Design and development of colon targeted drug delivery system for colonic diseases.

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DESIGN AND DEVELOPMENT OF COLON TARGETED

DRUG DELIVERY SYSTEM FOR COLONIC DISEASES

A SYNOPSIS OF THESIS SUBMITTED TO

The Tamilnadu Dr. M.G.R. Medical University, Guindy, Chennai-600032, Tamilnadu, India.

As a partial fulfillment of the requirement for the award of the degree of

DOCTOR OF PHILOSOPHY

(Faculty of Pharmacy

)

Submitted By

Mr. S . JEGANATH (Ref. No. EXII(1)/59327/2011)

Under the guidance of

Dr. K. SENTHILKUMARAN M.Pharm., Ph.D.

Professor and Head Department of Pharmaceutics

K.K. College of Pharmacy, Chennai, Tamilnadu, India.

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DESIGN AND DEVELOPMENT OF COLON TARGETED

DRUG DELIVERY SYSTEM FOR COLONIC DISEASES

A SYNOPSIS OF THESIS SUBMITTED TO

The Tamilnadu Dr. M.G.R. Medical University, Guindy, Chennai-600032, Tamilnadu, India.

As a partial fulfillment of the requirement for the award of the degree of

DOCTOR OF PHILOSOPHY

(Faculty of Pharmacy

)

Submitted By

Mr. S . JEGANATH (Ref. No. EXII(1)/59327/2011)

Under the guidance of

Dr. K. SENTHILKUMARAN M.Pharm., Ph.D.

Professor and Head Department of Pharmaceutics

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CERTIFICATE

This is to certify that Mr.S . JEGANATH (Ref. No. EXII (1) /

59327 / 2011) carried out research work on “DESIGN AND

DEVELOPMENT OF COLON TARGETED DRUG DELIVERY

SYSTEM FOR COLONIC DISEASES” for the degree of Doctor of

Philosophy in Pharmacy of The Tamilnadu Dr. MGR Medical University, Chennai for the requisite period under the regulation enforce and the Synopsis of thesis is a bonafide record of the work done by him under my supervision and guidance. This work is original and has not formed the basis of the award to the candidate of any degree, diploma, associateship, fellowship or other similar title.

Date:

Place: Chennai. Dr. K. Senthilkumaran (Guide)

Professor and Head

Department of Pharmaceutics K.K. College of Pharmacy,

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CERTIFICATE

This is to certify that Mr.S . JEGANATH (Ref. No. EXII (1) /

59327 / 2011) carried out research work on “DESIGN AND

DEVELOPMENT OF COLON TARGETED DRUG DELIVERY

SYSTEM FOR COLONIC DISEASES” at Department of Pharmaceutics,

Padmavathi college of Pharmacy, Dharmapuri for the degree of Doctor of Philosophy in Pharmacy of The Tamilnadu Dr. MGR Medical University, Chennai for the requisite period under the regulation enforce and the Synopsis of thesis is a bonafide record of the work done by him under the guidance and

supervision of Prof.Dr.K.SENTHIL KUMARAN during the period of

2011-2014.

Date:

Place: Dharmapuri. PRINCIPAL

Padmavathi College of Pharmacy, Periyanakalli,

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INTRODUCTION

Drug delivery system can be defined as the mechanisms to set up therapeutic agents into the body. Primitive approaches of delivering drugs lacked a very basic need in drug delivery; that is, consistency and uniformity which are safe, economical and efficient for means of providing the health and wellbeing of mankind. An perfect dosage regimen in the drug therapy of any disease is the one that instantly attains the preferred therapeutic concentration of drug in plasma and maintains it steady for the entire duration of treatment. This is possible through administration of a conventional dosage form in a particular dose and at a particular frequency resulting in a number of limitations. To overcome those limitations effective and safer use of existing drugs through concepts and techniques of modified release and targeted delivery system can result in increased interest. 1

COLON TARGETED DRUG DELIVERY SYSTEM (CTDDS)

CTDDS is considered to be beneficial in the local and systemic treatment of ileo-cecal and colon related diseases & disorders. Treatment might be more effective if the drug substances were targeted directly on the site of action in the colon.2 Lower doses might be adequate and, if so, systemic side effects might be reduced. The delivery of drugs to the colon has number of therapeutic implications in the field of drug delivery.3 These include the topical treatment of diseases

associated with the colon such as inflammatory bowel disease (IBD) as ulcerative colitis and crohn’s disease, inflammatory bowel syndrome (IBS), colon cancer, amebiasis etc. CTDDS are also of importance when delay in absorption is desired from therapeutic point of view in treatment of diseases showing peak symptoms in early morning a diurnal rhythm i.e. chronotherapy of diseases that are sensitive to circadian rhythms e.g. nocturnal asthma, rheumatic disease, ischemic heart disease and angina attack.4, 5

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their confined use, while enemas solution can offer only topical treatment to the sigmoid and descending colon. Thus, oral route is preferred but the absorption and degradation in upper part of gastrointestinal tract is the major obstacle and must be avoided for successful colonic delivery.6, 7

Formulations for CTDDS are, in general controlled release dosage forms which may be designed either to provide a pulsatile burst release or a sustain release once they reach to the colon. The proper selection of a formulation approach is dependent upon several important factors like pathology and pattern of the disease, physicochemical and biopharmaceutical properties of the drug and its desired release profile. 8

Several factors are to be studied and considered in designing CTDDS like anatomy and physiology of colon, pH of gastro intestinal tract (GIT), Gut Microbiota & their enzymes and GI transit time. The GI tract is constituted of stomach, small intestine and colon. In mammals, colon consists of four sections: the ascending colon, the transverse colon, the descending colon, and the sigmoid colon.

9 The pH is different in the stomach, small intestine and colon and it depends upon

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involving the mucosa and sub-mucosa of the colon. IBD is restricted to the GIT however; extra intestinal manifestations are far reaching.13, 14 In humans, the majority of IBD occurs in a variety of forms, the most common being Crohn’s disease (CD) and ulcerative colitis (UC). These two forms of diseases are clinically related and histologically distinct chronic inflammation of the bowel that is characterized by intermittent courses of acute attacks. UC is characterized by chronic inflammation in a continuous and confluent pattern which mostly affects rectum and colon. For effective treatment of UC the drug must release at site of proximal colonic region with slow release. The endoscopic features of UC include ulcers, erythema, and loss of vascular pattern, friability, spontaneous bleeding, and pseudopolyps. 15, 16

In contrast to UC, CD affects any region of the gastrointestinal tract and is characteristically segmental with areas of sparing throughout the gastrointestinal tract. The endoscopic features of Crohn’s disease include apthous, stellate, or linear ulcers, cobblestoning, and skip areas of normal mucosa and microscopic features include transmural inflammation, granulomas, and skip areas of normal uninflamed mucosa. 17, 18

Colorectal cancer usually develops slowly over a period of many years. A polyp is an abnormal protruding growth that develops in certain parts of the body. There are 2 types of polyps hyperplastic polyps and adenomatous polyps.19 Diverticulitis is defined as the inflammation of one or more diverticula. The inflammatory process may be limited to the immediate vicinity of the diverticula or may extend to surrounding structures and organs and can occur at any point in the gastrointestinal tract.20

Approaches or Strategies for CTDDS

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can effectively deliver to colon by various approaches based on release of drug at a predetermined time after administration. The employment of polymers as carrier matrices for colonic delivery often utilizes a time dependent, by utilizing pH change within the GI tract consist of pH sensitive polymers, using GI pressure differences, and by exploiting microflora and microbial enzymes predominantly present in the colonic region of the GI tract.8, 21, 22

Usually, time-dependent drug delivery systems are designed to deliver drugs after a lag time of five to six hours. This approach is based upon the theory that the lag time equates to the time taken for the dosage form to reach the colon. The lag time depends on the size of dosage form and gastric motility associated with the pathological condition of the individual. The residence times can vary from a few seconds to a number of hours. On the other hand the small intestine transit time is reported to be more consistent at three to four hours. Since the system is unable to sense and adapt to an individual’s condition, the approach clearly limits the utility.

Till date for the effective colonic delivery pH dependent polymers are commonly and effectively used as compare to approaches mentioned above. pH dependent polymers are insoluble at low pH gastric but become increasingly soluble as pH rises. The pH in the GIT varies between and within individuals and also between healthy and patients which could lead to the failure of the system in the treatment of IBD but may be overcome by modifying the technique. Most commonly used pH dependent coating polymers are copolymers of methacrylic acid and methyl methacrylate containing carboxyl groups like Eudragit S100 which is soluble above pH 7 and Eudragit L above pH 6 are polymers in targeted drug delivery to the colon. Eudragit S coating formulations have been used to target most of medicinal agents including anti inflammatory mesalamine formulations single as well as multiparticulate formulations to treat colon diseases.

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soon as a certain pressure limit is exceeded by an increase of the luminal pressure in the colon caused by peristaltic waves.

The use of GI microflora as a mechanism of drug release in the colonic region has been of great interest to researchers in recent times with some limitations. The colonic microflora produces a variety of enzymes that are not present or different from those in the stomach and the small intestine and could therefore be used to deliver drugs to the colon after enzymatic cleavage of degradable formulation components or drug carrier bonds.

IBD Treatment

Treatment must begin with accurate diagnosis. The diagnosis of IBD depends on the aggregate constellation of the clinical history, physical findings, and endoscopic, radiologic, and histologic features, as well as the results of routine laboratory tests. Typically, these features allow a clear diagnosis of IBD and distinction between UC and CD. Medicinal agents, such as antidiarrheal agents, that are directed primarily at relieving symptoms rather than controlling inflammation itself can be important. Effective anti-inflammatory therapy for the treatment of IBD began with the use of oral or topical preparations of mesalamine and corticosteroids. These therapies work primarily by targeting the inflammatory fall at various levels to reduce mucosal and peripheral inflammation.23, 24

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Corticosteroids and immunosuppressant are used for active disease or refractory disease. It is one of the highest used drugs in short term IBD treatment, although of their side effects, their efficacy cannot be refused and they are still accepted. Their effects have been shown to act through the inhibition of proinflammatory cytokine production. Oral corticosteroids, like budesonide 9 mg/day may be administered to patients with mild to moderate or severe forms of UC or CD. Finally parenteral administration may be utilized for hospitalized patients with severe stages of the IBD. Corticosteroids, with their popularity and efficiency in reducing immunologic and inflammatory reactions, produce severe side effects. Long term corticosteroid remedy will induce changes in ecchymoses, fat distribution, and abdominal strain. Some more severe conditions may occur include osteoporosis, hypertension, diabetes etc. Corticosteroids administered for a short period of time can also result in fluid and electrolyte imbalances, and metabolic abnormalities. Corticosteroid administration suppresses the immune system, thus exposing the patient to a greater risk of opportunistic infections.27-29

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AIM AND OBJECTIVE

The aim and need of this study was directed to develop, optimize and evaluate an efficient CTDDS in two parts. First part deals with budesonide burst release Crospovidone and pH dependent Eudragit L30D coated tablets for ileo caecal targeting to treat mild to moderate CD and in second part budesonide sustained release HPMC K4M and Eudragit L30D coated tablets developed for colonic delivery to treat mild to moderate IBD and for maintenance therapy during disease remission.

Budesonide were selected as model standard drugs to treat IBD. Budesonide is a potent, synthetic non-halogenated corticosteroid with high topical anti-inflammatory effect and little systemic effects. Additionally, budesonide has low incidence of adverse effects and high topical effects and has important suggestions in the pharmacotherapy of IBD, both in treatment of UC and CD. It was found that less than 5% of drug was available beyond the ileum and cecum, and hence, colonic delivery still needs to be optimized by a more reliable targeted system.

In the present research it was therefore decided to determine whether better formulations for colonic delivery could be formulated as coated with pH sensitive polymer with burst release of budesonide and sustained release budesonide tablets has advantages of bioavailability of high drug concentration at targeted site, reduction in dosage regimen and moreover controlling drug release respectively.

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Eudragit L30D and inner HPMC K100M control release polymer with budesonide for possible release in proximal colon to treat IBD efficiently.27-29

Currently in the market there is no immediate release budesonide formulation to target ileo-caecal region specifically and budesonide colon specific formulation to treatment of IBD. The type of formulations we studied has not been described and formulated or published earlier anywhere. Although the formulations are moderately complex, its manufacturing process is easy and might also be undertaken on an industrial scale.

The following objectives were outlined to achieve the aim and need of the study:

1. To select appropriate drug and polymers to formulate two colonic delivery systems to treat CD and IBD i.e. a burst immediate release and a sustained release system, both coated with pH dependant solubility profiles to achieve lag time of 5h respectively

2. To perform Preformulation studies

3. To develop budesonide loaded tablets with Crospovidone and based on coating with Eudragit L30D

4. To develop a budesonide loaded tablets with HPMC K4M and based on coating with Eudragit L30D

5. To perform in vitro release studies & other evaluation parameters for both formulations

6. Optimization of both formulations

7. Stability study of both optimized formulations

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EXPERIMENTAL PROTOCOL

1. Procurement of drugs, polymers and excipients for formulation development

2. Preformulation study

3. Characterization of drugs, excipients and its mixture using melting point determination, UV spectroscopy, Infrared spectroscopy, and Differential scanning calorimetry (DSC)

4. Preparation of calibration curve of drugs in distilled water, 0.1 N HCl and phosphate buffers of pH 7.4 and 6.8

5. Compatibility study of drugs, polymer and its mixture

6. Preliminary development of trial batches to establish the required profiles.

7. Selection of best formulation for optimization a) Physical evaluation and assay of tablet

b) Micromeritic properties: Bulk density, Tapped density, Angle of repose, and Hausner ratio

c) In vitro evaluation for dissolution profile & other evaluation

parameters to study optimized formulated tablets 8. Stability study of optimized formulations

9. In vivo evaluation of optimized formulations to assess site specificity

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MATERIALS AND METHODS

Materials

Budesonide was a kind gift from Ethypharma Pvt. Ltd. (Mumbai, India). Eudragit L30D was purchased from the Research-Lab Fine Chem Industries (Mumbai, India). Polyethylene Glycol was purchased from Clariant Pvt. Ltd. (Mumbai, India). Magnesium Stearate, lactose, polyvinyl pyrolidone (PVP K30), Methylene chloride were purchased from Signet India Pvt. Ltd, Mumbai. HPMC K4M, Crospovidone and Isopropyl alcohol (IPA) were purchased from Loba Chemicals (Mumbai, India). Other excipients used were of standard pharmaceutical grade

METHODS

PART I: Formulation of Budesonide Pulsatile Release Tablets for Ileo-cecal Targeting

Compatibility study of budesonide pure drug, excipients and its physical mixture was evaluated. Melting point of drug was determined using melting point apparatus using capillary method. The calibration curves of budesonide were measured in distilled water, 0.1N HCl and phosphate buffers of 6.8 and 7.4 pH.

Preparation of Budesonide Pulsatile Release Tablets

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Coating of Eudragit L30D Over Drug Containing Tablets

Eudragit L30D coating dispersion requires addition of polyethylene glycole as plasticizer and stirred the solution for few minutes with a magnetic stirrer. This solution was sprayed over the above processed tablets up to 5, 10, 15, 20, 25, 30 and 35% weight gain.

Preliminary batch formulation which showed acceptable lag time of 5h and 90% or more drug release within 90 min. after lag time was further selected for optimization study using 32 factorial design i.e. 3 levels and 2 factors as extent of Crospovidone and extent of Eudragit L30D coating as variable factors. All the other formulation aspects and processing variables were kept invariant throughout the process as mentioned above.

PART II: Formulation of Budesonide Sustained Release Tablets for Colon Targeting

Compatibility study of budesonide pure drug, excipients and its physical mixture was evaluated. Solubility determination and melting point of drug was determined using melting point apparatus using capillary method. The reported analytical method in methanol was tried by using UV spectrophotometer.

Preparation of Budesonide Pulsatile Release Tablets

The granules were prepared by wet granulation method. The drug budesonide, HPMC K4M and lactose were passed through sieve 40# separately and blended thoroughly. After proper mixing then slowly added the binding solution containing PVP K-30 in IPA till fine uniform granules were obtained. The wet mass was passed through sieve 16# and dried at 50°C for 30 minutes to get the moisture content less than one. Then lubricate the dried granules with magnesium stearate which were already passed through sieve 40#. Then lubricated granules were compressed on cadmach tablet punch machine for all formulations.31 Granules were

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Coating of Eudragit L30D Over Drug Containing Tablets

Eudragit L30D coating dispersion requires addition of polyethylene glycole as plasticizer and stirred the solution for few minutes with a magnetic stirrer. This solution was sprayed over the above processed tablets up to 5, 10, 15, 20, 25, and 30 % weight gain. Preliminary batch formulation which showed acceptable lag time of 5h and 90% or more drug release within 12h after lag time was further selected for optimization using 32 factorial design i.e. 3 levels and 2 factors as extent of HPMC K4M and extent of Eudragit L30D coating as variable factors. All the other formulation aspects and processing variables were kept invariant throughout the process as mentioned above.

Statistical Analysis of Datafor Part I and Part II

According to the results obtained from the dissolution profile of the preliminary experimental batches of part I and II, the batch that showed desirable lag time was selected for factorial studies to optimize effects of variables on formulation. Response Surface Methodology (RSM) is a widely practiced approach in the development and optimization of drug delivery devices. The technique requires minimum experimentation and time, thus proving to be far more effective and cost-effective than the conventional methods of formulating the dosage forms. A 32 full factorial design was constructed for part I, where the extent of Crospovidone and Eudragit L30D coating of were selected as the independent variables (factors) and extent of HPMC K4M and Eudragit L30D coating of were selected as the independent variables (factors) for part II formulation respectively. The levels of these factors were selected on the basis of initial studies and observations.

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Ease Inc., Minneapolis, MN, USA) with a manual linear regression technique. A significant term (p < 0.05) was chosen for final equations. Finally, response surface plots resulting from equations were drawn.

Y = b0 + b1X1+ b2X2 + b12X1X2+ b11X12+ b22X22 (1)

In above equation 1, Y is the dependent variable; b0 is the arithmetic

average of all the quantitative outcomes of nine runs. b1, b2, b12, b11, b22 are the

estimated coefficients computed from the observed experimental response values of Y and X1 and X2 are the coded levels of the independent variables. The interaction

term (X1X2) shows how the response values change when two factors are

simultaneously changed. The polynomial terms (X12, X22) are included to investigate

nonlinearity.

All nine batches of design have shown wide variation in lag time and percentage of drug release after lag time. Statistical validity of the polynomials was established on the basis of analysis of variance (ANOVA) provision in the software. Level of significance was considered at p < 0.05. The best-fitting mathematical model was selected based on the comparison of several statistical parameters, including the coefficient of variation (CV), the multiple correlation coefficient(R2), the adjusted multiple correlation coefficient (adjusted R2) and the predicted residual sum of squares (PRESS) provided by the software. The 3-D response surface graphs and the 2-D contour plots were also generated by the software. These plots are very useful to see interaction effects of the factors on responses.

In vitro and in vivo Evaluation Parameters Studied for Part I and part II Formulation

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Tablet Thickness and Diameter

Tablet thickness and diameter were accurately measured by using digital vernier caliper in mm.32 Results were expressed as mean values ± standard deviations (SD).

Hardness and Friability

Hardness of tablet was determined by Monsanto hardness tester. Friability test was done by Roche friabilator. Ten tablets were weighed and were subjected to the combined effect of attrition and shock by utilizing a plastic chamber that revolve at 25 rpm dropping the tablets at distance of 6 inch with each revolution. Operated for 100 revolutions, the tablets were dusted and reweighed.The percentage friability was calculated.33

Weight Variation

Twenty tablets were selected at random and average weight was determined. Then individual tablets were compared with the average weight.33

Drug Content Uniformity

For determination of drug content, weighed and powder 5 tablets, then weighed accurately a quantity of the powder equivalent to 9mg of budesonide were transferred to the conical flask and suitably diluted with 10mL phosphate buffer (pH 7.4) respectively. The solution was filtered through Whatman filter paper (no.41), and assayed at 245nm, using a JASCO V630, Japan UV- spectrophotometer.

In vitro Dissolution Study

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respectively. 5 ml aliquots of the dissolution fluid were removed at specified time intervals and replaced with fresh dissolution medium and assayed for the amount of budesonide by spectrophotometer (JASCO V630, Japan) at wavelength 245 nm. The dissolution data was analyzed to calculate % drug released at different time intervals.

Fourier-Transform Infrared Spectroscopy (FTIR)

Drug–polymer interactions of part I and II were studied by FTIR spectroscopy. The spectra were recorded for pure drug, polymer and optimized formulations using FTIR spectrophotometer (Jasco FTIR-410). Samples were prepared by KBr disc method and the scanned over the range of 400–4000 cm–1, and the resolution was 2/cm.

Differential Scanning Calorimetry (DSC)

The possibility of any interaction between drug, polymers, and its mixture of part I and II was assessed by DSC (Mettler Toledo Stare DSC 822c, Germany). The thermogram of the samples were obtained at a scanning rate of 10°C/min conducted over a range of 0–300°C under an inert atmosphere flushed with nitrogen at a rate of 20 ml/min.

Stability Study

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In Vivo Study

Animals

Standard laboratory conditions of temperature 24 ± 2 °C, relative humidity 55 ± 5 % and 12:12 h light dark cycle were maintained throughout all the experiments. Rabbits had free access of water filtered through Aquaguardâ and fed with a standard diet ad libitum. The rabbits were allowed to acclimatize for 1 week before experiment. Rabbits fasted for 24h before administration of formulation.

In vivo X-ray Radio Imaging Study

Rabbits were fasted overnight before start of the study. In part I and II optimized formulation were prepared by replacing drug with barium sulphate and further coated with Eudragit L30D, were administered through intubation tube followed by flushing of 25–30 mL of water. During the entire study, the rabbits had free access to water only. X-ray photographs were taken at different time interval to verify the site specificity of formulation.36

In Vivo Pharmacokinetics Study

The in vivo study of the optimized budesonide tablet formulation (part I

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The plasma sample were separated by centrifugation, drug was extracted and then assayed for budesonide by HPLC.37

Data Analysis

Data were generated by assuming the first order absorption and one compartment model with first order elimination. The maximum peak concentration (C max) and time of its occurrence (T max) were directly computed from the plasma

concentration vs. time plot. The elimination rate constant (Kel) was determined from the terminal phase of the log plasma concentration vs. time profile by least square regression analysis. From this Kel is calculated as Kel=slope×2.303. The elimination half life was calculated as t 1/2 =0.693/Kel. The area under the plasma concentration

time curve from 0→t (AUC 0→t), and area under first moment curve from 0→t

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RESULT

PART I

Compatibility study of budesonide pure drug, excipients and its physical mixture was evaluated and passed as per standards. The melting point of budesonide was determined by using capillary method and was found to be 241-245°C (Standard 245-255°C) which complies with the reported value. The calibration curves of budesonide were measured in distilled water, 0.1N HCl and phosphate buffers of 6.8 and 7.4 pH which showed good linearity with the regression coefficient (R2) as 0.997, 0.998, 0.999 and 0.999 respectively. Budesonide was loaded with Crospovidone and tablets were effectively coated with successive layers of Eudragit L30D on preliminary trial basis. The process had an efficiency of ~90% and ~90–95% in polymeric coating.

Angle of repose of all preliminary trial batches was found to be in the range of 20-25º which show that granules exhibit good flow properties. Hausner ratio closer to 1 indicates good flow property and packing ability. Interpretation of budesonide compared with optimized formulation IR spectra showed no evidence of the interaction between the drug and the excipients. All the major characteristic peaks of the drug were present viz. C=O (1720), C=C (1664), C-H Aliphatic (2949), C-H Aromatic (3170) etc were seen in the subsequent spectra

Micromeritic properties of all optimization formulations were found to be in the range which shows that granules exhibit good flow property. Angle of repose of all optimized formulations was found to be in the range of 21-25º which indicate that granules exhibit good flow properties. Hausner ratio was found closer to 1 indicates good flow property and packing ability.

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IP limit should not be more than 1 % w/w. The weight uniformity of tablet in all formulation was observed to be within the IP limit 10 %. All formulations were complying with the official test.

The release from drug containing tablets at pH 6.8 was observed more than 90% release in less than 5 min at pH 6.8. % drug release versus time was plotted to study in vitro dissolution study. All the optimized formulations showed no drug release in first 2 hours at pH 1.2. Only the three formulations showed cumulative percent drug release at pH 7.4 not exceeding 10% upto 5h and release at pH 6.8 buffer were found to be attaining the release within 90 min. after lag time of 5h. Various kinetic models such as Zero order, First order, Higuchi Matrix, Korsmeyer & Peppas were applied to the all optimization batches and values of coefficient of determination indicate that the release of drug from the formulated dosage forms follows zero order release kinetic model.

Among the results obtained from dissolution studies of preliminary trials, batch with 15.29 mg Crospovidone and 25.39% Eudragit L30D weight gain was found as the desired batch as it showed the cumulative % drug release more than 90% within 90 minutes after a lag time of 5h, hence it was selected for factorial studies to optimize effects of variables on formulation. In order to determine the levels of factors which yield optimum dissolution responses, mathematical relationships were generated between dependent and independent variables.

The equations of the responses are given below:

Final Equation in Terms of Coded Factors:

Drug release = +93.52 -1.93 * A -1.39 * B +1.06 * AB -1.09 * A2 -5.12 * B2 (2)

Final Equation in Terms of Actual Factors:

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Final Equation in Terms of Coded Factors:

Lag time = +4.67 -0.33 * A +0.25 * B (4)

Final Equation in Terms of Actual Factors:

Lag time = +4.75000 -0.066667 *Crospovidone +0.050000 *Eudragit (5) The above equation 2 and 4 represents the quantitative effect of independent variables (X1 and X2) upon the responses (Y1 and Y2). Analysis of variance (ANOVA) indicated the assumed regression models were significant and valid for each considered responses. The three dimensional (3D) response surfaces and two dimensional (2D) contour plot were plotted to estimate the effect of independent variables on each response. A numerical optimization technique by desirability approach was used to generate the optimum settings for the formulation. The process was optimized for the dependent (responses) variables. The optimum formulation was selected based on the criteria of attaining the maximum value of lag time and % drug release. The optimized formulation was evaluated for lag time and percentage drug release within 90 min. after lag time. The Model F-value for responses, implies the model is significant. The linear correlation plots drawn between the predicted and actual (experimental) values for all the batches of the optimization formulation, which demonstrated high values of R2.

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Stability Study

From the result the optimized formulation showed drug content and in vitro percent drug release were found to be in acceptable limit. Drug content of optimized formulation was found to 99.52, 98.99, 98.76 and 98.58% at end of 0, 1, 2 and 3 months respectively. The in vitro release profile of formulation shows decrease in release of budesonide tablets in slim descending manner at ambient condition as 94.92, 93.98, 93.55 and 93.31% and at 40ºC / 75%RH as 94.92,93.63,93.42 and 92.81% at 0,1,2 and 3 months respectively.

In vivo Study

In vivo X-ray Radio Imaging Study

From the radiographic images it was demonstrated that barium sulphate containing optimized tablet formulation administered orally in rabbits remain intact in its structural integrity in stomach at 2h and but diffusion observed at end of small intestine 5h. Upto 2h X-ray images shows no diffusion of barium sulphate from coated tablet. But at the end of 5h study barium sulphate start diffusing from tablets. Till end of 7th hour X-ray image indicated complete diffusion of barium sulphate from tablet and believed to be released in ileo-cecal region.

In Vivo Pharmacokinetics Study

The blood samples collected from the albino rabbits showed pharmacokinetic parameters for the optimized formulation started to appear in the systemic circulation at 4.82±0.05 h, after administration and the Tmax was found to

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PART II

Compatibility study of budesonide pure drug, excipients and its physical mixture was evaluated and passed as per standards. The melting point of budesonide was determined by using capillary method and was found to be 241-245°C (Standard 245-255°C) which complies with the reported value. The analytical method development of the drug was done as reported. The solvent used for the drug determination was methanol AR grade. Beer-Lambert’s law was seen to be obeyed within the concentration range of 2-20 µg/ml. The calibration curves of budesonide were measured in methanol, 0.1N HCl and phosphate buffers of pH 6.8 and 7.4 which showed good linearity with the regression coefficient (R2) as 0.997, 0.998, 0.997and 0.996 respectively. Interpretation of Budesonide compared with optimized formulation IR spectra showed no evidence of the interaction between the drug and the excipients. All the major characteristic peaks of the drug were present viz. C=O (1720), C=C (1664), C-H Aliphatic (2949), C-H Aromatic (3170) etc were seen in the subsequent spectra

In this part budesonide was mixed with HPMC K4M and with outer coating with pH dependent Eudragit L30D. pH dependent Eudragit L30D with coating weight gain of 5, 10, 15, 20, 25 and 30% to protect drug release in upper part of GIT. Angle of repose of granules was found to be excellent flow properties. Hausner ratio closer to 1 indicates good flow property and packing ability. Eudragit S100 coated pellets with 50% weight gain shows lag time of 5 hours but drug release was retarded due to excess of polymer coat. Therefore it was concluded that drug release is inversely proportional to polymer weight gain this is attributed due to increase in diffusional path length with increase in % coating. Tablets containing 35 mg of HPMC K4M and Eudragit L30D 25% weight gain showed optimum lag time & maximum drug release selected for further study. Hence the above said weight gain were further selected for 32 full factorial design statistical optimization.

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Final Equation in Terms of Coded Factors:

Drug release = +92.97 +2.10 *A +2.09 *B +0.21 *AB +0.26 *A2 -3.68 *B2 (6)

Final Equation in Terms of Actual Factors:

Drug release = -4.09444 -0.52300 *HPMC K4M +7.49133 *Eudragit L30D

+0.010467 *HPMC K4M2 -0.14733 *Eudragit L30D2 (7)

Final Equation in Terms of Coded Factors:

Lag time = +4.72 +0.17 *A +0.33 *B (8)

Final Equation in Terms of Actual Factors:

Lag time = +1.88889 +0.03333 *HPMC K4M +0.066667 *Eudragit L30D (9) The above equation 6 and 8 represents the quantitative effect of independent variables upon the responses. The effect of two formulation factors indicates that increase in coating level of Eudragit L30D rises lag time significantly. It was observed from the response curves and contour plots for both responses that increasing level of coating of Eudragit L30D retard the water uptake and thus prolongs drug release time while increase in level of HPMC K4M due to formation of swelled and thick viscous layer helps in releasing drug slowly and in controlled manner.

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batch of tablets with the predicted levels was prepared and evaluated. The optimum tablet formulation developed shows excellent flow properties and all passed as per standard. In vitro dissolution study by pH change method same as used in part I showed release at 2, 5, 6, 8, 10, 12& 16 h was with controlled manner with lag time of 5 hours. A value of coefficient of determination indicates that release of drug from checkpoint batch follows zero order release kinetic model.

Differential Scanning Calorimetry (DSC)

The DSC thermogram shows a sharp endothermic peak at 281°C for budesonide. While in final optimum formulation containing drug and polymer, the endothermic peak was observed at 262.45°C which was as same as close to pure drug. Evaluation and interpretation of the thermogram revealed no interaction between the drug and polymer in the optimized formulation.

Stability Study

From the result the optimized formulation showed drug content and in vitro percent drug release were found to be in acceptable limit. Drug content of optimized formulation was found to 99.02, 98.83, 98.54 and 98.08% at end of 0, 1, 2 and 3 months respectively. The in vitro release profile of budesonide pellets formulation shows decrease in release 12h after lag time in slight downward behavior at ambient condition as 95.36, 94.57, 94.43 and 93.52% and at 40ºC/ 75%RH as 94.19, 94.06, 93.68 and 93.75% at 0,1,2 and 3 months respectively.

In vivo Study

In vivo X-ray Radio Imaging Study

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indicated complete diffusion of barium sulphate from tablet and believed to be released in proximal colon region.

In Vivo Pharmacokinetics Study

The blood samples collected from the albino rabbits showed pharmacokinetic parameters for the optimized formulation started to appear in the systemic circulation at 5.08±0.05 h, after administration and the Tmax was found to

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DISCUSSION

UC and CD are two features of IBD. They are recognized by chronic relapsing inflammation in the whole GI tract from mouth to anus, but are two distinct entities. Recently researchers have shown an increased interest in investigating the effect of different anti-inflammatory drugs used for the treatment of IBD. Hence budesonide a first line therapy drug for long term treatment of CD and for effective short term remedy to treat UC, was selected in this research work.

In part I formulation after budesonide mixed with crosspovidone in order to bring rupture of the outer functional Eudragit L30D coat. When crosspovidone comes in contact of aqueous medium in GIT get swelled due to absorbing water, creates pressure thus leads to rupturing of outer coat. It was observed that the process parameters and solution composition used in Eudragit L30D coating worked with good efficiency. A numerical optimization technique 32 factorial design by the desirability approach was used to generate the optimum settings for the formulation. The process was optimized for the dependent (responses) variables selected based on criteria of attaining the %drug release anf lag time. It was observed from the response curves and contour plots responses that increasing coating weight gain of Eudragit L30D retard the water uptake and rises lag time significantly. Increasing level of crosspovidone creats more pressure over outer Eudragit L30D coat due to its wicking and swelling ability of disintegrant is best utilized and thus releases drug immediately by rupturing the outer membrane. According to the design the best area for formulation to obtain desired responses was found. The result shows that the observed responses were inside the constraints and close to predicted responses, and, therefore, factorial design is valid for predicting the optimum formulation. Various checkpoint formulations were predicted by design expert software and one is selected according to high desirability feature and further evaluated for in vitro and in vivo parameters.

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numerical optimization technique by the desirability approach was used to generate the optimum settings for the formulation. The process was optimized for the dependent (responses) variables. The optimum formulation was selected based on the criteria of attaining the desired lag time and sustained release of drug after lag time. The effect of two formulation factors on lag time and indicates that increase in ratio of Eudragit L30D rises lag time significantly. Increasing level of HPMC K4M forms thick swelled gel layer hepl in retarding the drug release. The predicted and actual values of the optimization formulation given by the Design Expert software. Thus the low magnitudes of error as well as the values of R2 in the present investigation prove the high prognostic ability of the optimization technique. Various checkpoint formulations were predicted by design expert software according to high desirability feature.

Part I and II formulations IR spectra of drug, polymer and its mixture showed prominent peaks of the drug were not affected indicating no interaction was observed between the drug and excipients. DSC study validate that there is no interaction between drug and excipients and no change in physical nature of drug and polymers respectively. Micromeritic study conducted in part I and II preliminary, optimization and optimized checkpoint batches passed as per standard limit. In vitro drug release study of part I and II optimized batches exhibits that, despite poor water solubility of drug, resulted in increased dissolution rate of drug. A value of coefficient of determination of part I and II checkpoint optimized batches indicates that release of drug follows zero order release kinetic model. Stability study of part I and part II optimized formulation respectively, showed no degradation of the drug and also similar dissolution profile between control samples and the samples exposed to ambient and 400c / 75%RH as per ICH guidelines.

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in vitro release pattern. This drug delivery system may not perform in a similar manner in humans due to some physiologic differences between the species and therefore needs further investigation in humans.

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SUMMARY AND CONCLUSION

From the present study it was concluded that, the budesonide pH dependant burst release might be successful preference for ileo-cecal targeting by achieving the desired lag time to treat CD effectively. Also budesonide pH dependant sustain released optimized formulation could be best choice for colon targeted drug delivery by achieving the desired lag time. Satisfactory results were found from evaluation of micromeritic parameters such as flow property, in vitro

dissolution study and kinetic study.

Radio imaging study of optimized formulation also concluded that formulation was found to be stable to acid environment of stomach and in small intestine and reached to targeted site i.e. Part I formulation in ileo-cecal region and Part II formulation in proximal colon. Lag time and target release was observed by good correlation between in vitro and in vivo study. Pharmacokinetic study revealed that budesonide optimized formulations was effectively protected in upper GIT and releases drug in ileo caecal region and proximal colon respectively. Pharmacokinetic data shows that drug availability in systemic circulation was very less, so it confirms that budesonide formulation possess good topical anti-inflammatory activity.

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