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The aim of the research was to develop a polysaccharide based compression coated tablet of Secnidazole. Core tablets of Secnidazole were compression coated with various proportions of Guar gum, Xanthan gum and Chitosan. Drug release studies were performed in simulated gastric fluid (SGF) for 2 h followed by simulated intestinal fluid (SIF) (phosphate buffer pH 7.4) up to 24 h. The dissolution data demonstrated that the rate of drug release is dependent upon the nature and concentration of polymer used in the formulation. The tablets coat containing guar gum or xanthan gum alone showed 30-40% drug release in 8 h. The tablets containing guar gum or xanthan gum with chitosan or combination of guar gum, xanthan gum and chitosan showed only 30-45% drug release in 8 h. Further, in vitro dissolution studies performed in the dissolution media with rat caceal contents showed 54.48±0.24-60.42±0.16% of drug release. The drug release from the prepared formulations followed super-case Ð†Ð† transport.
Amoebiasis is an infection of the large intestine caused by Entamoeba histolytica, a single celled protozoan parasite. The trophozoite form E. histolytica can invade the colonic epithelium; causing amoebic colitis. Secnidazole, tinidazole, ornidazole and metronidazole are preferred drugs used in the treatment of intestinal amoebiasis, giardiasis and tricomoniasis. These drugs are delivered to the colon for their effective action against E. histolytica. Secnidazole is completely absorbed after oral administration. The administration of this drug in conventional tablet dosage form provides minimal amount of Secnidazole for local action in the colon, still resulting in the relief of amoebiasis, but with unwanted side effects like metallic taste, glossitis, nausea, vomiting and abdominal pain[3,4]. Therefore, the targeting of Secnidazole to the colon for local action may be beneficial in avoiding the unwanted side effects as well as a lower dose of Secnidazole may be sufficient to treat amoebiasis.
The delivery systems intended to release drugs in the colon require protection of the drug from the hostile environment of stomach and small intestine. This target specific release is required for the topical treatment of diseases associated with colon such as amoebiasis, ulcerative colitis, crohn's disease and colon cancer. This is because the colon provides a less volatile environment for drugs caused by the low diversity and intensity of digestive enzymatic activities as well as near neutral pH.
Site-specific drug delivery to colon may therefore be achieved by different approaches. Which include covalent linkage of a drug with a carrier, coating with pH-sensitive polymers, time dependent release systems and the use of carriers that are degraded exclusively by colonic bacteria. Enteric coated systems are the most commonly used for colonic drug delivery, but the disadvantage of this system is that the pH difference between small intestine and colon is not being very pronounced. The limitation of time dependent release system is that it is not able to sense any variation in the upper gastro-intestinal tract transit time, any variation in gastric emptying time may lead to drug release in small intestine before arrival to colon. Apparently, the most convenient approach for site-specific drug delivery to colon is the use of carriers that are degraded exclusively by colonic bacteria[5,6].
To improve the specificity of drug release, several polysaccharides are investigated as carriers for colonic drug delivery like pectin, chondroitin sulphate[7,8], amylose, inulin, guar gum[11,12], locust bean gum, khaya gum, chitosan[15,16], xanthan gum[1,13] and others.
The present work was done by using the inexpensive, naturally and abundantly available guar gum (GG), xanthan gum (XG) and chitosan (CHN) for colon targeted delivery of secnidazole. Guar gum is a natural non-ionic polysaccharide derived from seeds of Cyamopsis tetragonolobus. Guar gum is reported as a colon-specific drug carrier in the form of a matrix tablets. Xanthan gum is high molecular weight extra vascular hetero polysaccharide produced by fermentation with gram negative bacterium Xanthomonas campestris. Xanthan gum is reported for colon specific drug delivery in the form of compression coated tablets[1,13]. Chitosan is a natural polymer obtained by alkaline deacetylation of chitin, is non-toxic, biocompatible and biodegradable. Chitosan could be promising for colonic delivery if its solubility is reduced in gastric acid conditions. This could be accomplished by combining with water-insoluble polymers.
MATERIALS AND METHODS
Secnidazole was obtained as gift sample from Magnus Pharma Pvt. Ltd, Nepal. Sodium starch glycollate was obtained as gift sample from Wockhardt research centre, Aurangabad. Guar gum and microcrystalline cellulose were purchased from SD fine chemicals, Mumbai. Xanthan gum and Chitosan (Mw 45 kDa, 87% DD) were purchased from Himedia laboratories Ltd, Mumbai. All other chemicals used were of analytical grade.
Preparation of fast disintegrating core tablets:
The core tablets of secnidazole were prepared by direct compression technique using the composition given in Table 1. Secniazole, sodium starch glycolate, micro crystalline cellulose, magnesium stearate and talc were thoroughly mixed in a polybag and passed through sieve no #80 (179 µm) to ensure complete mixing. Sodium starch glycolate was added to obtain a fast disintegrating tablet. The mixture was compressed in to tablet on a single station tablet punching machine (M/s Cadmach, India) using 9.5 mm round, flat-faced and plain punches.
Preparation of compression coated tablets:
The composition of compression coating material is shown in Table 2. All the ingredients of each coat formulation were weighed accurately and mixed in a polybag. 40% weight of total weight (250 mg) of coating mixture was placed in the die cavity of single station tablet punching machine, the core tablet was placed on it at centre, remaining 60% of coating mixture was added to the die cavity and tablets were compressed using 12.6 mm flat punches. The total weight of the compression coated tablet was about 400 mg.
Evaluation of tablets:
Thickness and Diameter:
The thickness and diameter of the tablets was determined by using dial thickness apparatus and vernier calipers respectively. Five tablets from each formulation were used and average values were noted.
Tablet hardness of all the formulations was determined by using Monsanto hardness tester. Five tablets from each formulation were used and average values were recorded.
Both core and compression coated tablets from all formulations were subjected for friability test using friabilator. Ten tablets were weighed (W0) and placed inside the Roche friabilator. The instrument was operated for 4 mins at 25 rpm. The resulting tablets after 100 falls from a height of six inches were collected; weighed (Wt) and percentage loss was calculated using following equations.
Wo - W t
Percent friability = ---------------- * 100
Weight variation test:
The weight variation studies of the prepared formulations was performed as per the standard procedure following Indian pharmacopoeia. The sample mean and standard deviation of each batch of tablets were reported.
Determination of drug content:
One tablet from each formulations of compression coated and the core tablets were powdered and transferred in to 100 ml volumetric flask. Initially 50 ml of phosphate buffer (pH 7.4) was added and allowed to rotate in a rotary shaker for 24 h; the final volume was made up with phosphate buffer (pH 7.4). The solution was filtered suitably and amount of secnidazole present in the solution was estimated by using UV- spectrophotometer (UV 1601, Shimadzu, Japan) at 320 nm against a suitable blank.
One tablet from each formulation was randomly selected, weighed individually (W1) and placed separately in a wire basket which was placed in a 100 ml beaker containing 0.1 N HCl for first 2 h and later pH 7.4 (24 h). After 2, 4, 6, 8 and 24 h the tablets were removed from wire basket and excess water was removed using filter paper. The swollen tablets were reweighed (W2) and swelling index of each tablet was calculated using the below equation.
% Swelling Index = --------------- * 100
Preparation of 2% rat cecal content:
Male wistar rats weighing 150-200 gm maintained on a normal diet were used for the study. Rats were asphyxiated using carbon dioxide. The abdomens were opened, ceci were traced, ligated at both ends, dissected immediately and transferred in to phosphate buffer (pH 7.4) previously bubbled with CO2. The cecal bags were opened and the contents were individually weighed pooled and then suspended in a phosphate buffer to provide 2 % (w/v) dilution, all of these operations were performed under constant supply of CO2 atmosphere.
In vitro drug release study:
Dissolution experiments were carried out in a USP basket type apparatus at 100 rpm at 37±1°C. Drug release studies were conducted in 900 ml of 0.1N HCl for the initial 2 h, followed by phosphate buffer (pH 7.4) up to 24 h. Samples of 10 ml were withdrawn at predetermined time intervals and were replaced with fresh dissolution medium to maintain sink conditions. Samples withdrawn were filtered and assayed spectrophotometrically at 277 nm and 320 nm in SGF and SIF respectively.
In order to assess the susceptibility of guar gum and xanthan gum, being acted upon by colonic bacteria, drug release studies were also carried out in presence of rat cecal content because of the similarity with human intestinal flora. In vitro drug release studies in the presence of rat cecal contents were same as mentioned above except that rat cecal content (2% w/v) was added only to phosphate buffer (pH 7.4), to simulate colonic condition.
In vitro release data were fit to first order, zero order and higuchi equations to analyse the kinetics of drug release from the tablets. Further, in vitro release results were fit to the following Koresmeyer-Peppas equation, to analyse drug release mechanism.
Mt/Mα = Ktn
Where Mt/Mα is the fraction of drug released at time t, K is kinetic constant and 'n' is release exponent that characterize the drug transport.
Stability of the formulations was assessed by storing formulations F1, F5 and F11 at 40°C/75% RH for 6 months. At the end of study period, formulations were observed for physical change, drug content and in vitro drug release.
The dissolution data, performed with and without rat cecal content was statistically analysed using students t-test. A value of P< 0.05 was considered statistically significant.
RESULTS AND DISCUSSION
The present study was aimed at developing oral colon targeted formulations for Secnidazole using different polysaccharides. Further, it was aimed to identify the most suitable polysaccharide either alone or in combinations for colonic delivery of secnidazole based on microbial degradation. Ideally, the drug delivery system targeted to colon should remain intact in the stomach and release the drug in the colonic region. Hence attempts were made to formulate the compression coated tablets using guar gum, xanthan gum, chitosan, either alone or in combination.
Results of drug content, hardness, thickness, diameter and friability of core tablets and all compression coated formulations are shown in Table 3. All the formulations showed a hardness value in the range of 5.67±0.15 to 6.23±0.15 kg/cm2; it indicates that the hardness is depending on the quantity and type of polysaccharide used in the tablet. The hardness of core tablets of secnidazole was found to be 2.8±0.01 kg/cm2. The percentage friability of all formulations was found in the range of 0.86±0.04 to 0.98±0.03%; indicating that the friability is within the acceptable limits. In case of core tablets the percentage friability was more and it was found to be 3.06±0.12%, because core tablets having lower hardness and fast disintegration characteristics. The results of weight variation of tablets for all formulations was found in the range of 396.67±3.51 to 402.33±2.08 mg and in case of core tablets it was found to be 152.67±3.51 mg; indicating that the weight variation is within the acceptable limits. The percentage drug content both for core tablets and compression coated tablets of all formulations was found in the range of 98.39±0.58 to 101.17±1.18%. The disintegration time was measured for core tablet and it was found to disintegrate within 60 sec.
Swelling study was performed for all formulations for 24 h in two different buffer media. Swelling studies was done for first 2 h in 0.1N HCl (pH 1.2) and later in phosphate buffer (pH 7.4). The swelling behavior was dependent upon the polymer or combination of the polymers used for coating the core tablet. In case of the formulations F1 (GG), F2 (XG) and F3 (CHN), the swelling index after 24 h were 478.09±14.02%, 1089.01±9.07% and 149.33±11.91%, respectively (Table 4). In case of combination of guar gum and chitosan the swelling index for the formulation F4, F5, F6 and F7 at 0.1N HCl after 2 h were 243.33±4.95%, 168.12±4.87%, 154.2±29.16% and 140.15±10.23%, respectively, and at pH 7.4 after 24 h were 388.79±8.11%, 394.05±14.96%, 422.62±21.55% and 470.01±13.44%, respectively. The data indicates that as chitosan proportion in the coating layer increases, the swelling index also increases in gastric condition (0.1N HCl), but it decreases in colonic conditions (pH 7.4). In case of combination of xanthan gum and chitosan, the swelling index for the formulation F8, F9, F10 and F11 at 0.1N HCl after 2 h were 262.53±6.06%, 150.42±6.30%, 141.46±4.35% and 127.52±5.59%, respectively, and at pH 7.4 after 24 h were 737.65±8.45%, 850.19±2.94%, 959.44±12.24% and 1009.18±10.75%, respectively. At the end of 24 h, formulation F11 with higher proportion of xanthan gum has shown highest swelling. This is potentially because of high swelling ability of xanthan gum, as the concentration of xanthan gum was increased swelling index was also increased. In case of combination of all three polymers, the swelling index for the formulation F12, F13, F14 and F15 after 24 h were 386.47±7.27%, 416.04±10.43%, 576.68±5.57% and 472.10±7.03%, respectively. The data indicates that the formulation F14 containing equal proportion of guar gum and xanthan gum (each at 75 mg) was more swollen compared with other formulations.
It was observed that as the proportion of guar gum and xanthan gum was increased, the percentage swelling index increased due to hydrophilic nature of the polymer. As the amount of polymer increased amount of water absorption was also increased, resulting in high swelling index.
In vitro release studies:
In case of formulations with single polymer, the amount of drug released from the formulation F1 (GG) and F2 (XG) after 8 h were 38.39±1.23% and 32.29±0.1% respectively, and after 24 h were 87.59±0.92% and 87.07±0.91%, respectively (fig. 1). Although, at the end of 8 h higher amount of drug was released from formulation containing guar gum (F1), at the end of 24 h there was no significant difference in amount of secnidazole released. This is probably because xanthan gum requires more time (≈ 50 h) to completely swell compared to guar gum (24 h). Formulation F3 containing chitosan alone was not possible to target the drug for colonic region, as 99.13±0.79% drug was released in gastric condition (0.1N HCl) itself.
In case of combination of guar gum and chitosan, the amount of drug released from the formulation F5, F6 and F7 at 0.1N HCl after 2 h were 5.86±0.25%, 5.86±0.07% and 5.14±0.17%, respectively, and at pH 7.4 after 8 h were 41.61±0.16%, 41.79±0.45% and 42.60±0.31%, respectively, and after 24 h were 97.89±0.62%, 94.59±0.93% and 93.57±1.24%, respectively (fig. 2). It is evident from fig. 2, that the formulation F4 containing chitosan is more than 50 mg in coating material resulted is 98.43±1.35% drug release in gastric condition (0.1N HCl) itself; so the formulation F4 was considered as not suitable for colonic delivery. From the release data it indicates that the formulation F5 containing 50 mg chitosan releases less amount of drug in simulated stomach condition and higher amount of drug release in simulated colonic condition.
In case of combination of xanthan gum and chitosan, the amount of drug released from the formulation F9, F10 and F11 at 0.1N HCl after 2 h were 4.17±0.11%, 3.95±0.15% and 3.51±0.15%, respectively, and at pH 7.4 after 8 h were 37.42±0.60%, 38.03±0.59% and 32.13±1.36%, respectively, and after 24 h were 96.25±1.81%, 92.83±1.20% and 91.25±2.72%, respectively (fig. 3). The release data indicates that the formulation F9 containing 50 mg chitosan in coating material gives higher drug release in colonic condition compared to other formulations. If the proportion of chitosan is more than 50 mg, as in case of F8, 97.45±0.61% of drug is released in gastric condition (0.1N HCl) itself, making it unsuitable for the desired colonic delivery systems.
In case of combination of guar gum, xanthan gum and chitosan, the chitosan quantity (50 mg) was same for all the formulation because the formulation which containing 50 mg of chitosan resulted in desired release profile. The amount of drug released from the formulation F12, F13, F14 and F15 at 0.1N HCl after 2 h were 3.49±0.05%, 4.12±0.07%, 3.06±0.29% and 4.56±0.15%, respectively, and at pH 7.4 after 8 h were 31.57±0.45%, 37.83±1.13%, 39.69±0.14% and 35.34±0.32%, respectively. At the end of 24 h the same set of formulations gave 95.95±1.37%, 98.83±0.06%, 99.95±0.28% and 98.56±0.78% drug release, respectively (fig. 4). The prepared formulations containing mixture of all three polymers (F12-F15) was also found to provide desired release profile of drug. As smaller amount of drug release was seen in gastric environment (0.1N HCl) in comparison to simulated colonic fluid.
On the basis of drug release data, formulation F5, F9 and F14 were selected to carryout dissolution studies in the presence of rat cecal contents (2% w/v). There was a significant difference (P < 0.05) in the drug release when compared to that of the release studies performed in the absence of rat cecal content. The rat cecal content in the release study was considered to mimic the human colonic environment as it contains microflora which releases many glycosidase and degrade the polysaccharide polymers.
When the in vitro dissolution studies were carried out in the presence of rat cecal content medium, the cumulative percentage drug released from the formulation F5, F9 and F14 after 8 h were found to be 58.62±0.42%, 54.48±0.24% and 60.42±0.16%, respectively, and without rat cecal content were found to be 41.61±0.16%, 37.42±0.60% and 39.69±0.14%, respectively (fig. 5).
In all three formulations almost 100% of drug was released after 24 h in presence of rat cecal content. Hence, in presence of rat cecal matter, drug was faster from formulations compared to dissolution medium without rat cecal content. This indicates that the drug release from formulations is mainly due to the presence of enzymes released by micro-organisms of rat cecal contents. Rat cecal matter 2% w/v was used in the study for the simple reason that the microbial load in the colon is 1011-1012 CFU/ml.
From the above two dissolution data (in the presence and in the absence of rat cecal content) significant changes in the release behavior was observed. From the data it can be concluded that polysaccharides alone neither can be used effectively for targeting the drug to the colon nor for sustaining the release of drug. Hence combination of various polysaccharides for compression coating is ideal for targeting the drug to colon.
In view of the potential utility of F5, F9 and F14 formulations for targeting of secnidazole to colon, stability studies were carried out at 40 °C/75% RH for 6 months to assess their long term stability. After storage, the formulations were observed for physical change and were subjected to assay of drug content and in vitro dissolution studies. At the end of storage period (40ËšC/75% RH for 6 months), there was no change either in physical appearance or in drug content. When the dissolution study was conducted in the simulated physiological environment as described in previous sections, no significant difference (P >0.05) was observed in the secnidazole release from F5, F9 and F14 in comparison to that released from the same formulations before storage.
To know the mechanism of drug release from the formulations, the data were treated according to first order, zero order and Higuchi equations. All formulations except F3, F4 and F8 showed the linearity with respect to zero order (R2=0.9634-0.9901) as compared to first order (R2=0.8858-0.9515) (Table 5). Formulation F3, F4 and F8 did not showed linearity with respect to first order equation (R2=0.9467-0.9655). In presence of 2% w/v rat cecal content as a dissolution medium, the formulation F5, F9 and F14 showed linearity with respect to first order equation (R2=0.9528-0.9683) (Table 6). Higuchi equation regression values for all formulations with and without rat cecal contents ranges from f (R2=0.8236-0.9342). Hence to confirm precisely the domination mechanism; the data was plotted according to Koresmeyer-Peppas equation.
The 'n' value (diffusional exponent) indicated the mechanism of drug release. For tablet systems, if n<0.45 it suggests the Fickian diffusion; if 0.46< n< 0.89, it suggests the anomalous (non-Fickian) transport, for n= 0.89, the zero-order release is possible and if n> 0.89, a super case-Ð†Ð† transport is operative. In order to predict and correlate the release behavior of drugs from the tablet, it is necessary to fit them in to release kinetics profiles (Fickian, anomalous or super-case Ð†Ð†). This will facilitate the understanding of mode of drug release such as whether the release is because of only diffusion or only erosion or caused by both diffusion and erosion. The 'n' values were calculated for all formulations. Except F3, F4 and F8, 'n' values were found in the range of 1.10-1.61, indicating a super case-Ð†Ð† transport. These observations confirmed that both diffusion and erosion is dominant mechanism for drug release. The 'n' values for formulation F3, F4 and F8 were found in the range of 0.481-0.492, indicating anomalous transport and these observations confirmed that the erosion is dominant mechanism for drug release. Hence natural polymer like guar gum, xanthan gum is ideal for site specificity especially in combination with chitosan to target the drug to colon.
The present investigation was performed to develop the colon targeted drug delivery systems of secnidazole for an effective and safe therapy of amoebiasis. The dissolution data obtained from the various formulation developed demonstrates that secnidazole release rate is dependent upon the nature and proportion of polysaccharides used as a carrier. Among the various formulations, it appears that compression coating with a mixture of guar gum and xanthan gum with chitosan (50 mg) in 250 mg coat or combination of guar gum and xanthan gum at equal proportion (each at 75 mg) combined with chitosan (50 mg) in 250 mg coat are most likely to provide targeted delivery of secnidazole to the colon. It can be concluded that a single polysaccharide can not be used for targeting the drug to the colon efficiently.