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Chapter 5 Experimental

SPP School of Pharmacy & Technology Management, SVKM‟s NMIMS, Mumbai 81

Chapter 5

Development and

Evaluation of formulation



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


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



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



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.



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.



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



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



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



with the buffer solution. From this stock solution 5 ml was further diluted with pH 4.5 acetate

buffer to 100 ml.



5.1.4.7 Preparation of stock and standard solutions with pH 6.8 phosphate buffer


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.


of 2.5 – 17.5 µg/ml. The linearity curves of gliclazide were prepared at 226 nm and 290 nm as


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


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.


of 2- 20 µg/ml. The linearity curves of gliclazide were prepared at 226 nm and 290 nm as


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


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.


of 2- 20 µg/ml. The linearity curves of gliclazide were prepared at 226 nm and 290 nm as


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.



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



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



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


(%)

Where, Ai - Area of single highest unknown impurity peak in solution (1)







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


Where,

AT - Area of all unknown impurity peak in solution (1)









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.



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



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.



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



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.



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.


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 √ √ √ √ √ √ √ √



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).



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)



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.



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



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



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

.



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.



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



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



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.



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.



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



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.



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


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


Average wt. 200 mg 200 mg 200 mg 200 mg





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



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.



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.



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


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


cellulose (HPMC K100 LV)

34 44 44 44 50 44 44 46 44 44


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



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



Figure 5.7: Gliclazide ER tablet (HDPE bottle with CRC cap and

induction sealing and Alu/Alu blister pack)



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



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



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.



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



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



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



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


2 Dicalcium phosphate, anhydrous Ph. Eur. 50 50


4 Hydroxypropylmethyl cellulose

(HPMC K100 LV)

Ph. Eur. 44 44


(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.



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



anhydrous

Ph. Eur. 50 500



(HPMC K100 LV)

Ph. Eur. 44 440


(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.



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:


Compression

speed In-process control of tablets:

Appearance

Weight of 20 tablets

Average mass

Thickness


Friability

Machine rpm

Appearance

Weight of 20 tablets

Average mass

Thickness


Friability

Dissolution profile

Assay



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


Friability

Dissolution profile

High and low speed

For data generation 50 tablets each



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.



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



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.


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


anhydrous Ph. Eur.

97

22 22 22 22


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



Table 5.20(B): Formulation composition of gliclazide extended release tablets – Hot melt granulation

Sr.

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


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



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.



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



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.



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).



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



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



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



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



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.



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.



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



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).



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.



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.



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'.



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.



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



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.



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).



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.



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.



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.



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.



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



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



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



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.



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.

chapter 5 experimental - inflibnetshodhganga.inflibnet.ac.in/bitstream/10603/9045/9/09_chapter...

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