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MATERIALS AND METHODS
6. MATERIALS AND METHODS
6.1. LIST OF CHEMICALS
1-Butanol (HPLC) : Merk, Mumbai, India
t- Butyl methyl ether (HPLC) : Merk, Mumbai, India
Acetic acid (HPLC) : Merk, Mumbai, India
Acetonitrile (HPLC) : Rankem Ltd., New Delhi, India
Anaesthetic ether (IP) : TKM Pharm. Ltd., Hyderabad, India
Calcium chloride (LR) : Central Drug House, Delhi, India
Chitosan (100cp) : CMFRI, Kochi, India
Clarithromycin USP : Ranbaxy Laboratories, Ltd. Gurgaon, India
Eudragit S100 : Degusa India Ltd., Mumbai, India
Heparin injection (IP) : Gland Pharma Ltd., Hyderabad, India
Hydrochloric acid (LR) : Merk, Mumbai, India
Isopropyl alcohol (LR) : Merk, Mumbai, India
Mehthanol (HPLC) : Merk, Mumbai, India
n-Hexane (HPLC) : Merk, Mumbai, India
Orthophosphoric acid (HPLC) : Rankem Ltd., New Delhi, India
Pantoprazole sodium sesquihydrate : M K Pharma (P) Ltd., Pune, India
Pectin (low methoxy) : Sigma Aldrich India Ltd., Delhi, India
Potassium dihydrogen phosphate (AR) : Rankem Ltd., New Delhi, India
Sodium acetate trihydrate (HPLC) : Rankem Ltd., New Delhi, India
Sodium alginate : Sigma Aldrich India Ltd., Delhi, India
Sodium chloride (AR) : Central Drug House, Delhi, India
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MATERIALS AND METHODS
Sodium hydroxide (AR) : Central Drug House, Delhi, India
Sulphuric acid (AR) : Merk, Mumbai, India
Trisodium citrate (LR) : Central Drug House, Delhi, India
Water (Type-I, Millipore® Water) : Millipore India (P) Ltd., Bangaluru, India
6.2. LIST OF EQUIPMENTS
Absorbant cotton : Pooja enterprises, New Delhi, india
Capillary tubes : Kalpatru Scientifics, New Delhi, India
Cooling centrifuge : Heal force, Neofuge15R, Shanghai, China
Cuvettes (Quartz) : Shimadzu, Japan
Deep freezer : Lektro lab equipment, Mumbai, India
Differential Scanning Colorimeter : DSC-60, Shimadzu, Japan
: DSC-6, Perkin Elmer, Japan
Digital balance : A and D Company Ltd, Japan
Eppendrof tubes (3 mL) : Kalpatru Scientifics, New Delhi, India
Filtration unit : Millipore, Bangalore, India
Glassware : Borosil Ltd., Mumbai, India
Gloves : Surgicare Pvt. Ltd., New Delhi, India
Heating plate : Icon Instruments, New Delhi, India
Hot air oven : Alcon, New Delhi, India
HPLC C18
column : Teknokroma, Spain
HPLC system : Waters, USA
Infra Red Spectrophotometer : JESCO-420, Japan
Laser light scattering : Mastersizer 2000, Malvern, UK
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MATERIALS AND METHODS
Lyophilizer : Allied Frost, New Delhi, India
Magnetic bead : Rama Scientific Works, New Delhi.
Magnetic stirrer : Remi Equipments Pvt. Ltd., Mumbai, India
Melting point apparatus : Scientific Systems, New Delhi, India
Micropipette disposable tips : Kalpatru scientifics, New Delhi, India
Micropipettes : Plastro crafts, Mumbai, India
Millipore filters (0.22 and 0.45 µm) : Millipore, Bangalore, India
pH meter : MicropH Analytical SDFCL, Mumbai
Refrigerator : LG electronics Ltd., India
Scanning Electron Microscope : Leo 435 VP, UK
Software (Factorial design) : DOE pack, PQ systems Inc., USA
Software (Statistics) : Microsoft XL, SigmaStat 3.5., USA
Syringe (2, 5, and 10 mL with 23G needle) : H S and Medical Device, Faridabad, India
Syringe filters (0.22 and 0.45µm) : Millipore India (P) Ltd., Bangaluru, India
Thermometer : GDP glass, India
U.V. Spectrophotometer : Hitachi, Japan
USP dissolution apparatus : Labindia (DS8000), Navi Mumbai, India
Vacuum desiccators : Poly lab, India
Vortex mixer : Nu Lab, India
Whatman no: 40# filter paper : Rathi Industries, Kanpur, India
X-ray diffractometer : Xpert PRO PANalytical, Netherlands.
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MATERIALS AND METHODS
6.3. ANIMALS
Wistar rats (Rattus norvegicus) of CDRI strain and albino rabbits (Oryctolagus
cuniculus) were bred in Animal House, Delhi Institute of Pharmaceutical Sciences and
Research (DIPSAR), New Delhi, India.
All animal experimentations were carried out as per the protocol number
(DIPSAR/IAEC/13/2009) approved by the „Institutional Animal Ethical Committee‟
(IAEC) formed as per the norms of Committee for Prevention, Control and Supervision
of Experiments on Animals (CPCSEA). The study was conducted in accordance with the
Declaration of Helsinki and “Animal Care and Facilities” in “Principles and Methods of
Toxicology” (Berger and Miller, 1989). All the animals were kept under identical
conditions at the Animal House, DIPSAR (Registration No. 215 CPCSEA 1 June 2000).
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MATERIALS AND METHODS
6.4. METHODS
6.4.1. Preformulation studies
6.4.1.1. Characterization of clarithromycin and pantoprazole
The clarithromycin (CL) powder USP obtained as a gift sample from Ranbaxy
Laboratories, Ltd. Gurgaon, India and the pantoprazole sodium sesquihydrate (PSS)
obtained as a gift sample from Murli Krishna Pharma (P) Ltd., Pune , Maharastra, India,
were characterized for the following parameters:
Physical appearance: Powder samples of CL and PSS were inspected visually to
check colour, appearance and other physical characteristics.
Solubility: Solubility of CL and PSS were studied in water and other organic
solvents.
Melting point: Melting points of CL and PSS samples were determined by
melting point apparatus (Scientific Systems, New Delhi) by capillary method.
UV analysis of clarithromycin: CL solution (100 µg mL-1
) in 1M H2SO4 was
scanned in UV spectrophotometer over wavelength range 200 to 400 nm to get
the wavelength of maximum absorption (λmax).
UV analysis of pantoprazole: PSS solution (100 µg mL-1
) in phosphate buffered
saline pH 7.4 was scanned in UV spectrophotometer over wavelength range 200–
400 nm to get the wavelength of maximum absorption (λmax).
Fourier Transform Infra Red (FTIR) analysis: An IR spectrum of the pure
drugs were obtained using KBr pellet technique and the peaks mentioned in
standards were compared with those obtained.
Differential Scanning Calorimetry (DSC): DSC analysis of pure CL was
performed on Perkin Elmer DSC 6 at IIT Delhi. DSC analysis of pure PSS was
performed on DSC- 60, Shimadzu.
X-ray diffraction (XRD) studies: XRD studies of pure drugs were carried out on
X-ray diffractometer (Xpert PRO PANalytical, Netherlands) at IIT Delhi.
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MATERIALS AND METHODS
6.4.2. Analysis of drugs and preparation of calibration curves
6.4.2.1. Analysis of clarithromycin by HPLC
HPLC method with a 210 nm PDA detector and 150 × 4.6 mm C18
Nuclosil
column was used for CL assay. Flow rate was about 1.0 mL min-1
, with column
temperature of 50oC and injection volume of 20 μL. Methanol and 67 mM monobasic
potassium phosphate (65:35) mixture was used as the mobile phase (adjusted to pH 4
with phosphoric acid) (Morgan et al., 1991; Chu et al., 1991; Rotsch et al., 1991; USP
26-NF 21, 2003). The method is validated for the performance characteristics of the
analytical method require accuracy, precision, specificity, detection limit, quantification
limit and linearity (USP 26-NF 21, 2003; Pav et al., 2002; Reiley and Fell, 1996).
Linearity: CL (50.0 mg) was weighed accurately and dissolved in 10 mL 0.1 M
sodium acetate buffer (pH 5) (5000 μg mL-1
stock solution). Six samples of
5-400 μL were taken from this stock solution and diluted to 1.0 mL with the
mobile phase (25-800 μg mL-1
solutions). CL peak responses of these samples
were determined. Regression equation and regression coefficients were calculated
(n=3).
Accuracy: It was calculated as the percentage of recovery by the assay of the
known amount of analyte which contained 50, 100 and 150 % active agent in the
sample, using the regression equation (n=6).
Precision: Three concentrations of CL solutions (25, 400, and 800 μg mL-1
) were
prepared using stock solution of CL. The peak responses of these samples were
measured. The relative standard deviation (coefficient of variation) of a series of
measurements was calculated. The same procedure was performed on consecutive
days (n=3).
Specificity: Method selectivity was assessed by the analysis of eight placebo
formulations at the same assay conditions. Sensitivity (detection and
quantification limits) the limit of detection is the lowest concentration of the
analyte which can be detected in a sample. Limit of quantification is the
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MATERIALS AND METHODS
parameter of quantitative assays for the low level compounds in sample matrices,
such as impurities in bulk drug substances and degradation products in finished
pharmaceuticals.
6.4.2.2. Preparation of calibration curve of PSS by UV spectrophotometry
Preparation of stock solution of PSS in phosphate buffered saline pH 7.4: Ten
milligrams of accurately weighed PSS was dissolved in sufficient quantity of
phosphate buffered saline pH 7.4, in a 10 mL standard flask and made up to the
volume. It produced a concentration of 10,000 µg mL-1
.
Preparation of calibration curve: From the above prepared stock solution 5mL
was taken and diluted to 100 mL with phosphate buffer pH 7.4 solution in the
volumetric flask. Various dilutions (5-40 µg mL-1
) were made from the stock and
the absorbance was measured at 290 nm wavelength using UV double beam
spectrophotometer.
6.4.3. Preparation of floating chitosan microbeads of CL
Chitosan microparticles containing CL were prepared by a capillary extrusion
procedure reported by Shiraishi et al., (1993) with slight modification. Briefly, CL was
dispersed in stirred solutions of 0.5, 1.0, and 2.0 % w/v chitosan in 2.0 % v/v acetic acid
until uniform dispersions were obtained. The drug to polymer ratios was fixed at 1:1, 1:2
and 1:3. In each formulation 300 mg CL was used. The microparticles were formed by
dropping the bubble free dispersion through a disposable syringe (23G) into 20 mL each
of gently agitated 0.5, 1.0, and 2.0 % solutions of the cross linking agent trisodium citrate
(TC), a poly electrolyte in de ionized water. The dropping rate was 30 drops per min. The
falling distance was 5 cm. The gelled microparticles were separated, after a reaction time
of 2 h, washed with de ionized water gently and transferred to round bottom flasks and
cooled to -28oC and then freeze dried at -60
oC in a freeze drier (Allied Frost, New
Delhi). Nine batches were prepared in triplicate. Placebo beads (without drug) were also
prepared.
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MATERIALS AND METHODS
Table 6.1. Formulations of clarithromycin loaded chitosan floating beads
Formulation
Code
Ratio of
CL:CH
TC
%
CLCH1 1:2 0.5
CLCH2 1:2 1.0
CLCH3 1:2 2.0
CLCH4 1:1 0.5
CLCH5 1:1 1.0
CLCH6 1:1 2.0
CLCH7 1:0.5 0.5
CLCH8 1:0.5 1.0
CLCH9 1:0.5 2.0
6.4.3.1. Product optimization of CLCH beads by factorial design
A 32 randomized full factorial design was used in development of the dosage
form. In this design, 2 factors were evaluated each at 3 levels and experimental trials
were performed using all possible 9 combinations. In the present investigation, the ratio
of clarithromycin (CL): chitosan (CH), as (X1) and percent of poly ionic cross linker
trisodium citrate used (TC) as (X2) were selected as independent variables. The
percentage drug released at 8 h as (Q8) was selected as dependent variable. For the
calculation of Q8, dissolution studies were carried out in a USP dissolution apparatus I. A
weight of floating microparticles equivalent to 250 mg of clarithromycin was placed in
the basket. Sodium acetate buffer (900 mL, 1M) at pH 5 (pH was adjusted with 0.1M
acetic acid) was used as dissolution medium, at a temperature of 37±0.5 °C with a stirring
speed of 50 rpm. Samples (5 mL) were withdrawn at time intervals, 0, 0.5, 1, 2, 4, 6, and
8 h. Fresh dissolution medium (5mL) was added to the flask after the withdrawal of each
samples. The samples were assayed by validated HPLC method. The percentage CL
released at 8 h were calculated (Q8).
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MATERIALS AND METHODS
The data was analysed in the DOE pack 3.0.22 software (PQ Systems Inc, USA)
for full factorial design. The polynomial equation was developed and which is reduced by
omitting the non significant interactive terms in the AONVA to explain the effect of
different independent variables on the drug release. The response / responses (Y ) is / are
measured for each trial and then either simple linear Y= b0 + b1X1 + b2X2 or interactive
Y= b0 + b1X1 + b2X2 + b12X1X2 + b11X1X1 + b22X2X2 or quadratic Y= b0 + b1X1 + b2X2 +
b12X1X2 + b11X12 + b22X2
2 model was fitted by carrying out multiple regression analysis
and F statistics to identify statistically significant terms. The reduced equation (an
equation containing only statistically significant terms) was then derived for drawing
response surface plots to visualize the impact of changing variables at a glance. The
effects and contour plots were also prepared. The optimum point was identified from the
plot and triplicate trials were run to verify the prediction of optimum formulation.
6.4.4. Preparation of Eudragit S100 enteric coated polymeric microbeads of PSS
The PSS loaded alginate formulations were prepared by ionotropic gelation
method described by Kikuchi et al (1997). Pantoprazole sodium was dissolved in
aqueous solution of sodium alginate. Drug to polymer ratios were maintained as 1:1, 1:2
and 1:3 (Table 6.2). In each formulation 100 mg PSS was used. PSS in aqueous sodium
alginate solutions were dropped into aqueous solution of calcium chloride (1 % w/v)
using a syringe fitted with 23 gauge needle, at a height of 10 cm. The medium was stirred
at a speed of 400 rpm using a magnetic stirrer. The gel beads formed due to ionotropic
gelation of alginate in presence of calcium ions were kept in the medium for 1 h. and
filtered using Whatman no: 40# filter paper. Recovered beads were washed with
demineralised water and dried at room temperature. After drying the beads were dip
coated in Eudragit S 100 solutions (1, 5 and 10 % w/v) in isopropyl alcohol. These coated
beads were dried at room temperature. Similarly the LM pectin (Table 6.3) and
combination of alginate LM pectin beads (Table 6.4) were also prepared and coated. In
pectin beads the drug and polymer ratios were fixed as in the case of alginate beads. The
polymer ratio was fixed as 1:1 in the case of alginate pectin combination beads.
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MATERIALS AND METHODS
Table 6.2. Formulations of PSS loaded Eudragit S100 coated sodium alginate beads
Formulation code
Ratio of
PSS:ALG
EUG coating
%
ALG1 1:3 1
ALG2 1:3 5
ALG3 1:3 10
ALG4 1:2 1
ALG5 1:2 5
ALG6 1:2 10
ALG7 1:1 1
ALG8 1:1 5
ALG9 1:1 10
Table 6.3. Formulations of PSS loaded Eudragit S100 coated LM pectin beads
Formulation
Code
Ratio of
PSS:PECT
EUG coating
%
PECT1 1:3 1
PECT2 1:3 5
PECT3 1:3 10
PECT4 1:2 1
PECT5 1:2 5
PECT6 1:2 10
PECT7 1:1 1
PECT8 1:1 5
PECT9 1:1 10
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Table 6.4. Formulations of PSS loaded Eudragit S100 coated sodium alginate and
LM pectin beads
Formulation
code
Ratio of
PSS:ALG&PECT
EUG coating
%
ALG-PECT1 1:3 1
ALG-PECT2 1:3 5
ALG-PECT3 1:3 10
ALG-PECT4 1:2 1
ALG-PECT5 1:2 5
ALG-PECT6 1:2 10
ALG-PECT7 1:1 1
ALG-PECT8 1:1 5
ALG-PECT9 1:1 10
6.4.4.1. Product optimization of PSS loaded Eudragit S100 coated sodium alginate,
LM pectin and combination of alginate and pectin beads using 32 full factorial
design
A 32 randomized full factorial design was used in development of the dosage
form. In this design, 2 factors were evaluated each at 3 levels and experimental trials
were performed using all possible 9 combinations. In the present investigation, the ratio
of pantoprazole sodium sesquihydrate (PSS): sodium alginate (SA), pectin, combination
of these two as (X1) and percent of Eudragit coating composition (EUG) as (X2) were
selected as independent variables. The percentage drug release at 8 h (Q8) for alginate
beads, for pectin beads and alginate pectin beads, were selected as dependent variables.
USP dissolution apparatus I was used for dissolution studies. A weight of
microbeads equivalent to 40 mg of PSS was placed in the basket. Phosphate buffered
saline pH 7.4 (900 mL, 1M) was selected as dissolution medium at a temperature of
37±0.5 °C with a stirring speed of 50 rpm. Samples (5 mL) were withdrawn at time
intervals, 0, 0.5, 1, 2, 4, 6, and 8 h. for alginate, pectin and alginate pectin combination
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MATERIALS AND METHODS
formulations. The samples were assayed by UV spectrophotometry at a λmax of 290 nm
and the percentage cumulative release of PSS was calculated (Q8).
The data was analysed in the DOE pack 3.0.22 software (PQ Systems Inc, USA)
for full factorial design. The polynomial equation was developed and which is reduced by
omitting the non significant interactive terms in the AONVA to explain the effect of
different independent variables on the drug release. The response / responses (Y ) is / are
measured for each trial and then either simple linear Y= b0 + b1X1 + b2X2 or interactive
Y= b0 + b1X1 + b2X2 + b12X1X2 + b11X1X1 + b22X2X2 or quadratic Y= b0 + b1X1 + b2X2 +
b12X1X2 + b11X12 + b22X2
2 model was fitted by carrying out multiple regression analysis
and F statistics to identify statistically significant terms. The reduced equation (an
equation containing only statistically significant terms) was then derived for drawing
response surface plots to visualize the impact of changing variables at a glance. The
effects and contour plots were also prepared. The optimum point was identified from the
plot and triplicate trials were run to verify the prediction of optimum formulation.
6.4.5. Drug excipients interaction studies
6.4.5.1. DSC Analysis
DSC analysis was performed by using DSC-6, Perkin Elmer, for clarithromycin,
chitosan, and trisodium citrate, alone and also the physical mixtures of these in
combinations were tested. The placebo as well as the drug loaded floating beads of
clarithromycin was also tested under same conditions. A quantity of 3 mg sample was
weighed and placed on the aluminum pan, which was then crimped. On the other side, an
empty crimped aluminum pan was placed as a reference standard. The sample was heated
between 40-400oC at the rate of 10
oC min
-1. Nitrogen gas was introduced at a pressure of
2 bars and a flow rate of 20 mL min-1
.
Similarly DSC analysis was performed for PSS and the various ingredients used
for the enteric coated PSS beads. The analysis was performed by using DSC-60,
Shimadzu. for PSS, sodium alginate, LM pectin, Eudragit S100, and calcium chloride
alone and also the physical mixture of these in combinations. The placebo as well as the
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MATERIALS AND METHODS
drug loaded (Eudragit coated and uncoated) beads of PSS with sodium alginate, LM
pectin, combination of sodium alginate and LM pectin were also tested under same
conditions. Data was analyzed by using TA-60 Collector ® software.
6.4.5.2. FTIR Analysis
FTIR spectra were obtained on JESCO-420, Japan. Spectra of samples were
obtained using the potassium bromide disc method. In each case, spectra in the region of
400 to 4000 cm-1
with a resolution of 4 cm-1
. Data was analyzed by Spectra Manager ®
software.
6.4.5.3. XRD analysis
The X-ray diffraction (XRD) patterns of PSS, ALG , PECT, and ALG-PECT
microbeads were recorded using a Xpert PRO PANalytical, diffractometer equipped
with Ni filtered Cu Ka radiation (k=1.5418A˚). Dried microbeads of uniform size were
mounted on a sample holder and the patterns were recorded in the 2θ range 10o
– 50o at
the speed of 5o/min to study the crystalline nature of the drug.
6.4.6. Entrapment efficiency or drug content
6.4.6.1. Entrapment efficiency in clarithromycin floating microbeads
An amount of floating microbeads, equivalent to 20 mg of Clarithromycin was
weighed and crushed in a mortar with a pestle. The crushed beads were stirred with 80
mL of methanol for 24 h, filtered and the volume was made up to 100 mL. The resultant
solution was assayed for CL content by validated HPLC method using Waters HPLC
system at λmax of 220 nm using a PDA detector.
6.4.6.2. Entrapment efficiency in Eudragit coated PPS microbeads
An amount of microbeads, equivalent to 20 mg of PSS was weighed and stirred
with 80 mL of 0.05 M NaOH for 24 h, filtered and the volume is made up to 100 mL.
The resultant solution was assayed for PSS content by spectrophotometric method at λmax
of 290 nm using Hitachi UV spectrophotometer.
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MATERIALS AND METHODS
The percentage drug contents of the entrapment efficiencies were determined using the
following equation
Weight of drug in the microbeads Drug Content (%) or entrapment efficiency = x 100
Theoretical loading
6.4.7. Scanning electron microscopy (SEM)
The morphology of the CL and PSS loaded microbeads were studied by scanning
electron microscopy Leo 435 VP which works in low and high vacuum mode and
equipped with digital imaging and 35 mm photography system). The microbeads were
coated for 120 sec with gold palladium under an argon atmosphere using a gold sputter
module in a high-vacuum evaporator. Coated samples were observed under scanning
electron microscope operated at an acceleration voltage of 10 kV. Cross sections of CL
microbeads were also prepared by cutting the freeze dried beads with a razor blade prior
to sputter coating
6.4.8. Micromeritics
The following micromeritic properties were determined as per the methods specified by
El-Gibaly et al., 2002; El-Kamel et al., 2001 and Sriamornsak et al., 2004.
6.4.8.1. Apparent particle density
The apparent particle density was determined by pyknometer (specific gravity bottle) by
water displacent method.
Apparent particle density=Mass of the particle/ apparent volume.
The apparent density of solid particles is the ratio of the mass density of solids to that
water. It is determined using the relation
M2-M1 ρ =
(M2-M1)- (M3-M4)
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MATERIALS AND METHODS
Where, M1 = mass of empty bottle, M2 = mass of the bottle and dry beads, M3 = mass of
bottle, beads and water, M4 = mass of bottle filled with water only. In this experiment 2 g
of microbeads were used.
6.4.8.2. Particle size
Particle size was measured by lasser diffraction method using Malveran mastersizer.
Malveran Mastersizer 2000 particle size analyzer which Measures materials size ranging
from 0.02 µm to 2000 µm. The microparticles were dispersed in 0.02 %of tween 80 and
introduced into the Malvern particle analyzer.
6.4.8.3. Particle size distribution
The particle size distribution also determined by Malveran Mastersizer 2000 particle size
analyzer which Measures materials size ranging from 0.02 µm to 2000 µm.
6.4.9. Buoyancy Test: (Ishak et al., 2007)
The floating ability of the CL beads was determined by USP dissolution apparatus
II. Fifty beads were introduced in to the vessels containing 900 mL 0.1 N HCl pH 1.2 and
rotate the paddles at 50 rpm, maintained at 37±0.5 °C. The floating ability of the beads
was measured by visual inspection and the percent of floating beads were calculated.
6.4.10. Measurement of in vitro drug release rate
The in vitro release studies were designed as per the method specified by El-Kamel et al.,
(2001)
6.4.10.1. Clarithromycin loaded floating chitosan microbeads: USP dissolution
apparatus I. was used for dissolution studies, a weight of floating microparticles
equivalent to 250 mg of clarithromycin was placed in the basket. Dissolution media
selected was 900 mL of 1M sodium acetate buffer at pH 5 (pH was adjusted with 0.1M
acetic acid), at a temperature 37±0.5 °C with a stirring speed of 50 rpm. Samples (5 mL)
were withdrawn at set time intervals, 0, 0.5, 1, 2, 4, 6, and 8 h. Fresh dissolution medium
(5mL) was added to the flask after the withdrawal of each samples. The samples were
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MATERIALS AND METHODS
assayed by validated HPLC method. The percentage cumulative release of clarithromycin
was calculated.
6.4.10.2. PSS loaded alginate, pectin, and combination of alginate and pectin beads
USP dissolution apparatus I was used for dissolution studies. A weight of
microparticles equivalent to 40 mg of pantoprazole was placed in the basket. Dissolution
media selected was 900 mL of phosphate buffered saline pH 7.4, at a temperature 37±0.5
°C with a stirring speed of 50 rpm. Samples (5 mL) were withdrawn at time intervals, 0,
0.5, 1, 2, 4, 6, 8, 12, 18, and 24 h for alginate formulations, 0, 0.5, 1, 2, 4, 6, and 8 h. for
pectin formulations and 0, 0.5, 1, 2, 4, 6, 8, and 12 h for alginate and pectin combination
formulations. The samples were assayed by validated UV method and the percentage
cumulative release of PSS was calculated for individual formulations.
6.4.11. Drug Release Kinetics
The clarithromycin and PSS release kinetics were studied by using different
imperical models such as zero, first order kinetics, Higuchi model kinetics and
Koarsmeyer-Peppas models.
6.4.12. Long Term Stability Studies
As Per ICH Guidelines under quality guideline number Q1A (R2) long term
stability studies were carried out at 30 °C ± 2 °C/65 % RH ± 5 % RH for 1 year. Samples
were withdrawn and assayed at every 3 months at an interval of 0, 3, 6 and 12 months.
The degradation constant during this period was determined.
6.4.13. In situ evaluation and comparative estimation of drug in the gastric
mucosa and blood of Wistar rats
The in situ evaluation and comparative estimation of drug in the gastric
mucosa and blood of Wistar rats were carried out as per the method reported
by Murata et al., (2000). This experimental protocol has been submitted for
approval by the Ethical Committee for animal experiments of DIPSAR.
All animal experimentations were carried out as per the protocol number
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MATERIALS AND METHODS
(DIPSAR/IAEC/13/2009) approved by the Institutional Animal Ethical Committee
(IAEC) formed as per the norms of Committee for Prevention, Control and
Supervision of Experiments on Animals (CPCSEA).
Wistar rats (72) of either sex weighing between 200-300 g were divided into 4
groups of 18 animals each. Animals were fasted for 24 h but water has been given ad
libitum. The following formulations were administered to group 1- 4 respectively using
oral tubing (oral catheter).
Group I : Clarithromycin suspension in 2 % CMC
Group II: Floating Micro beads of Clarithromycin
Group III: Floating beads of CL and Eudragit coated micro beads of PPI
Group IV: Control group (2 % CMC)
Animal dose: Clarithromycin (58.3 mg kg-1
)
: PPI - Pantoprazole (9.3 mg kg-1
)
At the end of 2 h, six animals from each group were anesthetized using
anaesthetic ether and the abdominal skin was opened. Blood smples (2 mL) were
collected from vena cava caudalis leading to death. The blood samples were centrifuged
in a refrigerated ultra centrifuge at 3600 rpm for 10 min. The serum samples were
deproteinized by adding methanol, followed by centrifugation. The supernatant was
removed and the clarithromycin concentration was estimated using validated HPLC
method (Amini and Ahmadiani 2005).
The rat stomach was removed, gently rinsed with Sorensen buffer (pH 7.4) three
times and spread on a glass plate. The mucosal surface was scraped gently with a glass
slide and the top layers separated from the muscular layers .The removed mucosa were
mixed with methanol in a glass tissue grinder. After being ground, the homogenate were
centrifuged in a refrigerated ultracentrifuge at 3000 rpm for 10 min. The supernatant was
removed and filtered through a 0.22 µm filter. The amount of clarithromycin contained in
the sample was determined using validated HPLC method (Noubarani et al., 2010).
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6.4.14. Pharmacokinetic studies: (Jain et al., 2005)
The in vivo studies were conducted in healthy albino rabbits of either sex or
weighing 2.2–2.5 kg. Rabbits were kept for one week in animal house to acclimatize
them and were provided with fixed standard diet. Rabbits (12) were divided into two
groups of six each and were fasted for 24 h. One group was fed with microbeads of
clarithromycin and the second group was administered with microbeads of PSS. At the
dose levels of clarithromycin (41.6 mg kg-1
) and proton pump inhibitor (6.6 mg kg-1
).The
rabbits were anaesthetized during or prior to the treatment and were administered the
formulation with an oral cannula. Blood samples (2 mL) were collected from the
marginal ear vein into heparinized centrifuge tubes just before dosing and at 0.5, 1, 1.5, 2,
3, 4, 5, 6, 8, 10, 12 and 24 and 48 h during the study. The blood samples were transferred
to a series of graduated centrifuge tubes contains heparin. The samples were centrifuged
at 2500 rpm for 5 min. The plasma samples were transferred into another set of sample
tubes and to be frozen until assayed. One drug free plasma sample was taken as blank.
The samples were filtered through 0.22 micron membrane filter (Millipore). The drug
concentrations in blood samples are to be analyzed using the validated HPLC method.
The following non compartment pharmacokinetic parameters were calculated.
Peak plasma concentration (Cmax)
Time to reach peak plasma concentration (Tmax)
Area under the curve (AUCo-∞)
6.4.15. HPLC analysis of blood samples
6.4.15.1. HPLC analysis of blood samples of clarithromycin formulations
Calibration standard for clarithromycin was prepared in with rabbit/rat plasma by
spiking a pool of plasma to a known concentration and then serially diluting it with blank
plasma to attain the desired concentration range (31.25–2000 ng mL-1
). The
concentrations of individual standards were 31.25, 62.5, 125, 250, 500, 1000 and 2000 ng
mL-1
. Extraction was performed by adding 20 µL of 1M NaOH and and 2.5 mL of n-
hexane/1-butanol (98:2, v/v) to 1 mL of plasma in 3 mL ependroff tube and shaking for 2
105
MATERIALS AND METHODS
min. After centrifugation at 12 000 rpm for 3 min, the whole organic layer was separated
and transferred into another 4 mL tube. Then, 50 µL of 0.1 % acetic acid was added. The
mixture was vortex-mixed for 2 min and then, some of the upper organic phase was
discarded and the remaining mixture (1 mL) was transferred into a 1.5 mL micro-
centrifuge tube. After centrifugation at 11 300 rpm for 2 min, the upper organic phase
was discarded completely. Finally, a volume of 40 µL of aqueous phase was injected into
the chromatograph (Amini and Ahmadiani 2005).
6.4.15.2. HPLC analysis of blood samples of pantoprazole formulations
Plasma samples (1 mL) were transferred to a 15 mL glass tube, and then 4 mL
aliquot of extraction solvent, 3o
butyl methyl ether was added. The sample was vortexed
for 3 min. The sample was then centrifuged for 3 min at 3000 rpm. The organic layer (3
mL) was quantitatively transferred to a 6 mL glass tube and evaporated at 37ºC Then, the
dried extract was reconstituted with 200 μL of mobile phase and a 50 μL aliquot was
injected into chromatographic system. To 950 μL of blank plasma, 50 μL of working
standard of PPS was added, yielding final concentrations of 20-4000 ng mL-1
PPS.
Calibration samples were prepared for analysis as described above. Each calibration
curve was analyzed three times with at least six different concentrations using the same
HPLC conditions as described above (Noubarani et al., 2010).
6.4.16. Statistical analysis
Analysis of variance (Kruskal-Wallis One-way ANOVA) along with multiple
comparison test (Student-Newman-Keuls Method) and t-test (for two samples) were
employed by SigmaStat® 3.5 software at P< 0.05. Student-Newman-Keuls method was
used as the post hoc test.
In factorial design one way analysis of variance followed by regression analysis
including quadratic and interactions were employed by DOE pack for Windows® 3.0.22
software at P<0.05.