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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.
2014
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.
2014
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,
NO. 1/161, Sankaralinganur Road,
Gerugambakkam, Chennai-600122.
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,
Dharmapuri - 635205.
1
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
Colonic delivery is considered better than other dosage form like rectal
delivery (suppositories and enemas) due to their lack of efficacy and a high
variability in distribution of drugs. Suppositories are effective only in rectum due to
2
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
factors such as diet, food intake, intestinal motility and disease states. The pH
gradient in GIT range from 1.2 in the stomach, 6.6 in the proximal small intestine to
a peak of about 7.5 in the distal small intestine.10 Drug release also depends upon
presence of intestinal enzymes that are derived from gut microflora residing in high
numbers in the colon. These enzymes are used to degrade coatings/matrices as well
as to break bonds between an inert carrier and an active agent resulting in the drug
release from the formulation.11 Gastric emptying time of formulation is variable and
primarily depends on fed or fasted condition of subject and on the characteristics of the
dosage form such as size and density. Colonic transit times (estimated from the
difference in mouth to caecum and whole gut transit times) ranged from 50 to 70 h. 12
Colon diseases
Inflammation is a central feature of many chronic diseases like IBD,
dermatitis, rheumatoid arthritis etc. IBD is an idiopathic inflammatory disorder
3
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
To achieve successful colonic delivery through oral route, the formulation
must prevent drug release in the stomach and small intestine but allow release after
their arrival in the colon. Although the concept looks quite simple, this is difficult to
achieve in practice as the colon is the most distal segment of the GI tract. CTDDS
4
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.
Pressure controlled drug delivery is novel delivery mechanism utilized to
initiate the release of the drug in the distal part of the gut. The muscular contractions
of the gut wall generate pressure, which is responsible for grinding and propulsion
of the intestinal contents. Pressure sensitive drug formulations release the drug as
5
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
Mesalamine group of drugs are first line therapy treatment for patients with
mild to moderate IBD, which include oral and rectal formulations of mesalamine
and oral pro-drugs like sulfasalazine, olsalazine and balsalazide are major drugs for
maintaining remission. These are capable of preventing the production of
prostaglandins and leukotrienes, thus preventing neutrophil chemotaxis. Doses of
1500–2400 mg per day of oral mesalamine will be effective in most patients, dose
escalation to between 3000 to 4800 mg per day or use of these higher doses initially
can result in an increase in absolute response rates of about 10%.25, 26
6
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
Some IBD treatment regimens are attempted to control the immune system,
thus affecting the extent and duration of disease. One very important compound in
this class is azathioprine, which is a thioguanine derivative with the active
metabolite being 6-mercaptopurine (6-MP). It is a highly successful drug, as two
thirds of IBD patients enter remission following treatment.29, 30
7
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.
CD can occur in any part of the GI tract from mouth to anus but
inflammation is mainly localized in the more distal regions of the small intestine and
starts of large intestine i.e. the ileo caecal region. So we tried to improve site
specificity of budesonide in relation to target ileo caecal region to treat CD through
incorporation of Crospovidone to achieve burst release in ileo caecal region with
drug and outer coated with Eudragit L30D to achieve lag time of 5 h. UC most often
affects a continuous segment of colon ranging from a limited short segment to
affecting the entire colon. In this formulation we studied with external coat of
8
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
8. To perform in vivo animal studies to perform site specificity by X-
ray imaging and establish pharmacokinetic parameters.
9
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
by X-ray radio imaging and establish pharmacokinetic parameters of
the formulation in rabbits.
10
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
The granules were prepared by wet granulation method. The drug
budesonide, crospovidone 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
evaluated for micromeritic properties such as bulk density, tapped density, angle of
repose and hausner ratio.
11
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
evaluated for micromeritic properties such as bulk density, tapped density, angle of
repose and hausner ratio.
12
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 Data for 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.
The effects of independent variables upon the responses were modeled
using a second order polynomial equation. The mathematical model of the effects of
independent variables upon the dependent variables was performed using Design
Expert® software (Design Expert trial version 9.0.3.1 for part I and part II; State-
13
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+ b22X2
2 (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, X2
2) 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
Preliminary batches and optimized budesonide tablet formulations were
subjected to evaluation for following micromeritic properties like angle of repose,
bulk density, tapped density and hausner ratio to ensure flow property of granules.
14
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
The test was carried out in a rotating basket method specified in the USP
XXIII dissolution tester (Electrolab, TDT-08L, India) at a rotation speed of 100 rpm
in 900 ml dissolution medium at 37 ± 0.5 °C in media with pH 1.2 (HCl 0.1 N),
pH 7.4 and pH 6.8 (phosphate buffer) for 2 h, 3 h, and till the end of the test,
15
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
Stability Study was carried out for part I and II formulations to assess its
stability, as per ICH guidelines. The optimized formulation were wrapped in the
laminated aluminum foils and was placed in the accelerated stability chamber
(6CHM-GMP, Remi Instrument Ltd., Mumbai) at elevated temperature and
humidity conditions of 400C/ 75% RH and a control sample was placed at an
ambient condition for a period of three months. Sampling was done at a
predetermined time of initial 0, 1, 2 and 3 months interval respectively. At the end of
study, samples were analyzed for the drug content, in vitro drug release and other
physicochemical parameters.34, 35
16
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
and part II) compared with marketed formulation for promising ileo caecal targeting
and colonic delivery respectively. Six male albino rabbits weighing approximately
1.5 kg and with the age of 12 months were selected for the part I and II study
respectively. The rabbits were divided into two groups of three in part I and three
animals for part II study. Each group was subjected to a single dose randomized
parallel design study. The animals were housed individually under environmental
conditions (23 ± 2 °C, 55 ± 5 % relative humidity, 12 hour light / dark cycle). The
rabbits were fasted overnight and allow free access to tap water only. The test
sample of optimized and the marketed formulation for part I and part II formulation
were administered to the rabbits by gastric intubation method, 0.5 ml of blood
sample were withdrawn from the marginal vein of rabbit at various time interval.
17
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
(AUMC 0→t) and mean residence time (MRT) were calculated using trapezoidal rule.
18
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.
There is no variation in tablet thickness and diameter between the
formulations. The hardness of the tablet were within the range and optimum for
controlled release, and ranging from 7.0-7.9 Kg/cm2 for all formulations. The
friability of all formulations was ranging from 0.095-0.237 % w/w and passes as per
19
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:
Drug release = -15.82889 +0.28950 *Crospovidone +9.10467 *Fudragit -0.042500 *Crospovidone *Eudragit -0.043467 *Crospovidone2 -0.20467*Eudragit2 (3)
20
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.
Twelve checkpoint formulations were found by software and the best
conditions to optimize drug release and because of it’s high desirability checkpoint
formulation with 15.29 mg Crospovidone and 25.39% Eudragit L30D weight gain
was selected to ensure the validity of the optimization procedure, checkpoint
formulation 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 drug release study shows lag time of 5h, and showed
burst release up to 94.51% release at pH 6.8 within 90 min. after lag time. A value of
coefficient of determination indicates that release of drug from checkpoint batch
follows zero order release kinetic model.
21
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
be 6.03±0.02 h, where the Cmax was observed 5.83±0.39 ng/mL.
22
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.
In order to determine the levels of factors which yield optimum dissolution
responses, mathematical relationships were generated between the dependent and
independent variables. The equations of the responses are given below:
23
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.
The linear correlation plots drawn between the predicted and actual
(experimental) values for all the batches of the optimized formulation, which
demonstrated high values of R2. 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. Eight checkpoint formulations were generated by software,
out of which the best conditions to optimize drug release and because of it’s high
desirability formulation with 39.76 mg of HPMC K4M and 26.78% weight gain of
Eudragit L30D. In order to check the validity of the optimization procedure, a new
24
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
From the radiographic images it was proved that barium sulphate
containing formulation administered orally in rabbits by same process applied in part
I. Up to 4h study X-ray images shows no diffusion of barium sulphate from tablets.
Barium sulphate starts diffusing from pellets from 5th h till end of 15thh. X-ray image
25
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
be 10.23±0.08 h, where the Cmax was observed 6.49±0.46 ng/mL.
26
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.
In part II formulation after budesonide mixed with HPMC K4M and
analyzed for weight gain in order to retard the drug release. Eudragit L30D coating
worked with good efficiency to avoid drug release in upper GIT as lag time of 5h. A
27
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.
The results for part I in vivo radio imaging study in rabbit conclusively
demonstrated complete release and diffusion of the delivery system within 7h.
Although we observed a 4.5h lag time in the in vitro as well in vivo radio imaging
studies in rabbits. Thus in vivo study confirms the finding of in vitro study. Part II
optimized formulation revealed complete diffusion of barium sulphate from the
delivery system at the end of 18h study images, which was close to same as that of
28
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.
Pharmacokinetic study for part I and part II budesonide formulation
showed that the drug was released only after 5 h indicating that the optimized
coating parameters has a capability of preventing the drug release in the stomach and
small intestine, which confirms that the formulation have ability to target drug
release in ileo caecal region and in proximal colon to treat IBD effectively.
29
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.
Thus, the designed formulations can be considered as one of the promising
formulation technique for ileo-cecal targeted pulsatile drug delivery of budesonide
to treat CD and budesonide for proximal colon targeting in management UC
respectively and for other colon inflammatory diseases.
30
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