Entric-Coated Pectin Microspheres of Indomethacin for Targeting Inflammation in Lower Gastro intestinal tract

 

 

A Chandy*1, Sudish Rai1, A Manigauha2 and Suresh Sahu3

*1School of pharmacy, Chouksey Engineering College, Lalkhadan, Bilaspur-495007, India

2NRI Institute of Pharmacy, Sajjan Singh Nagar, Raisen road, Bhopal - 462021, India

3S I R T, Ayodhya Bypass Road Bhopal 462041 India

 

ABSTRACT

An objective of the present investigation was to formulate and evaluate targeting of indomethacin (IND) using Methaacrylalte-coated pectin microspheres. Pectin microspheres were prepared by emulsion dehydration method using different ratios of IND and pectin (1:40 to 1:80), stirring speeds (400-1800 rpm) and emulsifier concentrations (0.75%-1.5% wt/vol). Microspheres prepared by using drug:polymer ratio 1:4, stirring speed 1000 rpm, and 1.25% wt/vol concentration of emulsifying agent were selected as an optimized formulation. Eudragit-coating of pectin microspheres was performed by oil-in-oil solvent evaporation method using coat:core ratio (5:1). Pectin microspheres and Eudragit-coated pectin microspheres were evaluated for surface morphology, particle size and size distribution, swellability, percentage drug entrapment, and in vitro drug release in simulated gastrointestinal fluids (SGF). The in vitro drug release study of optimized formulation was also performed in simulated colonic fluid, where higher release rate was observed than gastric fluid It is concluded from the present investigation that Eudragit-coated pectin microspheres are promising controlled release carriers for colon-targeted delivery of IND.


KEYWORDS: indomethacin, pectin, Eudragit coating, colorectal tumor 

 

INTRODUCTION

Cancer in lower GIT is the second leading cause of cancer deaths in the Western countries. Conventional cancer chemotherapy is not very effective for treatment of colorectal cancer, as the drug molecule does not reach the target site at therapeutic concentration. Therefore effective treatment of colon cancer by conventional therapy requires relatively large doses to compensate for drug loss during passage through the upper gastrointestinal (GI) tract.1 Indomethacin was used as model drug due to its suitable pharmacokinetic properties for colonic delivery and good absorption in the large intestine.2,3,4,5 Kapitanovic and coworkers had also studied about the effect of indomethacin on growth inhibition, induction of apoptosis, and alterations in the expression of several genes involved in Wnt signaling in HT-29 colon cancer cells3.The use of biodegradable polymers such as azo-polymer and polysaccharide (eg, chitosan and pectin) for colon targeting are also reported in the literature.6,7

 

The hydrophilic matrices are applied for the release of both hydrophilic and hydrophobic drugs and charged solutes.9,10 Recently, many controlled-release formulations based on hydrophilic polymer matrices have been developed.11-13 Pectin totally degraded by colonic bacteria but is not digested in the upper GI tract.8,14,15 The objective of the present investigation was to design a multiparticulate delivery system for site-specific delivery of indomethacin (IND)2,3,16using natural polysaccharides (pectin) and pH-sensitive polymer (Eudragit S100) for the treatment of colon cancer. Mura and coworkers, has developed system which was able


to suitably retard the onset of drug release and to provide a colon-specific delivery.17 The use of enteric polymers (ES) as protective coating on the microspheres makes them able to release the drug at the particular pH of colonic fluid. A combined mechanism of release is proposed, which combines specific biodegradability of polymer and pH-dependent drug release from the coated microspheres. Fude and coworkers were able to release aspirin in colon using such combined mechanism of release.18

 

Table 1. Mean Diameter and Percentage Drug Entrapment of Various Pectin Microspheres and Eudragit-coated Pectin Microspheres

Fabrication Variables

 

Pectin Microspheres

 

Mean Diameter (µm)

Percentage Drug Entrapment

 Polymer amount

(Drug: polymer)

1:40

1:50

1:60

1:70

32.41 ± 3.5

34.36 ± 3.6

37.67 ± 3.8

39.47 ± 4.2

71.85 ± 2.25

69.35 ± 1.95

63.55 ± 1.75

61.75 ± 1.35

Surfactant concentration (wt/vol)

0.80

1.00

1.20

1.40

39.47 ± 4.2

37.67 ± 3.8

34.36 ± 3.6

32.41 ± 3.5

61.75 ± 1.35

63.65 ± 1.75

69.35 ± 1.95

71.85 ± 2.25

*Values are average of 3 readings ± standard deviation.

 

MATERIALS AND METHODS:

Materials:

The drug, indomethacin (IND) was purchased from M/s Otto Kemi, Mumbai, India. Methacrylic acid copolymer (Eudragit S100) was supplied as a gift by Röhm Pharma (Weiterstadt, Germany) Commercial Pectin Span 85, acetone, isooctane, ethanol, n-hexane, and light liquid paraffin were purchased from CDH Lab reagent, Mumbai, India. All other chemicals used were of analytical reagent grade and were used as received.

 

Preparation of Eudragit-coated Pectin Microspheres:

The pectin microspheres were prepared by emulsion dehydration technique.19, 20 Pectin (3 g) and IND (80 mg) were dissolved in 20 mL of equivalent volume of distilled waterand ethanol and stirred overnight to solubilize completely. This drug-polymer solution was dispersed in 50 mL isooctane containing 1.25% wt/vol Span 85 and stirred at 1000 rpm continuously to obtain stable water/oil (w/o) emulsion. The solution was rapidly cooled to 15°C and then 50 mL of acetone was added in order to dehydrate the pectin droplets. This system was maintained under mechanical agitation with propeller stirrer at 1000 rpm at 25°C for 30 minutes to allow the complete solvent evaporation. The microspheres were Vaccum-dried overnight and kept in an airtight container for further studies. Pectin microspheres were prepared using different ratios of IND:pectin (ie, , 1:40, 1:50,1:60, 1:70, and 1:80).

 

Pectin microspheres were coated with ES using oil-in-oil solvent evaporation method. 21 Pectin microspheres (50 mg) were dispersed in 10 mL of coating solution prepared by dissolution of 500 mg of ES in ethanol:acetone (2:1) to give 4:1 (coat: core ratio). This organic phase was then poured in 70 mL of light liquid paraffin containing 1% wt/vol Span 85. The system was maintained under agitation speed of 1000 rpm at room temperature for 3 hours to allow for the evaporation of solvent. Finally, the coated microspheres were filtered, washed with n-hexane, and vacuum-dried overnight.

 

Scanning Electron Microscopy:

The shape and surface morphology of Uncoated and Eudragit coated pectin microspheres were studied using scanning electron microscopy (SEM). The samples were scanned and photomicrographs were taken with a scanning electron microscope (Jeol JSM-1600, Tokyo, Japan).

 

 

Figure 1. Scanning electron photomicrographs of (a) Pectin microspheres (magnification 400X) and (b) Eudragit-coated pectin microsphere (original magnification 200X). MS indicates microspheres. (c) Eudragit-coated pectin microsphere (original magnification 200X). MS indicates microspheres.

 

Swellability:

A known weight (100 mg) of various IND-loaded pectin microspheres and Eudragit-coated pectin microspheres were placed in enzyme-free simulated intestinal fluid (SIF, KH2PO4/NaOH buffer, pH 7.4) and allowed to swell for the required period of time at 37şC ± 0.5şC).22 The microspheres were periodically removed and blotted with filter paper; then their change in weight (after correcting for drug loss) was measured until attainment of equilibrium. The swelling ratio (SR) was then calculated using the following formula:

 

SR = (wg –wi)/wg×100            

Where, wg is final weight, wi is initial weight of formulation                                                                                                          

Percentage Drug Entrapment:

The microspheres (100 mg) were digested in 10 mL of pectinase solution (4% wt/wt) for 12 hours. The digested homogenate was centrifuged (Remi, Mumbai, India) at 3000 rpm for 5 minutes, and the supernatant was assayed for IND content using UV method. IND was detected at 244 nm using UV Spectrophotometer (SL-159, Elico Ltd.,India). A linear curve was constructed between the peak area and concentration and the equation of line was obtained, which is Y=15.63X-0.0021 with correlation coefficient of 0.9999. The linearity was observed in the range between 0.01 and 0.04 mg/mL. Validation and calibrations were performed before and during analysis.

 

In Vitro Drug Release Studies in Simulated Gastrointestinal Fluids:

Eudragit-coated pectin microspheres and uncoated pectin microspheres were evaluated for the in vitro drug release in simulated GI fluids (SGF). The drug dissolution test of microspheres was performed by the paddle method specified in USP XXIV. Microspheres (100 mg) were weighed accurately and gently spread over the surface of 500 mL of Stimulated gastric fluid,23 at 37°C ± 0.5°C and pH was kept 1.2 for 2 hours using 0.1 N HCl. Then mixture of KH2PO4 and Na2HPO4.2H2O in ratio of 1.7:2.2, were added to the dissolution mediumto obtain the pH to 4.5 with 1.0 M NaOH, and the release rate study was continued for an additional 2 hours. After 4 hours, the pH of the dissolution medium was adjusted to 7.4 with 0.1 N NaOH and maintained up to 24 hours.20he samples were withdrawn from the dissolution medium at various time intervals using a pipette fitted with a microfilter. The rate of IND release was analyzed using UV method. All dissolution studies were performed in triplicate.

 

 

Table 2. Degree of Swelling of Various Pectin Microspheres and Eudragit-coated Pectin Microspheres*

Serial Code

Degree of Swelling

MP

EP

1

0.91 ± 0.05

0.04 ± 0.01

2

1.31 ± 0.13

0.12 ± 0.02

3

1.37 ± 0.15

0.17 ± 0.02

4

1.42 ± 0.15

0.21 ± 0.03

*MP indicates pectin microspheres; EP, Eudragit-coated pectin microspheres. Values are average of 3 readings ± standard deviation.

 

 

Statistical Analysis:

The mean percentage of IND released in Systemic GIT fluids (at different pH) from both pectin microspheres and Eudragit-coated pectin microspheres was prepared by using various drug:polymer ratios and compared. The Student t test was used to find the statistical significance. A value of P less than 0.05 was considered statistically significant.

 

Kinetic analysis of dissolution data:

The rate and mechanism of release of Indomethacin from the prepared Eudragit coated Pectin microspheres were analyzed by fitting the dissolution data into the zero-order equation :

Q = k0t

Where, Q is the amount of drug released at time t, and k0 is the release rate constant.The dissolution data was fitted to the Higuchi’s equation

 

Q = k2 t1/2

Where, k2 is the diffusion rate constant.

 

RESULTS AND DISCUSSION:

Preparation of Eudragit-coated Pectin Microspheres:

Pectin microspheres of IND were successfully prepared by emulsion dehydration technique. Uniform, surface cross-linked and almost spherical microspheres were obtained as shown in scanning electron photomicrographs (Figure 1(a)). The pectin microspheres were coated with Eudragit S100 by oil-in-oil solvent evaporation method, using coat: core ratio 4:1. The coated microspheres were found to be of spherical shape as observed in SEM photomicrographs (Figure 1(a) and 1(b)). The method was optimized using different stirring rate and emulsifier concentration to produce microspheres of small size and narrow size distribution, high drug loading efficiency, and controlled drug release at the colonic pH.

 

Table 3.In-vitro dissolution kinetics of Indomethacin from microspheres

Formulation

Drug release kinetics (R2)

Zero-order

Higuchi type

MP1

0.9918

0.8635

MP2

0.9871

0.8832

MP3

0.9842

0.8769

MP4

0.9869

0.8716

EP1

0.8176

0.5441

EP2

0.8315

0.5631

EP3

0.7982

0.5519

EP4

0.8263

0.5635

 

The mean diameter of pectin microspheres varied from 32.41 ± 3.5 µm to 39.47 ± 4.2µm with varying pectin concentration from 2.8% wt/vol to 7.1% wt/vol. The percentage drug entrapment was found to be 72% ± 5% in all the microsphere formulations. The highest drug loading efficiency was found with 2.8% pectin (Table 1). A higher concentration of polymer produced a more viscous dispersion, which formed larger droplets and consequently larger microspheres as reported by Pongpaibul et al.25 Due to change in emulsifier concentration there was a change in the mean diameter of microspheres obsvered which vary from 39.47 ± 4.2 µm to 32.41 ± 3.5 µm on varying emulsifier concentration (Span 85) from 0.8% wt/vol to 1.4% wt/vol for pectin microspheres. Increasing Span 85 concentration from 0.8% to 1.4% wt/vol led to formation of particles with a lower mean diameter and stabilization of the emulsion droplets avoiding their coalescence, resulting in smaller microspheres.26 The drug loading efficiency varied from 61.75% ± 1.35% to 71.85% ± 2.25% with varying emulsifier concentration from 0.8% to 1.4% during preparation of pectin microspheres (Table 1). Swellability of different microspheres was determined. No significant swelling was observed with Eudragit-coated pectin microspheres as compared with pectin microspheres (Table 2), thus ensuring better resistance of Eudragit-coated microspheres in the upper GI tract to swelling and preventing subsequent drug release at the nontarget site.

 

Figure 2. Percentage cumulative in vitro IND release from pectin microspheres containing different drug:pectin ratios (1:40 to 1:70) in simulated gastrointestinal fluids of different pH. Values are average of 3 readings ± standard deviation. IND indicates indomethacin; MP, pectin microspheres.

In Vitro Drug Release Studies in Simulated Gastrointestinal Fluids:

The results of In vitro IND release study of pectin microspheres and Eudragit-coated pectin microspheres showed that the rate of release of IND from pectin microspheres was mainly influenced by polymer concentration. IND release from pectin microspheres (MP) in SGF followed the order MP1> MP2 > MP3> MP4 (Figure 2). The initial higher release of IND from microspheres might have resulted from the dissolution of drug crystals on the surface of microspheres. The cumulative percentage drug release from Eudragit-coated pectin microspheres showed the desired rate, as there was no measurable drug release observed up to 2 hours in SGF (pH 1.2), while at pH 4.5, the IND release was quite insignificant (<2%) up to 4 hours. IND release from Eudragit-coated pectin microspheres in SGF followed the order EP1> EP2 > EP3> EP4 (Figure 3).

 

CONCLUSION:

The designed site-specific delivery of IND from the system may reduce the side effects of the drug caused by its absorption from the upper part of the GI tract when given in conventional dosage forms such as tablets and capsules. The experimental results demonstrated that Eudragit-coated pectin microspheres have the potential to be used as a drug carrier for an effective colon-targeted delivery system.

 

Figure 3. Percentage cumulative in vitro IND release from different Eudragit-coated pectin microspheres in simulated gastrointestinal fluids of different pH. Values are average of 3 readings ± standard deviation. IND indicates indomethacin; EP, Eudragit-coated pectin microspheres.

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Received on 30.03.2009

Accepted on 14.05.2009     

© A&V Publication all right reserved

Research Journal . of Pharmaceutical Dosage Forms  and Technology. 1(1): July.-Aug. 2009, 29-32