COLON TARGETING

DEVELOPMENT OF ENTERIC COATED PECTIN MATRIX TABLETS OF METRONIDAZOLE FOR COLON TARGETING: 

DEVELOPMENT OF ENTERIC COATED PECTIN MATRIX TABLETS OF METRONIDAZOLE FOR COLON TARGETING DISSERTATION SUBMITTED BY Register no.26091071 Under the guidance of Dr. GRACE RATHNAM, M.Pharm ., Ph.D. Director and Head of the Department of Pharmaceutics

INTRODUCTION: 

INTRODUCTION ANATOMY AND PHYSIOLOGY OF COLON The colon comprises several segments: the cecum the ascending colon the transverse colon the descending colon the sigmoid colon the rectum

Rationale for Colon Specific Drug Delivery : 

Rationale for Colon Specific Drug Delivery Targeting of drugs to colon is done for various reasons. Local treatment of inflammatory diseases e.g. crohn’s disease . Oral delivery of peptide and protein drugs, which normally become inactivated in upper parts of the gastrointestinal tract. Oral delivery of drugs/therapeutic substances that have undesirable side effects in the stomach and small intestine. Colonic diseases can be treated (e.g. colorectal cancer and amoebiasis . ulcerative colitis, irritable bowel disease and infections) whereby high concentration can be achieved locally used to minimize side effects.

APPROACHES OF COLON TARGETED DRUG DELIVERY SYSTEM: 

APPROACHES OF COLON TARGETED DRUG DELIVERY SYSTEM Prodrug Approach Time-Dependent Approach pH-Dependent Approach Bacteria-Dependent Approach Pressure or Osmotically -Dependent Approach

Mechaism of action of pH-Sensitive system: 

Mechaism of action of pH-Sensitive system Enteric coated matrix tablet in upper GIT Colonic pH Drug core coated with pH sensitve polymer Drug release in colon Mechanism of Bacterially dependent system Enteric coated matrix tablet Enteric coat dissolves in colonic pH degradation of matrix starts by colonic bacteria Matrix degrades and drug released

Amoebiasis: 

Amoebiasis Amoebiasis is a GIT ( Gastro Intestinal Tract ) infection spreaded by different species of Entamoeba . The genus Entamoeba is usually found in the intestine of vertebrates and invertebrates. They are five different species of Entamoeba for which man is host are: 1. E. dispar 2. E. histolytica 3. E. coli. 4. E. gingivalis . 5. E. hartmanni .

Life-cycle of Entamoeba histolytica : 

Life-cycle of Entamoeba histolytica

Metronidazole: 

Metronidazole The most preferred drug for treatment of intestinal amoebiasis , Where trophozoites of E. histolytica reside in lumen of ceacum and large intestine and adhere to the colonic mucus and epithelial layers. PHARMACOKINETIC PROFILE Bioavailability : 100%(oral), 59–94% (rectal) Absorption : Absorbed readily and almost completely from GIT Distribution : Penetrates well in body tissues and fluids Metabolism : Hepatic Half-life : 6–7 hours Excretion : Renal (60-80%), biliary (6– 15%)

AIM AND OBJECTIVE: 

AIM AND OBJECTIVE AIM The aim of this study was to prepare and evaluate the Enteric Coated Pectin Matrix Tablets Of Metronidazole For Colon Targeting. OBJECTIVE The objective of the present study was to formulate pectin matrix tablets coated with eudragitS100 for site-specific delivery of metronidazole (MTZ) using natural polysaccharide) and pH-sensitive polymer (EudragitS100) for the treatment of Amoebiasis in colon. This system is anticipated to protect the drug loss in the upper GI tract, which results from the inherent property of eudragit S100, and deliver MTZ in the colon only. A combined mechanism of release is seen, which combines specific biodegradability of polymer and pH-dependent drug release from the coated matrix tablets.

PLAN OF WORK: 

PLAN OF WORK 1. Compatibility study. 1.1 Fourier transform infrared spectral analysis(FTIR) 2. Preparation of metronidazole tablets by wet granulation method. 2.1 Preparation of metronidazole loaded pectin matrix tablets. 2.2 Coating of matrix tablets with Eudragit S 100. 3. Evaluation of granules and tablets. 3.1 Granules evaluation 3.1.1 Bulk density 3.1.2 Tapped density 3.1.3 Carrs index 3.1.4 Angle of repose 3.1.5 Hausner ratio 3.2 Tablets evaluation 3.2.1 Weight variation test 3.2.2 Disintegration time 3.2.3 Hardness 3.2.4 Friability 3.2.5 Drug content 3.2.6 Dissolution profile 3.2.6.1 In vitro drug release study 3.2.6.2 In vitro drug release study in presence of pectinase enzyme 3.2.6.3 Drug release kinetics

Calibration Curves of metronidazole 0.1 M HCL, pH 6.8, pH 7.4 phosphate buffer : 

Calibration Curves of metronidazole 0.1 M HCL, pH 6.8, pH 7.4 phosphate buffer S.no Concentration (µg/ml) Absorbance in 0.1M HCL Absorbance in pH 6.8 PBS Absorbance in pH 7.4 PBS 1 0.5 0.053 0.115 0.150 2 1 0.084 0.180 0.321 3 1.5 0.132 0.260 0.511 4 2 0.204 0.375 0.606 5 2.5 0.227 0.501 0.899

Slide 12: 

Standard curve of metronidazole in 0.1 M HCL Linear regression analysis equation: y=0.00875x+0.971

Slide 13: 

Standard curve of metronidazole in 6.8 pH phosphate buffer Linear regression analysis equation: y=0.151x+0.065

Standard curve of metronidazole in 7.4 pH phosphate buffer : 

Standard curve of metronidazole in 7.4 pH phosphate buffer Linear regression analysis equation: y=0.3248x+0.0102

Compatibility study: 

Compatibility study The peak values obtained from spectra showed that under stressed condition the excipient does not influence any interaction with the drug. There was no significant change in the position of peak and relative intensities of the endothermic peak of the drug. This indicates that there is no interaction between drug and polymer .

FT-IR spectra of metronidazole: 

FT-IR spectra of metronidazole

FT-IR spectra of pectin: 

FT-IR spectra of pectin

FT-IR spectrum of metronidazole and pectin: 

FT-IR spectrum of metronidazole and pectin

Preparation of Matrix tablets : 

Preparation of Matrix tablets Ingredients F1 F2 F3 F4 F5 F6 Metronidazole 200 200 200 200 200 200 Pectin 200 100 200 200 100 100 MCC 50 100 100 200 25 50 Starch 36.5 86.5 36.5 36.5 161.5 136.5 Talc 9 9 9 9 9 9 Magnesium stearate 4.5 4.5 4.5 4.5 4.5 4.5 Composition of F1-F6 formulations per tablet in (mg)

Enteric coating:: 

Enteric coating: Enteric coating of tablets was performed by pan coating . The enteric coating solution was prepared by dissolving Eudragit S 100 in acetone at 12%w/v. Tablets were coated until the weight of the tablets were increased to10 % w/v. Evaporation of the solvent was performed by using a hot air gun.

Evaluation of granules : 

Evaluation of granules a) Bulk density Bulk Density = (Weight of the granules / Bulk volume ). b) Tapped density Tapped Density = (Weight of the granules / Tappedvolume ). c) Carrs /compressibility index/% compressibility Carr (%) = [(Tapped density – Bulk Density) / Tapped Density] × 100 d) Angle of repose : θ = tan -1 (h / r) Where, h = height of pile r = radius of the base of the peak θ = angle of repose e) Hausner ratio Hausner’s Ratio = Tapped density / Bulk Density

Granules evaluation: 

Granules evaluation Formulation Code Angle of Repose (θ) Bulk Density(g/cm 3 ) Tapped Density(g/cm 3 ) Carr’s Index(%) Hausner’s Ratio(%) F1 22.29 ± 2.25 0.350 ± 0.018 0.392 ± 0.008 10.7 ± 1.88 1.114 ± 0.031 F2 20.80 ± 1.85 0.361 ± 0.005 0.412 ± 0.011 12.3 ±1.90 1.141 ± 0.014 F3 23.12 ± 1.59 0.370 ± 0.011 0.420 ± 0.013 11.9 ± 1.67 1.135 ± 0.033 F4 24.84 ± 0.76 0.356 ± 0.005 0.404 ± 0.016 11.8 ± 1.85 1.134 ± 0.024 F5 23.98 ± 0.87 0.380 ± 0.007 0.436 ± 0.009 12.8 ± 1.78 1.147 ± 0.029 F6 23.17 ± 0.67 0.376 ± 0.015 0.431 ± 0.026 12.7 ± 1.72 1.146 ± 0.018 All values are expressed as mean ±SD, n=3

Tablet evaluation: 

Tablet evaluation General appearance Weight variation Hardness Friability Percentage drug content

Tablet evaluation : 

Tablet evaluation Formulation Code Weight variation(%) Hardness(kg/cm 2 ) Friability(%) Drug content(%) F1 Within the limits 4.6 0.20 95.905 F2 Within the limits 4.8 0.18 98.116 F3 Within the limits 4.6 0.16 95.211 F4 Within the limits 4.7 0.19 96.300 F5 Within the limits 4.65 0.21 95.812 F6 Within the limits 4.9 0.21 96.125

In vitro release study: 

In vitro release study Apparatus : USP basket type Medium : 0.1M HCL (2h),6.8 pH PBS (2h),7.4 pH PBS (6h) (presence and absence of pectinase enzymes) RPM : 100 Temperature : 37.5 ± 0.5ºC Time interval : 1,2,3,4,5,6,7,8,9,10 (hrs) Absorbance : 274 nm

Cumulative percentage release of metronidazole from F1-F6 in absence of pectinase enzymes: 

Cumulative percentage release of m etronidazole from F1-F6 in absence of pectinase enzymes S.NO Time (hrs) F1 F2 F3 F4 F5 F6 1 1 0 0 0 0 0 0 2 2 0 0 0 0 0 0 3 3 3.196 ± 0.76 14.63 ± 0.44 9.207 ± 0.45 14.63 ± 0.77 8.76 ± 0.66 11.69 ± 0.71 4 4 12.45 ± 0.45 26.40 ± 0.83 19.37 ± 0.93 29.21 ± 1.2 19.51 ± 0.87 26.13 ± 0.43 5 5 18.23 ± 0.98 33.91 ± 0.28 27.69 ± 0.87 41.11 ± 0.98 31.94 ± 0.76 39.17 ± 0.35 6 6 24.64 ± 0.38 42.88 ± 0.36 35.17 ± 0.33 42.36 ± 0.29 38.12 ± 0.23 41.08 ± 0.98 7 7 28.58 ± 0.57 54.70 ± 0.67 42.69 ± 0.28 51.83 ± 0.68 42.73 ± 0,43 50.17 ± 1.2 8 8 36.51 ± 0.78 63.69 ± 0.83 54.66 ± 0.35 57.68 ± 0.54 51.47 ± 0.76 61.07 ± 0.66 9 9 44.63 ± 0.87 80.39 ± 0.96 61.41 ± 0.45 69.14 ± 0.43 56.59 ± 0.87 69.40 ± 0.56 10 10 52.94 ± 0.78 88.20 ± 0.77 69.66 ± 0.88 80.51 ± 0.65 63.64 ± 0.43 75.94 ± 0.77 All values are expressed as mean ±SD, n=3

Cumulative percentage release of metronidazole from F1-F6 in presence of pectinase enzymes: 

Cumulative percentage release of m etronidazole from F1-F6 in presence of pectinase enzymes S.NO Time (hrs) F1 F2 F3 F4 F5 F6 1 1 0 0 0 0 0 0 2 2 0 0 0 0 0 0 3 3 2.60 ± 0.53 11.69 ± 0.75 10.38 ± 0.22 17.56 ± 0.33 6.421 ± 0.43 14.631 ± 0.65 4 4 13.61 ± 0.17 22.02 ± 0.38 20.99 ± 0.87 29.53 ± 0.41 20.97 ± 0.97 28.934 ± 0.35 5 5 20.73 ± 0.29 33.58 ± 0.56 32.39 ± 0.56 39.66 ± 0.22 33.10 ± 0.35 42.436 ± 0.54 6 6 27.73 ± 0.84 48.13 ± 0.29 36.09 ± 0.23 41.64 ± 0.37 40.91 ± 0.54 45.436 ± 0.22 7 7 33.31 ± 0.72 59.25 ± 0.63 45.23 ± 0.78 53.01 ± 0.38 46.27 ± 0.66 52.304 ± 0.36 8 8 41.12 ± 0.15 73.94 ± 0.87 59.41 ± 0.56 59.46 ± 0.42 56.35 ± 0.43 63.293 ± 0.46 9 9 46.77 ± 0.76 85.93 ± 0.38 66.48 ± 0.34 71.52 ± 0.89 60.47 ± 0.65 72.582 ± 0.78 10 10 56.55 ± 0.81 93.00 ± 0.43 72.85 ± 0.29 83.34 ± 0.91 68.81 ± 0.87 79.868 ± 0.81 All values are expressed as mean ±SD, n=3

Slide 28: 

Cummulative percentage Metronidazole release in presence and absence of pectinolytic enzymes from F1 Cummulative percentage Metronidazole release in presence and absence of pectinolytic enzymes from F2

Slide 29: 

Cummulative percentage m etronidazole release in presence and absence of pectinolytic enzymes from F3 Cummulative percentage m etronidazole release in presence and absence of pectinolytic enzymes from F4

Slide 30: 

pectinolytic enzymes from F3 Cummulative percentage m etronidazole release in presence and absence of pectinolytic enzymes from F5 Cummulative percentage m etronidazole release in presence and absence of pectinolytic enzymes from F6

Drug Release Kinetic Study: 

Drug Release Kinetic Study S.no Formulation Regression coefficient for selected release kinetic models Zero order First order Higuchi Kinetics Korsmeyer-Peppas 1 F1 0.9926 0.8551 0.9187 0.9736 2 F2 0.9944 0.9246 0.8833 0.9916 3 F3 0.9981 0.9761 0.9220 0.9980 4 F4 0.9935 0.9083 0.9530 0.9833 5 F5 0.9830 0.9749 0.9518 0.9826 6 F6 0.9829 0.9743 0.9515 0.9810 For most of the formulations good correlation coefficients(R values) were obtained for Korsmeyer-Peppas equation. The n values was found to be in between 0.85-1.26. The values are closer to 1 and hence it is concluded that the drug release was more dependent on the effect of polymer relaxation.

Discussion : 

Discussion In compatibility study there is no appearance or disappearance of any characteristics peaks was seen. This shows that there is no interaction between the drug and polymer used in the tablets. Bulk density for the granules was found to be in the range 0.350 -0.376. Tapped density for the granules was found to be in the range 0.392-0.436.All the granules are within specification limits. This shows values of Bulk density and tapped density indicates good packing characteristics. Angle of repose for granules was found to be in the range 20.8-24.84, which is well with in the specified limit of 20°-30°.The values of Angle of repose indicates good flow properties. Carr’s index and Hausner’s ratio for granules were found to be in the range 10.7-12.8 and 1.114-1.147. The value of Carr’s index and Hausner’s ratio indicates free flowing material. The hardness and percentage friability of all formulations ranged from 4.6-4.95 kg/cm 2 and 0.18 - 0.21% respectively. Drug content for six formulations was 95.90, 98.11, 95.21, 96.30, 95.81, 96.12. Results for the above parameters were found that all six formulations lies within the range.

Slide 33: 

Dissolution study was conducted for all the formulations by using buffer solutions 0.1M HCL (2h),pH 6.8 (2h), and pH7.4(6h) by means of dissolution apparatus (USP-II) with and without the presence of Pectinase enzyme ( Pectinex Ultra SP-L), whose pectinolytic activity is closely related with that of the Bacteroides ovatus , the main colon producer of pectinolytic enzymes. The drug release curves obtained from experiments in the presence of pectinolytic enzymes were clearly different from those obtained previously from same tablets in the absence of pectinolytic enzymes. The presence of pectinolytic enzymes resulted in all cases shows faster drug release rates from the tablets.

Conclusion: 

Conclusion Eudragit coated pectin matrix tablets of metronidazole were prepared for colon specific drug delivery. where Pectin act as a natural Polysaccharide carrier which is inexpensive and naturally occurring and also having hydrophilic and swelling properties. The IR spectras revealed that, there was no interaction between polymer and drug. All the polymers used were compatible with the drug. These properties and the viscous nature of the pectin retards release of the drug from the dosage form, making it more likely that degradation will occur in the colon. Pectin, which put hurdles in the development of colon targeted drug delivery systems. If used alone it swells when it contacts with the aqueous fluids of GIT and causes release of entrapped drug through diffusion. This problem can be prevented by coating the metronidazole loaded pectin matrix tablets by using Eudragit S 100. It is concluded that Eudragit coated pectin matrix tablets of metronidazole are effective for colon targeting.