6. materials and methods - inflibnetshodhganga.inflibnet.ac.in/bitstream/10603/9059/12/12...92...

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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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(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


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.

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