug extraction. After filtration and appropriate dilution with methanol, the absorbance was measured at 240 nm using a UV spectrophotometer (2600, Chemito). The drug loading capacity (LC) was calculated using the following
Table 2 Core–coat ratio of Eudragit coated chitosan microspheres. Core:coat ratio 1:4 1:6 1:8
Formulation code MC1 MC2 MC3
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equation: Loading Capacity (LC) =
Mass of DTZ in microspheres × 100 Mass of microspheres
The encapsulation efficiency (EE) was determined as the mass of DTZ encapsulated in the microspheres with respect to the total mass of DTZ initially weighted, using the following equation: Encapsulation Efficiency (EE) =
Mass of DTZ in microspheres × 100 Initial mass of DTZ
2.2.5. Thermal analysis The thermal analysis of pure DTZ, bulk chitosan, bulk Eudragit S-100 and of optimized microsphere formulation, were carried out by differential scanning calorimetry (DSC) equipped with a thermal analysis data system (PerkinElmer, USA). Samples weighting 3–5 mg were heated in flat-bottomed sealed aluminum pans over a temperature range of 40–300 ◦ C at a constant rate of 10 ◦ C/min under nitrogen purge (50 ml/min) using an empty aluminum pan as reference.
2.2.6. In vitro release of DTZ-loaded chitosan microspheres in simulated GI conditions The microspheres were tested for the in vitro DTZ release in simulated GI fluids [13]. An accurately weighed amount of microspheres, equivalent to 30 mg of DTZ, were added to 450 ml of dissolution medium and the drug release from microspheres was processed using USP rotating paddle dissolution apparatus at 100 rpm and at 37 ± 0.5 ◦ C. Perfect sink conditions prevailed during the drug dissolution study period. The simulation of GI pH variations was accomplished by modifying the pH of the dissolution medium at various time intervals. The pH of the dissolution medium was kept at 1.2 for 2 h with 0.1 N HCl. Then, 1.7 g of KH2 PO4 and 2.225 g of Na2 HPO4 ·2H2 O were added, adjusting the pH to 4.5 with 1.0 M NaOH. The release rate analysis was run for another 2 h. After 4 h, the pH of the dissolution medium was adjusted to 7.4 with 1.0 M NaOH and maintained up to 12 h. A sample volume of 2 ml was withdrawn from the medium at various time intervals and replaced with fresh dissolution medium. Samples were then subjected to UV analysis, as described previously. All release tests were performed in triplicate. The effects of drug–polymer ratio on in vitro drug release of DTZ-loaded chitosan microspheres were also evaluated.
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2.2.7. In vitro release of DTZ-loaded chitosan microspheres in the presence of rat caecal contents In vitro drug release from the optimized batch of Eudragit coated chitosan microspheres was performed in the presence of rat caecal contents to assess the biodegradability of chitosan by the colonic bacteria [13]. The study was approved by the Institutional Animal Ethical Committee (IAEC) under the section IAEC/MGU/CHE/M.Pharm/009/2009 dated 23/06/09. Four albino rats (Wistar strain) of uniform body weight (150–200 g) with no prior drug treatment were maintained on normal diet, and administered 1 ml of 2% dispersion of chitosan in water. This treatment was undertaken for 7 days to induce the enzymes that specifically act on the chitosan. Approximately 30 min before starting the study, each rat was sacrificed and the abdomen opened. The caecum was traced, legated at both ends, dissected, and immediately transferred into phosphate buffered saline (PBS) of pH 6.8, previously bubbled with CO2 . The caecal bag was opened, the contents were weighed, homogenized, and then suspended in simulated intestinal fluid of pH 7.4 to give the desired concentration of 2% caecal content, which was used as simulated colonic fluid. The experiment was carried out with a continuous supply of CO2 into dissolution media. Drug release studies for the initial 4 h were performed in simulated GI fluids as described above. After 4 h the studies were carried out in simulated intestinal fluid containing rat caecal contents. Aliquots of samples were withdrawn periodically and replaced with fresh buffer bubbled with CO2 . The samples were filtered through Whatman filter paper following determination of the DTZ content. 2.2.8. Estimation of kinetic model of DTZ-loaded chitosan microspheres release The in vitro drug release data of the optimized formulation MC2 was fitted to kinetic models, i.e. zero order, first order, Higuchi and Peppas models, as described by Costa and Sousa Lobo [14]. 3. Results DTZ-loaded chitosan microspheres prepared by emulsion cross-linking method were brownish-yellow colored free flowing particles. The processing variable parameters, e.g. polymer concentration, rotational speed, and emulsifier concentration, were changed to formulate different batches of DTZ-loaded chitosan microspheres (Table 1), and obtained mean particle size is given in Table 3. When varying the drug–polymer ratio from 1:2 to 1:10, the mean diameter of microspheres was in the range between 6.32 m and 9.44 m. The size increased with increasing polymer concentration, whereas it decreased from 12.72 to 5.20 m when increasing the rotational speed from 1000 to 2000 rpm. Span 80
Table 3 Average particle size, percentage yield, percent drug content and entrapment efficiency of uncoated and Eudragit coated chitosan microspheres (The results were subjected to one way ANOVA (Tukeys Test). (ns, non-significant; * mean ± SD (standard deviation); n = 3). Formulation code
Average particle size (m)
Percentage yield (%)*
P1 P2 P3 P4 P5 R1 R2 R3 S1 S2 S3 MC1 MC2 MC3
6.32 6.56 7.68 8.64 9.44 12.72 8.64 5.20 10.36 8.64 7.28 119 136 152
77.98 83.99 88.41 93.75 91.25 88.01 93.75 88.58 90.61 93.75 91.66 91.83 93.97 93.07
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.92 0.38 0.75 0.43 0.97 1.02 0.43 1.20 0.411 0.43 0.68 0.76 0.15 0.45
Loading capacity (%)*
Encapsulation efficiency (%)*
25.82 ± 0.31 16.08 ± 0.19 11.83 ± 0.07 9.79 ± 0.95 7.76 ± 0.11 9.80 ± 0.04 9.8 ± 0.07 9.61 ± 0.04 9.55 ± 0.06 9.79 ± 0.09 9.6 ± 0.05 8.9 9.22 9.18
77.48 ± 0.93 80.00 ± 0.96 82.76 ± 0.49 88.1 ± 0.86ns 89.25 ± 0.64ns 89.02 ± 0.32 88.12 ± 0.55 86.49 ± 0.32 86.57 ± 0.78ns 88.12 ± 0.79ns 87.51 ± 0.61ns 91.54 93.77 94.20ns
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Fig. 1. SEM image of DTZ-loaded chitosan microspheres.
concentration also influenced the microspheres mean diameter since it decreased from 10.36 m to 7.28 m when increasing the emulsifier concentration from 0.5% to 1.5% (w/v). The optimal Span 80 concentration was found to be 1.0% (w/v). SEM analysis revealed spherical-like shaped microspheres with a smooth surface (Fig. 1). The values of PY, LC and EE of several batches of DTZ-loaded chitosan microspheres are also listed in Table 3. The PY values varied between 77% and 93%. The EE increased with increasing drug–polymer ratio, and the highest value was observed for P5 which employed a drug–polymer ratio of 1:10. Other formulations depicted EE values between 76% and 89%. The EE was found to decrease from 89.0% to 86.5% upon increasing the rotational speed from 1000 to 2000 rpm. The influence of Span 80 concentration was not significant on EE values, as confirmed by ANOVA. DSC thermogram of pure DTZ exhibited a single sharp endothermic peak at 210 ◦ C, whereas no melting event of the drug was detected after loaded within chitosan microspheres (Fig. 2a and b). DTZ-loaded chitosan microspheres were subjected to in vitro dug release studies in the presence of simulated GI fluids using USP dissolution test apparatus. The studies were carried out in 450 ml of the dissolution medium, stirred at 100 rpm at 37 ± 0.1 ◦ C. The dissolution profiles for different batches were studied using acid buffer solution of pH 1.2 for 2 h (simulated gastric fluid), pH 4.5 for another 2 h (simulated duodenum), followed by pH 7.4 (simulated distal ileum and colon) for the remaining period of the study (Fig. 3).
Fig. 2. DSC thermogram of diltiazem hydrochloride (a); DSC thermogram of diltiazem hydrochloride loaded chitosan microspheres (b).
Eudragit S-100 coated chitosan microspheres prepared by emulsion solvent evaporation method were white colored free flowing particles. The average particle size of the formulations increased from 119 m to 152 m while increasing the core–coat ratio from 1:4 to 1:8. The coating efficiency varied between 91% and 94%. SEM analyses depicted discrete, uniform and spherical microspheres with a smooth surface as shown in Fig. 4. The in vitro release profiles of DTZ from the Eudragit coated chitosan microspheres in simulated GI fluids are depicted in Fig. 5. Results show that 9.5–12.7% of drug was released from the formu-
Fig. 3. In vitro release profiles showing the effect of drug to polymer ratio on DTZ release from chitosan microspheres (Formulations composition: Table 1). (Error bars ± SD (standard deviation); n = 3).
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Table 4 Correlation co-efficient value for drug release kinetics. Formulation code MC2
Correlation co-efficient R2 value Zero order
First order
Higuchi model
Peppas model
0.9765
0.8989
0.9935
0.9733
Table 5 Release exponent of the Peppas model plot.
Fig. 4. SEM image of Eudragit S-100 coated DTZ-loaded chitosan microspheres.
lations in the initial 4 h and thereafter the release rate increased. MC2 was selected as the optimized composition and later subjected to in vitro drug release in the presence of rat caecal contents. Comparative in vitro drug release profile of MC2 in the presence and absence of rat caecal contents is shown in Fig. 6. The different release plots obtained upon fitting the release data of MC2 are shown in Fig. 7. Comparing the correlation coefficients
Formulation code
Release exponent (n)
MC2
1.080
given in Table 4, a good fitting to Higuchi model could be anticipated for MC2. When analyzed according to the Peppas model, the release exponent was found to be greater than 1.0 (Table 5). DTZ-loaded chitosan microspheres were prepared by emulsion cross-linking method, where the drug was firstly dispersed in the chitosan solution and then emulsified in the oily phase in the presence of Span 80. The polysaccharide (chitosan) figuring in the internal phase was then cross-linked with glutaraldehyde saturated toluene. When aqueous glutaraldehyde was added to the dispersion of chitosan in paraffin oil, an instantaneous reaction occurred and the resultant product did not exhibit satisfactory spherical shape and smooth surface texture. Slow and uniform cross-linking of the droplets, par-
Fig. 5. Percentage DZT cumulative release from Eudragit coated chitosan microspheres in simulated GI fluids (Formulations composition: Table 3). (Error bars ± SD (standard deviation); n = 3).
Fig. 6. Percentage DZT cumulative release from Eudragit coated chitosan microspheres with and without rat caecal contents (Formulations composition: Table 3). (Error bars ± SD (standard deviation); n = 3).
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Fig. 7. Kinetic profiles obtained for MC2 following zero order (a), First order (b), Higuchi (c) and Peppas (d) models.
ticularly at the surface, was found desirable to form microspheres of appropriate sphericity. Thus, glutaraldehyde saturated toluene was used which, due to its solubility in oil medium, would help to uniformly cross-link the surface of the droplets. The average size of microspheres increased with increasing polysaccharide concentration, since a higher concentration of chitosan produced a more viscous dispersion, which formed larger droplets and consequently larger microspheres. The ultimate mean diameter of microspheres was determined by the size of dispersion of chitosan solution, which decreased with increasing mixer stirring speed. Higher stirring rates provided the shearing force needed to separate the oil phase into smaller droplets decreasing therefore the particle size. Span 80 tends to lower the interfacial tension between the two phases thus stabilizing the emulsion. An optimal concentration of emulsifier is required to produce the finest stable dispersion. Below this concentration, the dispersed droplets tend to fuse and increase their mean diameter because of insufficient lowering of interfacial tension. On the other hand, above the optimal concentration no significant decrease in particle size is observed, because a high amount of emulsifying agent increases the viscosity of the dispersion medium. The optimal concentration of Span 80 was found to be 1.0%. 4. Discussion For any drug delivery system aimed at targeting the colon, the release of the drug during its transit through the stomach and small intestine must be kept to the minimum to ensure that maximum dose reaches the colon. For in vitro release study, the pH condition was selected so as to mimic the GI conditions without enzymes. In vitro drug release of DTZ from chitosan microspheres showed a burst release in the initial hour (Fig. 3). Within 4 h 73–87% of drug was released from the formulations P1, P2, P3, P4 and P5. These profiles are not acceptable for systems aiming at releasing their load in the colon. The burst release may be due to the solubility of chitosan in the acidic pH. To overcome this shortcoming and avoid the drug release in the stomach and small intestine, the chitosan microspheres were coated with Eudragit S-100, which is a pH sensitive polymer having a threshold pH of 7.0. Based on the high EE and appropriate release pattern, formulation P4 was selected and subjected to a coating process with Eudragit S-100. Since this polymer exhibits solubility above pH 7 corresponding to the pH of the colon, it should prevent any significant DTZ release in the upper segments of the gut. The coating of chitosan microspheres was achieved using emulsion solvent evaporation method. The mean particle size of
the coated microspheres increased from 119 m to 152 m with increasing the core–coat ratio from 1:4 to 1:8, which may be due to the corresponding increase in the polymer concentration that resulted in larger emulsion droplets. The drug release studies indicate that Eudragit coating of chitosan microspheres offered a high degree of protection from the premature DTZ release in the stomach and small intestine. DTZ release rate increased after 4 h, since at that time formulations were exposed to pH 7.4, which is above the solubilizing pH of Eudragit S-100 polymer. It is expected that DTZ release from cross-linked chitosan microspheres occurred due to swelling of the polymer, resulting in the formation of a gel. This is followed by drug dissolution and further diffusion through the gel. Based on better EE (93.77%) and drug release pattern, MC2 was considered as the optimized formulation and therefore selected for in vitro drug release study in presence of rat ceacal contents, to assess the biodegradability of chitosan. A colon targeted drug delivery system should not only protect its load from being released in the physiological environment of the stomach and small intestine, but also ensure its delivery in the colon. Conventional dissolution testing is less likely to accurately predict the in vivo performance of CDDS particularly for which the release is triggered by colonic bacterial enzymes. Thus, release studies in the presence of rat ceacal contents were carried out from the 5th hour in the simulated intestinal fluid of pH 7.4. When carried out in the presence of rat caecal contents, drug release was significantly improved clearly indicating the biodegradability of chitosan by the colonic microflora and thereby the specific release in the colon. More than 98% of DTZ was released after 12 h when the dissolution study was carried out in the presence of rat caecal contents. Drug release mechanisms were determined by fitting drug release data of MC2 to various kinetic models. By comparing the correlation coefficient values (Table 4), from the applied models, the Higuchi model (R = 0.9924) was shown the most appropriate to describe the kinetics of MC2 formulation. Thus, the drug release mechanism was assumed to be controlled by diffusion. When analyzed according to the Peppas model, the release exponent was found to be greater than 1.0 (Table 5). Therefore, the diffusional release was found to follow super case II transport. 5. Conclusion Eudragit S-100 coated DTZ-loaded chitosan microspheres were successfully prepared. The microparticles were discrete, spherical
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and showed appropriate release patterns in simulated GI fluids and further enhanced release characteristics in the presence of colonic microflora. Eudragit S-100 proved to be effective in preventing a premature release of DTZ in the upper parts of the gut, while chitosan ensured a controlled release of DTZ in the colon. The biodegradability of chitosan was confirmed by the enhanced drug release in the presence of rat caecal contents. The release pattern of DTZ gave a good fit to Higuchi model indicating a diffusion controlled release with super case II transport. A novel CDDS for bedtime dosing has been presented here. From the obtained results, it can be anticipated that the developed Eudragit S-100 coated chitosan microspheres is a promising alternative for the treatment of early morning pathologies influenced by circadian rhythms by opening a ‘new lease of life’ to an existing drug molecule. References [1] W.J. Hrushesky, J. Grutsch, P. Wood, X. Yang, E.Y. Oh, C. Ansell, S. Kidder, C. Ferrans, D.F. Quiton, J. Reynolds, J. Du-Quiton, R. Levin, C. Lis, D. Braun, Circadian clock manipulation for cancer prevention and control and the relief of cancer symptoms, Integr. Cancer Ther. 8 (2009) 387–397. [2] V.D. Kadam, S.G. Gattani, Development of colon targeted multiparticulate pulsatile drug delivery system for treating nocturnal asthma, Drug Deliv. 17 (2010) 343–351. [3] Y. Lee, I.H. Kim, J. Kim, J.H. Yoon, Y.H. Shin, Y. Jung, Y.M. Kim, Evaluation of dextran-flufenamic acid ester as a polymeric colon-specific prodrug of flufenamic acid, an anti-inflammatory drug, for chronotherapy, J. Drug Target. (2010).
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Colon specific chitosan microspheres for chronotherapy of chronic stable angina S. Jose a , M.T. Prema a , A.J. Chacko a , A. Cinu Thomas a , E.B. Souto b,c,∗ a
Department of Pharmaceutical Sciences, Mahatma Gandhi University, Cheruvandoor Campus, Ettumanoor-686 631, Kerala, India Institute of Biotechnology and Bioengineering, Centre of Genomics and Biotechnology University of Trás-os-Montes and Alto Douro (CGB-UTAD/IBB), P.O. Box 1013, P-5001-801, Vila Real, Portugal c Faculty of Health Sciences, Fernando Pessoa University, Rua Carlos da Maia, 296, P-4200-150 Porto, Portugal b
a r t i c l e
i n f o
Article history: Received 17 September 2010 Received in revised form 23 November 2010 Accepted 23 November 2010 Available online 1 December 2010 Keywords: Chitosan Chronotherapeutics Colon specific drug delivery Eudragit S-100 Microspheres
a b s t r a c t In the present work, chitosan microspheres with a mean diameter between 6.32 m and 9.44 m, were produced by emulsion cross-linking of chitosan, and tested for chronotherapy of chronic stable angina. Aiming at developing a suitable colon specific strategy, diltiazem hydrochloride (DTZ) was encapsulated in the microspheres, following Eudragit S-100 coating by solvent evaporation technique, exploiting the advantages of microbiological properties of chitosan and pH dependent solubility of Eudragit S-100. Different microsphere formulations were prepared varying the ratio DTZ:chitosan (1:2 to 1:10), stirring speed (1000–2000 rpm), and the concentration of emulsifier Span 80 (0.5–1.5% (w/v)). The effect of these variables on the particle size and encapsulation parameters (production yield (PY), loading capacity (LC), encapsulation efficiency (EE)) was evaluated to develop an optimized formulation. In vitro release study of non-coated chitosan microspheres in simulated gastrointestinal (GI) fluid exhibited a burst release pattern in the first hour, whereas Eudragit S-100 coating allowed producing systems of controlled release diffusion fitting to the Higuchi model, and thus suitable for colon-specific drug delivery. DSC analysis indicated that DTZ was dispersed within the microspheres matrix. Scanning electron microscopy revealed that the microspheres were spherical and had a smooth surface. Chitosan biodegradability was proven by the enhanced release rate of DTZ in presence of rat caecal contents. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Chronotherapy refers to a therapeutic scheme in which in vivo drug availability is time-dependent on the circadian rhythm to produce the maximum health benefit and minimum harm to the patient [1]. Among cardiovascular diseases, hypertension, angina, acute myocardial infarction, and ischemic stroke, have a circadian pattern. Large database analyses and epidemiological studies, have demonstrated that acute myocardial infarction, sudden cardiac death, thrombotic stroke, and angina occur several-fold more frequently in the initial early morning (i.e., 6–12 h), compared with any other time of the day. Colon specific drug delivery systems (CDDS) have been developed as one of the site-specific delivery systems for several therapeutic agents for both local and systemic effects [2,3]. A CDDS could be of additional value when a delay in systemic absorption is desirable from a therapeutic point of view, as for diseases that have peak symptoms in the early morning and exhibit circadian
∗ Corresponding author at: Faculty of Health Sciences, Fernando Pessoa University, Rua Carlos da Maia, 296, P-4200-150 Porto, Portugal. Tel.: +351 225 074630; fax: +351 225 074637. E-mail address: [email protected] (E.B. Souto). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.11.033
rhythm, e.g. angina [4]. Angina pectoris is a clinical syndrome due to myocardial ischemia as a result of an imbalance in the myocardial oxygen supply-demand relationship and may be caused by an increase in myocardial oxygen demand (determined by heart rate, ventricular wall tension and ventricular contractility) or by a decrease in myocardial oxygen supply (primarily determined by coronary blood flow) or both. Diltiazem hydrochloride (DTZ), a calcium channel blocker, inhibits calcium influx into cardiac and vascular smooth muscle tissue and hence, acts as a potent dilator of coronary arteries, thereby reducing myocardial oxygen demand. A therapeutic system that would synchronize the drug delivery with the circadian variation in periods of increased risk is highly desirable for an anti-anginal regimen. This can be achieved by a bed time administration of a drug delivery system which, with a delayed start of drug release, can provide adequate protection in the early morning. In this context, CDDS have been used for chronotherapeutic drug administration [5,6]. CDDS based in microspheres show several advantages in comparison to single unit forms, because the former can: (i) provide more uniform drug dispersion in the gastrointestinal (GI) tract and a more homogeneous drug absorption, (ii) decrease the inter- and intra-individual variability, (iii) allow predicting the gastric emptying, (iv) increase the colonic residence time, and (v) decrease the local irritation. Furthermore, when compared to conventional
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dosage forms, microspheres-based CDDS provide more consistent and reproducible transit through the GI tract [7]. Following bedtime administration, microspheres are expected to maintain a low drug plasma concentration overnight when the cardiovascular risks are reported to be the minimum and release the optimal concentrations in the morning between 6 and 12 h when the ischemic risk is found to be the maximum. DTZ undergoes high pre-systemic metabolism (oral bioavailability is 40%) due to the inhibitory effects of cytochrome P-450 (CYP3A4) and P-glycoprotein, both found in the small intestine. By formulating CDDS, it is expected that DTZ bioavailability will increase as it escapes the interaction with CYP3A4 and P-glycoprotein [8]. In this study, DTZ-loaded chitosan microspheres for chronotherapy of chronic stable angina were produced for colon specific delivery and for that purpose, the systems were coated with Eudragit S-100. Being a pH-sensitive polymer and soluble above pH 7, Eudragit S-100 was used to prevent drug release from microspheres in the small intestine, until they reach the terminal ileum where chitosan ensures a controlled release of DTZ, following degradation by the abundant colonic microflora. 2. Materials and methods
Table 1 Processing variable parameters of the developed chitosan microspheres (rpm, rotations per minute; % (w/v), percentage weight per volume). Parameters
Process variables
Formulation code
Drug–polymer ratio (constant: 1500 rpm; 1%)
1:2 1:4 1:6 1:8 1:10
P1 P2 P3 P4 P5
Rotational speed (constant: 1:6; 1%)
1000 rpm 1500 rpm 2000 rpm
R1 R2 R3
Emulsifier concentration (constant: 1:6; 1500 rpm)
0.5% 1.0% 1.5%
S1 S2 S3
Immediately, 2 ml of ethanol was added drop-wise to form a stable emulsion. The emulsification was maintained for 3 h at 1000 rpm with mechanical stirrer. The Eudragit S-100 coated microspheres were collected, rinsed with petroleum ether and dried in hot air oven at 50 ◦ C. The processing variable parameters of the coated samples are shown in Table 2.
2.1. Materials Diltiazem hydrochloride (Sangrose Laboratories Ltd., Kerala, India), chitosan (Central Institute of Fisheries Technology, Cochin, India) and Eudragit S-100 (poly(methacylic acid-co-methyl methacrylate) 1:2) from Matrix Laboratories (Hyderabad, India) were kindly supplied as gift samples. Glacial acetic acid and Span 80 (sorbitan monooleate) were purchased from Central Drug House (New Delhi, India). All other reagents of analytical grade were supplied by Nice Chemicals (Cochin, India) which include petroleum ether, acetone, methanol, light liquid paraffin and heavy liquid paraffin. 2.2. Methods 2.2.1. Preparation of DTZ-loaded chitosan microspheres Chitosan microspheres were prepared by emulsion crosslinking method [9,10]. Briefly, 2% (w/v) chitosan solution was prepared in 1% aqueous solution of glacial acetic acid following dispersion of DTZ. A volume of 3 ml of resulting solution was then injected with a syringe (No. 23) into 20 ml of oil phase containing Span 80 (1%, v/v). Stirring was performed with a mechanical stirrer (Remi Motors, Mumbai, India) to form a w/o emulsion. The external oil phase was composed of heavy and light liquid paraffins in 1:1 ratio. After 30 min of homogenization, 1.5 ml of toluene-saturated glutaraldehyde (8:1) was added to the emulsion. It was then left for stabilization and cross-linking for a period of 7 h. The formed microspheres were centrifuged at 4000 rpm and the sediment washed with petroleum ether and acetone and dried in hot air oven at 50 ◦ C. The processing variable parameters were adjusted to prepare different batches, as shown in Table 1. For P1–P5 rotational speed selected was 1500 rpm and the emulsifier concentration was 1%. In case of R1–R3, drug–polymer ratio was maintained constant at 1:6, with the emulsifier concentration of 1%. For S1–S3 drug–polymer ratio was maintained constant at 1:6, with a rotational speed of 1500 rpm. 2.2.2. Polymeric coating of DTZ-loaded chitosan microspheres DTZ-loaded chitosan microspheres were coated with Eudragit S-100 by emulsion-solvent evaporation technique [11,12]. The previously obtained chitosan microspheres were suspended in 2.5 ml of ethanolic solution of Eudragit S-100 (10%, w/v) and then emulsified with 40 ml of light liquid paraffin containing Span 80 (1%, v/v).
2.2.3. Particle size and surface morphology analysis The particle size of the uncoated and Eudragit S-100 coated chitosan microspheres were first evaluated using an optical microscope fitted with a calibrated eyepiece micrometer under a magnification of 40×. The particle diameters of about 100 microspheres were measured randomly and the average particle size was determined using the Edmondson’s equation: Dmean =
nd n
where n stands for the number of counted microspheres, and d for the mean size range. The shape and surface morphology of the microspheres were studied using scanning electron microscopy (SEM). Microspheres were fixed with carbon tape, mounted on metal stubs and then coated with platinum, keeping the acceleration voltage at 10 kV. Photographs were taken using scanning electron microscope (6390, Jeol JSM). 2.2.4. Determination of percentage yield, loading capacity and encapsulation efficiency The percentage yield (PY) was calculated based on the dry weight of DTZ and the polymers, applying the following equation: Percentage Yield (PY) =
Obtained mass of microspheres × 100 Initial mass of drug + Initial mass of polymer
10 mg of microspheres were taken and triturated with 20 ml of methanol and kept for overnight drug extraction. After filtration and appropriate dilution with methanol, the absorbance was measured at 240 nm using a UV spectrophotometer (2600, Chemito). The drug loading capacity (LC) was calculated using the following
Table 2 Core–coat ratio of Eudragit coated chitosan microspheres. Core:coat ratio 1:4 1:6 1:8
Formulation code MC1 MC2 MC3
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equation: Loading Capacity (LC) =
Mass of DTZ in microspheres × 100 Mass of microspheres
The encapsulation efficiency (EE) was determined as the mass of DTZ encapsulated in the microspheres with respect to the total mass of DTZ initially weighted, using the following equation: Encapsulation Efficiency (EE) =
Mass of DTZ in microspheres × 100 Initial mass of DTZ
2.2.5. Thermal analysis The thermal analysis of pure DTZ, bulk chitosan, bulk Eudragit S-100 and of optimized microsphere formulation, were carried out by differential scanning calorimetry (DSC) equipped with a thermal analysis data system (PerkinElmer, USA). Samples weighting 3–5 mg were heated in flat-bottomed sealed aluminum pans over a temperature range of 40–300 ◦ C at a constant rate of 10 ◦ C/min under nitrogen purge (50 ml/min) using an empty aluminum pan as reference.
2.2.6. In vitro release of DTZ-loaded chitosan microspheres in simulated GI conditions The microspheres were tested for the in vitro DTZ release in simulated GI fluids [13]. An accurately weighed amount of microspheres, equivalent to 30 mg of DTZ, were added to 450 ml of dissolution medium and the drug release from microspheres was processed using USP rotating paddle dissolution apparatus at 100 rpm and at 37 ± 0.5 ◦ C. Perfect sink conditions prevailed during the drug dissolution study period. The simulation of GI pH variations was accomplished by modifying the pH of the dissolution medium at various time intervals. The pH of the dissolution medium was kept at 1.2 for 2 h with 0.1 N HCl. Then, 1.7 g of KH2 PO4 and 2.225 g of Na2 HPO4 ·2H2 O were added, adjusting the pH to 4.5 with 1.0 M NaOH. The release rate analysis was run for another 2 h. After 4 h, the pH of the dissolution medium was adjusted to 7.4 with 1.0 M NaOH and maintained up to 12 h. A sample volume of 2 ml was withdrawn from the medium at various time intervals and replaced with fresh dissolution medium. Samples were then subjected to UV analysis, as described previously. All release tests were performed in triplicate. The effects of drug–polymer ratio on in vitro drug release of DTZ-loaded chitosan microspheres were also evaluated.
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2.2.7. In vitro release of DTZ-loaded chitosan microspheres in the presence of rat caecal contents In vitro drug release from the optimized batch of Eudragit coated chitosan microspheres was performed in the presence of rat caecal contents to assess the biodegradability of chitosan by the colonic bacteria [13]. The study was approved by the Institutional Animal Ethical Committee (IAEC) under the section IAEC/MGU/CHE/M.Pharm/009/2009 dated 23/06/09. Four albino rats (Wistar strain) of uniform body weight (150–200 g) with no prior drug treatment were maintained on normal diet, and administered 1 ml of 2% dispersion of chitosan in water. This treatment was undertaken for 7 days to induce the enzymes that specifically act on the chitosan. Approximately 30 min before starting the study, each rat was sacrificed and the abdomen opened. The caecum was traced, legated at both ends, dissected, and immediately transferred into phosphate buffered saline (PBS) of pH 6.8, previously bubbled with CO2 . The caecal bag was opened, the contents were weighed, homogenized, and then suspended in simulated intestinal fluid of pH 7.4 to give the desired concentration of 2% caecal content, which was used as simulated colonic fluid. The experiment was carried out with a continuous supply of CO2 into dissolution media. Drug release studies for the initial 4 h were performed in simulated GI fluids as described above. After 4 h the studies were carried out in simulated intestinal fluid containing rat caecal contents. Aliquots of samples were withdrawn periodically and replaced with fresh buffer bubbled with CO2 . The samples were filtered through Whatman filter paper following determination of the DTZ content. 2.2.8. Estimation of kinetic model of DTZ-loaded chitosan microspheres release The in vitro drug release data of the optimized formulation MC2 was fitted to kinetic models, i.e. zero order, first order, Higuchi and Peppas models, as described by Costa and Sousa Lobo [14]. 3. Results DTZ-loaded chitosan microspheres prepared by emulsion cross-linking method were brownish-yellow colored free flowing particles. The processing variable parameters, e.g. polymer concentration, rotational speed, and emulsifier concentration, were changed to formulate different batches of DTZ-loaded chitosan microspheres (Table 1), and obtained mean particle size is given in Table 3. When varying the drug–polymer ratio from 1:2 to 1:10, the mean diameter of microspheres was in the range between 6.32 m and 9.44 m. The size increased with increasing polymer concentration, whereas it decreased from 12.72 to 5.20 m when increasing the rotational speed from 1000 to 2000 rpm. Span 80
Table 3 Average particle size, percentage yield, percent drug content and entrapment efficiency of uncoated and Eudragit coated chitosan microspheres (The results were subjected to one way ANOVA (Tukeys Test). (ns, non-significant; * mean ± SD (standard deviation); n = 3). Formulation code
Average particle size (m)
Percentage yield (%)*
P1 P2 P3 P4 P5 R1 R2 R3 S1 S2 S3 MC1 MC2 MC3
6.32 6.56 7.68 8.64 9.44 12.72 8.64 5.20 10.36 8.64 7.28 119 136 152
77.98 83.99 88.41 93.75 91.25 88.01 93.75 88.58 90.61 93.75 91.66 91.83 93.97 93.07
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.92 0.38 0.75 0.43 0.97 1.02 0.43 1.20 0.411 0.43 0.68 0.76 0.15 0.45
Loading capacity (%)*
Encapsulation efficiency (%)*
25.82 ± 0.31 16.08 ± 0.19 11.83 ± 0.07 9.79 ± 0.95 7.76 ± 0.11 9.80 ± 0.04 9.8 ± 0.07 9.61 ± 0.04 9.55 ± 0.06 9.79 ± 0.09 9.6 ± 0.05 8.9 9.22 9.18
77.48 ± 0.93 80.00 ± 0.96 82.76 ± 0.49 88.1 ± 0.86ns 89.25 ± 0.64ns 89.02 ± 0.32 88.12 ± 0.55 86.49 ± 0.32 86.57 ± 0.78ns 88.12 ± 0.79ns 87.51 ± 0.61ns 91.54 93.77 94.20ns
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Fig. 1. SEM image of DTZ-loaded chitosan microspheres.
concentration also influenced the microspheres mean diameter since it decreased from 10.36 m to 7.28 m when increasing the emulsifier concentration from 0.5% to 1.5% (w/v). The optimal Span 80 concentration was found to be 1.0% (w/v). SEM analysis revealed spherical-like shaped microspheres with a smooth surface (Fig. 1). The values of PY, LC and EE of several batches of DTZ-loaded chitosan microspheres are also listed in Table 3. The PY values varied between 77% and 93%. The EE increased with increasing drug–polymer ratio, and the highest value was observed for P5 which employed a drug–polymer ratio of 1:10. Other formulations depicted EE values between 76% and 89%. The EE was found to decrease from 89.0% to 86.5% upon increasing the rotational speed from 1000 to 2000 rpm. The influence of Span 80 concentration was not significant on EE values, as confirmed by ANOVA. DSC thermogram of pure DTZ exhibited a single sharp endothermic peak at 210 ◦ C, whereas no melting event of the drug was detected after loaded within chitosan microspheres (Fig. 2a and b). DTZ-loaded chitosan microspheres were subjected to in vitro dug release studies in the presence of simulated GI fluids using USP dissolution test apparatus. The studies were carried out in 450 ml of the dissolution medium, stirred at 100 rpm at 37 ± 0.1 ◦ C. The dissolution profiles for different batches were studied using acid buffer solution of pH 1.2 for 2 h (simulated gastric fluid), pH 4.5 for another 2 h (simulated duodenum), followed by pH 7.4 (simulated distal ileum and colon) for the remaining period of the study (Fig. 3).
Fig. 2. DSC thermogram of diltiazem hydrochloride (a); DSC thermogram of diltiazem hydrochloride loaded chitosan microspheres (b).
Eudragit S-100 coated chitosan microspheres prepared by emulsion solvent evaporation method were white colored free flowing particles. The average particle size of the formulations increased from 119 m to 152 m while increasing the core–coat ratio from 1:4 to 1:8. The coating efficiency varied between 91% and 94%. SEM analyses depicted discrete, uniform and spherical microspheres with a smooth surface as shown in Fig. 4. The in vitro release profiles of DTZ from the Eudragit coated chitosan microspheres in simulated GI fluids are depicted in Fig. 5. Results show that 9.5–12.7% of drug was released from the formu-
Fig. 3. In vitro release profiles showing the effect of drug to polymer ratio on DTZ release from chitosan microspheres (Formulations composition: Table 1). (Error bars ± SD (standard deviation); n = 3).
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Table 4 Correlation co-efficient value for drug release kinetics. Formulation code MC2
Correlation co-efficient R2 value Zero order
First order
Higuchi model
Peppas model
0.9765
0.8989
0.9935
0.9733
Table 5 Release exponent of the Peppas model plot.
Fig. 4. SEM image of Eudragit S-100 coated DTZ-loaded chitosan microspheres.
lations in the initial 4 h and thereafter the release rate increased. MC2 was selected as the optimized composition and later subjected to in vitro drug release in the presence of rat caecal contents. Comparative in vitro drug release profile of MC2 in the presence and absence of rat caecal contents is shown in Fig. 6. The different release plots obtained upon fitting the release data of MC2 are shown in Fig. 7. Comparing the correlation coefficients
Formulation code
Release exponent (n)
MC2
1.080
given in Table 4, a good fitting to Higuchi model could be anticipated for MC2. When analyzed according to the Peppas model, the release exponent was found to be greater than 1.0 (Table 5). DTZ-loaded chitosan microspheres were prepared by emulsion cross-linking method, where the drug was firstly dispersed in the chitosan solution and then emulsified in the oily phase in the presence of Span 80. The polysaccharide (chitosan) figuring in the internal phase was then cross-linked with glutaraldehyde saturated toluene. When aqueous glutaraldehyde was added to the dispersion of chitosan in paraffin oil, an instantaneous reaction occurred and the resultant product did not exhibit satisfactory spherical shape and smooth surface texture. Slow and uniform cross-linking of the droplets, par-
Fig. 5. Percentage DZT cumulative release from Eudragit coated chitosan microspheres in simulated GI fluids (Formulations composition: Table 3). (Error bars ± SD (standard deviation); n = 3).
Fig. 6. Percentage DZT cumulative release from Eudragit coated chitosan microspheres with and without rat caecal contents (Formulations composition: Table 3). (Error bars ± SD (standard deviation); n = 3).
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Fig. 7. Kinetic profiles obtained for MC2 following zero order (a), First order (b), Higuchi (c) and Peppas (d) models.
ticularly at the surface, was found desirable to form microspheres of appropriate sphericity. Thus, glutaraldehyde saturated toluene was used which, due to its solubility in oil medium, would help to uniformly cross-link the surface of the droplets. The average size of microspheres increased with increasing polysaccharide concentration, since a higher concentration of chitosan produced a more viscous dispersion, which formed larger droplets and consequently larger microspheres. The ultimate mean diameter of microspheres was determined by the size of dispersion of chitosan solution, which decreased with increasing mixer stirring speed. Higher stirring rates provided the shearing force needed to separate the oil phase into smaller droplets decreasing therefore the particle size. Span 80 tends to lower the interfacial tension between the two phases thus stabilizing the emulsion. An optimal concentration of emulsifier is required to produce the finest stable dispersion. Below this concentration, the dispersed droplets tend to fuse and increase their mean diameter because of insufficient lowering of interfacial tension. On the other hand, above the optimal concentration no significant decrease in particle size is observed, because a high amount of emulsifying agent increases the viscosity of the dispersion medium. The optimal concentration of Span 80 was found to be 1.0%. 4. Discussion For any drug delivery system aimed at targeting the colon, the release of the drug during its transit through the stomach and small intestine must be kept to the minimum to ensure that maximum dose reaches the colon. For in vitro release study, the pH condition was selected so as to mimic the GI conditions without enzymes. In vitro drug release of DTZ from chitosan microspheres showed a burst release in the initial hour (Fig. 3). Within 4 h 73–87% of drug was released from the formulations P1, P2, P3, P4 and P5. These profiles are not acceptable for systems aiming at releasing their load in the colon. The burst release may be due to the solubility of chitosan in the acidic pH. To overcome this shortcoming and avoid the drug release in the stomach and small intestine, the chitosan microspheres were coated with Eudragit S-100, which is a pH sensitive polymer having a threshold pH of 7.0. Based on the high EE and appropriate release pattern, formulation P4 was selected and subjected to a coating process with Eudragit S-100. Since this polymer exhibits solubility above pH 7 corresponding to the pH of the colon, it should prevent any significant DTZ release in the upper segments of the gut. The coating of chitosan microspheres was achieved using emulsion solvent evaporation method. The mean particle size of
the coated microspheres increased from 119 m to 152 m with increasing the core–coat ratio from 1:4 to 1:8, which may be due to the corresponding increase in the polymer concentration that resulted in larger emulsion droplets. The drug release studies indicate that Eudragit coating of chitosan microspheres offered a high degree of protection from the premature DTZ release in the stomach and small intestine. DTZ release rate increased after 4 h, since at that time formulations were exposed to pH 7.4, which is above the solubilizing pH of Eudragit S-100 polymer. It is expected that DTZ release from cross-linked chitosan microspheres occurred due to swelling of the polymer, resulting in the formation of a gel. This is followed by drug dissolution and further diffusion through the gel. Based on better EE (93.77%) and drug release pattern, MC2 was considered as the optimized formulation and therefore selected for in vitro drug release study in presence of rat ceacal contents, to assess the biodegradability of chitosan. A colon targeted drug delivery system should not only protect its load from being released in the physiological environment of the stomach and small intestine, but also ensure its delivery in the colon. Conventional dissolution testing is less likely to accurately predict the in vivo performance of CDDS particularly for which the release is triggered by colonic bacterial enzymes. Thus, release studies in the presence of rat ceacal contents were carried out from the 5th hour in the simulated intestinal fluid of pH 7.4. When carried out in the presence of rat caecal contents, drug release was significantly improved clearly indicating the biodegradability of chitosan by the colonic microflora and thereby the specific release in the colon. More than 98% of DTZ was released after 12 h when the dissolution study was carried out in the presence of rat caecal contents. Drug release mechanisms were determined by fitting drug release data of MC2 to various kinetic models. By comparing the correlation coefficient values (Table 4), from the applied models, the Higuchi model (R = 0.9924) was shown the most appropriate to describe the kinetics of MC2 formulation. Thus, the drug release mechanism was assumed to be controlled by diffusion. When analyzed according to the Peppas model, the release exponent was found to be greater than 1.0 (Table 5). Therefore, the diffusional release was found to follow super case II transport. 5. Conclusion Eudragit S-100 coated DTZ-loaded chitosan microspheres were successfully prepared. The microparticles were discrete, spherical
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and showed appropriate release patterns in simulated GI fluids and further enhanced release characteristics in the presence of colonic microflora. Eudragit S-100 proved to be effective in preventing a premature release of DTZ in the upper parts of the gut, while chitosan ensured a controlled release of DTZ in the colon. The biodegradability of chitosan was confirmed by the enhanced drug release in the presence of rat caecal contents. The release pattern of DTZ gave a good fit to Higuchi model indicating a diffusion controlled release with super case II transport. A novel CDDS for bedtime dosing has been presented here. From the obtained results, it can be anticipated that the developed Eudragit S-100 coated chitosan microspheres is a promising alternative for the treatment of early morning pathologies influenced by circadian rhythms by opening a ‘new lease of life’ to an existing drug molecule. References [1] W.J. Hrushesky, J. Grutsch, P. Wood, X. Yang, E.Y. Oh, C. Ansell, S. Kidder, C. Ferrans, D.F. Quiton, J. Reynolds, J. Du-Quiton, R. Levin, C. Lis, D. Braun, Circadian clock manipulation for cancer prevention and control and the relief of cancer symptoms, Integr. Cancer Ther. 8 (2009) 387–397. [2] V.D. Kadam, S.G. Gattani, Development of colon targeted multiparticulate pulsatile drug delivery system for treating nocturnal asthma, Drug Deliv. 17 (2010) 343–351. [3] Y. Lee, I.H. Kim, J. Kim, J.H. Yoon, Y.H. Shin, Y. Jung, Y.M. Kim, Evaluation of dextran-flufenamic acid ester as a polymeric colon-specific prodrug of flufenamic acid, an anti-inflammatory drug, for chronotherapy, J. Drug Target. (2010).
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