1. INTRODUCTION:

The oral route is considered to be most convenient for administration of drugs to patients. Oral administration of conventional dosage forms normally dissolves in the stomach fluid or intestinal fluid and absorb from these regions of the GIT depending upon the physicochemical properties of the drug. It is a serious drawback in conditions where localized delivery of the drugs in the colon is required or in conditions where a drug needs to be protected from the hostile environment of upper GIT. Dosage forms that deliver drugs into the colon rather than upper GIT offers number of advantages. Oral delivery of drugs to the colon is valuable in the treatment of diseases of colon (Ulcerative colitis, Chron’s disease, Carcinomas and Infections) whereby high local concentration can be achieved while minimizing side effects that occur because of release of drugs in the upper GIT or unnecessary systemic absorption^1,5.

Due to the lack of digestive enzymes and the long transit time, colon is considered as suitable site for the absorption of various drugs.

Colon drug delivery system is useful in administering drugs that are irritant to the upper GIT such as non-steroidal anti-inflammatory agents, or drugs such as peptides that are degraded by gastric juice or an enzyme present in the upper GIT².Various systems have been developed for colon-specific drug delivery. These include covalent linkage of a drug with a carrier, coating with pH-sensitive polymers, time dependent release systems, and enzymatically controlled delivery systems. 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.

Colon targeted drug delivery would ensure direct treatment at the disease site, lower dosing and less systemic side effects. In addition to restricted therapy, the colon can also be utilized as a portal for the entry of drugs into the systemic circulation. For example, molecules that are degraded/poorly absorbed in the upper gut, such as peptides and proteins, may be better absorbed from the more benign environment of the colon^3,7. Drug absorption in the colon can be influenced by colonic residence time. The colon shows variations in transit; the residence of a dosage form in the colon can be from ~1 h up to several days and this can affect the drug bioavailability⁴.

When hydroxylpropylmethylcellulose used for press-coated tablets with an outer shell then it functioned as a good time-release system. This press-coating technique is advantageous because the tablets can be prepared with the various characteristics suitable for a number of uses. Our system is also applicable as colon-targeting dosage form by prolonging the lag time to more than 6 h⁸.

Despite widespread use of pH dependent systems for colon targeted delivery of drugs, there has always been a controversy about their usefulness for the intended purpose, mainly because of high pH variability of the GIT among individuals and lack of proper coating materials that would dissolve at the desired pH of the colon. Although methacrylic acid copolymers such as Eudragit® L100 and Eudragit® S100 have commonly been used as pH dependent polymers for coating solid dosage forms, none of them is suitable for use alone for coating of dosage forms that would start releasing the drug specifically at pH 6.4 which is generally considered as the suitable pH for colon targeted drug delivery (Kun et al. 2005, Khan et al. 1999, Khan et al. 2000). A major drawback of Eudragit® coated pH dependent formulation is premature release of drug in the small intestine⁶.

The objective of this study is to develop, characterize, and evaluate prepared formulations using a combination of time and pH dependent system for delivering Mesalamine to the colon and to demonstrate its site specificity in the colon.

2. MATERIALS AND METHOD:

2.1 Materials:

Mesalamine was a kind gift from Watson Pharmaceuticals Ltd., (Goa, India), HPMC K4M and HPMC E5 was gift samples from Colorcon Asia Ltd., Eudragit S-100 from Roehm pharma Pvt. Ltd., (India), Magnesium stearate was procured from Research Lab, (Mumbai, India). All other reagents employed were of analytical or pharmaceutical grade.

Table 1: Formulation of core tablet

Ingredients (mg)	Formulation
Ingredients (mg)	MT1	MT2	MT3	MT4	MT5	MT6
Mesalamine	200	200	200	200	200	200
Croscarmellose sodium	15	16	17	-	-	-
Sodium starch glycolate	-	-	-	15	16	17
PVP: Isopropyl alcohol (5%w/w)	10	10	10	10	10	10
Microcrystalline cellulose	25	24	23	25	24	23
Total weight	250	250	250	250	250	250

2.2 Methods:

2.2.1 Preparation of Mesalamine core tablets:

Mesalamine is very moisture sensitive drug and its powder form exhibit poor flow properties. To improve flow properties granules were prepared by wet granulation method. Compositions of Mesalamine formulation is given in Table 1. Ingredients were passed through 60# mesh sieve size separately and collected. Then croscarmellose Na and Na-starch glycolate were mixed and granulated using a binder polyvinyl pyrrolidone K30 (PVP) in isopropyl alcohol. The wet mass was passed through 20# mesh sieve and the granules were dried in a tray dryer for 10-15min at 60ºC and evaluated for their properties [6]. Perfectly dried granules were mixed uniformly with 2% of talc and 1% magnesium stearate. Granules were compressed on a rotary tablet machine using 8 mm size punches.

2.2.2 Preparation of press-coated tablet:

The inner core tablet was press coated with HPMC (K4M and E5) to get desired lag time. Press coated tablets were prepared using a mixture of low and high viscosity polymer by placing 50% of press coat polymer layer in 13mm die. Inner core tablet was placed on it and remaining quantity of press coat was added to form the rapid release layer and finally compressed [9]. The press coated tablets were evaluated and used for further coating. A 3²randomized full factorial design was used in this study. Two factors were evaluated each at three levels, and experimental trials were performed at all nine possible combinations. The amounts of HPMC 4KM (X1) and HPMC E5 (X2) were selected as independent variable and the lag time was selected as dependent variable. Two variables at three levels and nine set of formulations were designed, which were assessed for lag period and drug release profile.

2.2.3 Preparation of enteric coating colon release tablet:

The outer coating was applied by using conventional coating pan method. First tablets were charged in coating pan for 30 min with drying temperature of 55 ºC and rotating speed of 14 rpm. The coating dispersion was intermittently applied using pilot type spray gun (model 630, Bollows Mumbai) fitted with 1mm spray nozzle. PEG-400 was added in isopropyl alcohol and stirred using magnetic stirrer for 10 min. While stirring, Eudragit L-100/ Eudragit S-100 were added. Stirring was continued until the completion of coating process^8,10.

2.3 Evaluation:

2.3.1 Evaluation of Mesalamine core tablet:

All the core tablets were evaluated for the hardness, thickness, diameter, in vitro disintegration time and drug content.

2.3.2 FTIR analysis:

FTIR spectra help to confirm the identity of the drug and to detect the interaction of the drug with the carriers. FTIR spectrum of pure drug and physical mixture of drug with polymers were obtained on FTIR (Shimadzu FTIR-8400S) instrument. The samples were mixed with KBr. The spectrum was scanned over the wave number range of 4000-400 cm^-1.

2.3.3 Swelling study of press–coated tablets:

The swelling index of press-coated tablet was carried out by using the medium phosphate buffer pH 6.8 and 7.2 at 37± 0.5 ºC. Study was carried out in dissolution test apparatus (U.S.P. Type-II)¹¹. Tablets were withdrawn and blotted with tissue paper to remove the excess water and weighed on the digital weighing balance (BL-220H Shimadzu) and swelling index was calculated by using the following equation,

2.3.4 In vitro dissolution study of enteric coated tablet:

The ability of tablets to retard drug release in physiological environment of the stomach and small intestine was assessed by conducting drug release studies in 0.1N HCl and phosphate buffer pH 6.8 and 7.2. Dissolution study was conducted in USP Type II apparatus at 100 rpm and at 37 ºC. Initially drug release was tested in 900mL 0.1N HCl for 2 h followed by in phosphate buffer pH 6.8 for next 3 h. Then dissolution was continued in phosphate buffer pH 7.2 till end of the test. 5ml sample was withdrawn at time interval of 1 h and filtered it was replaced with 5mL fresh medium to maintain the sink condition. The absorbance was recorded at 300 nm using UV- spectrophotometer¹².

2.3.5 PXRD and DSC study:

The cavity of the metal sample holder of x-ray diffractometer was filled with the ground sample powder and then smoothened with a spatula. X-ray diffractogram of Mesalamine and its dispersion samples were obtained using a Philips PW-3710, from Pune university. A scanning rate of 0.04 2θ s-1 over the range of 10-600 2θ by using CuK∝ as tube anode having wavelength 1.5418A0 was used to record each spectrum Powder x-ray diffraction studies have been widely used to understand crystallinity of solids. In PXRD it is considered that polymorphic changes have taken place or crystal habit has changed¹³. The Differential scanning calorimetry (DSC) of Mesalamine and optimized batch of formulation (F9) was carried out using SDT 2960TA instrument, USA. Samples were placed in a platinum crucible and DSC thermograms were recorded at a heating rate of 10 ºC per min in the temperature range of 50 ºC to 300 ºC, at a nitrogen flow of 20 ml/min.

2.3.6 Stability study:

Food and Drug Administration (FDA) and International Conference on Harmonization (ICH) specify the guidelines for stability testing of new drug products. The stability study was carried out on the optimized formulation. Short-term stability studies on promising formulation were carried out by storing the tablets at 40 ± 2 ºC, 75% ± 5% RH over a period of three months according to ICH guidelines. Tablets were evaluated for appearance, hardness, drug content and in vitro drug release studies for the period of one month¹⁴.

3. RESULT AND DISCUSSION:

3.1 Effect of Croscarmellose sodium and sodium starch glycolate level on drug release profile from core tablets:

In core compositions there is different concentration of sodium starch glycolate and croscarmellose sodium is present. In order to perform different release mechanism involved, effect of sodium starch glycolate and croscarmellose sodium level on drug release profile from uncoated tablet (Formulation MT1-MT6) were determined was given in Table 2. Formulations containing sodium starch glycolate (MT4-MT6) showed rapid drug dissolution than the formulation containing croscarmellose sodium (MT1-MT3) Fig. 1. The rapid increase in dissolution of Mesalamine with increased amounts of disintegrants may be attributed to rapid swelling and disintegration. Tablets prepared with sodium starch glycolate have maximum drug release with minimum disintegration time because higher concentration of sodium starch glycolate probably made larger pores with continuous network providing enough pressure for faster disintegration when contact with dissolution fluid. Studies on six formulations of Mesalamine inner core tablets revealed that sodium starch glycolate when used in concentration of 6.80% acts as a potential disintegrants.

Figure 1: Plot of percentage drug release Vs time from inner core tablets of Mesalamine

3.2 FTIR study:

In the IR study, it was found that there were no chemical interaction between Mesalamine and excipients used. The drug exhibits peaks due to the strong (C-H) stretch of the aromatic group, medium (C=C) stretch of the aromatic group, strong (C-C) stretching mode, strong (O-H) deformation of the hydroxyl groups, medium (C-O) stretching mode, strong (C-H) bond out of plane bending mode; ring deformation of the aromatic group, strong (O-H) stretching mode associated with the hydroxyl groups.

It was observed that there were no changes in these main peaks in the IR spectra of a mixture of drug and excipients, which showed that there were no physical interactions involving bond formation between drug and excipients Fig. 2.

Figure 2: Overlain of FTIR spectra of physical mixtures (Blue) and Mesalamine (Green)

3.3 In vitro dissolution profile of drug from press-coated tablets

A 3²randomized full factorial design was used in this study. The amounts of HPMC K4M (X1) and HPMC E5 (X2) were selected as independent variables. The lag time was selected as dependent variable. By combining high and low viscosity polymer HPMC K4M with HPMC E5 lag time increased with increasing weight ratio. The studies on drug release showed that 0% drug releases in initial 30 min (Table 3) for all formulation. In case of formulations F1, F4 and F7, the lag period decreased up to 30 min and % cumulative release for period of 10 h was 101.0±0.04, 100.24±0.62 and 99.98±0.57, respectively. The lag period of batches F1, F4, and F7 was retarded for only 30 min but this would not be sufficient to retard drug release for 2 to 4 h. The formulations F2, F5 and F8 showed the lag period up to 2 h and percent cumulative release for 10 h was 98.39±0.54, 99.72±0.03 and 98.08±0.43, respectively.

Figure 3: In-vitro drug release profiles of Mesalamine from batches F1-F9

The dissolution profile of batches F3, F6 and F9 revealed that as the thickness of the compression coat increases, the lag period increases. It was also observed that formulations F6, F9 and F3 showed lag time less than 4 h. and would not be sufficient for the tablet to reach intact to the colon Fig.3. Thus, an additional enteric coat of Eudragit S-100 was applied on the press coated tablets to prevent the premature release of drug in the upper GIT. From all the formulations, F9 formulation with grade of HPMC K4M and HPMC E5 (70% and 30%) was found to give the highest lag time and could be considered as best formulation for applying enteric coat and subjected to further evaluation.

Table 3: Coded levels as per 3²full factorial designs with observed responses

Batch codes	X1	X2	Lag time (min)	% Cumulative release (up to 10 h)
F1	-1	-1	30	101.0±0.04
F2	-1	0	60	98.39±0.54
F3	-1	+1	180	94.56±0.54
F4	0	-1	40	100.24±0.62
F5	0	0	60	99.72±0.03
F6	0	+1	240	84.01±0.45
F7	+1	-1	30	99.98±0.57
F8	+1	0	200	98.08±0.43
F9	+1	+1	240	98.19±0.52

3.4 Swelling study:

Increase in concentration of polymer ratio increased in swelling index and prolonged lag period and drug release, Fig. 4. The mechanism gives the idea regarding the water uptake study of various grades of polymer. This phenomenon attributes that swelling is maximum due to water uptake and then gradually decreased due to erosion. The formulation containing HPMC K4M: HPMC E5 (70: 30%) showed higher swelling index.

Figure 4: Graph of swelling indices of press coated tablet of batches F7- F9

3.5 Evaluation of tablets containing combination of time and pH dependent systems for colon targeted Mesalamine:

The core tablets of Mesalamine were compression coated with combination of HPMC E5 and K4M (30% and 70 %w/w). These were further coated with Eudragit S100 and Eudragit L100 (5-8 %w/w) polymer so as to prevent the drug release in stomach and small intestine. Table 4, Table 5 shows specifications for combination of time and pH dependent system. The lag time and drug release profile of Mesalamine from press coated tablets using different ratio of HPMC E5 and HPMC K4M mixture are given if Fig. 5.

By combining HPMC E5 with HPMC K4M lag time increases with increasing weight ratio HPMC E5/ HPMC K4M in formulation F1 to F9. As the coated tablet was placed in the dissolution medium, it was observed that the hydrophilic polymeric layer started erosion, which underwent progressive modification in terms of thickness and consistency. In the second phase of the dissolution procedure, the coating layer gradually starts to erode up to a limiting thickness. All of this process corresponded to a lag time capable of exhibiting a colonic release of the drug. The delay duration clearly depends on the kind and amount of hydrophilic polymer which was applied on the rapid release tablet.

Figure 5: Lag time of batches CT1-CT8

Eudragit S100 can prevent the drug release in stomach and small intestine and better than Eudragit L100 and can be used as enteric coating polymer for colon delivery of Mesalamine. By keeping the coat level of HPMC (K4M and E5) constant and by increasing the coating level of Eudragit S100, the lag time for drug release can be increased. All the formulations were stable at simulated gastric fluid with no drug release for first 4 h. The result of in vitro testing shows that fulfill the selection criteria for delivering Mesalamine to the colon through oral targeted tablets. Formulation containing Mesalamine was selected as the optimized one as it could prevent the drug release up to 6 h.

Table 4: Specification of batches CT1-CT4 for time and pH dependent systems

Batches	CT1	CT2	CT3	CT4
Eudragit S-100 coating level (%w/w)	5	6	7	8
Lag time (min)	5	5.5	5.5	6
Amount of drug release (%) in first 6 h	25.56 ± 1.56	18.45 ± 1.39	09.14 ± 1.24	No drug release

All readings are average ± (SD)

Table 5: Specification of batches CT5-CT8 for time and pH dependent systems

Batches	CT5	CT6	CT7	CT8
Eudragit L-100 coating level (%w/w)	5	6	7	8
Lag time (h)	3.5	4.5	5	5
Amount of drug release (%) in first 6 h	50.46 ± 1.16	45.43 ± 1.43	33.46 ± 1.31	30.10 ± 1.28

All readings are average ± (SD)

3.6 Powder x-ray diffraction (PXRD) Study:

Press coated enteric coating optimized tablet of Mesalamine were subjected to PXRD study by using X-ray diffractometer (Philips PW-3710). PXRD studies were carried out in order to study change in the nature of the pure drug and its physical mixture of optimized formulation. The overlay of PX-RD patterns of Mesalamine and the optimized formulation is shown in the Fig. 6. Major peaks and peak intensities observed in the diffractogram of pure drug and its physical mixture of optimized formulation are given in Fig 5. The diffraction pattern of the pure drug and physical mixture showed remarkable difference. The pure drug showed sharp peaks at optimized formulation when drug was incorporated into the polymer the intensities of the peaks were decreased. The reduced peak intensities in X-ray pattern clearly show that the Mesalamine appears amorphous. Similar observations have been already reported for solid dispersion of Mesalamine (kneading method) (AV Yadav et al., 2008).

Figure 6: Overlain of PX-RD patterns of Mesalamine and physical mixture of optimized batch

3.7 DSC study:

Thermograms of Mesalamine and its physical mixture with formulation components are shown in Fig. 7, the thermogram of pure drug showed a sharp endotherm at 283.37°C which represents its melting point. In the second layer physical mixture of optimized batch the endotherm was observed at 281.04 °C with the loss of its sharp appearance. The thermograms of the physical mixture showed same peak at 281.04 ºC indicating their compatibility of drug with polymers.

Figure 7: Overlain of DSC thermograms of Mesalamine and its physical mixture with other components

3.8 Stability studies:

Stability study samples of Mesalamine tablet formulation kept at 40 ± 2 ºC, 75% ± 5% RH over a period of three months according to ICH guidelines. The results reported in Table 6 indicate no significant changes in physical parameters; lag time and % drug release at the end of three months period.

Table 6: Stability of optimized formulation

Parameters	Before stability	After stability
Thickness (mm)	3.47 ± 0.05	3.49 ± 0.03
Diameter (mm)	13.07 ± 0.06	13.17 ± 0.09
Hardness (kg/cm²)	8 ± 0.02	8 ± 0.04
Lag time (h)	6	6
% Drug release (up to 12h)	98.19 ± 0.52	97.86 ± 0.06

4. CONCLUSION:

The objective of the present study was to formulate a novel pH- and time-dependent system for delivering drugs after oral administration to the colon. The combination of high and low viscosity polymers in the ratio 70:30 achieved a lag period of 4h. Superdisitegrants and Eudragit S 100 were appropriate for novel press coated colon release tablet that regulated lag time with prolonged drug release in colon. The in vitro study revealed drug release at a predetermined time can be controlled by application of coating layers of novel pH and time dependent system.

5. ACKNOWLEDGEMENT:

The authors wish to thank Watson Pharmaceuticals Ltd., (Goa, India) for providing Mesalamine as gift sample for this research work.

6. REFERENCES:

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Received on 11.08.2011 Modified on 25.08.2011

Research J. Pharm. and Tech. 4(11): Nov. 2011; Page 1751-1756

Batch Codes	Thickness (mm)	Diameter (mm)	Hardness (kg/cm²)	In vitro disintegration time (sec)	Drug content (%)
MT1	1.87 ± 0.10	8.12 ± 0.12	5 ± 0.13	281	98.9 ± 0.02
MT2	1.84 ± 0.06	8.11 ± 0.13	4 ± 0.14	263	100.1 ± 0.10
MT3	1.88 ± 0.09	8.13 ± 0.08	4 ± 0.12	229	100.8 ± 0.13
MT4	1.83 ± 0.10	8.12 ± 0.11	5 ± 0.11	252	99.1 ± 0.06
MT5	1.86 ± 0.11	8.14 ± 0.09	5 ± 0.10	195	102.0 ± 0.08
MT6	1.87 ± 0.07	8.15 ± 0.09	4 ± 0.11	180	103.4 ± 0.09