1. INTRODUCTION:

Amoebiasis is a ubiquitous disease particularly in areas of Asia, Africa and South America, where it is endemic. The disease affects over 10000000 people worldwide and is the third leading cause of death due to parasite disease. Different classes of drugs e.g. Tissue (Metronidazole, Tinidazole, Chloroquine, Emetine, Dehydroemetine) and luminal (Diloxanide furoate, Iodoquinol, Paromomycin) are needed to treat the infection, many of them has serious side effect (1).

Secnidazole (SNZ), 1-(2-methyl-5-niroimidazole-1-yl) propan-2-ol is a relatively new antiprotozoal agent used in the treatment of amoebiasis and approximately equipotent to metronidazole (2).The common reported adverse effect are nausea, vomiting, glossitis, anorexia epigastric pain and metallic taste due to its absorption in upper Gastro Intestinal Tract (GIT), which can be minimize by site specific delivery (3).

Recently greater emphasis has been placed on site specific delivery because of safety, efficacy and effectiveness. Colon targeted drug delivery has benefits of local and systemic treatment of many chronic disease like irritable bowel syndrome, colitis, Crohn’s disease, colon cancer and infections etc. (4). Colon provides a less hostile environment for drug due to low diversity and intensity of digestive enzymatic activities and near neutral pH and transit time of 4 to 78 h, that increase the time of drug availability for action and absorption (5). Different systems are being developed for purpose of site-specific drug delivery to the colon, like pH dependent, time dependent, pressure dependent, enzyme dependent systems (6).One or a combination of above approaches is utilized to achieve colon targeted drug delivery system (7).The poor site specificity of pH dependent system, because of large variation in pH of the GIT, failure the time dependent system due to variation in gastric emptying time of different age, sex etc of patient (8). The best alternative approach for colon specific drug delivery is the use of carriers that are degraded exclusively by colonic bacteria since large intestine contains bacterial count 10¹¹ cfu/mL compared to 10⁴cfu/mL in small intestine, Carbohydrates like pectin, chondroitin sulphate, amylase, inulin HP and guar gum (9).

Pectin is a polysaccharide, found in the cell walls of plants, is a predominately linear polymer of mainly α-(1-4)- linked D-galacturonic acid residues interrupted by 1, 2-linked L-rhamnase residues. It is totally degraded by colonic bacteria but is not digested in the upper gastrointestinal tract. Pectin has a few hundred to about 1000 building blocks per molecule. Because pectin is soluble in water, it is not able to shield its drug load effectively during its passage through the stomach and small intestine. This can however be adjusted by changing its degree of methoxylation, or by preparing it polyionic complex such as calcium /zinc pectinate. It has been shown in studies that pectin-coated tablets disintegrate in the colon during transit (10, 11).

Aim of study was development of Secnidazole microspheres with negligible to no release in upper G.I.T. and control release in lower GI Region for achievement of better local effect and minimize side effect like nausea, vomiting, glossitis, anorexia epigastric pain by using economic, easily available, nontoxic, biocompatible polymers.

2. EXPERIMENTAL:

2.1 Material

Pectin from apples (M_r 30000-100000; degree of esterification 70-75%), (CDH India), Secnidazole (purity > 99%) was obtained as gift sample of unichem lab. (Mumbai india), Pectinase (from Aspergillus niger, 25 u/mg at pH 4, and 25°C Sigma Co., St. louis, MO, USA), KH₂PO₄, Na₂HPO₄, NaOH, Acetonitril, Methanol, Hydrocholoric acid were purchased from CDH mumbai india. Sprague–dawley albino rats (male and female average weight 210-265g) were procured by Animal house Institute of Pharmaceutical Sciences, G. G Central University, Bilaspur, C.G. India. The animals were allowed water and laboratory chw ad libitum. The animals were maintained in a 12 h light-dark circle. All animal procedures were performed in accordance with protocols approved by Institutional animal ethical committee.

2.2 Preparation of microspheres of Secnidazole

Microspheres were prepared by spray-drying method (12, 13). Briefly pectin (1.25 g; 2.50 g; 3.75 g; 5.00 g) was dissolved in 250 mL of de-ionized water to obtain different concentration of polymers and then drug was added with vigorous shaking for overnight. The prepared solution was spray dried to get microspheres through the nozzle (0.2 mm diameter) of the spray-dryer (Labultima Laboratory Spray Dryer, LU-228, India) having pressure of 200kPa to 300 kPa. The inlet temperature was 110°C fixed to get outlet air between 45-50° C.

2.3 Hardening of Microspheres as Ionic Crosslinking with Zinc–acetate

The hardening microspheres were done by Zinc–acetate (14). Microspheres were dispersed in different concentration of hardening agent for 30 min and to avoid particle swelling a mixture of ethanol and water 9:1(w/w) was used as solvent in place of water.

2.4 Drug polymer interaction study

Drug and Polymer and mixture of drug and polymer were analyzed by Fourier Transform Infrared Spectrometry (FT-IR) (Shimadzu model C) and DSC (Mettler DSC-30-S). On the basis of spectra obtained by FT-IR and DSC, the interaction between drug and polymer were studied.

2.5 Study of particle size and morphology

The particle size of microspheres was determined by using laser particle size analyzer (Shimazu SALD 2201). The morphology and appearances of microspheres were studied by scanning electron microscopy (SEM). Briefly, the prepared microspheres were freeze-dried at -30°C for 48 h and coated by coater (SCD004, BALTEC, Liechtenstein) with gold palladium under an argon atmosphere for 150 psi to achieve a 20 nm film. The coated microspheres were examined using a scanning electron microscope (Jeol JSM-1600, Japan).

2.6 High- Performance liquid chromatography (HPLC) analysis of Secnidazole.

The quantitative estimation of Secnidazole in microspheres and dissolution fluid were performed using HPLC as per findings of Bakshi et al. (15). The HPLC System consisted a 600E pump, photodiode array (PDA) Detector (A-996), Autoinjector (A-717), A degasser module (waters, milford, USA). Data acquisition and processing was performed by use of Water Millennium software 2.1. Chromatograms were recorded using CLASS-VP software (Shimadzu, Kyoto, Japan) using C-18 columns (250mm) 4.6 mm internal dia with particle size of 5µm. The mobile phase used was a mixture of water:acetonitril (86:14). The filtered mobile phase was pumped at flow rate 1ml/min. The analytical wavelength was 310 nm. A standard curve was constructed for Secnidazole in range of 10-500µg/ml in methanol using metronidazole as internal standard. The linearity (R² = 0.994) response was shown by Secnidazole in concentration range 50-500 µg/ml.

2.7 Determination of loading efficiency in microspheres

Determination loading efficiency was done per previous reported work (16). Briefly An accurately weighted quantity of drug containing microspheres placed in phosphate buffer saline (PBS, pH 7.4) containing pectinase solution (4% w/w) for 48h at 37°C with vigorous stirring. The concentration of Secnidazole was analyzed HPLC as described previously. Each determination was made in triplicate.

2.8 Water uptake studies

Water uptake capacity of microspheres was determined as per method reported by Jain (17)Briefly, weighed quantity of microspheres (100mg) were immersed in simulated gastric fluid (0.1 HCl, pH 1.2) and simulated intestinal fluid (pH 7.4) and at regular intervals of time, the microspheres were reweighed after carefully wiping off excess of liquid with a tissue paper. The water uptake was determined from the expression (W_t-W₀)/ W_0,

Where, W_tand W₀are the weight of the microsphere at time ‘t’ and under dry state, respectively.

2.9 In vitro Drug Release Studies.

An accurately weighted amount of microspheres equivalent to 100 mg of Secnidazole was added to 900 ml of dissolution medium and the release of Secnidazole from microspheres was investigated, using USP rotating XXIII dissolution rate test apparatus (DT-06, Erveka, Germany) at 100 rpm and 37 ± 0.5°C as per method reported by Yang (18)and Wakerly (19). The simulation of gastrointestinal transit condition was achieved by altering the pH of the dissolution medium at various time intervals. Briefly, the dissolution medium consisted of 900 mL 0.1 mol/L HCl for 1h, replaced by phosphate buffer (pH 4.5) for 2 h and phosphate buffer (pH 7.4) in 900 mL dissolution medium, as the average small intestinal transit time is 3 h and to assess the susceptibility of the prepared microspheres delivery system to enzymatic action of colonic bacteria drug release studies were continued with the pH 7 phosphate buffer absence (control) and presence of rat cecal bacteria (2% w/v) up to 24 h. The final volume in all cases was 900 mL. The samples were withdrawn from the dissolution medium at various time intervals using a pipette fitted with a microfilter, and the filtrate was subjected to HPLC analysis as describe previously. All dissolution studies were performed in triplicate.

2.10 In vivo organ Distribution study

In vivo GIT distribution study (16, 20) was performed to evaluate microspheres in Sprague –dawley albino rat (male and female average weight 210-265 g) were randomly selected and divided into four groups of twenty four animals each. The first group control received no drug. The second group received plain drug (20 mg/kg body weight). Third group received uncoated and fourth group recevied coated microspheres. The dosages were given orally with help of cannula. Heparinized whole blood was sampled from the abdominal aorta of sacrificed rats at 2, 4, 6, 8, 10, 12, 18 and 24 h after oral administration, respectively. Meanwhile, the stomach, small intestine and colon were isolated. Theses content of GIT were homogenized along with a small amount of PBS (pH7.5) and then centrifuged at 10,000 rpm for 5 min, supernatant was separated, in the separated supernatant 1 ml methanol was added and kept for 30 min and filtered. These filtrates were assayed for drug content by the HPLC analysis as describe previously. The heparinized whole blood was centrifuge at 10000 rpm for 3 min, 0.2 ml supernatant plasma was taken, added 1 mL methanol and agitated for 2 min. Then 0.02 mL supernantant were taken for HPLC analysis.

2.11 In vivo evaluation of colon specific delivery of Microspheres by X-ray imaging using Barirum-sulphate (BaSO₄)

2.11.1. Formulation of microsphere with Radio opaque material

The core microspheres were prepared using the same method and ingredients as reported in previous section, but drug was replaced with BaSO₄, as opaque agent. The prepared microsphere were coated by same method and agent reported in previous section. The prepared microspheres were collected, washed three times with distilled water, dried and kept in a desiccators. Optimized Formulation was selected for the in vivo studies in rabbits.

2.11.2. In Vivo behavior of microspheres

The X-ray studies were carried out using three male albino rabbits applying the procedure previously reported by Yassin (4). In each experiment, the animals were allowed to fast overnight with free access to water and a radiograph was made just before the administration of the unit to ensure the absence of radio-opaque material in the GIT. The microspheres were administered by natural swallowing followed by 50 mL of water. The radiographic imaging were taken to animals in a standing position using X-ray system (Siemens, Model No.3064581-B-5310, Germany) and print on X-ray film. The distance between the source of X-rays and the animal were kept the same for all imaging, thus the observations of the microspheres movements could be easily noticed. Standing radiographs of the abdomen were taken at 0 h, 0.25 h, 2.5 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, and 24 h after the ingestion

2.12 Statistical analysis

Experimental data have been represented as the mean with standard deviation (SD) of different independent determinations. Statistical evaluation was carried out using one way analysis of variances (ANOVA followed by Dunnett’s post hoc test). The programme used for the statistical analysis was GraphPad Prism (version 6). The significance of differences was considered statistically significant at p < 0.005.

3. RESULT:

3.1 Drug polymer interaction study

To confirm the interaction of secnidazole with pectin sample were analyzed by Fourier Transform Infrared Spectrometry (FT-IR). Fig. 1 secnidazole, shows the characteristic band of amide 1654cm^-1.. Fig.2 shows characteristic pectin band of methyl ester group at 1747cm^-1and band of carboxyl group at 1615cm^-1and in Fig.3 similar peak observed in mixture of drug and polymer show no interaction. In DSC study (Fig. 4) the melting point of pure drug and polymer 116º C and 90º C shows peak which was to change in spectra of mixture of drug and polymer.

3.2 Study of particle size and morphology

The average size of microspheres found 148 ± 12.5 µm, 180 ± 13.0 µm, 240 ± 12.0 µm, 260 ± 13.4 µm and 283 ± 15.2 µm with 0.5, 1, 1.5, and 2% of polymer concentration. Effects of polymer concentration on microspheres size, swelling index, drug loading capacity shown in Table 1. SEM study (Fig.5) of microsphere show spherical smooth shape in coated and small fractures in uncoated microsphere surface. The size of the microspheres varied between 140 to 250 µm, while 92% of the particles had a size range between 100-300 µm. However, about 50% of the microspheres fall within the 140 and 268 µm size range. These results were not much different for the three microspheres preparation conditions applied. Fig. 6 shows the particle size distribution of the prepared microspheres.

Table 1. Average particle size, entrapment efficiency and water uptake uncoated pectin microspheres

Formulation code	Pectin concentration	Drug concentration	Average Particle size (± SD*)	Entrapment efficiency@ (± SD*)	Swelling index (± SD*)
PSM1	0.5	100mg	148±12.5 µm	62.13±0.8	10.77±2.25
PSM2	1	100mg	180±13.0 µm	66.80±0.6	13.23±4.11
PSM3	1.5	100mg	240±12.0 µm	68.02±0.3	15.63±2.71
PSM4	2^	100mg	260±13.4 µm	67.82±0.5	18.77±5.21
PSM5	3	100mg	350±15.7µm	67.95±1.8	21.86±5.91

* data are expressed as mean ± SD of at least triplicate

@ encapsulation efficiency was calculated based on the initial drug loading

^ indicate optimized polymer concentration.

Table. 2 Result of concentration of coating material on percentage drug release

Formulation code	Concentration of crosslinking agent (Zn-acetate)w/v	Cumulative drug released after 6 h of dissolution (± SD^#)
PSM4a	0.5	14±1.15
PSM4b	1	10±0.81
PSM4c	1.5	7.5±0.69
PSM4d	2.0^##	5±0.35
PSM4e	3	5±0.62

^# Data are expressed as mean ± SD of at least triplicate. ^## indicate optimized crassliking concentration.

Graph1 Result of concentration of coating material on cumulative percentage drug release after 6h of dissolution. Formulation code PSM4a contains 0.5 %, PSM4b contains 1.0 %, PSM4c contains 1.5 %, PSM4d contains 2.0 % and PSM4e contains 3.0 % of Concentration of Crosslinking agent (Zn acetate) w/v. Data are expressed as mean ± SD of at least triplicate. ^## indicate optimized crass liking concentration.

Graph 2. In -Vitro Cumulative Percentage Drug release in Various Simulated Gastric Fluids (SGF) and Simulated Intestinal Fluids (SIF) dissolution conditions. Formulation code PSM0 without coating and PSM4d contains 2.0 % (optimized) Concentration of crosslinking agent.

3.3 Determination of loading efficiency in microspheres

Optimum loading efficiency of pectin microsphere found in 2% (w/v) of pectin concentration. Initially result data show (Table 1) increment in entrapment efficiency as polymer concentration increase but beyond 2% of polymer concentration, there was no valuable increment in entrapment efficiency seen and result suggest maximum 68.88 ± 0.2% drug entrapment with spray drying method.

3.4 Water uptake studies

Water uptake (Table 1 and Graph 1) of microsphere having 0.5 to 2 % (w/v) pectin was in the range of 10 to 23 % W/w of initial volume taken.

3.5 In vitro Drug Release Studies.

In-vitro release profile data (Graph 2) showed that coated microsphere have no drug release in gastric pH 1.3 and 05 % up to 6 h with pH 7.4, the release rate abruptly increase up to 28% of 12^th h in presence of 2% rat cecal content, but in uncoated Pectin microsphere more than 80 % drug release in first 6^th h (Table 2). Effect of zinc acetate concentration (Crosslinking agent) on drug release pattern shown in Table 2. Only 1.3% cumulative drug release found in 2 % in compare to 3% concentration of crosslingking agent in first 6 h of release.

3.6 In vivo organ Distribution study

The Vivo organ distribution study Result suggest (Graph 3, 4, 5 and 6) that only 10 % drug reached to colon when pure drug was given. But in coated microspheres about 54 % drugs protected by formulation up to colon in comparison to only 15 % in uncoated microsphere. In case pure drug less than 1% drug reached to colon.

3.7 In vivo evaluation of colon specific delivery of Microspheres by X-ray imaging using BaSO4

Fig. 8 shows the presence of the microsphere in the Rabbits’s stomach. After 15 min, it was seen that the microsphere remained in the stomach, Fig.8 show a clear description of the different parts of albino rabbit GI anatomy. Fig. 8 taken 6 h post dose, showed small white cloud in the proximal part of the small intestine that show presence of formulation. Fig. 9 shows the colonic distribution of opaque material in rabbits. The images were selected based on clarity.

Graph 3. Concentration profile of pure Secnidazole, uncoated microspheres and coated microspheres in blood (n=3)

Graph 4. In-vivo organ distribution of coated Diloxanide furate (SNZ) microspheres (n=3)

Graph 5. In-vivo organ distribution of uncoated Diloxanide furate (SNZ) microspheres (n=3)

Graph 6. In-vivo organ distribution of Secnidazole, (SNZ) (n=3)

Figure 1. FTIR spectra of Secnidazole.

Figure 2. FTIR spectra of pectin.

Figure 3. FTIR spectra of Secnidazole and petin mixture

Figure 4. DSC spectra of Secnidazole, pectin and mixture pectin and polymers

Figure 5. SEM pictures of pectin Microspheres coated with Zn-acetate (2%) with Magnification X50

Figure 6. Particle size distribution of Zn- Pectinate microsphere

Figure 7. Egg-box structure of Pectine Zn acetate polyionic complex

Figure 8. X-ray photograph 15 min after administration of PSM4d to albino rabbit.

Figure 9. X-ray photograph after 6 h administration of PSM4d to albino rabbit.

Figure 10. X-ray photograph after 24 hrs administration of PSM4d to albino rabbit.

4. DISCUSSION:

The basic aim of the present study were that the development of site specific formulation suitable for colonic environment, biodegradable and high drug loading capacity which prevent the releasing of drug in stomach and small intestine. Previous works suggest that pH and time dependent system are failed to protect formulations due variation in pH and transit time of GIT (2, 17, 18). For this present research was performed using pectin in different concentration as a polymer to formulate targeted microspheres and coated as Polyionic complex made by zinc-acetate and pectin. since pectin is polysaccharide absorb water and convert in water soluble gel and drug can easily release form this gel that can be minimized by crosslinking of pectin that make it stable in lower acidic pH and maintain its integrity at least 3 to 7 h time. The release of drug from microspheres was dependent upon concentration of crosslinking agent, as concentration from 1 to 2% w/v, cause slower drug release patterns (Graph 1), especially with 2% w/v concentration found more stable over a wider range of pH values presumably due to the formation of an insoluble Polyionic complex between pectin and Zinc acetate called egg box dimer (22) (Fig. 7), which tends to restrict the rate of drug/water permeation through mixed pectin and zinc-acetate complex which retard the drug release as result found from release data and also enhances the mechanical properties of microspheres. The FT-IR (Fig. 1, 2, 3) and DSC (Fig. 4) graphs of drug, polymer and mixture of polymer shows no polymer drug intraction in microspheres. Findings suggest that concentration of polymers increase which makes spherical shape of microspheres. The size and shape of pectin microspheres depend on aggregation of polymer chain and aggregation effected by coulombic repulsion between two carboxylate anions present in the pectin that can be minimize by forced dehydradation of polymeric solution that was achieved by spray drying in place of emulsion dehydration or inotropic gelation method of microspheres manufacturing method. In Earlier reported work (16, 17, 23) microspheres were prepared by ether emulsion dehydration method or ionotropic gelation method, all these methods were showed time consuming, multi factors dependency, unfit to scale up or bulk production, irregular shape size and less drug entrapment capacity. But in spray dry method (14) having advantage of higher entrapment, compatible to scale up and limited factor affecting process, in present research work founded 72.88 ± 4.1 % drug entrapment with spray drying method. The SEM study of cross-linked microspheres shows that crasslinking fill up the crack, found in without crasslinked microspheres, that make surface smooth. It also provides protection from upper G.I. tract. In-vitro release profile (Graph 2) Secnidazole microspheres shows lag time of 3 h where negligible drug release found, this lag time was due to water insoluble and pH resist polyionic Zn/pectinate complex found in microspheres. This Zinc acetate polyionic coating mixture improves the physiochemical properties like toughness and elasticity (18), which increase as we increase the concentration 1to 2%. But after 8 h release rate was change due to erosion of surface polyionic pectin/zn complex. Due to presence of rat cecal content simultaneously attack of water and enzymes that increase the pore size and erosion of complex coating films start, which increase the drug release that was 15 % in 9 h but without enzymes only less than 5 % cumulative drug release. After 12 h more than 50 % cumulative drug release found and 99 % cumulative drug release found after 17 h of dissolution study. Dissolution containing enzymes shows faster drug release rate with compare to without enzyme. These result shows pectin microspheres degraded by enzymes and release the drug since enzyme pectinase found in colon. After administration of secnidazole, uncoated and coated microspheres, SNZ distributed in stomach, small intestine and in blood at different concentration depend on formulation. Coated microspheres show low and negligiable concentartion of drug in stoamch and intestine in first 10 h but after 12 h found in high in colon. Result (Graph 3, 4, 5 and 6 ) shows that only 10 % drug reached to colon if pure drug was given. But in the coated microspheres about 54 % drugs protected by formulations. Blood concetration of first group suggest that secnidazole well absorbed in upper G. I. tract and since secnidazole has long half life (20), that make it remains in blood that show it action as antiamoebic. But in case of formulations uncoated and coated, absorption of drug depend on drug release which was reduced by polymers and coating of polymer shield the drug to reach colon for its action on amoebae. The release behaviour of the Barium sulphate loaded microspheres was investigated by taking a series of X-ray photographs of the GIT. Initially 15 min. of administration no cloud was seen stomach (Fig. 8) that show resistant to erosion of coating of microsphere by Gastric fluid, El-Gibaly (14) showed zn–pectinate gel has complete protection against acidic media. But the existence of microsphere in terminal ileum of rabbis after 6 h post administration show by small white cloud that conform the surface erosion of formulation. Previous reported works (15, 16) used X -ray imaging to monitor the colon targeted formulation indicated that the formulation began to disintegrate after 10 h most compatible for colonic delivery and In fig 08 and 09 show presence of formulation in large intestine and after 24 h of administration the lower GIT was seen full of white cloud confirmed the disintegration of microspheres that conformed targeted approach of microspheres.

5. CONCLUSION:

Present finding conclude that microspheres formulated by Zn:pectinate polyionic complex has a promising approach to target drug to colon which is site of infection. Further research is going on to reduction of dose of Secnidazole by above delivery system.

6. REFERENCES:

1. Sinha YR, Kaimal P and Bhatt RM. The antiamoebic effect of a crude drug formulation of herbal extract against E. histolytica in vitro and in vivo. J. Ethano. (1995) 45: 43-52.

2. El Wally MAFM, Adbine H, Razak OM, Zamel S. Spectrophotometric and HPLC determination of secnidazole in Pharmaceutical tablets. J. Pharm. Biomed. Anal. (2000) 22: 887-97.

3. Gillis JC and Wiseman LR. Secnidazole A Review of its antimicrobial Activity Pharmacokinetic Properties and therapeutic use in the management of protozoal infection and Bacterial Vaginosis. Drug (1996) 51: 621-638.

4. Yassin AE, Alsarra IA, Alanazi FK and Al-Mohizea AM. New targeted colon delivery system:invitro and in vivo evaluation using X-ray Imaging. J. Drug Target. (2010) 18: 59-66.

5. Mrsny J R. The colon as a site for drug delivery. J. Controlled Release. (1992) 22: 15-34.

6. Yang L, Cu JS and Fix JA. Colon specific drug delivery: new approaches and in vitro/invivio evaluation. Int. J. Pharm. (2002) 35: 1-15

7. Macleod GS, Fell JT, Collett JH, Sharma HL and Smith AM. Selective drug delivery to colon using pectin:chitosan: hydroxypropyl methyl cellulose film. Int. J. Pharm. (1999) 187: 251-57

8. Krishnaiah YSR, Satyanarayan V, Kumar BD, Krthikeyan RS and Reddy PR. Studies on the development of oral colon targeted drug delivery systems for metronidazole in the treatment of Amoebiasis. Int. J. Pharm. (2002) 236: 43-55.

9. Krishnaiah YSR, Satyanarayan V, Kumar BD, Krthikeyan RS and Bhaskar P. In Vivo pharmacokinetics in human volunteers: oral administered guar gum based colon target 5-fluorouracil Tablets. Eur. J. Pharm. Sci. (2003) 19: 355-62.

10. Beneke CE, Viljoen AM and Hamman JH. Polymeric Plant derived Excipients in Drug Delivery. Molecules. (2009) 14: 2602-2620.

11. Sinha VR and Kumria R. Binder for colon specific drug delivery: An in vitro evaluation. Int. J. Pharm. (2002) 249: 23-31.

12. lee-M, Change, Kim DW, lee HC, lee KY. Pectin microspheres for oral colon delivery; preparation using spray drying method and in vitro release of indomethacin. Biotech. and Bio. Engg. (2004) 9: 191-95.

13. Bigucci F, luppi B, Monaco L. Pectin-based microsphere for colon-specific delivery of Vancomycin. J. Pharm. Pharmacol. (2003) 55: 1623-33.

14. El-Gibaly I. Oral delayed-release system based on Zn-pectinate gel microparticles as an alternative carrier to calcium pectinate beads for colonic drug delivery. Int. J. Pharm. (2002) 232: 199-211.

15. Bakshi M and Singh S. ICH guidance in practice: establishment of inherent stability of Secnidazole and development of validated stability indicating high-performance liquid chromatographic assay method. J. Pharm. Biomed. Anal. (2004) 36: 767-75.

16. Vaidya A, Jain A, Khare P, Agrawal RK and Jain SK. Metronidazole loaded pectin Microspheres for Colon targeting. J. Pharm. Pharma. Sci. (2009) 98: 4229-36.

17. Jain SK, Jain A, Gupta Y and Ahirwar M. Design and development of hydrogel beads for targeted drug delivery to colon. AAPS Pharm. Sci. Tech. (2007) 8: E1-9.

18. Yang L, Cu JS and Fix JA. Colon specific drug delivery: new approaches and in vitro/invivio evaluation. Int. J. Pharm. (2002) 35: 1-15.

19. Wakerly Z, Fell JT, Attwood D and Parkins DA. In vitro evaluation of pectin-based colonic drug delivery systems. Int. J. Pharm. (1996) 129:73-77.

20. Xi MM, Zhang QS, Wang YX and Fang QK. Study of characteristic of Pectin-Ketoprofen for colon targeting in rat. Int. J. Pharm. (2005) 298: 91-97.

21. Kwakye OK and Fell JT. Biphasic drug release the permeability of films containing pectin chitosan and HPMC. Int. J. Pharm. (2001) 226: 139-45.

22. Novosel'skaya IL, Voropaeva NL, Semenova LN and Rashidova SSH. Trends in the science and applications of pectins. Chemistry of Natural Compounds (2000) 36: 1-10.

23. Paharia A, Yadav AK, Rai G, Jain SK, Pancholi SS and Agrawal GP. Eudragit- Coated Pectin microspheres of 5- fluorouracil for colon targeting. AAPS Pharma. Sci. Tech. (2007) 8: E1-7.

24. Omar S, Aldosari B and Refai H. Colon-specific drug delivery for mebeverine hydrochoride. J. Drug Target. (2007) 15: 691-700

Received on 07.12.2012 Modified on 10.12.2012

Research J. Pharm. and Tech. 5(12): Dec. 2012; Page 1588-1595