INTRODUCTION:

The concepts of the chronopharmacokinetics and chronotherapy of drugs have been extensively utilized in clinical therapy for improving drug efficacy and preventing side effects and tolerance of drugs ^1-3. In order to emulate innate circadian rhythms, a reasonable and generally accepted rationale is a delivery system capable of releasing drugs in a pulsatile fashion rather than continuous delivery at predetermined time or site following oral administration ^4-7. Pulsatile drug delivery system is characterized by a lag time that is an interval of no drug release followed by rapid drug release ⁸. Such systems are advantageous for drug with an extensive first pass metabolism, targeting of drugs to a specific site in the intestinal tract, the drugs that undergo degradation in gastric medium, irritate the gastric mucosa and adaptation of the therapy to chronopharmacological need. Pulsatile drug delivery systems are usually of reservoir type, where by a drug reservoir is surrounded by a diffusional barrier. This barrier erodes, dissolves or ruptures after a specified lag time, followed by a rapid drug release ^9-10.

In general, pulsatile drug delivery system can be classified in to time controlled and site specific delivery systems. Drug release from the former group is primarily activated by plug ejection or a barrier coating that dissolves, erodes or ruptures after a certain lag time, while release from the latter group is primarily regulated by the biological environment in the gastrointestinal tract such as the pH or the presence of enzyme. Based on the plug ejection mechanism, various capsular delivery systems were designed ^11-12. Capsular delivery system intended for pulsatile release generally consist of an insoluble body, in which the drug formulation is incorporated, a soluble cap and swellable hydrogel plug was used to seal the drug contents in to the capsule body. When this capsule came in contact with the dissolution fluid, it swelled, and after a lag time, the plug pushed itself outside the capsule and rapidly released the polymer it is formed from, the plug delays the onset of release through its erosion or swelling processes until timed removal from the capsule body and resulting release of the drug content in to the aqueous medium ^13-14. Samantha MK, et al. designed and evaluated Pulsincap drug delivery system of Salbutamol sulphate for drug targeting to colon in disease conditions like asthma. Empty gelatin capsule was coated with ethylcellulose keeping the cap portion as such. A hydrogel plug made of gelatin was suitably coated with Cellulose Acetate Phathalate in such a way that it was fixed to the body under the cap.

DM-1 DM-1

DM-2 DM-2

DM-3 DM-3

DM-4 DM-4

Fig 1: Scanning Electron micrographs of Diclofenac sodium microcapsules for DM-1, DM-2, DM-3 and DM-4 formulations

Eudragit microcapsules containing Salbutomol sulphate were prepared and incorporated into this specialized capsule shell. The in-vitro dissolution studies indicated that the onset of drug release was after 7 to 8 hr of the experiment and revealed its better sustaining efficacy over a period of 24 hrs ¹⁵. Howard Stevens NE, et al designed Pulsincap^®formulations to deliver a dose of drug following5 h delay and evaluated the capability of the formulation to deliver dofetilide to the lower GIT. The combination of scintigraphic and pharmacokinetic analysis permits identification of the site of drug release from the dosage form and pharmacokinetic parameters to be studied in man in a non-invasive manner¹⁶. Bussemer T, Bodmeier, et al. developed and evaluated a rupturable pulsatile drug delivery system based on the soft gelatin capsule with or without a swelling layer and an external water insoluble but permeable polymer coating, which release the drug after a lag time. Soft gelatin capsule based systems showed shorter lag times compared to hard gelatin capsules. Typical pulsatile release profile was obtained at lower polymer coating levels, while the release was slower and incomplete at higher coating levels. CAP coated capsules resulted in a more complete release than EC coated capsule ¹⁷.

Fig 2: Comparative zero order plots.

Fig 3: Comparative in-vitro release profile of formulations F1, F2 and F3.

The purpose of the present study was to produce a hard gelatin capsule – based pulsatile release system for the delivery of drug contents. Diclofenac sodium, an acid–insoluble NSAID, was used as model drug.

MATERIALS AND METHODS:

Materials: Diclofenac sodium was obtained from Cipla India Ltd., Mumbai. Methacrylic acid co-polymers (Eudragit® RS- 100 and L-100) were supplied as gifts by Degussa India Pvt. Ltd., Mumbai. Hydroxypropylmethylcellulose (HPMC-K4M, HPMC-E50 PREM LV and HPMC-E15 PREM LV) was obtained as gift from Colorcon, Goa. Ethyl Cellulose was obtained from S.D. Fine chem. Ltd. Mumbai. The rest of the chemicals were obtained from the following commercial supplier: heavy liquid paraffin (Ranbaxy fine chemicals Ltd., New Delhi), Span 80 (Research Lab. Mumbai), acetone, petroleum ether (S.D. Fine Chem. Ltd., Mumbai) were of analytical grade.

Fig 4: Comparative in-vitro release profile of formulations F4, F5 and F6.

Preparation of microcapsules:

Four batches were prepared with different proportion of drug and polymer. Accurately weighted Eudragit RS-100 was dissolved in 10 ml of acetone to form a polymer solution. Core material, i.e. Diclofenac sodium was dispersed in it and mixed thoroughly. This organic phase was slowly poured into liquid paraffin (100 ml) containing 1%w/w of Span-80 with stirring at 1000 rpm to form a smooth emulsion. stirring was continued until residual acetone evaporated and smooth-walled, rigid and discrete microcapsules were formed. The microcapsules were collected by decantation and the product was washed with petroleum ether (40-60^oC), four times and dried at room temperature for 3 hrs. The microcapsules were then stored in a dessicator ¹⁸. Four batches were prepared with different proportions of core to coat materials (drug: polymer = 1:0.5, 1:1, 1:1.5 and 1:2 (w/w) named DM-1–4, respectively).

Evaluation of microcapsules:

Particle size and External morphology:

Determination of average particle size of DM microcapsules was carried out by optical microscopy. SEM studies were carried out by using JEOL JSM T-330 a Scanning microscope (Japan).

Study of Micromeritic Properties:

Angle of repose, Bulk density, Carr’s index and Hausner’s ratio of the formulations were carried.

Drug content:

In a 100 ml volumetric flask, 25 mg of crushed microcapsules were taken, and volume was made up to mark with pH 6.8. The flask was shaken for 12 hrs using an orbital shaker. Then the solution was filtered and from the filtrate appropriate dilutions were made and absorbance was measured at 276 nm by using UV absorption spectroscopy.

In-vitro dissolution studies:

In-vitro dissolution profile of each formulation was determined by employing USP XXIII rotating basket method (900 ml pH 6.8 phosphate buffers, 100rpm, 37^o±0.5^oC). Microcapsules equivalent to 75 mg of DM was loaded into the basket of the dissolution apparatus. 5ml of the sample was withdrawn from the dissolution media at suitable time intervals and the same amount was replaced with fresh buffer. The absorbance of the filtrate was determined at wavelength of 276 nm and cumulative percent of drug release was calculated. Data obtained was also subjected to kinetic treatment to obtain the order of release and release mechanism.

Preparation of Cross-Linked Gelatin Capsules:

Hard gelatin capsules of 100 in number were taken. Their bodies were separated from the caps. 25 ml of 15% v/v formaldehyde was taken into desiccator and a pinch of potassium permanganate was added to it, to generate formaldehyde vapors. The wire mesh containing the bodies of the capsule was then exposed to formaldehyde vapors. The desiccator was tightly closed. The reaction was carried out for 12 hrs after which the bodies were removed and dried at 50^oC for 30 min.

Test for formaldehyde treated empty capsules:

Solubility studies for treated capsules:

Solubility tests were carried out for normal capsules and formaldehyde treated capsules for 24hrs. Ten capsules were randomly selected and then subjected to solubility studies in buffers of pH 1.2, 7.4 and 6.8. A single capsule was placed in the buffer solution and stirred for 24hrs. The time at which the capsule dissolves was noted.

Chemical test for free formaldehyde:

Standard solution used is formaldehyde solution (0.002, w/v) and sample solution is formaldehyde treated bodies (about 25 in number) were cut into small pieces and taken into a beaker containing distilled water. This was stirred for 1 h with a magnetic stirrer, to solubilize the free formaldehyde. The solution was then filtered into a 50ml volumetric flask, washed with distilled water and volume was made up to 50 ml with the washings. One milliliter of resulting solution was taken into a test tube and mixed with 4ml of water and 5ml of acetone reagent. The test tube was warmed in a water bath at 40⁰C and allowed to stand for 40 min. Standard solution was prepared in the same manner using 1ml of standard solution in place of the sample solution. The comparison should be made by examining tubes.

Formulation of Pulsatile drug delivery system:

Formaldehyde treated hard gelatin capsules of were chosen for the formulation. Microcapsules equivalent to 75mg of Diclofenac sodium were accurately weighed and filled into the treated bodies by hand filling. The capsules containing the microcapsules were then plugged with different grades of hydroxypropylmethylcellulose polymers (K₄M, E50 LV, and E15 LV) and, at different concentration from F1-F9. The joint of the capsule body and cap was sealed with a small amount of the 5% ethyl cellulose ethanol solution. The sealed capsules were completely coated with 5% eudragit L 100 (pH sensitive polymer) to prevent variable gastric emptying ^19-21. Coating was repeated until an 8-12% increase in weight is obtained.

In vitro release profile:

In-vitro dissolution profile of each formulation was determined by employing USP XXIII rotating paddle method. In order to simulate the pH changes along the GI tract, 900ml of three dissolution media with pH 1.2 for 2hrs, pH 7.4 for 3hrs, 6.8 were sequentially used ^22-23, referred to as pH change method. Rotation was 100rpm and temperature was maintained at 37^o±0.5^oC. 5ml of the sample was withdrawn from the dissolution media at suitable time intervals and the same amount was replaced with fresh buffer. The Withdrawn samples were analyzed at 276 nm, by UV absorption spectroscopy and the cumulative percentage release was calculated.

RESULTS AND DISCUSSION:

Particle size:

The mean particle size of the microcapsules significantly increased with increase in polymer concentration and was ranged between 79.70μm to 144.79 μm.

External morphology:

Scanning electron microscopy was performed to characterize the surface of the formed microcapsules. Particle from DM-1 and DM-2 were rough surfaced but spherical, where as DM-3 and DM-4 were found to be spherical, smooth and discrete. Scanning electron photomicrographs of all the four formulations are shown in Fig. 1.

Micromeritic properties:

The value of angle of repose between 20-25^oindicates free flowing nature of the formed microcapsules. Bulk density and tapped density showed good packability of the microcapsules. Carr’s index ranges from 16.33% to 23.53%, indicating excellent compressibility. Hausner’s ratio ranges from 1.19 to 1.30 indicate that all preparation showed that they had good flow properties. Micromeritic properties of all the four formulations are shown in Table.1.

Drug Content:

The drug content was found to be high in all the formulation. Amount of microcapsules to be taken for in vitro release studies and further development of pulsincap was calculated based on content of drug present in each formulation. Drug content of all the four formulations are shown in Table.1.

In-vitro release studies for microcapsules:

In-vitro release studies were carried out using USP-XXIII dissolution assembly. Cumulative % drug release after 12 hrs was 95.15%, 92.30%, 91.40%, and 86.50% for DM-1, DM-2, DM-3 and DM-4 respectively. It was observed that the drug release from the formulations decreased with increase in the amount of polymer added in each formulation. The release data obtained for all the four formulations are depicted in fig. 2.

The release data was fitted in to various mathematical models to know which mathematical model will best fit the obtained release profile. The regression coefficients for formulation DM-1 to DM-4 of zero-order plot were found to be 0.7880, 0.7716, 0.9020, and 0.9217, respectively. The regression coefficients for formulations DM:1–4 of first-order plot were found to be 0.9696, 0.9803, 0.9811 and 0.98332, respectively. Higuchi matrix plot regression coefficients of formulations DM:1–4 were found to be 0.9786, 0.9869, 0.9936, and 0.9943 respectively. Based on highest regression value (r) the best-fit model for DM-1, DM-2, DM-3 and DM-4 was Higuchi matrix, indicating that the release is by diffusion from these formulations.

Formaldehyde treatment of hard gelatin capsules:

The bodies of hard gelatin capsules were made insoluble by formaldehyde treatment by exposing the bodies of the capsules to vapors of formaldehyde; the caps were not exposed leaving them water-soluble. Exposure to formalin vapors results in an unpredictable decrease in solubility of gelatin owing to the crosslinkage of the amino groups in the gelatin molecular chain with aldehyde groups of formaldehyde.

Test for formaldehyde treated empty capsules:

The solubility tests were carried out for normal capsules and formaldehyde treated capsules for 24 h. It was observed that in all the case of normal capsules, both cap and body dissolved within 15 min where as in formaldehyde treated capsules, only the cap dissolved within 15 min, while the capsule body remained intact for about 24 h and hence indicates the suitability for colon targeting.

The formaldehyde capsules were tested for the presence of free formaldehyde. The sample solution was not more intensely colored than the standard solution inferring that less than 20 g of free formaldehyde per 25 capsules, taken for test.

Formulation and Evaluation of modified pulsincap:

On the basis of drug content, particle size morphology, in vitro release and release kinetics, formulation DM-3 was selected as better formulation for designing pulsatile delivery system.

In-vitro release studies:

During dissolution studies, it was observed that, the enteric coat of the Eudragit L-100 was intact for 2hrs in pH 1.2, but dissolved in pH 7.4, and then the exposed polymer plug absorbed the surrounding fluid, swelled and released the drug through the swollen matrix. After complete wetting of the plug, it formed a soft mass, which was then easily ejected out of the capsule body; releasing the Eudragit microcapsules into simulated colonic fluid (pH 6.8 phosphate buffer). The results obtained for all the nine formulations (F1–F9) are shown in Fig. 3, 4, 5.

Fig 5: Comparative in-vitro release profile of formulations F7, F8 and F9.

Formulations with HPMC K4M (F1-F3):

In case of F1 and F2 it was observed that polymer concentration was sufficient to retard the drug release in small intestinal fluid and the plug ejected out in colonic fluid, releasing the entire drug in colonic pH, in a controlled manner. At 24 hours, 94.04 % and 90.42 % of drug release was found in F1 and F2 respectively. With F3, a decrease in expelling power of plug was observed which might be due to inadequate wetting of the polymer. It was observed that at the end of 24hrs 86.42% of drug release was observed.

Formulation with HPMC E50 LV (F4-F6):

At the end of 24^th hour F4 formulation had released 96.04 % of drug, whereas F5 formulation released 93.22 % of drug up to 24 hours in controlled manner. In case of F6, decrease in expelling power of plug, due to inadequate wetting of polymer at higher concentration. At the end of 24^th hr 89.18 % of drug was released.

Formulations with HPMC E15 LV (F7-F9):

With formulation F7 released 94.23 % of drug within 24 hrs where as F8, F9 released 98.97 % and 92.26 % of drug at the end of 24^th hour.

The release of drug from the pulsatile capsule was found to be proportional to the concentration of different grades of HPMC. The formulations F7 & F8 there is no significant difference in controlling release of the drug. Formulations F1-F3 released in controlled manner, when compared to other formulations.

CONCLUSION:

The present study demonstrates that the Diclofenac sodium microcapsules could be successfully colon targeted by design of time and pH dependent modified chronopharmaceutical formulation. Pulsatile drug release over a period of 3–24 hrs, consistent with the requirements for chronopharmaceutical drug delivery was achieved from insoluble gelatin capsules, in which microencapsulated Diclofenac sodium was sealed by means of a suitable hydrogel plug. Thus the designed formulation can be considered as one of the promising formulation for preparing colon-specific drug delivery systems.

ACKNOWLEDGEMENTS:

The authors are thankful to Degussa India Pvt. Ltd., Mumbai for providing Eudragit and Colorcon asia, goa for providing different grades of HPMC.

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Received on 17.09.2009 Modified on 23.11.2009

Research J. Pharm. and Tech. 3(1): Jan.-Mar. 2010; Page 234-238

Code	Angle of repose	Bulk density	Tapped density	Carr’s index (ci) (%)	Hausner’s ratio	Drug content (%)
DM-1	20.25 + 0.586	0.507 + 0.013	0.606 + 0.028	16.33 + 1.965	1.195 + 0.035	90.03± 0.43
DM-2	21.73 + 0.543	0.511 + 0.019	0.703 + 0.039	27.31 + 1.405	1.375 + 0.030	91.20± 0.79
DM-3	20.78 + 0.621	0.491 + 0.008	0.673 + 0.014	12.24 + 0.829	1.370 + 0.015	99.70± 0.48
DM-4	24.08 + 0.590	0.562 + 0.012	0.735 + 0.020	23.53 + 1.248	1.307 + 0.020	95.33± 0.52