Chapter 5 Experimental
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Chapter 5
Development and
Evaluation of formulation
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Experimental Work
Various materials and equipments were used to carry out the experimental work. The list of
materials and equipments used in formulation is given below
Table 5.1: List of drug and excipient used in the formulation development
Sr.
No.
Ingredients Spec. Brand name Manufacturer Function
1 Gliclazide Ph. Eur. NA Bal Pharma Ltd. Drug
2 Lactose monohydrate Ph. Eur. Supertab 30 DMV Fonterra Diluent
3 Diclacium phosphate Ph. Eur. A-Tab granular Innophos, USA Diluent
4 Microcrystalline
Cellulose
Ph. Eur. Avicel PH 101
& 102
FMC
Biopolymer
Diluent
5 Maltodextrin Ph. Eur. Glucidex IT 12 Roquette, France Diluent
6 Hydroxypropylmethyl
Cellulose
Ph. Eur. HPMC K 100
LV
DOW
Chemicals
Polymer
7 Hydroxypropylmethyl
Cellulose
Ph. Eur. HPMC K 4 M DOW
Chemicals
Polymer
8 Polyethylene oxide Ph. Eur. Polyox WSR
coagulant
DOW
Chemicals
Polymer
9 Xanthan gum Ph. Eur. Xanthural 75 C P Kelco Polymer
10 Co-polymer of
Polyvinyl alcohol and
Povidone
Int. spec. Kollidone SR
BASF Polymer
11 Ethyl cellulose 7 cps
& 20 cps
Ph. Eur. Ethocel DOW
Chemicals
Polymer
12 Hydroxypropyl
cellulose
Ph. Eur. Klucel LF Hercules
Polymer
13 Light magnesium
Carbonate
Ph. Eur. NA Dead sea, Israel
Alkaliser
14 Magnesium stearate Ph. Eur. NA Ferro, Spain Lubricant
15 Colloidal anhydrous
Silica
Ph. Eur. Aerosil 200 Degussa
Glidant
16 Hydrogenated
castor oil
Ph. Eur. Cutina HR PH Cognis Release
modifier
17 Stearic acid Ph. Eur. Stearin Stearinerie
Dubois Fils
Release
modifier
18 Polyethylene glycol
8000 (Macrogol)
Ph. Eur. NA DOW chemicals
Release
modifier
19 Aminomethacrylate
Copolymer
Int. spec. Eudragit RSPO Evonik
Release
modifier
20 Polyacrylate 30%
Dispersion
Int. spec. Eudragit N 30 D
Evonik
Release
modifier
21 Talc Ph. Eur. Luzenac Rio Tinto, Italy Lubricant
22 Crospovidone Ph. Eur. Kollidone CR BASF Disintegrant
23 Sodium Alginate Ph. Eur. Keltone HVCR FMC
Biopolymer
Polymer
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List of Equipment used for the formulation development
All equipments in the development lab. are cleaned after use as per the SOP for cleaning.
This ensures the proper handling and safety of the operating personnel. All the instruments
are calibrated at a pre-determined frequency based on the usage and sensitivity of the
operations. pH meters and balances are compulsorily calibrated on daily basis. Standard pH
buffers are also renewed every week and if there is any odd observation then also fresh
solutions are prepared. All the instruments and machines are covered by annual maintenance
contract (AMC) so preventive and breakdown maintenance is assured.
Table 5.2: List of equipments used in the development of gliclazide ER tablets
Sr. No. Equipment name Manufacturer Function
1 Microbalance Metteler Toledo Weighing
2 Container blender R.P Products Blending
3 Tablet compression machine Cadmach
Machinery
Tablet press
4 Hardness tester Dr. Schlevniger Testing hardness
5 Roche friabilator Electrolab Testing friability
6 Electromagnetic sieve shaker
(Vibrosifter)
Pharma Fab
Engg.
Sieve analysis
7 Bulk density tester Electrolab Density of blend
8 Vernier caliper Miyototo, Japan Measurement
9 Multi mill Gansons Milling
10 Induction cap sealing
Machine
Electronic
devices
Induction sealing
11 Moisture balance Sartorious Moisture content
12 Extruder and Spheronizer Omung pharma
Tech.
For pellets
13 Retchz Dryer Retchz Drying
Table 5.3: List of analytical equipments used in the analysis
Sr. No. Equipment name Manufacturer Function
1 Microbalance Metteler Toledo Weighing
2 Digital pH meter Thermolab Determine pH
3 Dissolution apparatus Labindia
instruments
Dissolution testing
4 HPLC apparatus Waters Ltd. HPLC Analysis
5 UV-Visible
spectrophotometer
ID-1700
Shimadzu Spectrophotometric
Analysis
6 Microscope Nikon Microscopy
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IIG limits for excipients used in the formulation
Materials have been used within the specified (CDER USFDA) IIG limits ensuring the human
safety levels as already approved by regulatory authorities.
The inactive database provides information on inactive ingredients present in FDA approved
drug products. This information can be used by industry as an aid in developing drug
products. For example if a particular inactive ingredient has been approved in a certain dosage
form at certain potency, a sponsor could consider it safe for use in a similar manner for a
similar type of product.
Table 5.4: IIG limits for excipients in the development of gliclazide ER tablets
Sr. No. Excipients IIG limits (mg)
1 Lactose Monohydrate 427.26
2 Dibasic calcium phosphate 850.00
3 HPMC K 100 LV 480.00
4 HPMC K 4 M 480.00
5 Maltodextrin 292.00
6 Xanthan gum 109.50
7 Polyethylene oxide 543.90
8 Kollidone SR 122.58
9 Light magnesium carbonate 175.00
10 Colloidal anhydrous silica 56.80
11 Magnesium stearate 256.40
The IIG limits mentioned are the maximum quantity of excipients reported to be safe in any
oral formulation.
5.1 Analytical method validation
Analytical method development and validation play an important role in the discovery,
development and manufacture of pharmaceuticals. A well developed method should be easy
to validate and should be developed with the aim to rapidly test preclinical samples,
formulation prototypes and commercial samples. The input obtained during the process of
method validation is applied for the improvement and fine tuning of the method.
In case of newly developed dosage forms, analytical method development may require
development of totally new method. Sometimes, an already existing official pharmacopeial
method of analysis for the API or another dosage form of the same API already outlined in
the pharmacopeia needs to be modified suitably to get acceptable analytical parameters.
Excipient interference is extremely critical to deal with and the method selected should be
able to distinguish between the drug and excipient and be able to selectively analyse the drug.
The method selected should be robust to support both analysis as well as stability studies and
should be stability indicating.
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In the present study, selected drug gliclazide and its formulation is official in European
pharmacopeia and Indian Pharmacopeia respectively, the analytical method is given in the
pharmacopeia. The given method was verified and modified suitably for estimation of the
formulations made by different technologies. It was further partially validated. UV
spectrophotometry was investigated for dissolution profile and Assay and HPLC method for
the related substances studies.
5.1.1 Analytical method verification of gliclazide
5.1.2 Methods
5.1.2.1 Calibration curve of gliclazide in methanol
30 mg of gliclazide was dissolved in methanol and volume was made up to 100 ml. 5 ml
of this solution was further diluted to 100 ml with methanol. This diluted solution was
scanned from 200 – 400 nm and UV spectrum was recorded. Figure 6.1 represents the UV
spectrum of gliclazide in Methanol.
5.1.2.2 Calibration curve of gliclazide in distilled water
30 mg of gliclazide was dissolved in 10 ml of methanol, the solution was further diluted upto
100 ml with distilled water. 5 ml of this solution was further diluted to 100 ml with distilled
water. This diluted solution was scanned from 200 -400 nm and UV spectrum was recorded.
figure 6.2 represents the UV spectrum of gliclazide in distilled distilled water.
5.1.3 Method Validation
The UV method was validated for linearity, accuracy or recovery, precision, intermediate
precision and stability of analytical solution.
5.1.3.1 Range and Linearity
Range of a method is the concentration interval over which the method is precise, accurate
and linear. Linearity of a method is defined as the proportionality of measured value to
concentration. It determines if the method is able to measure the concentration of an analyte
in a directly proportional ratio with a proportional increase in concentration.
Preparation of stock and standard solution
Stock solution of gliclazide was prepared by dissolving 30 mg of gliclazide in 10 ml methanol
and the volume was made up to 100 ml. The stock solution was diluted with water to obtain
the final working standards having concentration of 2.5, 5.0, 7.5, 10, 12.5, 15 and 17.5 µg/ml.
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Calibration curve of gliclazide was prepared using the working standard solutions in the range
of 2.5 – 17.5 µg/ml. The linearity curves of gliclazide were prepared at 226 nm and 290 nm as
reported maxima (λmax) and as observed in the spectrum. Each curve was fitted by linear
regression analysis and r2 was calculated. Results of the linearity curve of gliclazide in water
is given study are given.
5.1.3.2 Accuracy
Accuracy is defined as the closeness between the measured and the real value.
15 mg gliclazide API was dissolved in 10 ml of methanol and finally diluted with water, 5 ml
of this solution was further diluted upto 100 ml. This solution was spiked with 50% (7.5 mg),
100% (15 mg) and 150% (22.5 mg) of gliclazide. The drug content was determined by VU
spectrophotometric method and the % recovery calculated.
5.1.3.3 Precision
The precision of an analytical method describes the closeness of individual measures of an
analyte when the procedure is applied repeatedly to multiple aliquots of a single homogenous
volume of matrix.
30 mg gliclazide API was dissolved in 10 ml of methanol and volume was made up to 100 ml, 5
ml of this solution was further diluted to 100 ml, to obtain a stock solution of 15 ppm. The drug
content was determined by measurement on UV spectrophotometer and the % RSD was
calculated.
5.1.3.4 Stability of analytical solution
Stability study of analytical solution of gliclazide was performed at room temperature.
30 mg drug was dissolved in10 ml of methanol and further diluted with water. 5 ml of this
solution was diluted further with water upto 100 ml and initial drug content was determined.
The solution was kept at room temperature for 8 and 24 hrs. Drug content after 8 and 24 hrs.
was determined by UV spectrophotometer and solution was scanned for any degradation
peaks. All measurements were performed on six determinations on three independent
samples.
5.1.4 Analysis of gliclazide extended release tablet formulations
Estimation of gliclazide in extended release tablets for dissolution profile by UV
spectrophotometry. In-vitro dissolution was performed in pH 6.8 phosphate buffer, pH 4.5
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acetate buffer and pH 7.4 phosphate buffer, standard curve were prepared in all the
dissolution media.
5.1.4.1 Partial validation of analytical method for analysis of gliclazide extended release
tablets
5.1.4.2 Precision
Six determinations were carried out on 20 tablets of gliclazide ER tablets. Drug content was
determined by crushing 20 tablets and taking the blend equivalent to 30 mg and further
sonicating in 100 ml of water and diluting 5 ml of the solution to 100 ml with water and
taking the reading.
5.1.4.3 Estimation of gliclazide in extended release tablets for in-vitro dissolution profile
by UV spectrophotometry Method
Samples obtained from in–vitro analysis were diluted as 5 ml sample to 10 ml and making up
the volume with the buffer solution. This diluted sample was measured on UV
spectrophotometer. The equation obtained in standard curve was applied for calculations.
5.1.4.4 Preparation of standard curve in pH 6.8 phosphate buffer
Standard curve was prepared using UV spectrophotometer. 30 mg of gliclazide was dissolved
in 10 ml of methanol in 100 ml volumetric flask and the volume was made up to the mark
with the buffer solution. From this stock solution 5 ml was further diluted with pH 6.8
phosphate buffer to 100 ml.
5.1.4.5 Preparation of standard curve in pH 7.4 phosphate buffer
Standard curve was prepared using UV spectrophotometer. 30 mg of gliclazide was dissolved
in 10 ml of methanol in 100 ml volumetric flask and the volume was made up to the mark
with the buffer solution. From this stock solution 5 ml was further diluted with pH 7.4
phosphate buffer to 100 ml.
5.1.4.6 Preparation of standard curve in pH 4.5 acetate buffer
Standard curve was prepared using UV spectrophotometer. 30 mg of gliclazide was dissolved
in 10 ml of methanol in 100 ml volumetric flask and the volume was made up to the mark
with the buffer solution. From this stock solution 5 ml was further diluted with pH 4.5 acetate
buffer to 100 ml.
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5.1.4.7 Preparation of stock and standard solutions with pH 6.8 phosphate buffer
Stock solution of gliclazide was prepared by dissolving 30 mg of gliclazide in 10 ml methanol
and the volume was made up with pH 6.8 phosphate buffer to 100 ml. The stock solution was
diluted with pH 6.8 phosphate buffer to obtain the final working standards having
concentration of 2.5, 5.0, 7.5, 10.0, 12.5, 15.0 and 17.5 µg/ml.
Calibration curve of gliclazide was prepared using the working standard solutions in the range
of 2.5 – 17.5 µg/ml. The linearity curves of gliclazide were prepared at 226 nm and 290 nm as
reported maxima (λmax) and as observed in the spectrum. Each curve was fitted by linear
regression analysis and r2 was calculated. Linearity curve in results section represents the
linearity of gliclazide API in pH 6.8 phosphate buffer.
5.1.4.8 Preparation of stock and standard solutions with pH 7.4 phosphate buffer
Stock solution of gliclazide was prepared by dissolving 30 mg of gliclazide in 10 ml methanol
and the volume was made up with pH 7.4 phosphate buffer to 100 ml. The stock solution was
diluted with pH 7.4 phosphate buffer to obtain the final working standards having
concentration of 2.5, 5.0, 7.5, 10.0, 12.5, 15.0 and 17.5 µg/ml.
Calibration curve of gliclazide was prepared using the working standard solutions in the range
of 2- 20 µg/ml. The linearity curves of gliclazide were prepared at 226 nm and 290 nm as
reported maxima (λmax) and as observed in the spectrum. Each curve was fitted by linear
regression analysis and r2 was calculated. Results of the linearity study are given as figure 6.9
which represents the linearity curve of gliclazide API in pH 7.4 phosphate buffer.
5.1.4.9 Preparation of stock and standard solutions with pH 4.5 acetate buffer
Stock solution of gliclazide was prepared by dissolving 30 mg of gliclazide in 10 ml methanol
and the volume was made up with pH 4.5 acetate buffer to 100 ml. The stock solution was
diluted with pH 4.5 acetate buffer to obtain the final working standards having concentration
of 2.5, 5.0, 7.5, 10.0, 12.5, 15.0 and 17.5 µg/ml.
Calibration curve of gliclazide was prepared using the working standard solutions in the range
of 2- 20 µg/ml. The linearity curves of gliclazide were prepared at 226 nm and 290 nm as
reported maxima (λmax) and as observed in the spectrum. Each curve was fitted by linear
regression analysis and r2 was calculated. Results of the linearity study are given as figure
6.10 which represents the linearity curve of gliclazide in pH 4.5 acetate buffer.
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After diluting the gliclazide standard solution with buffer, ensure that the solution remains
clear and gliclazide content was not affected, it was ensured by conducting stability study of
gliclazide in buffers.
5.1.4 Estimation of related substances in gliclazide extended release tablet by HPLC
method
5.1.5.1 Method - HPLC system with UV detector
The following chromatographic conditions were maintained during the study
Instrument
HPLC system with UV detector
Reagents
Triethylamine AR grade
Trifluoroacetic acid
Acetonitrile HPLC grade
Water HPLC grade
Chromatographic system
Column : Lichrospher C8, (4.6 X 250) mm, 5 μm
Flow rate: 0.9 ml per minute
Wavelength: 235 nm
Injection volume: 20 μl
Mobile phase
A mixture of Triethylamine, Trifluoroacetic acid, Acetonitrile and water (0.1: 0.1 : 40 : 60 ),
was prepared, degassed and filtered..
Test solution
20 tablets were selected randomly and powdered. Weighed and transferred accurately the
tablet powder equivalent to about 30 mg of gliclazide to a 100 ml volumetric flask. 45 ml of
acetonitrile was added to the volumetric flask, the flask was sonicated for 15 minutes with
intermittent shaking and volume was made up with water and mixed again for 5 minutes.
Standard solution
Weighed accurately about 30 mg of working standard of gliclazide and transferred to 100 ml
voumetric flask, it was dissolved in acetonitrile and volume was made up. 2 ml of this stock
solution was transferred to 100 ml volumetric flask and diluted using a mixture of 45 volumes
of acetonitrile and 55 volume of water and mixed. Further 0 ml of the resulting solution was
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diluted to 100 ml with a mixture of 45 volumes of acetonitrile and 55 volumes of water and
mixed.
Impurity (working standard) Solution
Dissolved 5 mg of working standard and 15 mg of 1-(3-azabicyclo [3.3.0] oct-3-yl)-3-o-
tolylsulphonylurea CRS in 25 ml of acetonitrile, diluted to 50 ml with water. Diluted one
volume of the resulting solution to 20 volumes with a mixture of 45 volumes of water.
Blank solution
Prepared a mixture of 45 volumes of acetonitrile and 55 volumes of water and mix.
System suitability
Injected seperately each of the blank solution and solution (3) (in single), after filtering
through 0.45 µm (Millipore HVLP type) filter and the peak responses were recorded.
The system is not suitable until the following parameters are met:
Resolution between the 1-(3-azabicyclo [3.3.0] oct-3-yl)-3-o- tolylsuponylurea and
gliclazide peaks, in the chromatogram obtained with the solution (3), is not less than 1.8.
Inject solution (2) (six replicate injections), into the chromatograph, after filtering
through 0.45µm membrane filter (Millipore HVLP type) and record the chromatograms.
The system is not suitable until the following parameters are met:
The column efficiency as determined on gliclazide peak is not less than 5000 theoretical
plates.
The tailing factor determined on gliclazide peak is not more than 2.0.
The relative standard deviation for gliclazide peak, in terms of area, for six replicate
injections is not more than 5.0%.
Procedure
Solution (1) (single injection) was injected in HPLC after filtering through 0.45µm membrane
filter (Millipore HVLP type). Chromatograms were recorded and the peak responses were
compared. Peaks (if any) due to blank and peaks due to placebo were disregarded at relative
retention time of about 0.1 and 0.25 with respect to gliclazide peak. Chromatograms is run for
twice the retention time of gliclazide peak.
Calculation: (%)
Percentage of single highest impurity (specified RRT about 0.3) was calculated as given
below
Single highest impurity = AR x WS x 2 x 10 x 100 x P x AV 100
(Specified RRT about 0.3) As 100 100 100 WT 100 C
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Where,
AR - Area of single highest impurity (specified RRT about 0.3) peak in
solution(1)
AS – Average area of peak due to gliclazide in solution (2)
Ws – Mass of gliclazide WS taken (in mg)
WT – Mass of test sample taken (in mg)
Av – Average mass of the tablet (in mg)
P – Potency of gliclazide WS (in % w/w)
C – Assay value of gliclazide in tablets (in mg)
Percentage of single highest unknown impurity (specified RRT about 0.3) was calculated as
given below
Single highest impurity = Ai x WS x 2 x 10 x 100 x P x AV 100
(Specified RRT about 0.3) As 100 100 100 WT 100 C
(%)
Where, Ai - Area of single highest unknown impurity peak in solution (1)
AS – Average area of peak due to gliclazide in solution (2)
Ws – Mass of gliclazide WS taken (in mg)
WT – Mass of test sample taken (in mg)
Av – Average mass of the tablet (in mg)
P – Potency of gliclazide WS (in % w/w)
C – Assay value of gliclazide in tablets (in mg)
Calculate the percentage of total unnknown impurities as given below:
Single highest impurity = AT x WS x 2 x 10 x 100 x P x AV 100
(Specified RRT about 0.3) As 100 100 100 WT 100 C
Where,
AT - Area of all unknown impurity peak in solution (1)
AS – Average area of peak due to gliclazide in solution (2)
Ws – Mass of gliclazide WS taken (in mg)
WT – Mass of test sample taken (in mg)
Av – Average mass of the tablet (in mg)
P – Potency of gliclazide WS (in % w/w)
C – Assay value of gliclazide in tablets (in mg)
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Percentage of total impurities is calculated as given below
Total impurities (%) = Percentage of single highest impurity (specified RRT about 0.3) +
Total unknown impurities.
Peaks less than 0.05% are disregarded.
5.1.6 Forced degradation studies
Sample of gliclazide have been subjected to the following conditions and the products were
analysed.
Acid catalysed degradation
Base catalysed degradation
Oxidative degradation studies using hydrogen peroxide
UV light induced degradation
Thermal degradation
5.1.6.1 Acid Catalysed degradation
Gliclazide (50.0 mg) was dissolved in acetonitrile 23 ml and 5 ml 1 N HCl was added to the
solution. The solution was heated at 50oC for 30 minutes, cooled to room temperature, diluted
to 50 ml and analysed by HPLC method following Ph.Eur. procedure for related substances.
The results of the study are presented in next chapter.
5.1.6.2 Base catalysed degradation
Gliclazide (50.0 mg) was dissolved in acetonitrile (50 ml), 5 mL of 1N sodium hydroxide
solution was added and the solution was heated at 50oC for 30 minutes. Resulting solution was
cooled to room temperature diluted to 50 ml and then analysed by HPLC method following Ph.
Eur. Procedure for related substances analysis.
5.1.6.3 Degradation under oxidation conditions
Gliclazide (50 mg) was dissolved in acetonitrile (23 ml). 5 mL of 3% hydrogen peroxide was
added to the solution and heated at 50oC for 30 minutes. The resulting solution was cooled to
room temperature, diluted to 50 ml with water and analyzed by HPLC method.
5.1.6.4 Thermal stability
Gliclazide was placed in an oven at 105oC for 24 hrs. Portion of gliclazide (50mg) was
dissolved in acetonitrile (23ml). Solution was further diluted to 50 ml with water and analysed
by HPCL method.
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5.1.6.5 Photo stability
Sample of gliclazide was exposed to UV radiation at 254 nm for 24 hrs. Gliclazide (50 mg)
was dissolved in acetonitrile (50 mL) and the solution was further diluted to 50 mL and
analyzed by HPLC method.
5.1.7 Analytical method and Validation of gliclazide in the plasma
Analytical method based on liquid chromatography with positive ion electrospray ionization
(ESI) coupled to tandem mass spectrometry detection was developed for the determination of
gliclazide in human plasma using nifedipine as internal standard. The analyte and the internal
standard were extracted from the plasma samples by protein precipitation and
chromatographed on a C 18 analytical column. The mobile phase consisted of acetonitrile -2
mM ammonium acetate (80:20). The method had a chromatographic total run time of 2.5
minutes and was linear within the range of 5.0 – 150.0 ng.ml. Detection was carried out on an
API 3000 triple quadrapole instrument (Applied Biosystems, Concord, Ontario, Canada)
tandem mass spectrometer by multiple reaction monitoring (MRM). The precision (CV %)
and accuracy, calculated from limit of quantification (LOQ) samples (n=8), were 9.97 and
101% respectively. The method was successfully applied to characterise the pharamacokinetic
profiles of Gliclazide in 12 healthy volunteers after an oral dose of Gliclazide 30 mg.
Equipments and Materials
Table 5.5: List of chemicals
Reagents Grade Supplier
Acetonitrile HPLC Burdick and Jackson
Methanol AR Burdick and Jackson
Ammonium acetate ACS Fischer Scientific
Water was prepared In-house from Purite Select – analyst HP purifier
Table 5.6: Reference Compound
Name Supplier
Gliclazide Sigma
Nifedipine Sigma
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Chromatographic conditions
HPLC System: Shimadzu, Kyoto, Japan
Column : Hypurity C 18 analytical column (50 mm X 4.6 mm) operating at 40oC
Mobile phase: Acetonitrile-2mM, ammonium acetate (80:20) at a flow rate of 0.5 ml/ min
Volume : 50 µL/min
Auto sampler temperature : 10oC
Run Time : 2.5 min
Mass spectrophotometry system
System: API 3000 triple quadrapole instrument (Applied Biosystems, Concord,
Ontario, Canada)
Sample : Gliclazide and Nifedipine prepared in a mixture of Methanol/Water (50: 50 v/v)
Flow volume : 0.5 mL/min
Flow rate : 20 µL/min
5.2 Preformulation studies
Optimization of drug substances and excipients through the determination of some physical
and chemical properties is mandatory in the development of new dosage forms.
Preformulation is described as a phase of research and development process where the
physicochemical properties of drug substances and excipients used are characterized in order
to achieve success in developing stable and bioavailable formulation.
5.2.1 Characterisation of gliclazide
5.2.1.1 Organoleptic properties
The colour and odour of Gliclazide was determined and reported by visual analysis.
5.2.1.2 Particle size determination
Particle size of the drug was determined by Malvern Technique. A dispersed gliclazide
sample is passed through the measurement area of the optical bench where the laser beam
illuminates the particles and particle size is measured by the intensity of the light scattered by
detectors.
5.2.1.3 Sieve analysis
Average size of gliclazide was determined using vibratory sieve shaker. 25 g of gliclazide was
weighed and placed on an ultrasonic sieve shaker. Percentage retained on 20#, 40#, 60#, 80#
and 100# and fines was determined.
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5.2.1.4 Water content
Water content of the gliclazide was determined using Karl Fischer Technique. Appropriate
powder sample was weighed and transferred to K.F. titrator. The readings obtained were used
to calculate water content.
5.2.1.5 Flow properties
Flow properties of gliclazide were determined by calculating parameters such as bulk density,
tapped density, compressibility index and hausners‟s ratio and excipients for bulk density and
tapped density.
Bulk density and tapped density
10 g powder was placed in 100 ml measuring cylinder. Volume occupied by the powder was
noted down as V0, without disturbing the cylinder. Then cylinder was fitted in instrument and
tapped for 500 times. Measure the difference between the initial volume and the final volume
(after 500 taps). If the difference is more than 2%, then again tap the cylinder for 750 times
more and measure the difference. Bulk density and tapped density was calculated using
following formula:
Bulk density (g/ml) = Weight of sample in grams
V0
Tapped density (g/ml) = Weight of sample in grams
Vb
Compressibility index
Tapped and apparent bulk density measurements can be used to estimate the compressibility
of the material
Compressibility index = 100* (Bulk volume – True volume)
Bulk volume
Hausner’s ratio
It is the ratio of bulk volume to tapped volume or tapped density to bulk density.
Hausner‟s ratio = Tapped density
Bulk density
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5.2.1.6 Standardization of gliclazide
Gliclazide was sourced from Bal Pharma Bangalore, India, and standardized as per the
Certificate of Analysis (COA) and Ph. Eur. Gliclazide and other excipients were analysed and
verified as per specifications set for the materials.
5.2.1.6.1 Identification
Spectra obtained from infrared absorption spectrophotometer for test substance should be
concordant with that of reference spectrum of Gliclazide (standard).
Gliclazide identification was carried out using FT-IR spectroscopy. IR spectrum of drug was
recorded in the stretching frequency range of 450-4000 cm-1
. The samples were prepared by
potassium bromide pellet technique.
5.2.1.6.2 Determination of melting point
The drug was finely powdered and charged in thin glass capillary tube, one end of which was
sealed. Sufficient amount of drug was filled in the glass capillary, to form a column at the
bottom of the tube (2.5-3.5 mm height), when packed down closely as possible by moderate
tapping on solid surface. The capillary tube was placed in a melting point apparatus and the
range of temperature when the drug just starts melting and till it completely melted was noted.
5.2.1.6.3 Solubility
pH solubility was investigated by measuring solubility of gliclazide using buffers of various
pH 1.5, 2.5, 3.5, 4.5, 5.5, 6.5 and 7.5. Weighed sample of about 80 mg of gliclazide was
added to 250 mL of buffer solution and stirred magnetically in a water bath at 37oC. After one
hour 5 mL sample was withdrawn and diluted suitably. Gliclazide absorbance was recorded in
different buffer solution was measured at 226 nm and 290 nm on UV spectrophotometer and
concentration of Gliclazide in each buffer solution was calculated.
5.2.1.6.4 Assay
Assay of gliclazide was done using UV method. The limit is 98.0 – 102% on dried basis.
5.2.1.6.5 Analysis of gliclazide
Complete analysis of Gliclazide was done as per the European Pharmacopeia and vendors‟
certificate of analysis.
5.2.1.6.6 Microscopy of gliclazide
Microscopy of gliclazide was done to know the shape of the gliclazide.
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 97
5.2.2 Solid state stability – Compatibility studies
Excipients are included in dosage form to aid manufacture, administration or absorption.
Other reasons for inclusion of excipient are for product differentiation, appearance,
enhancement or retention of quality. They rarely possess pharmacological activity and are
accordingly loosely characterized as „inert‟. However excipient can initiate, propagate or
participate in chemical and physical interaction with an active, possibly leading to
compromised quality or performance of the medication. Chemical interaction can lead to
degradation of the active ingredient, thereby reducing the amount available for the therapeutic
effect. Physical interaction can affect rate of dissolution, uniformity of dose or ease of
administration. Understanding physical and chemical nature of the excipient, impurities and
residue associated with them and how they may interact with other materials, or each other,
forewarns the pharmaceutical technologist of possibilities for understanding development.
Drug substance along with the excipient was kept at different temperature and humidity
conditions to check the stability in solid state.
5.2.2.1 Method
Drug and excipients were taken in the ratio as mentioned in table 6.1 of drug-excipient
compatibility study protocol. Glass vials were filled with drug-excipients individually which
act as a control and drug - excipients were blended in the ratio as given in table 6.1(A) and
6.1(B). The drug and excipients ratio is calculated based on the contents as present in the
formulation. These vials were kept at different conditions as mentioned in table no. 6.1(A)
and 6.1(B), (Initial, 40oC/75% RH in open condition for 4 weeks and 8 weeks, 60
oC for one
week and 25o C/65% RH in open and closed condition for 4 weeks and 8 weeks.
All samples were withdrawn at the specific time intervals and were evaluated for their
physical as well as chemical parameters.
Physical parameters
Different physical parameters like physical appearance, color, consistency, flow were
recorded by visual inspection as mentioned in table 6.2(A) and 6.2(B).
Chemical parameters
Samples were analysed for assay and related substances at specified time points. Results are
given in table 6.3(A), 6.3(B) and 6.3(C) and their graphical presentation in Figure 6.1 – 6.4.
Chapter 5 Experimental
SPP School of Pharmacy and Technology Management, SVKM‟s NMIMS, Mumbai 98
Table no. 5.7: Drug-excipient compatibility study protocol
4 weeks 8 weeks 4 weeks 4 weeks 8 weeks 8 weeks
Sr.
No.
Gliclazide + Excipients Ratio Initial 60oC
(closed)
40oC/75
%RRH
(open)
40oC/75
% RH
(open)
25oC/60
% RH
(open)
25oC/60
% RH
(closed)
25oC/60
% RH
(open)
25oC/60
%RH
(closed)
1 Gliclazide 1 √ √ √ √ √ √ √ √
2 Gliclazide +Lactose
monohydrate
1:2 √ √ √ √ √ √ √ √
3 Gliclazide +Dibasic calcium
phosphate, anhydrous
1:2 √ √ √ √ √ √ √ √
4 Gliclazide + HPMC
K100LV
1:1.5 √ √ √ √ √ √ √ √
5 Gliclazide + HPMC K4 M 1:0.5 √ √ √ √ √ √ √ √
6 Gliclazide + Maltodextrin 1:2 √ √ √ √ √ √ √ √
7 Gliclazide+ Hydroxypropyl
cellulose
1:1 √ √ √ √ √ √ √ √
8 Gliclazide + Magnesium
stearate
1:0.2 √ √ √ √ √ √ √ √
9 Gliclaizide + Colloidal
anhydrous silica
1:0.2 √ √ √ √ √ √ √ √
10 Gliclazide + Ethyl cellulose 1:1 √ √ √ √ √ √ √ √
11 Gliclazide + Light
magnesium carbonate
1:0.5 √ √ √ √ √ √ √ √
12 Gliclazide + PEG 8000 1:1 √ √ √ √ √ √ √ √
13 Gliclazide + Purified talc 1:1 √ √ √ √ √ √ √ √
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SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 99
5.2.3 Drug and excipient characterisation studies by DSC
5.2.3.1 Introduction
To characterise the drug and excipient, differential scanning calorimetry studies were carried
out. Differential scanning calorimetry is a thermo analytical technique in which the amount
of heat required to increase the temperature of a sample is recorded.
The basic principle underlying this technique is that, when sample undergoes a physical
transformation such as phase transitions, more (or less) heat will need to flow into it than the
temperature is recorded. Differential scanning calorimeters are able to measure the amount of
heat absorbed or released during such transitions.
Method
Approximately 100 mg of sample was taken to be analyzed and placed in sample cup, which
was kept in a sample holder. Temperature was gradually increased by 10oC from room
temperature to 250oC. Thermograms were obtained.
5.2.3 Drug and Excipients Compatibility Studies by XRD
5.2.3.1 XRD studies
To investigate any drastic change in thermal behaviour of either drug or excipients in
combination, x-ray diffraction studies were carried out. X-ray powder diffraction patterns
were obtained at room temperature using a PW1710 X-ray diffractometer (Philips, Holland)
with Cu as anode material and graphite monochromator, operated at a voltage of 35 kV and
20 mA current. The samples were analysed in the 2o angle range of 5°–70° and the process
parameters were set as scan step size of 0.02° (2o), and scan step time of 0.5s. XRD was
performed only for the excipients used in the final formulations made by three technologies.
Method
As mentioned in table 6.1 of drug-excipients compatibility studies, glass vials were filled with
drug-excipients individually which acts as a control and drug excipients blended as binary
mixtures in the ratio as present in the tablet composition. These all vial were kept at different
conditions as mentioned in table no. 6.1. The samples kept at 60oC for one week in closed
condition were analysed for XRD. (Excipients used in the final formulation of gliclazide ER
tablets were subjected for XRD studies).
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SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 100
5.3 Formulation development
5.3.1 Innovator product (brand product)
As gliclazide extended release tablets were to be developed as generic product for regulated
market, the first step in the development was the evaluation of the innovator product. The
chemical properties needs to be matched. Physical properties can be different as it will only
affect the asthetics of the formulation. Following are the properties of innovator product.
Gliclazide MR tablets (ROW/EP/IN)
EU approval date for MR tablets 30 mg – Dec. 2000
Gliclazide is not approved in US.
Diamicron MR tablet 30 mg – Servier Lab. Ltd.
Approved form - Gliclazide
Approved indication – Non-insulin dependent diabetes mellitus (Type II).
Diamicron MR tablets are white, oblong tablets engraved on both faces, DIA 30 on
one side and company logo on the other side.
Dosage : For adult use only
Daily dose : May vary from 1-4 tablets per day that is 30-120 mg taken orally in a
single intake at breakfast.
Figure 5.1: Diamicron MR tablet with external carton pack (front)
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SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 101
Figure 5.2: Diamicron MR tablet primary pack (Blister) and external carton pack
Figure 5.3: Diamicron MR tablet (Innovator)
5.3.1.1 Evaluation of Diamicron tablets 30 mg
Diamicron MR tablets (Gliclazide MR tablets) [Reference: SPC emea]
Table 5.8: Diamicron MR 30 mg tablets formulation.
Description Diamicron tablets are white oblong uncoated tablets with
a „DIA 30 engraved on one face and „company‟s logo‟
on the other face.
Manufactured by Servier lab. Ltd. France
Tablet dimensions
Average weight – 160 mg
Length – 10 X 5.5 mm
Thickness – 3.4 mm
Inactive ingredients Calcium hydrogen phosphate, Maltodextrin,
Hypromellose, Silicon dioxide and Magnesium stearate.
Uses and Indication
For the treatment of Type II diabetes along with the
dietary measures to control blood glucose level.
Storage Store below 30oC.
Pack profile 1 X 14 PVC/PVdC blister card in a carton.
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SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 102
Dose justification and rationalization
The equivalency of the dosage of Gliclazide 30 mg as ER formulation and 80 mg IR tablets
has been established in several clinical studies and to avoid unnecessary dosing and wasting
huge amount of drug being excreted without active usage has prompted to commercialize 30
and 60 mg strengths as ER tablets formulations. With 30 mg being the most popular, well
tolerated and having minimal possibilities of hypoglycemic situations, has been the chosen
product for the evaluation of technological applications for the ER profiling.
The present study had a prime focus to develop the formulations for ER profile using different
technologies viz. matrix, hot-melt and MUPS. The dose 30 mg is a therapeutic dose hence
dose has never been the point of concern as we had not to establish the dose level or dose
frequency. Servier, the innovator company has enough data to prove the superiority of the
drug in ER formulation and we had plans to work on the generic version (which is a normal
way of developing the product and applying the non-infringing IPR avenues) and evolve a
stable and bioequivalent formulation by different ER technologies. Since the excipients
utilized are different and the blood level profiling coupled with the accelerated stability are
the criteria for suitability, the study becomes innovative and elaborate.
Following instances and the studies clearly reveal the significance of the ER formulation over
the IR dosage which is otherwise more than 2 fold and is being repeated more frequently. A
single ER formulation maintains the desired blood level for extended period of time by way of
controlling the release at different hours, without necessitating any initial build up which was
conventionally considered in the beginning. These days the therapy starts off with the ER
dose only and the quantitative releases are at times maintained by way of pH control or
solubility cum swelling behavior of the polymers. The Cmax and Tmax in case of ER
formulations are getting extended (the effective concentration levels are achieved almost
instantly) thus providing the therapeutic levels for much longer duration with much reduced
amounts of drug at a reduced dosing frequency. Even though Cmax may be higher in case of
higher IR dose but it is really not required and vanishes soon by forcing a greater amount of
drug to eliminate from the system, asking for a new dose and giving rise to lag phase for
achieving the therapeutic levels.
Oral drug delivery system is the most popular route, which is due in part to the ease of
administration and to the fact that gastrointestinal physiology offers more flexibility in dosage
form design than most other routes (Gupta and Robinson, 1992). Development of oral
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SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 103
controlled release dosage forms of a given drug involves optimization of the dosage form
characteristics within the inherited constrains of the gastrointestinal physiology. Controlled
release delivery systems have added advantages over immediate release dosage form. These
include reduction of dosing frequency by administering the drug once or twice a day (Hayashi
et al., 2005). Since the frequency of drug administration is reduced, patient compliance can be
improved, and drug administration can be more convenient (Nokhodchi et al., 2002) due to
reduction of gastrointestinal side effects (Hosny, 1996). Also causes less fluctuation of plasma
drug level and leads to more uniform drug effect and lesser total dose.
Gliclazide is indicated for control of hyperglycemia. Gliclazide responsive in diabetes
mellitus of stable, mild, non-ketosis prone, maturity onset or adult type which cannot be
controlled by proper dietary management and exercise, or when insulin therapy is not
appropriate. Determination of the proper dosage for gliclazide for each patient should be
made on the basis of frequent determinations of blood glucose during dose titration and
throughout maintenance. (Ref Product Monograph)
Oral absorption of gliclazide is similar in patients and healthy volunteers, but there is inter-
subject variation in time to reach peak plasma concentrations (tmax). Ages related differences
in plasma peak concentrations (Cmax) and tmax, have been observed. A single oral dose of 80
to 320 mg of gliclazide results in a Cmax of 2.2 to 8.0 ug/ml within 2 to 8 hours. Tmax and
cmax are increased after repeated gliclazide administration. Steady state concentration is
achieved after 2 days administration of 80 to 320 mg of gliclazide. Gliclazide has low volume
of distribution (13 to 24L) in both patients and healthy volunteers due to its high protein
binding affinity (85 to 97%) (Najib et al., 2002). The elimination half-life (t1/2) is about 8.1 to
20.5 hr in healthy volunteers and patients after administration of 80 to 320 mg orally.
Moreover, its plasma clearance is 0.78 L/h (13 ml/min). It is extensively metabolized to 7
metabolites and excreted in urine therefore renal insufficiency has no effect in
pharmacokinetic of gliclazide. The variability in absorption of gliclazide could be related to
its early dissolution in the stomach leading to more variability in the absorption in the
intestine (Delrat et al., 2002). This process resulted in low bioavailability of the conventional
dosage forms.
To maintain normal plasma glucose levels, patients have to take one or more doses of
conventional or sustained release gliclazide tablets. The physiological requirement is to
provide constant plasma glucose levels over an extended period of time to meet the basal
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SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 104
needs between meals and during the night. If there was a formulation of gliclazide that could
provide adequate control of glucose level for an extended period of time without any
hypoglycaemic symptoms (Crepaldi and Fioretto, 2000), patients could be relived from the
necessity of taking 1- 4 tablets of 80 mg of gliclazide daily.
Such a formulation would be a helpful not only to improve the patients‟ conditions and
convenience but also to reduce the risk of prevalence of other diseases associated with
diabetes mellitus. Diamicron MR 30 mg is a formulation of gliclazide with modified release
characteristics, allowing a once daily dose regimen. Its release profile with more than 50% of
the gliclazide being released within the first 4 – 6 hours, was chosen to properly address
morning hyperglycemia and to avoid excessive release during night, with the aim of keeping
the good safety/efficacy profile of the standard formulation. Gliclazide ER tablets 30 mg is
insensitive to pH, so that the plasma concentration is not affected by food or by treatment with
drugs that modify gastrointestinal pH. This resulted in 97 – 110% of drug bioavailibility thus
enables to reduse the dose. Based on the bioavailibility and the metabolism 30 mg ER dose is
equivalent in effect to the 80 mg of IR dose.
To support, clinical trial data also reported on rationale of Gliclazide MR formulation over
Gliclazide IR formulation. Large phase III, multinational, comparative, randomized, double-
blind trial aimed at demonstrating the therapeutic equivalence of gliclazide MR 30 mg
compared to the gliclazide 80 mg immediate release formulation. A total of 664 patients were
randomized in two parallel groups, one assigned to gliclazide 80 mg (336 patients) and one to
gliclazide MR 30 mg (328 patients). After a 4-month dose escalating period allowing patient-
tailored titration, patients entered a maintenance period of 6 months. Gliclazide 80 mg was
administered at 80, 160, 240 or 320 mg/day, with doses above 80 mg given twice daily;
gliclazide MR 30 mg was always administered once daily at breakfast time at 30, 60, 90 or
120 mg/day. The study demonstrated that after 10 months of treatment, gliclazide MR 30 mg
is at least as effective as gliclazide 80 mg in controlling HbA1c and FPG levels of type 2
diabetic patients. The therapeutic equivalence was actually achieved with lower daily doses of
the MR formulation, 30 mg of gliclazide MR producing a similar effect as 80 mg of gliclazide
immediate release formulation. The general safety of both formulations was good with no
difference in type and incidence of adverse events. With regard to hypoglycemia, the number
of patients experiencing hypoglycemic episodes was almost the same in both groups.
However, the number of hypoglycemic episodes was lower in the gliclazide MR group than in
the gliclazide 80 mg group. (Ref. Scientific discussion by Servier labs.)178, 179,180
.
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SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 105
The recommended starting dose of gliclazide MR is 30 mg daily, i.e. one half tablet of
gliclazide MR 60 mg or one tablet of gliclazide MR 30 mg, even in elderly patients (over 65
years old). A single daily dose provides effective blood glucose control. The single daily dose
may be between 30 mg and 90 mg, or even 120 mg.
Thus gliclazide extended release formulation is and efficient and well tolerated treatment for
Type II diabetes. Once daily dosing should improve patient compliance, an important factor in
long term glycemic control.
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SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 106
Experimental Design- Gliclazide ER tablets by Matrix Technology- Process flow chart
Figure 5.4: Process flow chart of gliclazide ER tablets containing polymers Polywox WSR
coagulant, Sodium alginate, Kollidone SR and Xanthan gum
Process flow chart
Figure 5.5: Flow chart of formulation with combination of HPMC
as the rate controlling polymer
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SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 107
5.3.2 Experimental design – Matrix technology
For developing a non infringing formulation of gliclazide extended release tablet, the first
step was to develop a formulation with the polymer other than those covered in the patent that
is cellulose. Another way to develop non infringing strategy is to change the concentration of
polymer. Innovator (Servier labs.) patent had covered the cellulose polymer in ratio not more
than 15%. Another claim of innovator was the manufacturing process using wet granulation
method.
The first development strategy was to use polymers other than cellulose. The polymers
studied were Polyethylene oxide (PEO WSR Coagulant), Copolymer of Polyvinyl alcohol and
Povidone (Kollidone SR), Xanthan gum (Xanthural 75), Sodium alginate. The first step was
to match the dissolution profile of formulation with Diamicron MR tablets. Further
experiments were also carried out using combination of Hydroxypropylmethyl cellulose
(HPMC K 100 LV and HPMC K 4 M) by keeping the concentration of the polymer other than
claimed in the patent and changing the manufacturing process.
The formulation was prepared using dry granulation method that is slugging and deslugging.
Initial batches of gliclazide extended release tablets were formulated based on the fact that
hydrophilic polymers rapidly swell in contact with water and provide sustained release effect.
The polymers used in the extended release formulation of hydrophobic drug gliclazide are the
hydrophilic polymers such as polyethylene oxide (PEO WSR Coagulant), Xanthan gum,
Copolymer of polyvinyl alcohol and povidone (Kollidone SR), sodium alginate and
hydroxypropylmethyl cellulose (HPMC K 100 LV and HPMC K 4M) which efficiently
control drug release rate. Hydrophilic polymers are generally used to enhance the release of
hydrophobic molecules. A special consideration was given to the sequence of addition of
excipients, where drug along with the diluents and the lubricant were compressed into slugs
then milled to get desired particle size to be compressed into final tablets. The prepared
batches were evaluated for various physical and chemical parameters. The optimized batches
were subjected to stability studies as per ICH guidelines.
LABEL CLAIM
Each extended release tablet contains
Gliclazide Ph.Eur / IP..................... 30 mg
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 108
5.3.2.1 Formulation with polyethylene oxide (PEO WSR coagulant) as the polymer
The formulation of gliclazide extended release tablets by matrix dissolution system is the
most commonly used system in pharmaceutical industry. In matrix system drug is
homogeneously distributed throughout the polymer matrix which dissolves and drug
molecules are released. For matrix dissolution system, drugs may release through diffusion
mechanism as well as erosion mechanism which depends on the properties of drugs and
polymers used in the formulation. In the matrix dissolution system, since the size of the
matrix decreases as more drugs are released, the amount of drug released is also decreased,
that results in a non zero order release.
The formulation development was initiated using Polywox WSR coagulant (PEO) polymer as
it has been reported as the best alternative to the cellulose polymer for the development of
extended release formulations. Polyethylene oxide (Polywox WSR Coagulant) had a viscosity
range of 5500 -7500 cps (1% solution at 25oC) which was used mainly as the rate retardant. A
homogenous mixture of drug along with the polymer and the diluents was prepared. Polywox
WSR coagulant has good flow, the composition of slugs was drug and diluents. Polymer was
added in the lubrication stage. As polyethylene oxide is reported to undergo oxidative
degradation, Butylated hydroxy toluene was added in all the formulation to protect the
polymer from oxidation. The quantity of butylated hydroxy toluene was kept within the IIG
limits.
Experimental batch B. No. P01/09 was made with the PEO concentration of 47.5%. The
physical parameters for the tablet formulation were found to be satisfactory. But the
dissolution profile showed drug release of 75% after 10 hours. Further batches were prepared
(B. No.P02/10 to P07/09) by reducing polymer concentration till satisfactory dissolution
profile was obtained. The formulation was optimised on the basis of its dissolution profile
which was compared with the innovators dissolution profile to prove equivalency of the
products. Another factor which was considered for the formulation was drug release and f2
calculation which indicated the similarity of two products if f2 value is more than 50.
f2 = 501 g {[ 1 + 1/n Wt (R1-T1)2] -0.5 X 100}
Where n is the number of dissolution sample times and R1 and T1 are the individual
percentages dissolved at each time points, t for the reference and test dissolution profiles
respectively.
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SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 109
Formulation P08/10 was prepared using polymer in the concentration of 22.5% which showed
satisfactory physical and chemical parameters. Further batches were prepared to verify its
reproducibility. After achieving the desired release profile and satisfactory physical
parameters, formulations were packed in Alu/Alu blister pack and kept on stability to check
short term stability of the formulation.
The composition of various batches taken with PEO WSR coagulant is mentioned in table 5.9.
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 110
Table 5.9: Composition of gliclazide ER tablets 30 mg with polyethylene oxide as release rate controlling polymer
B. Size: Each batch of 1000 tablets
Sr. No.
Ingredients Spec.
B. N. P01/
09
B. N. P02/
09
B. N. P03/
09
B. N. P04/
09
B. N. P05/
09
B. N. P06/
09
B. N. P07/
09
B. N. P08/
09
B. N. P09/
09
B. N. P10/
10
B. N. P11/
10
B. N. P12/
10
B. N. P13/
10
B. N. P14/
10
mg/tablet
1 Gliclazide Ph.
Eur. 30 30 30 30 30 30 30 30 30 30 30 30 30 30
2 Lactose
monohydrate Ph.
Eur. 54.6 67.6 - - - - - - - - - - - -
3 Dicalcium
phosphate, Anhydrous
Ph.
Eur. - - 77.6 82.6 87.6 97.6 87.6 77.6 72.6 77.6 78.6 77.6 77.6 77.6
4 Maltodextrin Ph.
Eur. 18 20 20 20 20 20 30 40 45 45 45 45 45 45
Lubrication
5 Polyethylene
oxide (PEO)
In.
Spec. 95 80 70 65 60 50 50 50 50 45 45 45 45 45
6 BHT (sifted
through # 100 mesh sieve)
Ph.
Eur. 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4
7 Magnesium Stearate
Ph.
Eur. 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 1.0 2.0 2.0 2.0
Average wt.
200
mg 200
mg 200
mg 200
mg 200
mg 200
mg 200
mg 200
mg 200
mg 200
mg 200
mg 200
mg 200
mg 200
mg
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SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 111
Method of tablet preparation
Gliclazide, dicalcium phosphate anhydrous and maltodextrin were sifted through # 20 mesh
sieve. The whole blend was sifted again through # 20 mesh sieve. The above blend was mixed
in blender (container blender) for 10 minutes followed by blending with magnesium stearate
(presifted through # 40 mesh sieve) for 2 minutes. The lubricated blend was compressed on
20 station rotary tablet press machine using 16 mm round flat punches to form slugs with a
hardness of 50- 70 N and machine speed of 20-25 rpm. The slugs obtained were milled using
multimill with 8 mm screen followed by 1.5 mm screen. The sized granules were then sifted
through # 20 mesh sieve. The granules were lubricated in the container blender with
polyethylene oxide (presifted through # 30 mesh sieve) for 15 minutes followed by
magnesium stearate (presifted through # 40 mesh sieve) for 2 minutes. The final blend was
compressed on 20 station rotary tablet press using 8.00 mm round standard concave punches
plain on both sides.
5.3.2.2 Formulation with Xanthan gum as polymer
Gliclazide extended release tablets were made using Xanthan gum with viscosity of 1200-166
cps as the rate controlling polymer. Xanthan gum is a high molecular weight polysaccharide
gum. Xanthan gum has been used to prepare sustained release matrix tablets. It has been
reported to sustain the drug release in a predictable manner and the drug release profiles of
these tablets was not affected by pH and agitation rate. The formulation was made with the
similar process as described earlier in the formulation with polyethylene oxide. The polymer
was used in concentration of 29% and analyzed for dissolution profile but could achieve the
required profile. In further experiments, Xanthan gum concentration was reduced from 29%
upto 7.5%. The physical attributes of the extended release tablets were satisfactory. But the
comparative dissolution profile could not be achieved even after reducing the concentration of
polymer to 7.5%. Hence, this approach was discontinued, because as a general rule to achieve
uniform distribution of polymer in the matrix blend, the concentration of polymer should be
around 5%.
Various experiments with reducing concentration of Xanthan gum (B.No. X01/09 to X04/09)
were carried out to match the dissolution profile. The composition of the formulation using
Xanthan gum as the rate controlling polymer is given in Table 5.10. As the comparative
dissolution profile was not achieved, the formulations were not subjected to stability studies.
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SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 112
Table 5.10: Composition of formulation with Xanthan gum as retarding polymer
B. Size – 1000 Tablets
Sr.
No.
Ingredients Spec. B. No.
X01/09
B. No.
X02/09
B. No.
X03/09
B. No.
X04/09
mg/tablet
1 Gliclazide Ph. Eur. 30 30 30 30
2 Dicalcium phosphate,
anhydrous
Ph. Eur. 58 76 86 101
3 Maltodextrin Ph. Eur. 50 50 50 50
4 Magnesium stearate Ph. Eur. 2.0 2.0 2.0 2.0
Lubrication
5 Xanthan gum Ph. Eur. 58 40 30 15
6 Magnesium stearate Ph. Eur. 2.0 2.0 2.0 2.0
Average weight 200 mg 200 mg 200 mg 200 mg
The batch was formulated using the same procedure mentioned earlier for formulation
containing polyethylene oxide.
5.3.2.3 Formulation with Sodium alginate as polymer
Alginate is one of the most studied and applied natural polysaccharide polymers in oral controlled
delivery system. The ability of sodium alginate to rapidly form viscous solutions in contact with
aqueous media and to form gels in contact with acid or di – or trivalent cationic ions is ideal for its
use as hydrophilic matrix in oral controlled release dosage forms. Formulation with sodium
alginate as polymer was made using the process as described earlier. Formulation development
started with B. No. SA01/09 with polymer concentration of 29%, but the desired release could
not be achieved. Hence the concentration was increased upto 50% in the subsequent batches. In
spite of increasing the polymer concentration to 50% the required retardation could not be
achieved. This approach was therefore discontinued for further optimisation and stability. The
composition of the formulations with sodium alginate as polymer is given in table 5.11
Table 5.11: Composition of formulation with sodium alginate as retarding polymer
Sr.
No. Ingredients Spec. B. No.
SA01/09 B. No. SA02/09
B. No. SA03/09
B. No. SA04/09
mg/tablet
1 Gliclazide Ph. Eur. 30 30 30 30
2 Dicalcium phosphate, anhyd. Ph. Eur. 60 48 53 43
3 Maltodextrin Ph. Eur. 50 50 25 25
Lubrication
4 Sodium alginate Ph. Eur. 58 70 90 100
5 Magnesium stearate Ph. Eur. 2.0 2.0 2.0 2.0
Average wt. 200 mg 200 mg 200 mg 200 mg
The batch was formulated using the same procedure mentioned earlier for formulation
containing polyethylene oxide.
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SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 113
5.3.2.4 Formulation with Kollidone SR as polymer
Extended release tablets of gliclazide were also made using Kollidone SR which is the
copolymer of polyvinyl alcohol and povidone as the rate retarding polymer by matrix
technology. It has excellent flowability and compressibility therefore suitable for sustained
release tablets by direct compression technology. It was added during the lubrication stage of
the formulation. Kollidone SR was blended uniformly with the gliclazide granules. The
required content of Kollidone SR in tablet depends on particle size and the solubility of the
active ingredient. The finer the particle size faster is the dissolution. The composition of the
formulation with Kollidone SR is given in table no. 5.12. To check the feasibility two
experiments were conducted with the polymer concentration of 25%, but the desired
dissolution parameter could not be achieved. Hence another batch with lower concentration
was prepared, but the required dissolution profile was not achieved and then the approach was
discontinued as there was no scope to match the dissolution profile with the innovator by
altering the concentration of the polymer.
Table 5.12: Composition of formulation with Kollidone SR as retarding polymer
Sr. No. Ingredients Spec. B. No.
K01/09
B. No.
K02/09
mg/tablet
1 Gliclazide Ph. Eur. 30 30
2 Lactose monohydrate Ph. Eur. 77 87
3 Maltodextrin Ph. Eur. 40 40
Lubrication
4 Kollidone SR Ph. Eur. 50 40
5 Colloidal anhydrous silica Ph. Eur. 1 1
6 Magnesium stearate Ph. Eur. 2 2
Average wt. 200 mg 200 mg
The batch was formulated using the same procedure mentioned earlier for formulation
containing polyethylene oxide.
5.3.2.5 Formulation with combination of Hydroxypropylmethyl cellulose as polymer
Hydroxypropylmethyl cellulose (HPMC) or hypromellose refers to soluble methylcellulose
ethers. HPMC has good compressibility and is particularly suitable for direct compression
HPMC matrix systems are considered as simple, low cost, and easy to make sustained release
systems.
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The formulations were initiated with a single grade of HPMC K 100 LV (B. No. S01/09). The
physical parameters of the tablets were satisfactory, when analysed for dissolution profile, it
showed a complete drug release in few hours. Further experiments were conducted with
combination of HPMC of various viscosities as polymer to retard the drug release profile. To
develop a non-infringing formulation using cellulose polymer the concentration required was
more than 15%. The formulations were made taking into consideration the concentration of
the polymer in the tablet. Therefore the combination of low viscosity and high viscosity
grades were used. The method used for the manufacturing was dry granulation method /
slugging/deslugging method as manufacturing process was also covered in the patent by
innovator. The formulations were made using the combination of polymer HPMC K100 LV
and HPMC K4 M to get the concentration of polymer more than 15% as HPMC K 100 LV
has very low viscosity and increase in the concentration of HPMC K 100 LV did not retard
the drug release profile. The composition of the formulation containing HPMC is given in
table 5.13.
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Table 5.13: Composition of formulation with Hydroxypropylmethyl cellulose as retarding polymer
B. size: Each batch of 1000 tablets
Sr. No.
Ingredients B. No. S01/09
B. No. S02/09
B. No. S03/09
B. No. S04/09
B. No. S05/09
B. No. S06/09
B. No. S07/10
B. No. S08/10
B. No. S09/10
B. No. S10/10
mg/tab
1 Gliclazide 30 30 30 30 30 30 30 30 30 30
2 Dicalcium phosphate,
anhydrous 79.9 51.5 60 55 49 50 50 48 50 50
3 Maltodextrin 15 72.5 57 57 57 57 57 57 57 57
4 Hydroxypropylmethyl
cellulose (HPMC K100 LV)
34 44 44 44 50 44 44 46 44 44
5 Hydroxypropylmethyl
cellulose (HPMC K 4 M) 5 10 10 15 15 15 15 15
6 Magnesium stearate 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
Lubrication
7 Magnesium stearate 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
Average wt. (mg) 200 200 200 200 200 200 200 200 200 200
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Method of preparation
Gliclazide, Dicalcium phosphate anhydrous, hydroxypropylmethyl cellulose (HPMC K100
LV) and (HPMC K4M) and maltodextrin were sifted through # 20 mesh sieve. The whole
blend was resifted through # 20 mesh sieve. The above blend was mixed in blender for 10
minutes followed by blending with magnesium stearate (presifted through # 40 mesh sieve)
for 2 minutes. The lubricated blend was compressed on 20 station rotary tablet compression
machine using 16 mm flat punches to get slugs at the hardness of 70 -80 N and machine speed
of 20 -25 rpm. The slugs obtained were milled using multimill with 8 mm screen followed by
1.5 mm screen with knife forward and slow speed. The sized granules were sifted through #
20 mesh sieve. The above sized granules were lubricated again in the container blender with
magnesium stearate (presifted through # 40 mesh sieve) for 2 minutes. The final blend was
compressed on 20 station rotary tablet compression machine using 8.00 mm („B‟ tooling)
biconvex punches plain on both sides at hardness of 50 -70 N and machine speed of 20 -25
rpm.
Gliclazide extended release tablets with HPMC as polymer
Figure 5.6: Gliclazide extended release tablet prepared using matrix technology
Chapter 5 Experimental
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Figure 5.7: Gliclazide ER tablet (HDPE bottle with CRC cap and
induction sealing and Alu/Alu blister pack)
Chapter 5 Experimental
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Gliclazide ER tablets 30 mg in pH 6.8 phosphate buffer at 0-10 hours - Drug release by
erosion and diffusion
0 hour 2nd hour
4th hour 6th hour
8th hour 10th hour
Figure 5.8: Gliclazide ER tablet with HPMC polymer in pH 6.8 phosphate buffer
Chapter 5 Experimental
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Diamicron 30 mg MR tablets in pH 6.8 phosphate buffer at 0-10 hours - Drug release by
erosion and diffusion
0-10 hrs.- Drug release by erosion and diffusion
0 hour 2nd hour
4th hour 6th hour
8th hour 10th hour
Figure 5.9: Diamicron MR tablet in pH 6.8 phosphate buffer
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 120
5.3.2.6 Evaluation of gliclazide extended release tablets 30 mg
5.3.2.6.1 Pre- compression parameters
Macroscopic appearance
The optimised formulation was observed for colour and appearance.
Loss on drying
Weighed amount of powder was placed on the sample holder of LOD apparatus and the
amount of moisture in the granules was determined.
Acceptance criteria: The LOD of the granules should be between 2-3%.
Density analysis
Bulk density and tapped density
10 g powder was placed in 100 ml measuring cylinder. Volume occupied by the powder was
noted down as V0, without disturbing the cylinder. Then cylinder was fitted in instrument and
tapped for 500 times. Now measure the difference between the initial volume and the final
volume (after 500 taps). If the difference is more than 2%, then again tap the cylinder for 750
times more and measure the difference. Bulk density and tapped density was calculated using
following formula:
Bulk density (g/ml) = Weight of sample in grams
V0
Tapped density (g/ml) = Weight of sample in grams
Vb
Compressibility index or Carr’s index
Compressibility index is indicative of the flow properties of the material. An index of below
15 indicates good flow properties while an index of greater than 25 is indicative of poor flow
of the material.
Compressibility index = 100* (Tapped density – Bulk density)
Tapped density
Hausner’s ratio
It is the ratio of bulk volume to tapped volume or tapped density to bulk density.
Hausner‟s ratio = Tapped density
Bulk density
Hausner‟s ratio is indicative of the flow properties of the material. A value less than 1.25 is
indicative of good flow and more than 1.25 indicates poor flow.
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5.3.2.6.2 Post compression parameters
Description
Colour and shape of the tablets were observed by visual observation.
Acceptance criteria: White coloured circular biconvex tablets.
Average weight of tablets
Twenty tablets were dedusted and weighed accurately.
Acceptance criteria: Average weight of Gliclazide ER tablets
1 Matrix technology 200 mg ± 3%
2 Hot melt granulation technology 160 mg ± 3%
3 Pellet technology 360 mg ± 3%
Thickness
Ten tablets were randomly selected and thickness of the tablets was measured by
previously calibrated vernier calliper.
Acceptance criteria: Thickness of Gliclazide ER tablets
1 Matrix technology 3.2 ±0.5 mm
2 Hot melt granulation technology 3.0 ±0.5 mm
3 Pellet technology 3.5 ±0.5 mm
Hardness test
Ten tablets were randomly selected. One tablet at a time was placed in the hardness tester
which was already set at zero. Pressure was applied by pressing start button of the apparatus,
till the tablet breaks. Reading on the tester, that is, hardness of tablets was noted down in
newtons.
Acceptance criteria: The tablet passes the test if it falls in the range of 40-80 N. The lowest
hardness at which the tablets pass the friability test was used to decide the hardness range.
Friability test
Sample size of tablets corresponding to 6.5 gm was taken (recorded as weight X). These
tablets were loaded to the friability test apparatus which was set to 25 rpm and after
completion of 100 revolutions, tablets were removed, dedusted and weight of the tablet was
noted down as Y.
% friability = X-Y x 100
X
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Acceptance criteria: Friability of tablets should be less than 1% as per Ph.Eur.
Uniformity of weight
20 tablets were randomly selected, dedusted and weighed individually. % weight variation
from actual average weight of tablet was calculated using following formula.
% weight variation from 100 X (Individual tablet weight – Average weight)
actual average weight of tablet = Average weight of tablet
Acceptance criteria: The tablet passes the test if not more than two tablets are outside the
percentage limit and if no tablet differs by more than two times the percentage limit. The
following percentage deviation in weight variation is allowed according to Ph.Eur.
Table 5.14: Weight variation limit as per average weight of tablet (Ref. Ph. Eur.)
Average weight of tablet Percentage deviation
80 mg or less 10%
More than 80 mg, less than 250 mg 7.5%
250 mg or more 5%
Assay of tablets
20 tablets were weighed and crushed to obtain powder equivalent to 30 mg of gliclazide. The
assay of tablets was carried out by UV method as per IP.
Acceptance criteria: Assay should be 90 – 110% of the labelled amount of Gliclazide.
Dissolution profile (IP method)
Dissolution testing for the amount of gliclazide with different polymers and varied
concentrations was studied using the following dissolution parameters:
Dissolution parameters
Apparatus: USP type II, paddle
Speed: 100 rpm
Dissolution medium: pH 6.8 phosphate buffer
Sampling time point: 1, 2, 4, 6, 8 and 10 hours
Temperature: 37± 0.5oC.
One tablet was transferred to each vessel containing 900 ml of dissolution medium. 10 ml
sample was withdrawn at each time interval and filtered through 10 micron filter. Then 5 ml
of withdrawn sample was diluted up to 10 ml with dissolution medium and % gliclazide
Chapter 5 Experimental
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released was calculated by estimating drug in dissolution medium using UV
spectrophotometer.
Standard solution
30 mg of gliclazide was dissolved in 10 ml of methanol and volume was made upto 100 ml
with pH 6.8 phosphate buffer. 5 ml of this solution was further diluted to 100 ml with
dissolution media.
Calculation
Test reading X test sample dilution X standard sample dilution x 100
Std. Reading
This gives the drug released in percentage per withdrawal.
Acceptance criteria: % drug release of labelled amount of drug
Time in hours % drug release
1st hour 5 – 20
4th
hour 25 – 50
10th
hour Not less than 80
Multiple media dissolution profile
Multiple media dissolution study was carried out to check the comparative dissolution of test
product with the innovator product according to the physiological systems in the human body.
The media selected were pH 4.5 acetate buffer and pH 7.4 phosphate buffers as the drug
gliclazide has poor solubility in acidic pH. Dissolution testing for the amount of gliclazide
with different polymers and varied concentrations was studied using the following dissolution
parameters as mentioned earlier.
Acceptance criteria: Dissolution profile should be comparative with innovator product.
Related substances
Test for related substances was performed using HPLC system with UV detector.
Reagents
Triethylamine AR grade
Trifluroacetic acid AR grade
Acetonitrile HPLC grade
Water HPLC grade
Chromatographic system
Column: Lichrospher C8, (4.6 x 250) mm, 5 µm
Flow rate: 0.9 ml per minute
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Wave length: 235 nm
Injection volume: 20 µl
Mobile phase: A mixture of triethylamine, trifluroacetic acid, acetonitrile was prepared,
filtered and degassed.
The procedure as described under analytical method verification section was used.
Composition of the final formulation for further studies
Gliclazide extended release tablets formulation B.no. S07/10 showed dissolution profile
comparable to Diamicron MR tablet (innovator brand). To check the reproducibility, of the
formulation, B.no. S09/10 was prepared with the same composition and a batch size of 2,000
tablets.
Table 5.15: Final composition of gliclazide extended release tablets 30 mg
(Matrix technology)
Sr.
No.
Ingredients Specification B. No.
S07/10
B. No.
S09/10
mg/tablet
1 Gliclazide Ph. Eur. 30 30
2 Dicalcium phosphate, anhydrous Ph. Eur. 50 50
3 Maltodextrin Ph. Eur. 57 57
4 Hydroxypropylmethyl cellulose
(HPMC K100 LV)
Ph. Eur. 44 44
5 Hydroxypropylmethyl cellulose
(HPMC K 4 M)
Ph. Eur. 15 15
6 Magnesium stearate Ph. Eur. 2.0 2.0
Lubrication
7 Magnesium stearate Ph. Eur. 2.0 2.0
Average wt. (mg) 200 200
5.3.2.7 Scale up studies
Scale up studies is an important step towards extrapolating the lab scale experiments to
production scale. This study is an important part of product development and a formulation is
considered robust only after successful scale up.
Since formulation with combination of HPMC in concentration of 29.5% with dibasic
calcium phosphate, maltodextrin and magnesium stearate was found to give satisfactory
results in terms of physical and chemical parameters, the same composition was finalised for
scale up studies to understand the feasibility of scale up of the proposed composition.
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SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 125
The earlier optimised batches were of 1000 tablets each. The scale up batch size was of
10,000 tablets. Since the process of tablet manufacturing is slugging/deslugging, the
equipment capacity was not a constraint in deciding the batch size.
5.3.2.7.1 Cleaning of the equipments (GMP batch for bioequivalence studies)
In line with the compliance to good development practises, complete control on GLP‟s and
GMP‟s has been maintained. Since the research work has been conducted in the
pharmaceutical company indulging in the development and manufacturing of products for
highly regulated markets, the SOP‟s are in force to ensure proper cleaning before any new
work is initiated.
The equipments used for the manufacture of the batch to be used specifically for
bioequivalence studies included – Sieve no. 20 and 40, container blender 5 ltrs., multimill
with 8.00 mm and 1.5 mm screens, tablet compression machine 20 station and blister packing
machine. All these were cleaned as per SOP‟s applicable.
The previous product handled using these equipment was Pioglitazone Hydrochloride tablets.
As per the MACO calculation (Maximum allowable carryover) the permissible content is
44mg/batch which is calculated using safety factor i.e. NMT 1/1000th
part of product A
presence allowable to Product B and using 10 ppm criteria.
Then Pioglitazone hydrochloride content was determined by HPLC method. The analysis of
rinsing revealed the levels of Pioglitazone hydrochloride below the detection limits.
Scale up batch : B.No. S10/10 was scaled up with a batch size of 10,000 tablets
Table 5.16: Composition of scale up batch of gliclazide ER tablets
Sr. No. Ingredients Spec. mg/tablet Qty in gms. /
10,000 tablets
1 Gliclazide Ph. Eur. 30 300
2 Dicalcium phosphate,
anhydrous
Ph. Eur. 50 500
3 Maltodextrin Ph. Eur. 57 570
4 Hydroxypropylmethyl cellulose
(HPMC K100 LV)
Ph. Eur. 44 440
5 Hydroxypropylmethyl cellulose
(HPMC K 4 M)
Ph. Eur. 15 150
6 Magnesium stearate Ph. Eur. 2.0 20
Lubrication
7 Magnesium stearate Ph. Eur. 2.0 20
Average wt. 200 mg 2000 gms.
Method of preparation- It is the same as mentioned earlier section.
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5.3.2.7.2 Critical processing steps: The following process steps and their respective process parameters have been indentified to be critical during
manufacturing of gliclazide ER tablets 30 mg.
Table 5.17: Critical processing steps during manufacturing
Process step Critical parameters Preventive action Testing during scale up
Dry blending (Initial) Blender: Blending time and blender speed
Parameters recorded: Blending time and blender speed
Unit dose sample taken after blending for determination of content uniformity of gliclazide
incase of failed content uniformity in final blend. Sizing of slugs Sifter and Multimill:
Sieve and Screen size
Position of blade
Speed of multimill
Parameters recorded:
Sieve and screen size
Position & Speed of Multimill
Nil
Blending (Final granules)
Mixing time
Blender speed
Parameters recorded:
Mixing time
RPM of blender
Blend uniformity
Sieve analysis
Bulk and tapped density
Assay
Compression Tablet press: Feeding of final bend
Demixing
Incomplete die filling Compression parameters:
Resistance to crushing
Sticking /capping
Insufficient physiochemical properties of the tablets
Preset compression parameters:
Resistance to crushing
Compression
speed In-process control of tablets:
Appearance
Weight of 20 tablets
Average mass
Thickness
Resistance to crushing
Friability
Machine rpm
Appearance
Weight of 20 tablets
Average mass
Thickness
Resistance to crushing
Friability
Dissolution profile
Assay
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SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 127
Table 5.18: Sampling plan for the scale up batch according to ICH guidelines
Stage Test parameter Specifications Sample qty.
Gliclazide Bulk density
Particle size Distribution
For data
d(0.9)- less than 30 microns
50 gm
50 gm
Dry mixing/
Blending
Uniformity of mixed
Blend
To be analyzed in case the
content uniformity of final blend
fails.
1X -3X (X- is qty. equivalent
to one dose)
Blending Blend uniformity of final blend
Bulk and tapped density
Sieve analysis
Assay
LOD
90 – 110% of labeled amount of
gliclazide
1X -3X (X- is qty. equivalent
to one dose)
Compression High, low and optimum resistance to
crushing:
Appearance
Thickness
Resistance to crushing
Friability
Dissolution profile
High and low speed
For data generation 50 tablets each
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5.3.2.8 Effect of alkaline excipient on the dissolution profile of gliclazide extended
release tablets
Gliclazide is a second generation sulfonylurea which acts as insulin secretion enhancer used
in the treatment of diabetes mellitus. The major drawback in the therapeutic application and
efficacy of gliclazide as oral dosage form is its very low aqueous solubility because of its
hydrophobic nature. It is characterized by low dissolution rate in purified water. Because of
these reasons, its in-vitro dissolution profile shows intra tablet release variations which
ultimately leads to the inter individual variations in its bioavailability, therefore poses
problems in design of extended release tablets.
Gliclazide being hydrophobic weak acid is insoluble in water and acidic pH and soluble
towards neutral to alkaline pH 6-8. The solubility increases with the increase in pH. Various
techniques have been adopted to improve the dissolution profile variability. During
development when lactose monohydrate was used as the filler in the tablets, dissolution
profile of the tablets showed intra tablets variability. Hence to overcome this problem,
experiments were carried out using alkaline excipient in the formulation. The major objective
of this study was to prepare Gliclazide extended release tablets with matrix technology, to
reduce the intra tablets variation in drug release profile
Formulation of oral extended release tablets
Extended release tablets containing 30 mg of Gliclazide were formulated with varied
compositions as listed in Table 5.19, Gliclazide and excipients were mixed and passed
through 40 mesh screen. The blend was processed using conventional non aqueous
granulation method by incorporating combination of hydroxypropylmethyl cellulose (HPMC
K4 M and HPMC K100LV), lactose monohydrate, colloidal silicon dioxide and magnesium
stearate and alkaline excipients used were light magnesium carbonate and sodium hydrogen
phosphate. Tablets were also formulated using Dibasic calcium phosphate which is water
insoluble and has alkaline pH.
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 129
Table 5.19: Formulations of Gliclazide extended release tablets with alkaline excipient
Ingredients B. No. T1 B. No. T2 B. No. T3 B. No. T4
% quantity
Gliclazide 15 15 15 15
HPMC K4M 7.5 7.5 7.5 7.5
HPMC K 100 LV 22 22 22 22
Light magnesium carbonate 1.25 2.5
Lactose monohydrate 23.75 25 18.0
Maltodextrin 28.5 28.5 28.5 28.5
Dibasic calcium phosphate 22.5
Sodium hydrogen carbonate 7
Magnesium stearate 2 2 2 2
Chapter 5 Experimental
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5.4 Gliclazide extended release tablet by hot melt granulation technology Process
flow chart
Figure 5.10: Process flow chart of gliclazide ER tablets by Hot melt granulation technology
Figure 5.11: Gliclazide ER Tablets by melt granulation
Chapter 5 Experimental work
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS , Mumbai 131
5.4.1 Gliclazide extended release tablets development with hot melt granulation technology
Table 5.20(A): Formulation composition of gliclazide extended release tablets - Hot melt granulation
B. size – 1000 tablets
Sr. No.
Ingredients Spec. B. No.
M01/09
B. No.
M02/09
B. No.
M03/09
B. No.
M04/09
B. No.
M05/09
B. No.
M06/09
B. No.
M07/10
B. No.
M08/10
B. No.
M09/10
B. No.
M10/10
B. No.
M11/10
mg/tablet
1 Gliclazide Ph. Eur. 30 30 30 30 30 30 30 30 30 30 30
2 Hydrogenated castor oil Ph. Eur. 30 32
3 Stearic acid Ph. Eur.
10
4 Polyethylene glycol 8000 Ph. Eur.
30 40 40 40 30 30 30 30 30
5 Dicalcium phosphate, anhydrous
Ph. Eur.
65 57
52 30 35 27.5
6 Lactose monohydrate Ph. Eur.
57 57 52 52
7 Maltodextrin Ph. Eur.
30 30 30 30 15 15 15 15 15 15
Lubrication
8 HPMC 100 LV Ph. Eur.
20 30 30 30 25 30
9 HPMC K 4 M Ph. Eur.
20 10 10
10 Dicalcium phosphate,
anhydrous Ph. Eur.
97
22 22 22 22
11 Colloidal anhydrous
silica Ph. Eur.
1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5
12 Magnesium stearate Ph. Eur. 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5
Average wt. 160 mg 160 mg 160 mg 160 mg 160 mg 160 mg 160 mg 160 mg 160 mg 160 mg 160 mg
Chapter 5 Experimental
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Table 5.20(B): Formulation composition of gliclazide extended release tablets – Hot melt granulation
Sr.
No.
Ingredients Spec. B. No.
M12/10
B. No.
M13/10
B. No.
M14/10
B. No.
M15/10
B. No.
M16/10
B. No.
M17/10
B. No.
M18/10
B. No.
M19/10
B. No.
M20/10
B. No.
M21/10
mg/tablet
1 Gliclazide Ph.Eur. 30 30 30 30 30 30 30 30 30 30
2 Polyethylene glycol
8000
Ph.Eur. 30 30 30 30 30 30 30 30 30 30
3 Dicalcium
phosphate,
anhydrous
Ph.Eur. 33.5 32.5 44.5 33.5 12 33.5 32 32 32.5 32 .5
4 Maltodextrin Ph.Eur. 15 15 15 15 15 15 15 15 15 15
5 HPMC K 15 M Ph.Eur. 16.5
6 Light magnesium
carbonate
Ph.Eur. 2.5 2.5 5 5 5 5 5 5
Lubrication
7 HPMC K 100 LV Ph.Eur.
8 HPMC K 4 M Ph.Eur. 25 15 25 45 20 25 25 25 25
9 Dicalcium
phosphate,
anhydrous
Ph.Eur. 32 22 20 22 20 22 20 20 22 22
10 Colloidal anhydrous
silica
Ph.Eur. 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5
11 Magnesium stearate Ph.Eur. 1.5 1.5 1.5 3.0 1.5 3.0 1.5 1.5 1.5 1.5
Average wt. 160 mg 160 mg 160 mg 160 mg 160 mg 160 mg 160 mg 160 mg 160 mg 160 mg
Chapter 5 Experimental
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Method of tablet preparation
Gliclazide, Dicalcium phosphate anhydrous and maltodextrin were sifted through # 20 mesh
sieve. The blend was transferred to rapid mixer granulator and mixed for 10 minutes at 125
rpm. Polyethylene glycol 8000 was melted in a beaker at 50-60oC. The above blend was
granulated using the binder in rapid mixer granulator for 5 minutes at 125 rpm. After
granulation allow the blend to cool to room temperature. Blend is then sized using multimill
with 1.5 mm screen followed by 1.0 mm screen. The sized granules were then sifted through
#20 mesh sieve. The granules were lubricated in the container blender with
Hydroxypropylmethyl cellulose, Dibasic calcium phosphate and colloidal anhydrous silica
(presifted through # 30 mesh sieve) for 15 minutes followed by magnesium stearate (presifted
through # 30 mesh sieve) for 2 minutes. The lubricated blend was compressed on 20 station
rotary tablet press machine using 7.00 mm round flat punches plain on both sides at the
machine rpm of 25- 30 and hardness of 60 -70 N.
1.4.1 Evaluation of gliclazide ER tablets
Gliclazide ER tablets made by hot melt granulation technology were evaluated for the
parameters as mentioned in the earlier section.
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1.5 Gliclazide extended release tablets by Multiparticulate unit dose technology
Process flow chart – Pellets coating and final tablets
Figure 5.12: Process flow chart- extruded pellets and ER coating
Figure 5.13: Coated pellets to final tablets
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 135
5.5.1 Formulation of gliclazide ER tablets using multiparticulate unit dose technology
Formulation development of gliclazide extended release tablets by multiparticulate unit dose
technology was started with the development of gliclazide extended release matrix pellets. B.
No. P01/10 was prepared with a combination of diluents microcrystalline cellulose and
Dicalcium phosphate and Eudragit dispersion as binder. Due to presence of dicalcium
phosphate, extrudes were found to be brittle without elasticity which on spheronization gave
pellets of very low particle size. The extrusion screen used was #8 screen and spheronisation
plate was no. 2. The speed of the spheronisation plate was optimized to get the desired sized
pellets. The next batches P02/10 and P03/10 were prepared using microcrystalline cellulose as
the diluents and Eudragit dispersion as the binder. This batch also resulted in the pellets with
very small size. Dissolution was performed which showed almost 100% drug release in two
hours.
Based on the results, it was decided to prepare gliclazide immediate release pellets and
coating the pellets suitably to achieve the desired drug release profile. Further batches were
prepared as B.No. P04/10 with microcrystalline cellulose and hydroxypropyl cellulose low
substituted. The concentration of binder used was 6%. Extrudes obtained were very hard. Due
to higher binder quantity extrudes when spheronized, resulted in the dumbbells and larger
sized pellets on spheronisation. Further batches were prepared by reducing the binder
quantity, B.No. P06/10 with 3% binder gave immediate release pellets with satisfactory
physical parameters. Reproducible batches were taken to confirm the results.
Next step was to coat pellets to retard the drug release from pellets. Hydrophobic polymer
ethyl cellulose with the viscosity grade of 7cps and 20 cps were used with non aqueous
solvents such as dichloromethane and isopropyl alcohol in the ratio of 40:60 concentration.
Initial experiments showed dissolution profile comparative to innovator profile at the later
time points. Main problem was to match the initial release profile.
Functional coating containing eudragit NE 30 D with talc and magnesium stearate was
studied. Aqueous dispersion containing magnesium stearate and talc was made by
homogenization. To this mixture eudragit NE 30 D was added, mixed and used as coating
dispersion. Pellets were coated in fluidized bed processor with inlet temperature of 50-60oC
and product temperature of 40-45oC. Dispersion was kept under continuous stirring for
uniformity. As the dissolution profile obtained was not satisfactory for the batches prepared
using these ingredients, the experiments were not continued further.
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 136
To match the dissolution release profile further trials were continued using ethyl cellulose as
release retardant. Ethyl cellulose with the viscosity of 7 and 20 cps were used. Non-aqueous
solvent system was used for ethyl cellulose polymer. The solvents used were dichloromethane
and isopropyl alcohol in the ratio 40:60. The experiment with 7cps viscosity grade showed the
initial dissolution profile faster than the Diamicron MR tablet. Experiments were carried out
to match the initial dissolution profile. Experimental B. No. P08/10 was conducted with
coarser grade of gliclazide (d0.9 -110 microns). This experiment was taken considering that
coarser grade of API should show slow dissolution. But no improvement in the dissolution
profile was observed and hence the experiments were continued further with the finer grade
of the drug with polymer coating.
It was decided to base coat the pellets so as to get smooth base and which may also show
improvement in the initial drug release profile. For this, base coating of
hydroxypropylmethyl cellulose (Opadry white) was given to the pellets and further coating
with the polymer for drug retardation. This experiment showed dissolution release profile
comparable to Diamicron MR tablet profile. The final batch B.No.9/10 was prepared with
combination of microcrystalline cellulose and maltodextrin as diluent and hydroxypropyl
cellulose as binder. The blend was granulated, further extruded and spheronised which gave
the desired shape and size of pellets. The pellets were sized through #18 mesh sieve and
pellets retained on #24 mesh sieve were taken for polymer coating. Sized pellets were coated
using opadry as the base coat and ethyl cellulose 7 cps as the polymer coat. Pellets were
further compressed into tablets. For this microcrystalline cellulose was used as diluent. The
quantity used was double the quantity of the pellets. Disintegrant crospovidone was added to
facilitate the immediate disintegration of the tablet so as to release the coated pellets to give
the extended release drug profile. Magnesium stearate was used as lubricant.
Gliclazide extended release tablets with pellets showed slightly dappled appearance due to the
presence of pellets in the blend for compression. Thus for asthetic appeal the tablets were
further coated with non functional coating that is opadry white with HPMC as the polymer
and distilled water was used as the medium for coating dispersion. The composition of
various batches prepared is listed in table 5.21(A, B, C).
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 138
Gliclazide extended release coated pellets
Figure 5.14: Gliclazide coated pellets
Figure 5.15: Gliclazide extended release tablet in pH 6.8 phosphate buffer turning into loose coated pellets
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 139
Table 5.21(A-1): Composition of the gliclazide pellets and extrusion parameters
Sr.
No.
Ingredients Spec. B.No.
P01/10
B.No.
P02/10
B.No.
P03/10
B.No.
P04/10
B.No.
P05/10
B.No.
P06/10
B.No.
P07/10
mg / qty. of pellets per tablet
1 Gliclazide Ph.Eur. 30 30 30 30 30 30 30
2 Microcrystalline cellulose Ph.Eur. 47.5 52 55 49 51.5 52 52
3 Maltodextrin Ph.Eur. 15 15 15 15 15 15
4 Dicalcium phosphate Ph.Eur. 47.5
5 Eudragit RS PO Int. Spec. 9 33
6 Eudragit N 30 D Int. Spec. 100
(30)
7 Hydroxypropyl cellulose Ph.Eur. 6 4.5 3 3
Distilled water Ph.Eur. 30 ml 30 ml 70 60 60 60
Wt. of pellets/tablet (mg) 134 130 130 100 100 100 100
Equipment process parameters for extrusion and spheronization
Extrusion screen 0.8 0.8 0.8 0.8 0.8 0.8 0.8
Extrusion speed (rpm) 40-45 40-45 40-45 40-45 40-45 40-45 40-45
Spheronisation plate (mm) 2 2 2 2 2 2 2
Spheronisation speed
(rpm)
1000 1000 1200 1250 1200- 1min
1350-1min
1200- 1min
1350-1min
1200- 1min.
1350-1 min.
Spheronisation time 30 sec. 30 sec. 1.0 min 1.5 min 2.0 min 2.0 min 2.0 min
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 139
Table 5.21 (A- 2): Composition of the gliclazide pellets and extrusion parameters
Sr.
No.
Ingredients Spec.
B.No. P08/10
(d0.9 -110.87
µ)
B.No.
P09/10
B.No.
P10/10
B.No.
P11/10
mg/100 mg of pellets
1 Gliclazide Ph.Eur. 30 30 30 30
2 Microcrystalline cellulose Ph.Eur 52 52 52 52
3 Maltodextrin Ph.Eur 15 15 15 15
4 Hydroxypropyl cellulose Ph.Eur 3 3 3 3
Distilled water Ph.Eur 60 60 60 60
Wt. of pellets/tablet (mg) 100 100 100 100
Equipment process parameters for extrusion and spheronization
Extrusion screen 0.8 0.8 0.8 0.8
Extrusion speed (rpm) 40-45 40-45 40-45 40-45
Spheronisation plate (mm) 2 2 2 2
Spheronisation speed (rpm) 1200- 1min.
1350-1 min.
1200- 1min.
1350-1 min.
1200- 1min.
1350-1 min.
1200- 1min.
1350-1 min.
Spheronisation time 2.0 min 2.0 min 2.0 min 2.0 min
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 140
Table 5.21(B-1): Composition of the gliclazide pellets extended release coating
Table 5.21(B-2): Composition of the gliclazide pellets extended release coating
Sr. No. Ingredients B.No. P07/10C B.No. P08/10 B.No. P09/10 B.No. P10/10 B.No.P11/10
Coating mg/ qty. of pellets per tablet
1 Base coating – Opadry White 10 10 10 10 10
2 Ethyl cellulose 7 cps 10 10 10 10 10
3 Ethyl cellulose 20 cps
4 Dichloromethane and Isopropyl alcohol (40: 60)
q.s q.s q.s q.s q.s
Average wt. of the pellets after coating
120 mg 120 mg 120 mg 120 mg 120 mg
Sr. No.
Ingredients B.No.
P01/10 A B.No. P02/10 A
B.No. P03/10 A
B.No. P04/10 A
B.No. P05/10 A
B.No. P06/10 A
B.No. P06/10 B
B.No. P06/10 C
B.No. P07/10A
B.No. P07/10B
Coating mg/ qty. of pellets per tablet
1 Ethyl cellulose 7 cps As the binder was used for the drug retardation, Coating was not done on the following batches
Extrudes obtained were very hard, when
spheronized resulted in hard and bigger sized pellets. Coating was not done on following batches.
5 10
2 Ethyl cellulose 20 cps 5 7
Dichloromethane and
Isopropyl alcohol (40: 60)
q.s q.s q.s
3 Eudragit NE 30 D + Mg stearate + Talc + Purified water
10
Average wt. of pellets after coating (mg)
105 105 107 110 110
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SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 141
Table 5.21(C): Composition of the gliclazide extended release tablets
Sr.
No.
Ingredients B.No.
P06/10 A
B.No.
P06/10 B
B.No.
P06/10 C
B.No.
P07/10A
B.No.
P07/10B
B.No.
P07/10C
B.No.
P08/10
B.No.
P09/10
B.No.
P10/10
B.No.
P11/10
Tabletting
1 Pellets (mg) 100 100 100 100 100 100 100 100 100 100
Avg. wt. pellets
with coating
105 105 107 110 110 120 120 120 120 120
2 Microcrystalline
cellulose
(Avicel PH 102)
233 233 231 228 228 218 218 218 218 218
3 Crospovidone 10 10 10 10 10 10 10 10 10 10
4 Magnesium stearate 2 2 2 2 2 2 2 2 2 2
Average wt.
tablet (mg)
350 350 350 350 350 350 350 350 350 350
Coating
5 Opadry white 10 10
Final average weight 360
mg
360
mg
B.No. P01/10 to P05/10 was not compressed as tablets, as the desired physical parameters of the pellets could not be achieved.
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 142
Method of preparation
Gliclazide, Microcrystalline cellulose and maltodextrin were sifted through # 20 mesh sieve.
The blend was transferred to rapid mixer granulator and mixed for 10 minutes at 125 rpm.
Purified water was transferred to a s.s vessel and hydroxypropyl cellulose was added. Stirring
was continued to get clear solution. Blend was granulated using the binder in Rapid mixer
granulator for 5 minutes at 125 rpm. Granulated blend was then extruded using # 8 screen of
the extruder at 40 -50 rpm and spheronized using plate no.2, to achieve required shape and
size of pellets. Pellets were dried in rapid dryer (Retsch dryer) at temperature of 50 - 60oC
with fluidisation of 30 - 40 cfm. Dried pellets were sifted through #18 mesh sieve followed by
# 24 mesh sieve. Pellets retained on the # 24 mesh sieve were used for coating.
Base coating dispersion and coating: Dichloromethane and Isopropyl alcohol were
transferred to a s.s vessel. To it opadry white was added slowly, stirred for 30 minutes to get
clear dispersion. Sized pellets were then coated using coating dispersion in fluidised bed
processor, with inlet temperature of 40 - 45oC, fluidisation of 30-40 and product temperature
of 35-40oC. Coated pellets were dried for 10 minutes after achieving the weight gain. Pellets
were sifted through #18 mesh sieve.
Extended release coating dispersion and coating: Dichloromethane and Isopropyl alcohol
were transferred to a s.s vessel. To it ethyl cellulose was added slowly, stirred for 30 minutes
to get clear dispersion.
Sized pellets were then coated using coating dispersion in fluidised bed processor, with inlet
temperature of 40 - 45oC, fluidisation of 30-40 cfm and product temperature of 35-40
oC.
Coated pellets were dried for 10 minutes after achieving weight gain. Pellets were sifted
through #18 mesh sieve. Coated pellets were lubricated in the container blender with
microcrystalline cellulose and crospovidone (presifted through # 30 mesh sieve) for 15
minutes followed by magnesium stearate (presifted through # 30 mesh sieve) for 2 minutes.
Lubricated blend was compressed on 20 station rotary tablet press machine using 10.00 mm
round biconvex punches plain on both sides at the machine rpm of 25- 30 and hardness of 50 -
70 N.
Final coating dispersion and coating: Purified water was added to s.s vessel. To it opadry
was added slowly, stirred for 30 minutes to get clear dispersion.
Core tablets were then coated using the coating dispersion in Neocota coating pan, with inlet
temperature of 50 - 60oC, pan rpm of 6-7 and product temperature of 40 - 50
oC. Coated tablets
were dried for 10 minutes after achieving the weight gain.
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 143
Table 5.22: Final composition of gliclazide extended release tablets by pellet technology
Sr. No. Ingredients mg/tablet,
B.No 10/10
Gliclazide pellets
1 Gliclazide 30.0
2 Microcrystalline cellulose 45
3 Maltodextrin 15
4 Hydroxypropyl cellulose 3
5 Purified water q.s
Average weight of pellets 100 mg
Base coating
6 Opadry white 10
7 Dichloromethane q.s
8 Isopropyl alcohol q.s
Polymer coating
9 Ethyl cellulose 7 cps 10
10 Dichloromethane q.s
11 Isopropyl alcohol q.s
Average wt. of coated pellets 120 mg
Tablet blend
12 Coated Pellets 120
13 Microcrystalline cellulose (Avicel
PH 102) 218
14 Crospovidone 10
15 Magnesium stearate 2
Average wt. tablet (mg) 350 mg
Final coating
Opadry white 10
Average wt. of the tablets 360 mg
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 144
5.6 Stability studies
Stability testing is an integral part of the pharmaceutical product development process. It is
routinely performed on the drug substance and the drug products. The primary purpose of
stability testing in an industrial set up is to provide supporting evidence on stability behaviour
of chemical or biological entities as well as to study the stability behaviour of pharmaceutical
products. Stability testing is function of time against environmental factors on an otherwise
satisfactory product. Stability can be defined as the extent to which a product retains, within
specified limits and throughout its period of storage and use, the same properties and
characteristics (i.e. chemical, physical, microbiological and biopharmaceutical) at the time of
its manufacture. The purpose of stability studies is to gather evidence on the variation in
quality of the drug substance or drug product with time under the influence of a number of
environmental factors, such as temperature, humidity and light, and to assign a retest period
for the drug substance or a shelf life for the drug product and recommended storage
conditions.
Stability testing is conducted so as to determine storage conditions, retest period, expiry dates
and shelf life of pharmaceutical products. Primary stability studies are intended to show that
the drug product stored in the proposed container/closure will remain within specifications if
stored under the conditions that support the proposed shelf life.
The ICH guidelines provides a general indication on the requirement for stability testing
leaving sufficient flexibility to encompass a variety of different practical situations required
for specific situations and characteristics of the materials being evaluated. According to the
ICH guidelines which are also adopted by practically all the regulatory bodies around the
globe, accelerated testing for 6 months should be carried out at a temperature at least 15oC
above the designated long term testing storage temperature combined with appropriate
humidity conditions for that temperature. Long term testing should be done for a minimum
period of 12 months. For instance, if a product needs a long term storage under refrigerated
condition, accelerated testing should be conducted at 25o± 2
oC/60% RH. The designated long
term testing conditions will be reflected in the labelling and expiration date. For products that
require storage at 25o± 2
oC/60% RH, an accelerated testing at 40
o± 2
oC/75% RH for 6 months
must be conducted. Where “significant change” occurs during 6 months storage under
condition of accelerated stability testing at 40o± 2
oC/75% RH, an additional testing at
intermediate condition such as 30o± 2
oC/65% RH should be conducted for 12 months period
(Stability testing of active substances and pharmaceutical products).
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 145
In the present work, optimised and scaled up formulation made by matrix technology, hot
melt granulation technology and pellets technology were subjected to the following storage
conditions as recommended in the ICH guidelines.
Samples were refrigerated as control (5oC ± 3
oC) for 12 months.
25o ± 2
oC/60% ± 5% RH for 24 months, Long term stability condition
30o ± 2
oC/65% ± 5% RH for 12 months, Intermediate stability condition
40o ± 2
oC/75% ± 5% RH for 6 months, Accelerated stability condition
Pack profiles
Stability for the final formulations made by the three technologies were tested in the
following pack profiles
Alu/Alu blisters
PVC/PVdC blisters
30 cc HDPE bottles with CRC cap and induction sealing
5.6.1 Gliclazide extended release tablets 30 mg by matrix technology
The optimised and scaled up formulation B.No.S10/10 was chosen for the stability studies as
per the ICH guidelines. The tablets were evaluated for parameters like appearance, drug
content, related substances and dissolution profile. Tablets were additionally tested for the
moisture content. Formulation was also exposed to 60oC for one week and was checked for
any change in the XRD graph pattern. Formulation was packed in PVC/PVdC blisters,
Alu/Alu blister pack and HDPE bottles 30cc containing one silica sachet with induction
sealing with CRC cap. B.No.S 10/10 was packed in Alu/Alu Blister and batch labelled as
B.No. S10/10 A was packed in PVC/PVdC blister. Samples were analysed for all the
parameters above mentioned at timepoints 1, 2, 3, and 6 months at accelerated conditions of
40oC/75% RH and 3, 6, 12 and 24 months at long term conditions.
Gliclazide ER tablets labelled as B.No.S10/10 B were packed in HDPE bottles with silica bag
and induction sealing and the samples were analysed at 1, 3, 6 months at accelerated
conditions of 40oC/75% RH and 3, 6 and 12 months at long term conditions.
Formulation B. No. S10/10A packed in PVC/PVDC blister showed a faster drug release at
one months 40oC/75% RH stability condition when compared to the initial dissolution profile
of the formulation. However, formulation packed in Alu/Alu blisters showed a comparative
dissolution profile as the initial. Therefore the formulation stability study in PVC/PVDC pack
was discontinued.
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 146
Alu/Alu blister was chosen as the pack of choice for the formulations made by other
technologies also. Formulation packed in HDPE bottles with silica sachet, with induction
sealing and CRC caps, showed satisfactory results.
5.6.2 Gliclazide extended release tablets 30 mg by melt granulation technology
Optimised and scaled up formulation B.No.M21/10 was packed in Alu/Alu blisters for
stability studies as per the ICH guidelines. Tablets were evaluated for parameters like
appearance, drug content, related substances and dissolution profile. Formulation was also
exposed at 60oC for one week and was checked for any change in the XRD pattern.
5.6.3 Gliclazide extended release tablets 30 mg by multiparticulate unit dosage
technology
Optimised and scaled up formulation B.No.P10A/10 was packed in Alu/Alu blister for
stability studies as per the ICH guidelines. Tablets were evaluated for parameters like
appearance, drug content, related substances and dissolution profile. Formulation was kept at
60oC for one week and was checked for any change in the XRD pattern.
5.7 In-vivo Studies
Gliclazide extended release tablets were developed using three different technologies. The
technologies employed were matrix technology using combination of HPMC as the rate
modifying polymer, hot melt granulation technology using combination of polyethylene
glycol 8000 and HPMC as the drug rate modifier and pellet technologies using ethyl cellulose
as functional coating. Out of three techniques used, it was found that ease, reproducibility,
convenience, cost were best with the product made with matrix technology. This turned out to
be the best amongst all the techniques tried. From the point of view of scale- up, commercial
and the formulation complexities, this technique was chosen to develop a patent non –
infringing product.
In the development of any generic product, the main criterion for the product is that the
generic drug has to pass limited clinical trials in the form of comparative bioequivalence
study. For this the extended release products with similar form of drug release study is the
ideal way of selection of the formulation. Based on these criteria, formulation made by matrix
technology was the product of choice for the bioequivalence studies as the drug release for
innovator as well as the formulation by matrix technology are by erosion.
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 147
5.7.1 Study title
A balanced, open label, analyst blind, single centre, two treatment, two Period, two sequence,
comparative bioavailability study of “Gliclazide ER Tablets 30 mg” of SPP School of
Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai with “Diamicron MR
tablet 30 mg” (containing Gliclazide 30 mg) of Servier Laboratories Ltd., France in healthy,
adult, male, human subjects under fasting conditions in a randomized, crossover design.
Table 5.23: Table of contents for bioequivalence studies
Test Reference
Gliclazide ER tablets 30mg
Mfg. by: School of
Pharmacy & Technology
Management, SVKM‟s
NMIMS, Mumbai
DIAMICRON® MR tablets 30 mg
(containing Gliclazide 30 mg)
Mfg. by: Servier Laboratories Ltd.,
France
Study design A balanced, open label, analyst blind, single centre, two treatment, two period,
two sequence, bioequivalence study in healthy, adult, male, human subjects under fasting
conditions in a randomized, crossover design.
Study Code S087/10/GLIC Protocol No. DMRI-PR10-205-GLIC
Phase of Study Bioequivalence study Version 00,
Study Initiation Date 27/09/10 Date 06/09/10
Date of Report 24/10/10 Study Completion Date 23/10/10
Sponsor’s Representative Principal Investigator
Ms. Monika Srivastav Dr. Prashant Bodhe
SPP School of Pharmacy & Technology Drug Monitoring Research Institute
Management, R-374, MIDC, TTC Industrial Area
SVKM‟s NMIMS, Rabale, Navi Mumbai – 400 701
Vile Parle (W), Mumbai – 400 056 Maharashtra, India
Table 5.24: Declaration
Investigators' Declaration
We, the undersigned, have read and understood this report and hereby assure that the
study was conducted in accordance with the approved protocol (DMRI-PR10-205-
GLIC, Version 00, Dated: 06/09/10) and in compliance with all the requirements
regarding the obligations of investigators and all other pertinent requirements of the
ICH (Step 5) 'Guidance on Good Clinical Practices & Good Laboratory Practices'.
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 148
Principal Investigator QA, Head
Name Dr. Prashant Bodhe Mr. Nelson Varghese
Signature
Table 5.25: Synopsis
Title of the
study
A Balanced, Open Label, Analyst Blind, Single Centre, Two
Treatment, Two Period, Two Sequence, Comparative Bioavailability
Study of “Gliclazide ER Tablets 30 mg” of School of Pharmacy &
Technology Management, SVKM‟s NMIMS, Mumbai with
“Diamicron MR tablet 30mg” (containing Gliclazide 30mg) of
Servier Laboratories Ltd., France in healthy, adult, male, human
subjects under fasting conditions in a randomized, crossover design.
Food does not have any effect on the absorption of gliclazide,
considering this the bioequivalence a study was done as fasted
studies.
Study center Clinical, Bioanalytical, Statistical and QA Services Drug Monitoring Research Institute
R-374, MIDC, TTC Industrial Area, Rabale,
Navi Mumbai – 400 701
Tel: +91– 22 – 2764 2746/47, Fax: +91– 22–2764 2748
Principal
Investigator Dr. Prashant Bodhe
Study period
Study initiation date (check-in for period I) 27/09/10
Bioanalysis initiation date 18/10/10
Statistical analysis initiation date 23/10/10
Study completion date 23/10/10
Phase of development Bioequivalence study
Objectives To assess the oral bioavailability of Gliclazide ER tablets 30mg of
School of Pharmacy & Technology Management, SVKM‟s NMIMS,
Mumbai with “Diamicron MR tablet 30 mg” (containing Gliclazide
30mg) of Servier Laboratories Ltd., France in 12 healthy, adult, male,
human subjects under fasting condition.
Study design To monitor the safety and tolerability of a single dose of the test
product as compared to the reference product in healthy, male,
human subjects.
A balanced, open label, analyst blind, single centre, two treatment,
two period, two sequence, bioequivalence study in healthy, adult,
male, human subjects under fasting conditions in a randomized,
crossover design.
Number of
subjects
12 subjects were planned as per the protocol. 12 subjects completed
both the periods of the study and included in the bioanalytical and
statistical analysis.
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 149
Main
inclusion
criteria
Subjects aged between 18 and 45 years (including both) weighing at
least 45 kg with BMI 18.50 – 24.99kg/m2, in normal health as
determined by medical history, clinical examination, laboratory or
other tests.
Exclusion
criteria
Subjects having contraindications or hypersensitivity to
investigational drugs.
Subjects with history of hypoglycemic episodes
History or presence of any medical condition or disease according to
the opinion of physician
Major illness during 03 months before screening
Participation in any clinical study in 03 months before the study
5.7.2 Rationale and aim of the bioequivalence study
Many diseases are still major culprits for morbidity and mortality. We do not have effective
and sufficient drugs to control these diseases. However, to launch new molecules as drugs is
very much costly. Hence, to recover cost of development new molecule is allowed to be
patented for some time. After patent expires, other companies are allowed to launch same
molecule as generic version. To make drug therapy more cost effective, it is necessary to
reduce cost of drug development while taking care of safety and efficacy of drug product.
Thus, generic drugs have to pass limited clinical trial in the form of comparative
bioavailability or bioequivalence study. This fastens the process of development and takes
care of safety issue. This view is also supported by regulatory authorities.
SPP School of Pharmacy and Technology Management, SVKM‟s NMIMS, Mumbai has
developed a generic product of Gliclazide ER tablet 30 mg. Gliclazide is used to control blood
glucose (sugar) in patients with Type II diabetes mellitus.
This study was designed to evaluate the bioequivalence of Gliclazide ER tablets 30 mg of SPP
School of Pharmacy and Technology Management, SVKM‟s NMIMS, Mumbai with
DIAMICRON®
MR tablet 30 mg (containing Gliclazide 30 mg) of Servier Laboratories Ltd.,
France.
Target Population for study - Healthy adult human volunteers.
Treatment - Two
Test Product - Gliclazide ER tablets 30mg
Reference Product - DIAMICRON® MR tablets 30 mg (containing Gliclazide 30 mg)
Duration of clinical study - 19 days
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 150
The study was designed and conducted as per
1. ICMR guidelines
2. Schedule Y 2005 of Drugs & Cosmetics Act and Rules Govt. of India
3. The World Medical Association Declaration of Helsinki, Seoul, 2008.
4. The ICH (Step V), “Guidance on Good Clinical Practice” (GCP).
5.7.5 Study objective
Primary objective
The primary objective of this study was to assess the oral bioavailability of Gliclazide ER
tablets 30 mg of SPP School of Pharmacy and Technology Management, SVKM‟s NMIMS,
Mumbai with DIAMICRON® MR tablets 30 mg (containing Gliclazide 30 mg) of Servier
Labs Pvt. Ltd., France in 12 healthy, adult, male, human subjects under fasting condition.
Secondary objective
The secondary objective was to monitor the safety profile of the test product as compared to
the reference product.
5.7.6 INVESTIGATIONAL PLAN
Overall study design and plan
A balanced, open label, analyst blind, single centre, two treatment, two period, two sequence,
bioequivalence study of Gliclazide ER tablets 30 mg of SPP School of Pharmacy and
Technology Management, SVKM‟s NMIMS, Mumbai with DIAMICRON®
MR tablet 30 mg
(containing Gliclazide 30 mg) of Servier Laboratories Ltd., France in healthy, adult, male,
human subjects under fasting conditions in a randomized, crossover design.
A copy of the approved protocol is attached as appendix.
A copy of the sample case record form is attached as appendix.
The study was designed with 12 subjects as a single-dose crossover study.
Number of subjects
12 subjects enrolled in the study as per the IEC approved protocol. 12 subjects completed
both the periods of the study and included in the bioanalytical and statistical analysis.
Duration of study
All subjects underwent a screening procedure within 21 days before the first day of their
dosing. Upon entering into the study, the subjects were housed in the clinical facility of DMRI
from-11 hours pre-dose till up to 24 hours post-dose in each period.
Chapter 5 Experimental
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 151
Washout period
A washout period of at least 10 days was kept between the two dosing. As it was more than
five half lives of the drug, it eliminated chances of carry over effects.
Randomization
Administration of test or reference product was decided by the randomization schedule
generated by SAS® software version 9.2. The randomization was balanced. The record of
randomization was kept under controlled access with the in-house statistician and the PI. The
dispensing sheets were kept in the pharmacy under controlled access. The study personnel
involved in dispensing and the PI were accountable for ensuring compliance to randomization
schedule. The randomization schedule was kept ready to be made available to the treating
physician and the Principal Investigator in case of any serious adverse event/s to ascertain the
treatment allocation.
Dose of drug
Following an overnight fast of at least 10 hrs, subjects were scheduled for dosing as per the
randomization in each period. A single dose of the test product; one Gliclazide ER tablet 30
mg was administered orally along with 240 ml of 20% glucose solution in water in sitting
posture. A single dose of the reference product; one DIAMICRON®
MR tablet 30 mg
(containing Gliclazide 30 mg) was administered orally along with 240 ml of 20% glucose
solution in water in sitting posture.
Termination of the study
The sponsor had the right to discontinue the study at any time. The Principal Investigator had
the right to discontinue the study for safety reasons at any time. The IEC had the right to
terminate the study, if there were any major violations of the ethical considerations or due to
any serious adverse event (s). The study was completed as per schedule and approved
protocol by IEC.
5.7.7 Selection of study population
On the following inclusion and exclusion criteria, healthy, adult subjects were selected. For
selection, they had passed through the screening process.
Inclusion criteria
The subjects were included based on the following criteria
1. Healthy males within 18-45 years of age ( both inclusive)
2. Weigh at least 45 kg and BMI in the range of 18.5 – 24.99/m2kg (both inclusive).
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3. Normal health as determined by medical history and clinical examination, laboratory
or other tests (mentioned in sections 8.4.2, 8.4.3) within normal range.
4. Willingness to provide written informed consent to participate in the study, ability to
comprehend the nature and purpose of the study
5. Willingness to comply with the requirement of the protocol including all the
restrictions.
6. Availability of subject for the entire study period
Exclusion criteria
The subjects were excluded based on the following criteria.
1. History of allergy or hypersensitivity to Investigational Product.
2. Subjects with history of hypoglycemic episodes.
3. Abnormalities in vital signs (systolic blood pressure < 90 or > 140 mm Hg or diastolic
blood pressure < 50 or > 90 mm Hg or heart rate < 50 bpm or > 100 bpm) at
screening, at pre-entry and at pre-dose physical examination.
4. Clinically significant cardiovascular, gastrointestinal, liver, renal, pulmonary,
hematological, neurological, endocrinal disease.
5. History of epilepsy or psychiatric disorder.
6. Any illness within 21days or hospitalized or a major illness within the 3 months prior
to the first dosing.
7. Any other clinical condition, which may affect the absorption, distribution,
biotransformation or excretion of the study drug. (e. g. diarrhoea, vomiting in 3 days
prior to or at dosing).
8. Use of any prescribed medication during last two weeks or OTC medical products
during the last one week preceding the first dosing.
9. Participated in any other clinical investigation requiring repeated blood sampling / a
blood donation program / have blood loss of more than 350 ml in the past three
months.
10. History of consumption of alcohol for more than two years & drink more than two
alcoholic drinks per day or consumed alcohol within 48 hours prior to first dosing
[one drink is equal to one unit of alcohol (one glass wine, half pint beer, and one
measure i.e. one fluid ounce of spirit)].
11. Smoke more than 10 cigarettes / day or Unable to abstain from smoking during the
study.
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12. Consumption of products containing xanthine & nicotine within 48 hours before
dosing.
13. Intake of grapefruits or products containing grapefruits within 72 hours prior to
receiving the dose of study medication in each period.
14. An unusual diet, for whatever reason (e. g. low-sodium or high protein) for four weeks
prior to receiving the study medication.
15. Use of any recreational drug or a history of drug addiction.
16. Participation in any clinical study within the past 3 months.
17. History of difficulty in accessibility of veins in arms.
Only the subjects, fulfilling the inclusion and exclusion criteria defined, were enrolled in the
study.
Removal of the subject from assessment
The subjects were free to withdraw from the study at any time without having to give any
reasons thereof. The investigator had right to withdraw a subject from the study for any of the
valid reasons which he would feel appropriate in view of the safety and well-being of subject,
GCP principles or objectives of the project, in particular for:
Any serious side effect
Any abnormal laboratory test considered to be of clinical significance
Any serious protocol violation
Lack of co-operation
Inter-current illness requiring treatment / inter-current surgery
If the subject vomits at or before 2 times median Tmax.
5.7.8 Treatments
Treatments administered
Following an overnight fast of at least 10 hrs, subjects were scheduled for dosing as per the
randomization schedule in each period.
A single dose of the test product; one Gliclazide ER tablet 30 mg was administered orally
along with 240 ml of 20% glucose solution in water in sitting posture.
A single dose of the reference product; one DIAMICRON®
MR tablet 30 mg (containing
Gliclazide 30 mg) was administered orally along with 240 ml of 20% glucose solution in
water in sitting posture.
Subjects were asked to swallow the tablet as whole and not chew or crush it. Details of the
dosing procedure were captured in the respective forms. Compliance for the oral dosing was
assessed by a thorough mouth check using torch and spatula immediately after dosing.
Secondary compliance was assessed by estimation of plasma concentration.
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6 subjects were administered test product and 6 subjects were administered reference product
for period I.
6 subjects were administered reference product and 6 subjects were administered test product
for period II.
Identify of investigational products
Table 5.26: Details of the investigational products administered
to the subjects during the study
Investigational products
Test Product A Gliclazide ER tablets 30 mg
Active ingredient Each extended release tablet contains Gliclazide 30 mg
Manufactured by SPP School of Pharmacy & Technology Management,
SVKM‟s NMIMS, Mumbai
Batch No. S10/10
Date of Manufacture 08/2010
Date of Expiry 07/2012
Dose One tablet
Method of Administration Per oral
Reference Product B DIAMICRON®
MR Tablets 30 mg
Active Ingredient Each modified release tablet contains Gliclazide 30 mg
Manufactured By Servier Laboratories Ltd., France
Batch No. 30044
Date of Manufacturing -
Date of Expiry 07/2011
Dose One tablet
Method of Administration Per oral
Method of assigning subjects to treatment groups
The order of receiving the test and reference products for each subject during each period of
the study was determined using statistical software SAS® 9.2. Equal allocation of treatments
or balanced randomization was ensured. The study personnel involved in dispensing and the
PI were accountable for ensuring compliance to randomization schedule. A copy of
Randomization Sheet is attached as an appendix.
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Selection of dose
Single dose of Gliclazide ER tablet 30mg is the normally accepted dose for bioequivalence
study.
Selection and timing of dose for each subject
All subjects fasted for at least 10 hours (overnight) before the scheduled time of dosing.
Subjects were not allowed to drink water from 1 hour before dosing and until 1 hr post-dose
except 240 mL water at the time of dosing. At all other times drinking water was given ad-
libitum. All subjects were dosed the Investigational Product (test-B or reference-A) in a
sequential order. 12 subjects were dosed from 08:00 to 08:10 hours on 28/09/10 for the
period I and 12 subjects were dosed from 08:00 to 08:10 hours on 12/09/10 for the period II
of the study respectively.
Table 5.27: Details of the dosing schedule for subject no. 01 to 12
Date and time of
drug administration
Sub. No. Period I Period II
28/09/10 12/10/10
01 08.00 08.00
02 08.00 08.00
03 08.02 08.02
04 08.02 08.02
05 08.04 08.04
06 08.04 08.04
07 08.06 08.06
08 08.06 08.06
09 08.08 08.08
10 08.08 08.08
11 08.10 08.10
12 08.10 08.10
Subjects remained in seated position for 2 hours after dosing in each period. Thereafter the
subjects continued to sit or lie down or were allowed to engage only in normal activities while
avoiding severe physical activities. Standard meals were provided at 4, 8, 12 and 24 hours
after dosing. All these meal plans were identical in both the periods.
Blinding
The study was an open-label study in terms of the drug and dose. However, analyst was
blinded for allocation of test and reference. Dispensing record was kept under controlled
access in pharmacy. Randomization schedule was not accessed by bio analysts.
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Prior and concomitant medication procedure
Subjects were instructed not to take any prescribed medications (either prescribed for at least
14 days or OTC for at least 07 days) prior to the dosing in first period and during the study. If
drug other than that specified in the protocol was urgently required during study or in the
washout period, decision to continue or discontinue the subject was taken by the Principal
Investigator and / or the sponsor, and was based on the following:
Safety and well being of subject
Pharmacology and pharmacokinetics of the non-study medication.
Likelihood of a drug interaction, which may affect the pharmacokinetic comparison of
the study medications.
The time of administration of the non-study medication, and likelihood of interference
in bio-analysis.
If any of the subjects had to take any medication during the course of the study, he was
instructed to inform the investigator. All occasions of the drug intake were recorded. No
subjects have taken concomitant medication during study periods.
5.7.9 Pharmacokinetic parameters and safety variables
Pharmacokinetic and safety measurements assessed and flow chart
The following parameters were calculated for each subject-product wise using the non-
compartmental model by using statistical package WinNonlin® 5.2:
Table 5.28: Primary parameters
Primary
Parameters
Cmax Maximum measured concentration of drug in plasma.
AUC0-t Area under the plasma concentration - time curve measured to
the last quantifiable concentration, using the trapezoidal rule.
AUC0-inf
AUC0-t plus additional area extrapolated to infinity, calculated
using the formula AUC0-t + Ct/Kel, where Ct is the last
measurable drug concentration and Kel is the elimination rate
constant.
Table 5.29 Secondary parameters
Secondary Parameters
Tmax - Time to reach the maximum concentration of drug in plasma
Kel
Apparent first – order terminal elimination rate constant
calculated from a semi-log plot of the plasma concentration
versus time curve, using the method of least square regression
t1/2 Terminal half-life as determined by quotient 0.693/Kel
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Auxiliary temperature was measured and recorded at check in, before dosing and at check out.
Sitting blood pressure, radial pulse rate was measured and recorded at check in, before dosing,
at 2, 5, 12 hours post dosing and at check out.
Tests for breath sample for alcohol consumption were done at the check in of each period.
Drugs of abuse were done at the check in of period I. Clinical examination of the subjects was
done at the time of check in and check out at each period. After dosing, adverse event
monitoring was done throughout the study.
Appropriateness of measurements
The blood sampling time points were planned to provide an adequate estimation of Cmax, and
terminal half life. For determining the bioavailability characteristics of the drug and
bioequivalence of the test with reference product, the following measures obtained from the
plasma concentration time profile were compared. Maximum plasma concentration (Cmax),
area under the plasma concentration vs. time curve till last measurable concentration (AUC0-t)
and area under the plasma concentration vs. time curve extrapolated to infinity (AUC0-inf) for
the test and reference products.
The choice of timings for subject-safety measurements (clinical examination and vital signs)
were planned to assess the well being for the subject.
5.7.9.2 Primary pharmacokinetic variables
The pharmacokinetic parameters Cmax, AUC0-t and AUC0-inf were taken as primary efficacy
variables for establishing the bioequivalence of the test product; Gliclazide ER tablets 30 mg of
SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai with the
reference product; Diamicron MR tablet 30 mg (containing Gliclazide 30 mg) of Servier
Laboratories Ltd., France.
5.7.10 Analytical method validation
Plasma samples were analyzed to quantify the concentration of Gliclazide using a validated
LC/MS/MS method. The bioanalytical method was validated at the analytical facility for
sensitivity, specificity, linearity, accuracy and precision (repeatability and reproducibility),
percent recovery and stability of samples (freeze-thaw stability, bench-top stability,
autosampler stability, short-term and long-term stability of stock solution and internal
standard).
The linearity range for the analytical method was 5.0 to 150.0 ng/mL for Gliclazide and the
Limit of quantification was 5.0 ng/mL for Gliclazide. During analysis, standard and quality
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SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 158
control samples were distributed throughout each batch study samples analyzed. The analyst
didn't have access to the randomization schedule as well as the dispensing records. The
method validation report, representative chromatographic data have been detailed in the
Analytical report presented along with the report.
Complete analytical study report and method validation is given in separate report.
5.7.10.1 Data of quality assurance
The study underwent quality assurance inspections at various stages (study initiation phase,
study in process phase and reporting phase) for conformance to the study protocol and the
governing SOPs. QA auditors at the end of the study checked completed CRFs and QA filled
all internal audit observation sheets. A statement that the relevant Standard Operating
Procedures as well as the pertinent requirements of the ICH 'Guidance on Good Clinical
Practices (GCP- Step 5) & Good Laboratories Practices (GLP)' for quality assurance duly
signed by the Quality Assurance person is attached as appendix.
5.7.11 Statistical methods planned in the protocol and determination of sample Size
15.7.11.1 Statistical and analytical plans
Calculation of pharmacokinetic parameters was performed by using the non-compartmental
model by using statistical package WinNonlin® 5.2 or higher version and statistical analysis
for establishing bioequivalence was performed using the statistical package SAS® 9.2. PROC
GLM was used for the estimation of least square mean differences (test-reference) of the test
and reference products on the log-transformed pharmacokinetic parameters Cmax, AUC0-t and
AUC0-inf and the corresponding standard errors of the differences were computed.
Descriptive statistics
The plasma concentrations at each sampling time points and pharmacokinetic parameters
were tabulated for each subject and product combination, together with descriptive statistics
including mean, standard deviation, coefficient of variation, median and range for each
product at each scheduled sampling time point.
Analysis of variance
Analysis of variance (ANOVA) was performed (=0.05) on the log-transformed
pharmacokinetic parameters Cmax, AUC0-t and AUC0-inf. The analysis of variance model
included sequence, subjects nested within sequence, period and treatment as factors. Each
analysis of variance included calculation of least-square means, adjusted differences between
formulation means and the standard error associated with these differences. The significance
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of the sequence effect was tested using the subjects nested within the sequence as the error
term.
Ratio and confidence intervals
Ratio analysis was performed on geometric mean of test and reference for log transformed
data. Consistent with the two one-sided tests for bioequivalence, 90% confidence intervals
were constructed for geometric mean of test and reference of the log-transformed Cmax, AUC0-
t and AUC0-inf.
Sample size
This study was designed with 12 subjects as single-dose, crossover study. 12 subjects were
enrolled and completed both the periods of the study and were included in the bioanalytical
and statistical analysis.
Changes in conduct of the study or planned analyses
There were no changes in the conduct of the study or planned analysis. 12 subjects were
enrolled in the study as planned and 12 subjects completed both the periods of the study and
were included in pharmacokinetic and statistical analysis. There were no protocol deviations
during the study.
5.7.12 Study subjects
Disposition of subjects
29 volunteers were screened out of them 23 passed and 12 volunteers reported to the facility
for study ICF presentation on 27/09/10. 12 healthy, adult, human subjects eligible for
participation were enrolled in the study as per the inclusion and / or exclusion criteria. 12
subjects completed the clinical phase of the study and were included in bioanalytical and
statistical data of the study.
Protocol deviations
No sampling point deviations and missing samples.
5.7.13 Pharmacokinetic evaluation
5.7.13.1 Data sets analyzed
Samples from 12 subjects completing both the periods were analyzed and the data were
included in statistical analysis. All concentration values below the limit of quantitation (BLQ)
were set to "zero" for all pharmacokinetic and statistical calculations.
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5.7.13.2 Demographic and other baseline characteristics
12 subjects were enrolled into the study and their mean age, height, weight and BMI was
27.88 yr, 168.43 cm, 62.83 kg and 22.13 kg/m2 respectively.
All the subjects were normal based on their BMI.
The individual and mean demographic data is tabulated in chapter 6
The mean demographic data of all the subjects who have been included in the study is
tabulated in chapter 6.
5.7.13.3 Measurements of treatment compliance
A thorough check of the oral cavity of the subjects was carried out immediately after dosing.
The duplicate label of the dosing container was stuck on the respective dosing record of the
respective subject and this ensured the correct allocation of the investigational product as per
the randomization schedule. Compliance was also ensured by estimation of drug in the
plasma.
5.7.14 Pharmacokinetic results
Pharmacokinetic analyses
Non-compartmental analysis was applied for the estimation of PK parameters Cmax, AUC0-t
and AUC0-inf, Tmax, Kel, and t½ of Gliclazide concentration time using WinNonlin® version 5.2.
The untransformed mean pharmacokinetic parameters viz, Cmax, AUC0-t and AUC0-inf, T max,
Kel and t ½ from plasma concentration time profile of Gliclazide for test and reference product
are tabulated, given in chapter 6.