FORMULATION AND INVITRO EVALUATION OF COLON SPECIFIC DRUG DELIVERY

SYSTEMBY USING METRONIDAZOLE 

By

ALOK PRAKASH KAR M.PHARM (P.A &Q.A) EMAIL – alokprakashkar@gmail.com

INSTITUTE OF PHARMACY AND TECHNOLOGY

SALIPUR , CUTTACK, ODISHA 2016

INTRODUCTION In the field of medical therapy the disorders of colon requires a morden method of

treatment are receiving the attention of medical experts.The colonic disorders like constipation, diarrhea, inflammatory Bowel diseases ,Gastro enteritis needs a new approach of delivery of drugs to specific desired site.

Colon specific drug delivery system offers several potential therapeutic advantages Prolonged residence time, Relatively low enzyme secretion which makes it a particular site for absorption.

Colon drug delivery can be achieved by two routes 1.By oral route 2.By rectal

route.

Factors to be considered in designing of colon specific drug delivery system. pH in the colon: pH of gastrointestinal tract has highest pH levels (7.5±0.5) are

found to be in the terminal illium in the column. Gastrointestinal Transit: Gastric emptying of dosage form its highly variable and

depends on whether the subject is feed or fasted and on the properties of dosage form such as size and density.Small intestinal transit is const. at 3 to 4 hours.

INTRODUCTION Conlinc Micro Flora: The concentration of bacteria in the human colon is 1011-1212 . A large no of anaerobic and aerobic bacteria present through entire length of Git.the

most important anaerobic bacteria are bacteriodes,Bifidobacterium,Eubacterium.Various Approaches to colon-specific drug delivery. Coating with pH Dependent Polymers . Timed released dosage form Delivery system based on the metabolic activity of colonic bacteria. 1. Coating with biodegradable azopolymer. 2. Prodrug. 3. Polysaccaride as carrier. Bioadhesive system. Osmotic controled drug delivery.

OBJECTIVE

To develop formulation using various Biodegradable and

biocompartable polymer as carrier for colon targeting

Pathology of disease specially the affected parte of lower GIT

Physicochemical and biopharmaceutical properties of drug such

as solubility stability and permeability at the intended site of

delivery

Then preferred realease data of drug

Plan of Work Degradation and swelling potential of the various polymer are to be tested .

After selection of after natural polymer suitable formulations are to be design

and drug to be loaded

Preformulation studies, drug entrapment ,drug release kinetic, stability studies

are to be carried out.

Different in-vitro and in-vivo methods are used to evaluate different carrier

system to deliver drugs to colon.

Another in-vitro method involvs incubation of the drug delivery system.

More research is focused on the specificity of drug at colon site is necessary.

DRUG PROFILE Molecular Formula: C6H9N3O3

Molecular Weight :171.16 Melting range :125-163ºC

(Metronidazole Structure ) Chemically Metronidzole is 2-methyl-5-nitromidazole-1-ethanol Property; white or yellowish odour less powder and slightly soluble in water and

ethanol(95)

DRUG PROFILEPharmacology and Therapeutic Uses: Metronidazole is active against a wide variety of anaerobic protozoal parasite and

an aerobic bacteria. The mechanism of action is reflected in a selective toxicity to anaerobic micro

organism.once the drug has diffused into the cells, the nitro group accepts electrons from electron transport protein.

These intermediate destroys cells probably by the reaction with the cellular macromolecule such as DNA, proteins and membranes.

Usually the drug is completely absorbed after oral administration resulting in plasma concentration of about 10µg/ml. The half life of Metronidazole in plasma is about 8h and Vd is about 10% drug is bound to plasma protein.

Metronidazole and several metabolites are excreted in various proportion in the urine after oral adminstration of the parent compound.The liver is the main site of metabolism and more than 50% of the systemic clearnce of Metronidazole.

DRUG PROFILE

Pharmacokinetics: Absorption : Oral absorption is rapid and almost all is absorbed from stomach. Distribution : Vd = 0.74±0.10 L/Kg. Metabolism : Metabolised mainly in the liver by oxidation of side chain resulting in

two principle ,metabolites having antitrichomonical activity. Bioavailability : 99% Oral. Half life : 8.5±2.9 h. Elimination : Excreted in unchanged from upto 10±2% through urine.Adverse Drug Reaction: The most common side effects are headache, metallic taste, diarrhea,glossitis and

stomatitis may occur during therapy. And associated with a sudden intensification . Metronidazole has a well documented disulfiarm like effect and should be used with

caution in the patient with active disease of the CNS due to its neurological toxicity.

POLYMER PROFILEEthyl Cellulose: Proprietary name : Ethyl cellulose. Functional Category : Tablet binder, coating agent Viscosity: it was found that the viscosity is 29.2cps at 31ºC Solubility: insoluble in water , glycerin, propylene glycol. Stability : resistance to alkalis, more sensitive to acidic material.Eudragit L100: Solubility: Dissolves in methanol, ethanol, isopropyl alcohol in 1N NaoH to give

clear to cloudy solution. Particle size: Atleast 95% less than 0.25 mm . Viscosity: It was found that 18 cps at 31Cº. Storage: Protect from warm temperature. Film Formation: when the polymer solution is prepared onto a glass plate, a clear

film forms of on evaporation of the solvent.

POLYMER PROFILEEudragit S100: Solubility: Dissolves in methanol, ethanol, isopropyl alcohol in 1N NaoH to give

clear to cloudy solution. Particle size: Atleast 95% less than 0.25 mm . Viscosity: It was found that 18 cps at 31Cº. Storage: Protect from warm temperature. Film Formation: when the polymer solution is prepared onto a glass plate, a clear

film forms of on evaporation of the solvent.Cellulose Acetate Butyrate: Viscosity: 14.5 cps at 31ºC. Density: 1.18. Tensile Strength: 35, water absorption(%): 1.3, oxygen index(%): 17. Max. operating temperature (ºC): 60. Melting Temperature (ºC): 170-140.

Material Used:NAME SOURCEAcetone S.D.Fine chemical , Mumbai

Cellulose Acetate Butyrate M/S Triomed Formulation PVT,LTD,Kakinada

Dibasic Calcium Phosphate Himedia Laboratories , Mumbai

Ethanol S.D.Fine chemical , Mumbai

Ethyl Cellulose Himedia Laboratories , Mumbai

Eudragit L 100 M/S Triomed Formulation PVT,LTD,Kakinada

Eudragit S 100 Genuine chemicals,Mumbai

Magnesium stearate Himedia Laboratories , Mumbai

Metronidazole M/S Triomed Formulation PVT,LTD,Kakinada

Monobasic Potassium Phosphate Himedia Laboratories , Mumbai

Poly Vinyl Pyrrolidone Himedia Laboratories , Mumbai

Propylene Glycol Loba chemical,Mumbai

Sodium Hydroxide Himedia Laboratories , Mumbai

Starch Shiles chemicals,Mumbai

Talc Genuine chemicals,Mumbai

Tetrazine M/S Triomed Formulation PVT,LTD,Kakinada

Titanium Dioxide Loba chemical,Mumbai

Formulation of metronidazole tablets Metronidazole tablets were prepared by using P.V.P as binder. The formulation

details are given in the table 5.3.

Table No 5.3: Formulation Data for Metronidazole Tablets.

Ingredients F1Metronidazole(mg) 100 mg

D.C.P (mg) 65 mg

P.V.P(%w/w) 1 mg

Starch(mg) 15 mg

Mg.sterate (mg) 5 mg

Talc(mg) 5 mg

Procedure: All the ingredients except Talc and Magnesium Sterate were passed through through

sieve no 60 and transferred into a mortar. Isopropyl alchol was added and mixed well and the wet mass was passed through

sieve no 22 to wet the granules. The granules were dried in a tray drier at 50ºC for 30 min. Then dried granules

were passed through 20 number sieve and mixed with talc Magnesium sterate. The granules were compressed in to tablets using cadmach single punch tablet

compression Machine.The prepared metronidazole tablets were subjected to in-vitro evaluation.

In Vitro Evaluation of Metronidazole Tablets1. Size and thickness of tablets: The average size of tablet is 7.9mm and the average

thickness of tablet is 3mm 2. Uniformity of weight: The test was carried out by using 10 tablets. It was observed

that the % deviation was within the specified limits.3. Friability test : The friability test was performed with 10 tablets by using 4. Roche Friabilator: The apparatus was revolved for 100 revolutions diff. in weight

of 10 tablets before and after the test was recorded and % of friability was computed.The % deviation is with in 1%

5. Hardness Test 6. Disintigration test: There are two methods for disintigration of uncoated and

enteric coated tablets.7. Dissolution test: Dissolution test is carried out for evaluation of the formulation

and process variables of the drug and to ensure that the preparation comply with product.

In-vitro parameters of uncoated Metronidazole Tablets

Parameter F1

Disintegration Time (in min) 5±0.8

Friability test (%) 0.72

Uniformity of wt. (Max. Dev in %) 196.3±0.6 mg (3%)

Hardness test (kg/cm2) 3.5±0.2

Assay (%) 98±0.19

Formulation of Coating Suspension with Ethyl Cellulose :

Formulation code

Ethyl Cellulose(In gm)

Acetone(in ml)

Ethyl Alcohol(in ml)

Talc (in mg)

Titanium Dioxide (in mg)

EC1 0.75 100 3 200 100

EC2 1.5 100 3 200 100

EC3 3 100 3 200 100

Formulation of Coating Suspension with Cellulose Acetate:

Formulation code

Cellulose Acetate Butyrate (in gm)

Acetone (in ml)

PropyleneGlycol (in ml)

Talc(in mg)

Titanium Dioxide (in mg)

C1 0.5 100 3 200 400

C2 1.75 100 3 200 400

C3 3 100 3 200 400

C4 4 100 3 200 400

Tablet Coating Process

Different Batches of Metronidazole tablets were coated with different coating suspension using conventional coating pan and chromatographic spray gun.

Process details were as follows: Tablet coating process operating data: Coating unit: Round shaped, Stainless steel tablet coating pan with hot air blower,

German Filter machinary, . Inclination of the pan – 30º , Pan size: 25cm in diameter Distance: Nozzle to tablet is 10cm. Spray System: Chromatographic Spray gun. Tablet: Tablets were prepared by wet granulation method using P.V.P (2%) as binder Tablet size: 7.9mm (diameter), Thickness: 3mm, Weight: 195mg, Hardness: 4.5 (kg/cm2).

Evaluation of Coated Tablets

Film Thickness: Film thickness was measured by observing the difference of thickness between the coated and uncoated tablets by using screw gauge.

% of weight gained upon coating: The difference in weight of tablets was determined and % of weight gained.

Hardness test: The hardness test was carry out. Assay: Assay test was carried out. Disintegration test: The test was carried out. Dissolution test: The test was carried out by using U.S.P six stage dissolution rate

test apparatus-1. The medium was 0.1N HCL for the first two hours and the RPM was set at 100 and temperature at 37±0.5ºc.

Discussion : Data for calibration curve of metronidazole in 0.1N HCL (at 227nm).

Concentration(µg/ml) Absorbances

0 0

5 0.19

10 0.377

15 0.56

20 0.752

25 0.948

Analytical Methods used for the Estimation of Metronidazole: Metronidazole occours as white or yellowish crystaline powder and is slightly

soluble in water and ethanol. For estimatio of Metronidazole in pharmaceutical dosage forms and bilogical

fluoids U.V Visible Spectrophtometer method was used Calibration Curve of Metronidazole in 0.1N HCL: 100mg of metronidazole was accurately weighed and dissolved in 0.1N HCL in a

100 ml volumetric flask. And volume was made made upto 100ml with 0.1N HCL. The absorbance was measured at 227nm by using U.V Visible Spectrometer

againest blank. Calibration Curve Of Metronidazole in Phosphatr Buffer Solution: 10µg/ml solution of metronidazole in phosphate buffer solution was prepared and

absorbance was observed in range of 200nm to 400nm usingvU.V Spectrometer. For construction of calibration curve for metronidazole in phosphate buffer

solution, 100mg of metronidazole was accurately weighed and dissolved in 100 ml of PBS

Invitro Parmeter:

Formulation code

Film thickness(mm)

% of weight gained

Hardness(kg/cm2)

D.T(min) Assay(%) Means s.d

EC1 0.12±0.02± 0.5±0.1 4.8±0.5 11±0.5 97.8±0. ±98

EC2 0.12±0.02 0.5±0.09 5±0.4 11±0.35 97.6±0.33

EC3 0.16±0.03 2±0.2 5.48±0.6 12±0.9 97.6±0.25

C1 0.08±0.01 0.05±0.02 4.5±0.05 11±0.08 97±0.96

C2 0.19±0.02 1.5±0.01 4.7±0.02 12±0.09 97.4±109

C3 0.17±0.020 2±0.04 5.02±0.3 12±0.09 97.6±1.39

C4 0.18±0.02 2.6±0.3 5.2±0.4 13±0.34 97±0.33

EL1 0.11±0.01 1±0.03 4.9±0.09 13±0.33 97±0.99

EL2 0.14±0.04 1.7±0.08 5.2±0.07 14±0 97.2±0.54

EL3 0.02±0.02 2.8±0.03 5.36±0.03 15±1.2 97. ±0.25

EL4 0.23±0.02 3.2±0.03 5.5±.0.9 16±0.8 97.6±0.19

RESULTS AND DISCUSSION It indicates that the drug release from the tablet coated with EC following “Zero

order kinetics”. The correlation coefficient values are greater than 0.9852, indicating that the drug

released is Diffusion mechanism. The plots of cum % drug released Vs time and plots of log % drug remained Vs

time of EC coated Tablet were found linear and the correlation coefficient values of cum % drug released Vs time are greater than that of log % drug remained Vs time indicating that drug release from the tablets coated with C.A.B, EL, ES followiing zero order kinetic.

The tablets coated with LS (1:1), and LS (1:2) were evaluated of drug released using different Ph media like Phosphate buffer solutions at pH 2.2, 3.0, 4.2, 5.8 and 6.2 respectively.

No drug release was found at these lower pH media . These formulations were also evaluated for drug release in 0.1N HCL upto 6 hours

and no drug release was found.

RESULTS AND DISCUSSION

Metronidazole tablets were preparaed by wet granulation method using P.V.P as binder .The formulation were prepared and evaluated for in vitro behavior.

All the in-vitro parameters like Friability ,Assay(%purity) and Dissolution rate were with in the official limits

Different batches of the tablets were coated with different polymers like EC,CAB,EL100 and combinations of EL100 and ES100.

It was observed that the Hardness,Thickness of film ,Disintigration time were increased upon increasing the polymer concentration in all formulation.

All the tablets coated with ES100 exibited the dissolution rate which is inversly propostional to thickness of coat as well as conc. of polymer .

All the tablets coated with combination of EL100 and ES100 in different ratios exhibited the drug release rate, which is inversly proportional to the concentration of ES100 in the formulation.

The plots of cum % drug released Vs time and plots of log % drug remained Vs time of EC coated Tablet were found linear and the correlation coefficient values of cum % drug released Vs time are greater than that of log % drug remained Vs time.

CONCLUSION The prepared formulation were subjected to various invitro evaluation studies.the

data obtained from the dissolution studies was obtained by statistical and graphical model to study the drug release “kinetics and mechanism”.

The formulation were also subjected to IR study and further evaluated for drug release in different pH to ensure that the formulations are much resistance at lower pH levels.

From the observation it was found that the tablets coated with combinations of LS (1:1) and LS (1:2) showed good resistant to acidic environment in 0.1 N HCL and drug was released in PBS at pH 6.8 and showed no drug release at lower pH.

The study reveals that the formulation prepared with LS (1:1) and LS (1:2) exhibited certisfactory drug release profiles comply with the desired requirements of colon specific drug delivery and the said polymers are recommended in the reported concentrations for designing of suitable colon specific drug delivery system.

Further studies can be carried out with these selected polymers using other specific drugs wgich required colon targetting , besides this studies on invivo efficasy of the reported method can be taken up by intrested research group.

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