Materials and methods - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/28261/12/12_chapter...

Materials and methods

Dept of Pharmaceutics, JSSCP, Mysore 84

MATERIALS

Drugs and polymers

Methotrexate (MTX) Sigma Aldrich, Bangalore, India.

Cyclophosphamide (CYP) Sigma Aldrich, Bangalore, India.

Capecitabine (CAP) Dr. Reddy’s Laboratories, Hyderabad,

India.

Anastrozole (ANS) Sigma Aldrich, Bangalore, India

Imatinib Mesylate (IM) Natco Pharm Ltd, Hyderabad, India.

Hydroxypropyl methylcellulose

E 5 LV (HPMC E 5 LV), viscosity: 4 - 6

cps for 2 % aqueous solution

Loba chemie Pvt. Ltd.,Mumbai, India.

Hydroxypropyl methylcellulose

K4M (HPMC K4M), viscosity: 4000 cps

for 2 % aqueous solution

Colorcon, Goa, India.

Sodium Carboxy Methyl Cellulose

(Na CMC), 200 - 300 cps (1 % w/w)

Sd Fine Chem. Ltd., Mumbai, India.

Sodium Carboxy Methyl Cellulose (Na

CMC), 1100 - 1900 cps (1 % w/w)

Merck Limited, Mumbai

Sodium Alginate (Na Alginate),

Low viscosity: 1 % w/v solution had 5.5 ±

2 cps


Sodium Alginate (Na Alginate),

High viscosity: 1 % w/v solution had 1300

cps

FMC Biopolymer, India.

Silk cocoons CSRTI, Mysore

HydroxyPropyl Cellulose (HPC) Himedia, Mumbai, India

Xanthum Gum (XG), viscosity: 1 %

aqueous solution-1340 cps.


Polyvinyl Alcohol (PVA), viscosity: 4 %

aqueous solution – 25 to 32 cps.


Chemicals

Crospovidone (CP) Micro Labs, Bangalore, India.

Sodium Starch Glycolate (SSG) Malpe biotech Pvt. Ltd., Pune, India.

Croscarmellose Sodium (CCS) Malpe biotech Pvt. Ltd., Pune, India.

Potassium dihydrogen phosphate Merck Limited, Mumbai

Sodium hydroxide CDH (P) ltd. New Delhi.

Sucralose J.K. Sucralose India Ltd., Delhi, India.

Magnesium Stearate Lobachemie Pvt. Ltd., Mumbai, India.



Microcrystalline cellulose (AV 102) Micro Labs, Bangalore, India.

Strawberry Flavor SK florescences Pvt. Ltd., Delhi, India.

D-Mannitol Lobachemie Pvt. Ltd., Mumbai, India.

Spray dried mannitol

(SD mannitol; Pearlitol SD 200)

Micro Labs, Bangalore, India.

Propylene Glycol (PG) Lobachemie Pvt. Ltd., Mumbai, India.

Glycerol Rankem, New Delhi.

Neusilin ® US2 Fuji Chemical Industry, Toyama, Japan.

Acetonitrile Merck Ltd., Mumbai, India.

Methanol Merck Ltd., Mumbai, India.

Citric acid monohydrate Lobachemie, Mumbai, India.

Methyl paraben Sd Fine Chem. Ltd., Mumbai, India.

Propyl paraben Sd Fine Chem. Ltd., Mumbai, India.

Equipments

FT-IR Spectrophotometer Shimadzu, 8400s, Japan

KBr Press Techno search instruments, India

Digital Weighing Balance Shimadzu, AW 120, Japan.

Digital pH-Meter Systronics pH meter 335.

Hot Air Oven Memmert, UNB – 400.

Magnetic stirrer Tarsons, Spinit model – MC 02.

Refrigirator Whirlpool

Differential scanning calorimeter SDT Q600 (V20.9 Build 20)

Thermal gravimetric analyzer SDT Q600 (V20.9 Build 20)

Stress controlled rheometer Anton Paar, MCR 300.

In vitro dissolution apparatus USP XXIV Electrolab TDT-08L, Mumbai.

Stability Chambers Thermolab humidity chambers, India.

Micrometer screw gauge Mitutoyo Manufacturing corporation Ltd.,

Japan

Tablet hardness tester Inweka India Pvt Ltd, IHT 100,

Ahmedabad , India

Scanning electron microscopy (SEM) Scanning electron microscope (SEM),

Model QUANTA-200 FEI Netherlands

Tablet punching machine Rimek, Ahmedabad, India

Centrifuge Remi, Rajendra Electrical Industries Ltd,

Vasai, India.

Vacuum oven Memmert Vacuum oven VO 200.

Vortex mixer IKA, Vortex Genius 3.

Tensile Testing Machine Model 1121, Instron Ltd., Japan

Trinocular microscope Coslab, Model HL-10



High-Performance Liquid Chromatography

(HPLC)

The integrated HPLC system (SHIMADZU

LC-2010A HT, Kyoto, Japan) equipped

with low pressure quaternary gradient

pump along with dual wavelength UV

detector (SPD-20A), column oven and auto

sampler has been used for the analysis. The

chromatographic data was processed using

LC solution version 1.25 software.

Column Phenomenex Luna 5μ C18 or C8 (2) 100A,

(250 X 4.60 mm i.d., 5 μm particle size)

Disintegration apparatus Electrolab, Mumbai, ED – 2AL.

X – ray diffraction (XRD) analysis Rigaku Miniflex II desktop X-Ray

diffractometer (Japan)

Friability Apparatus Electrolab, Mumbai, EF – 2.

Tap density tester (USP) Electrolab, ETD – 1020, Mumbai

Statistical Analysis GraphPad prism-5

E-Tongue Sensory analyzer α Astree liquid and taste analyzer

connected with LS16 autosampler unit,

taste sensors and reference electrode was

purchased from Alpha MOS Inc., and the

system was equipped with a data

acquisition and analysis software package.



PREFORMULATION, ANALYTICAL AND BIOANALYTICAL

STUDIES

Description

Melting point

Drug excipient compatibility studies

Analytical and Bioanalytical methods

Prior to the development of a dosage form, it is essential to investigate physico-

chemical properties of the drug that could affect drug performance and development of

an efficacious dosage form.

The approved material of certain chemical identity and purity can have varied

pharmaceutical properties that can have an impact over formulation and drug release

patterns. So any batch-to-batch variations in these characteristics of the material and

their effect on the performance of the dosage form are to be established.

Description

Physical appearance (color, amorphous or crystalline) of the drug was carried out

by visual appearance.

Melting point

Melting point for the drugs was determined by capillary fusion method; one sided

closed capillary filled with drug and inserted into the melting point apparatus.

Temperature was noted at which solid drug changed into liquid.



Drug excipient compatibility studies

The successful formulation of a stable and effective solid dosage form depends on

the careful selection of the excipients that are added to facilitate administration, promote

the consistent release, improve bioavailability and protects from degradation.

FTIR was used to investigate any interactions between the drug and components

in a formulation. Samples were taken in 1:100 ratio of mixture of drug and potassium

bromide. The mixture was compressed to prepare a pellet using KBr press. The prepared

pellet was placed in the sample holder and analyzed. Twenty scans were acquired in the

4000 – 600 cm-1

range with a resolution of 4 cm-1

using FT-IR spectrophotometer. FT-IR

spectrum of physical mixture was compared with that of drug 91

. The results of the

compatibility studies are presented in Results and Discussion under each dosage forms.

Analytical Methods

A rapid and sensitive RP-HPLC method was carried out for the quantification of

drugs in various dosage forms as per individual chromatographic conditions. The

chromatographic separation was achieved using a stationary column and a suitable

mobile phase. The mobile phase was filtered through a 0.22 µm filter (Millipore,

Bedford, USA) and degassed in an ultrasonic bath prior to use. The analysis was

performed at room temperature by isocratic elution at a specified flow rate using a UV

detector to obtain well resolved peaks of the drugs. The chromatographic data obtained

were processed using LC solution version 1.25 software. The calibration curve was

constructed by plotting the peak area on ordinate as a function of drug concentration on

abscissa. The results were expressed as mean of six determinations. Validation was

carried out to ensure performance of the chromatographic method. The method was

validated as per International Conference on Harmonization (ICH) for specificity, Limit

of Quantification (LOQ), linearity, accuracy, precision 92, 93

.



· Specificity

For chromatographic methods, developing a separation involves demonstrating

specificity, which is the ability of the method to accurately measure the analyte response

in the presence of all potential sample components. The developed method is considered

to be specific when there is no interference by placebo solution at the retention time of

the analyte.

· The limit of quantification (LOQ)

The limit of quantification (LOQ) is defined as the lowest concentration of an

analyte in a sample that can be determined with acceptable precision and accuracy under

the stated operational conditions of the method. The limit of quantification (LOQ) was

calculated using the equation LOQ = 10 (S.D./S) where S.D. is the standard deviation and

S is the slope of the calibration curve.

· Linearity

It is the ability of the method to elicit test results that are directly proportional to

analyte concentration within a given range. Acceptability of linearity data is often judged

by examining the correlation coefficient and y-intercept of the linear regression line for

the response versus concentration plot. A correlation coefficient of 0.999 is generally

considered as evidence of acceptable fit of the data to the regression line. The y-intercept

should be less than a few percent of the response obtained for the analyte at the target

level.

· Accuracy and precision

Accuracy is the closeness of the test results obtained by the analytical method to

the true value. Precision is the measure of the degree of repeatability of an analytical

method under normal operation.



The intra-batch accuracy and precision were determined by analysis of six

replicates each of low, medium and high concentration quality control samples; while

inter-batch accuracy and precision were determined by the analysis of these quality

control samples on three separate occasions. The precision of the method at each

concentration was determined by comparing the coefficient of variation (CV), obtained

by calculating the standard deviation (SD) as a percentage of the calculated mean

concentration. The accuracy estimated for each spiked control was obtained by

comparing the nominal concentration with the assayed concentration. The % C.V has to

be within 20 % for the lowest concentration and 15 % for the upper concentration levels

94.

[CV (%) = 100 (SD/M); Accuracy (%) = 100 (M/T), where M is the mean, SD is the

standard deviation of M, and T is the theoretical concentration].



Chromatographic Conditions

1. ANASTROZOLE (ANS) 86

Mobile Phase

Mobile phase consisted of Acetonitrile:Phosphate buffer in the ratio of

60:40 v/v. Phosphate buffer was prepared using 10.0 mM of potassium

dihydrogen phosphate and pH was adjusted to 3.0 ± 0.2 using dilute ortho

phosphoric acid.

Column : C18

Flow rate : 1.0 ml/min

Maximum absorbance : 210 nm

Column temperature : 25 °C

Stock and standard solutions

The primary stock solution of ANS standard was prepared in methanol (1

mg/ml). Secondary stock solution (0.1 µg/ml) was prepared by diluting primary

stock solution with methanol. Series of standard solutions were prepared by

diluting appropriate quantity of secondary stock solution with mobile phase to get

the concentrations of 0.5, 1, 2, 4, 8, 16 and 32 ng/ml.

Linear range : 0.5 - 32 ng/ml (r2 = 0.998)

Retention time : 8.663 ± 0.15 min



2. METHOTREXATE (MTX) 95

Mobile Phase

Mobile phase consisted of Acetonitrile: Solution ‘A’ in the ratio of 1:9

v/v. Solution ‘A’ was prepared using 0.2 M dibasic sodium phosphate and 0.1 M

citric acid (63:37) and pH of the solution was adjusted to 6.0 using 0.1 M citric

acid or 0.2 M dibasic sodium phosphate.

Column : C18





Primary stock solution of MTX was prepared by dissolving the drug in

mobile phase to yield concentration of 1 mg/ml. Calibration standard solutions

were prepared from primary stock solution by serial dilution with mobile phase

to yield final concentration of 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 and

12.8 µg/ml

Linear range : 0.025 – 12.8 µg/ml (r2 = 0.999)

Retention time : 6.659 ± 0.021 min



3. CAPECITABINE (CAP) 96

Mobile Phase

Mobile Phase consisted of Acetonitrile: phosphate buffer in the ratio of

50:50 v/v. Phosphate buffer was prepared using 0.05 M potassium dihydrogen

phosphate and pH was adjusted to 3.0 ± 0.05 using dilute ortho phosphoric acid.

Column : C8





The primary stock solution of CAP was prepared in mobile phase (1

mg/ml). Calibration standard solutions were prepared from stock solution by

diluting primary stock solution with mobile phase to yield final concentration of

0.5, 1, 2, 4, 8, 16, 32 and 64 µg/ml

Linear range : 0.5 - 64 µg/ml (r2 = 0.999)

Retention time : 4.849 ± 0.008 min.



4. CYCLOPHOSPHAMIDE (CYP) 97

Mobile Phase

Mobile Phase consisted of Phosphate buffer: Acetonitrile in the ratio of

80:20 v/v. Phosphate buffer was prepared using 10 mM Potassium dihydrogen

phosphate and pH was adjusted to 6.8 using 0.5M NaOH

Column : C18





Primary stock solution was prepared by dissolving the drug in deionized

HPLC water to yield concentration of 1 mg/ml. Calibration standard solutions

were prepared from primary stock solution by serial dilution with deionized

HPLC water to yield final concentration of 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4

and 12.8 µg/ml.


Retention time : 10.821 ± 0.031



5. IMATINIB MESYLATE (IM) 98

Mobile Phase

Mobile phase was prepared using buffer: Acetonitrile: methanol in the

ratio of 55:25:20 v/v/v. Phosphate buffer was prepared using 0.5 % potassium

dihydrogen phosphate and pH was adjusted to 3.5 using 50 % phosphoric acid.

Column : C18





Primary stock solution of IM was prepared by dissolving the drug in

methanol to yield concentration of 1 mg/ml. Calibration standard solutions were

prepared from primary stock solution by serial dilution with methanol to yield

final concentration of 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 and 12.8 µg/ml.


Retention time : 6.597 ± 0.045



Bioanalytical Methods

Methods

Methods employed to determine plasma drug concentration are as per the

procedures given in previous studies. Validation was carried out to ensure performance of

the chromatographic method. The methods were validated as per International

Conference on Harmonization (ICH) guidelines for specificity, accuracy, precision and

linearity. Blood drawn from jugular vein of wistar rats was collected in heparinized tubes

and centrifuged at 10000 rpm for 5 min. Separated plasma was collected and stored at –

50 °C until further analysis.

1. ANASTROZOLE (ANS)

86

ANS primary stock solution of 1 mg/ml was prepared using methanol. From the

primary stock solution, working standard solutions were prepared in the concentration

range of 0.01 – 0.32 µg/ml. Blank plasma was brought to room temperature prior to

analysis. Aliquots of plasma (100 μl) was transferred into series of micro-centrifuge

tubes, spiked with 100 μl of different working standard solutions and made up to 2 ml

with protein precipitating reagent [Acetonitrile: Methanol (1:1)] to get final

concentrations of 0.5 - 16 ng/ml of ANS in rat plasma. The tubes were vortex-mixed for 5

min, then centrifuged at 10000 rpm for 5 min at 4 °C. The clear supernatant liquid was

separated, filtered (0.45 μ membrane filter) and injected into the HPLC system to

determine concentration of drug in the plasma. The chromatographic conditions were set

similarly as given under ANS analytical method.

The calibration curve was constructed by plotting the peak area on ordinate as a

function of ANS concentration on abscissa. The bio-analytical method for ANS

estimation showed retention time of 8.89 ± 0.12 min. Linear relationship was observed

between the concentration range of 0.5 – 16 ng/ml (r2 = 0.998). The results were

expressed as mean of six determinations.



2. METHOTREXATE (MTX) 99

Simple and reliable bio-analytical method is used to determine MTX

concentration in plasma.

MTX primary stock solution (1 mg /ml) was prepared by dissolving drug in 0.1 N

sodium hydroxide solution. From the primary stock solution, working standard solutions

were prepared in the concentration range of 0.25 - 128 µg/ml. Drug free plasma samples

were brought to room temperature prior to analysis. 100 µl of the plasma was transferred

into series of micro-centrifuge tubes and spiked with 100 μl of different working standard

solutions. The volume was made up to 2 ml with protein precipitating reagent (10 %

perchloric acid; v/v) to get final concentrations of 0.0125 – 6.4 µg/ml of MTX in rat

plasma. The tubes were briefly vortex-mixed for 5 min then centrifuged at 10000 rpm for

8 min. The clear supernatant liquid was injected into a C18 column to determine

concentration of drug in the plasma. The mobile phase was composed of a mixture of 50

mM ammonium acetate buffer (pH 6.0) and methanol (77:23, v/v) with a flow rate of 1.0

ml/min. The ultraviolet absorbance of the effluent was monitored at a wavelength of 313

nm.

The calibration curve was constructed by plotting the peak area on ordinate as a

function of MTX concentration on abscissa. Linearity was obtained in the concentration

range of 0.0125 – 6.4 µg/ml with drug eluting at a retention time of 8.883 ± 0.12 min.

The results were expressed as mean of six determinations.



3. CAPECITABINE (CAP) 100

CAP primary stock solution was prepared by dissolving 10 mg of capecitabine in

10 ml of acetonitrile:water mixture (50:50 v/v). Working standard solutions were

prepared in the concentration range of 1.0 - 256 µg/ml. Blank plasma samples were

brought to room temperature prior to analysis. 100 µl of the plasma was transferred into

series of micro-centrifuge tubes followed by 100 μl of different working standard

solutions and 100 μl of the internal standard (1 µg/ml; d11-capecitabine). The volume

was made up to 2 ml with protein precipitating reagent (acetonitrile) to get final

concentrations of 0.05 – 12.8 µg/ml of CAP in rat plasma. The tubes were vortex-mixed

for 5 min then centrifuged at 10000 rpm for 5 min at 4 ºC. The clear supernatant liquid

was injected into the HPLC system to determine concentration of drug in the plasma.

HPLC was performed with C18 column at a flow rate of 1.0 ml/min. The analysis

was performed using mobile phase of acetonitrile/water in the ratio of 1:1 v/v with a UV

detection wavelength of 310 nm. The column operated at room temperature while the

temperature of the auto sampler was maintained at 8 ± 2 ° C.

The calibration curve was constructed by plotting ratio of the peaks of CAP and

d-11 CAP versus the concentrations of CAP. Linearity was obtained in the concentration

versus area curve for standard (0.05 – 12.8 µg/ml) from spiked plasma samples with

drugs eluting at 2.625 ± 0.020 min and 2.995 ± 0.026 min for IS and CAP respectively.



4. CYCLOPHOSPHAMIDE 101

Primary stock solution of CYP was prepared by dissolving 10 mg of drug

substance in water (10 ml). From the primary stock solution, working standard solutions

were prepared in the concentration range of 8 - 512 µg/ml using mobile phase. Blank

plasma samples were brought to room temperature prior to analysis. 100 µl of the plasma

was transferred into series of micro-centrifuge tubes followed by 100 μl of different

working standard solutions and 100 μl of the internal standard (ifosfamide dissolved in

water). The volume was made up to 2 ml with protein precipitating reagent

(trichloroacetic acid) to get final concentrations of 0.4 – 25.6 µg/ml of CYP in rat plasma.

The tubes were briefly vortex-mixed for 5 min then centrifuged at 10000 rpm for 5 min.

The clear supernatant liquid was directly injected into the HPLC system to determine

standard concentration of drug in the plasma.

HPLC was performed with C18 column at a flow rate of 1.3 ml/min. The analysis

was performed using mobile phase of Acetonitrile – 0.05 mol/l KH2PO4 buffer, pH 4.8,

24:76 v/v with a UV detection wavelength of 195 nm. The column was operated at room

temperature.

The calibration curve was established by plotting the ratio of the peak areas of

cyclophosphamide and ifosfamide versus the concentrations of cyclophosphamide

samples. A good linearity (0.4 – 25.6 µg/ml) was obtained in the between peak ratio and

cyclophosphamide concentration with a correlation coefficient value of 0.998. The

retention time of the ifosfamide and CYP were found to be 9.935 ± 0.034 and 12.421 ±

0.11 min respectively.



5. IMATINIB MESYLATE 102

Primary stock solution of IM was prepared by dissolving 10 mg of drug in 10

ml of HPLC water. From the primary stock solution, working standard solutions were

prepared in the concentration range of 1.0 - 256 µg/ml. Blank plasma samples were

brought to room temperature prior to analysis. 100 µl of the plasma was transferred

into series of micro-centrifuge tubes followed by 100 μl of different working standard

solutions. The volume was made up to 2 ml with protein precipitating reagent

(methanol) to get final concentrations of 0.05 – 12.8 µg/ml of IM in rat plasma. The

tubes were vortex-mixed for 5 min then centrifuged at 10000 rpm for 5 min. The clear

supernatant liquid was injected into the HPLC system to determine standard

concentration of drug in the plasma.

HPLC was performed with C8 column at a flow rate of 1.0 ml/min. The

analysis was performed at room temperature using mobile phase of 0.02 M potassium

dihydrogen phosphate – acetonitrile (7:3, v/v) with a UV detection wavelength of 265

nm.

The calibration curve was constructed by plotting the peak area on ordinate as

a function of IM concentration on abscissa. A good linearity was obtained in the

concentration (0.05 – 12.8 µg/ml) versus area curve for spiked plasma samples with

drug eluting at a retention time of 7.368 ± 0.06 min. The results were expressed as

mean of six determinations.



FORMULATIONS

Fast disintegrating Films

Fast disintegrating tablets

Eatable Gels

FAST DISINTEGRATING FILMS

Method of preparation for fast disintegrating films (FDF)

Fast-disintegrating films for selected drugs were prepared by solvent-casting

method.

Aqueous solution I was prepared by dissolving the required quantities of polymer

and plasticizer in distilled water. The solution was stirred (using magnetic stirrer at 800

rpm) until the polymer dissolves. The polymeric solution was kept aside until entrapped

air bubbles disappear. Plasticizer was added at three different concentrations (10 %, 15 %

and 20 % w/w of the total film weight).

Aqueous solution II was prepared by dissolving drug, sucralose and strawberry

flavor in a suitable soluble solvent. Aqueous solution I was added to solution II and

stirred well until it forms clear polymeric drug solution. After disappearance of air

bubbles, the polymeric solution containing drug was casted onto a glass petridish of

specified dimension (25 cm2) and dried in the vacuum oven. The film was carefully

removed from the petridish, observed for any imperfections and cut according to the size

required for testing. The samples were wrapped in a butter paper followed by aluminum

foil, placed in an aluminum pouch and were heat-sealed. The packed films were stored in

a desiccator until further use 103,104

.



1. Anastrozole FDF

Solution of ANS was prepared in mixture of distilled water/alcohol (Alcohol was

used in small quantity to dissolve the drug). The polymeric drug solution after casting

were dried in a vacuum oven at 27 °C for 22 h. Each 5 cm2 (2.0 cm x 2.5 cm) optimized

film contained 1 mg ANS, 200 mg HPMC E 5 LV polymer, sucralose (5.7 mg),

propylene glycol (15 %) and strawberry flavor (12 mg).

2. Methotrexate FDF

Solution of MTX was prepared in distilled water (pH was adjusted to 7.4 using 1

N NaOH and 1 N HCl). The polymeric drug solution after casting were dried in a vacuum

oven at 28 °C for 27 h. Each 6.25 cm2 (2.5 cm x 2.5 cm) optimized film contained 22.5

mg MTX, 380 mg HPMC E 5 LV polymer, sucralose (5.7 mg), glycerol (15 %) and

strawberry flavor (12 mg).

3. Cyclophosphamide FDF

For 25 mg CYP FDF:

Solution of CYP was prepared in distilled water. The polymeric drug solution

after casting was dried in a vacuum oven at 26 °C for 24 h. Each 6.25 cm2 (2.5 cm x 2.5

cm) optimized film contained 25 mg CYP, 190 mg HPMC E 5 LV polymer, sucralose

(5.7 mg), propylene glycol (15 %) and strawberry flavor (12 mg).

For 50 mg CYP FDF:

Solution of CYP was prepared in distilled water. The polymeric drug solution

after casting was dried in a vacuum oven at 26 °C for 26 h. Each 6.25 cm2 (2.5 cm x 2.5

cm) optimized film contained 50 mg CYP, 380 mg HPMC E 5 LV polymer, sucralose

(5.7 mg), propylene glycol (15 %) and strawberry flavor (12 mg).



4. Imatinib mesylate FDF

For 50 mg IM FDF:

Solution of IM was prepared in distilled water. The polymeric drug solution after

casting was dried in a vacuum oven at 28 °C for 24 h. Each 6.25 cm2 (2.5 cm x 2.5 cm)

optimized film contained 50 mg IM, 180 mg HPMC E 5 LV polymer, sucralose (5.7 mg),

propylene glycol (15 %) and strawberry flavor (12 mg).

For 100 mg IM FDF:

Solution of IM was prepared in distilled water. The polymeric drug solution after

casting was dried in a vacuum oven at 28 °C for 26 h. Each 6.25 cm2 (2.5 cm x 2.5 cm)

optimized film contained 100 mg IM, 380 mg HPMC E 5 LV polymer, sucralose (5.7

mg), propylene glycol (15 %) and strawberry flavor (12 mg).



EVALUATION OF FDF’S

Appearance and pH

Thickness

Content uniformity

Mechanical properties

In vitro disintegration test

Differential scanning calorimeter (DSC) & Thermal gravimetric analysis (TGA) studies

X-Ray Diffraction studies (XRD)

Film morphology

Dissolution studies

In vivo evaluation

Stability studies

Appearance and pH of the films

The films were visually tested for any imperfections, transparency or semi-

transparency nature and air bubble in the films. Films exhibiting gritty appearance,

containing air bubble, nicks or tears were excluded from further studies.

The pH of the oral cavity is 6.8 105

. An acidic or alkaline pH may cause irritation

to the buccal mucosa. Hence surface pH of the formulation should be close to oral pH.



The film to be tested was placed in the pertridish and was moistened with 0.5 ml

distilled water and kept for 30 s. The pH was noted by bringing the electrode of the pH

meter in contact with the formulation and allowing it to equilibrate for 1 min 106

.

Thickness

The thickness of each film was measured using a Screw gauge (Mitutoyo

Manufacturing Corporation Ltd., Japan) at five different locations (center and four

corners). Data are represented as mean ± S.D. for each film. Mean thickness variation

greater than 5 % were excluded from further analysis 104

.

Uniformity of the drug content

A piece of FDF film (unit dose) was dissolved in suitable solvent by sonication.

The solution was filtered using 0.45 μm millipore filter and after appropriate dilution, the

sample was injected into the HPLC and the amount of drug was determined by using

standard calibration curve. A blank solution containing equivalent amount of excipients

(without drug) was treated in similar manner as that of the sample. Ten FDF were

examined and the acceptance value (AV) was calculated using the following equation:

AV = | M – X | + ks

Where ‘M’ is the label claim (100 %), ‘X’ is the measured content of drug;‘s’ is the

standard deviation and ‘k’ is the acceptability constant (2.4). The AV value in the

preparation should be within the range of ≤ 15 % 107

.

Measurement of mechanical properties of the FDF

Mechanical properties of the films were evaluated using universal testing machine

(Model 1121, Instron Ltd., Japan) with a 2-kilogram load cell. FDF of known dimension



were positioned between two clamps at a distance of 5 cm. The FDF were pulled by the

clamp at the rate of 50 mm/min. Measurements were run in triplicate for each film. The

mechanical properties like tensile strength, elastic modulus and % elongation were

calculated 103

.

Tensile strength (N/mm2) is the maximum stress applied to a point at which the

film breaks and can be computed from the applied force at rupture as a mean of three

measurements and the cross-sectional area of the fractured film.

Force at break (N)

Tensile strength = ---------------------------------------------------

Initial cross sectional area of the film (mm2)

Elastic modulus (N/mm2) is the ratio of applied stress and corresponding strain in

the region of approximately linear proportion of elastic deformation on the load

displacement profile and calculated using the following equation-

Force at corresponding strain (N) 1

Elastic modulus = ------------------------------------------ x -----------------------

Cross-sectional area of the film Corresponding strain

Percentage elongation was calculated by the following equation-

Increase in length

Percent Elongation = ------------------------ x 100

Original length

In vitro disintegration test

In vitro disintegration time was determined visually by placing the FDF

(equivalent dose) in glass dish (6.5 cm diameter) containing 25 ml distilled water at 37

°C and swirling the medium every 10 s. The disintegration time was recorded as the time



at which the film starts to break or disintegrate. The disintegration time of prepared films

was measured in triplicate 104

.

Formulations were optimized such that it produces shortest DT, good mechanical

property (high tensile strength, low modulus and high % elongation) without any problem

of blooming and brittleness. The final optimized formulation was evaluated for further

evaluation parameters.

Differential scanning calorimeter (DSC) & Thermal gravimetric analysis (TGA)

studies

Measurements were performed using a SDT Q600 (V20.9 Build 20). Samples

weighing approximately 2 - 5 mg were placed in an aluminum crucible cell which was

firmly crimped around the lid to provide an adequate seal. The analysis was done under

purge of dry nitrogen gas at a flow rate of 25 ml/min.

The DSC of the pure drug and film was performed by heating it from ambient

temperature (°C) to certain temperature above melting point of drug with heating rate of

10 °C/min.

The objective of TGA is to measure the change in mass of a sample, as the sample

is heated. TGA of the optimized film was carried out by heating from ambient

temperature to a temperature at which the weight of the film was reduced 20 % or until

complete degradation with heating rate of 10 °C/min.

X-Ray Diffraction studies (XRD)

X - ray diffraction (XRD) analysis of optimized film, in comparison with pure

drug and placebo, was performed using Rigaku Miniflex II desktop X-Ray diffractometer

(Japan) using a monochromator addition that captures X-rays other than Cu Kα for use in



analysis. The samples were scanned over a 2θ range of 10° to 70° at a scan speed of

10°/min and a step size of 0.01° 20

.

Film morphology

The surface morphology of the FDF was evaluated using scanning electron

microscope (SEM), Model QUANTA-200 FEI Netherlands. The samples were attached

to the slab surfaces with double-sided adhesive tapes and the scanning electron

photomicrograph was taken at 5000 X magnification.

E-tongue sensory analysis

To assess the palatability of a pharmaceutical formulation and the efficiency of

the masking strategy, human sensory evaluation can be used, but this method raises

safety concerns and cost issues. In our study, an attempt was made to evaluate the taste

similarity of film formulation by comparing with sucralose, a sweetener using αAstree

liquid and taste analyzer (e-Tongue) connected with LS16 autosampler unit, taste sensors

(7 no’s) and reference electrode (Alpha MOS Inc.). The system was equipped with a data

acquisition and analysis software package. Each sensor consists of a silicon transistor

with an organic coating that determines the sensitivity and selectivity of the sensor. This

system was found to permit good characterization and to allow differentiation between

the majority of food groups and pharmaceutical products 108

.

Sample preparation and analysis

The films to be tested were taken in a beaker containing 25 ml of purified water.

When the reference electrode and sensors (7 no’s) were dipped into a beaker containing a

test solution, a potentiometric difference between each individually coated sensor with

the Ag/AgCl reference electrode was measured and recorded by the e-Tongue software.

Each sample (unit highest dose) was analyzed for 120 s. This was followed by sequential

immersion into two rinsing beakers containing fresh purified water for 60 s each to



prevent any cross contamination or carry-over residues from previous samples. Each

sample was tested for five times. The first two replicate measurements of the test solution

were for sensor training purposes and the readings from the last three replicates were

used for data analysis. Figure 1 shows the schematic representation of the ASTREE

electronic tongue system.

The analysis was carried out for optimized film, sucralose (5.7 mg), pure drug and

placebo film (without drug and sucralose). The potentiometric difference created between

each individual sensor and the reference electrode was measured and recorded by the e-

Tongue Alphasoft software. All samples were analyzed at room temperature.

Due to the complexities of analyzing the output data from several sensors for

more than two samples, all data were processed and analyzed using the αAstree software

provided by Alpha M.O.S. Data reduction allows responses of the seven sensors to be

processed and displayed in two - dimensional maps. This map shows the similarities

between the different samples and groups. PCA is a multivariate statistical method used

by the Astree E-Tongue system software to extract useful information from the sensor

responses, and give a representative map of the various sample groups. Using PCA, data

points for one sample or set of samples are compared by measuring the distance between

them. The distance is the Euclidean distance between the calculated center of the cluster

of one sample set to the center of the cluster of another sample set.

The smaller the Euclidean distance between two samples, the more similar their

taste. Accordingly, the difference between the taste of optimized film and that of the

sucralose was evaluated by calculating the Euclidean distances between them 108,109

.



Figure 1. Schematic representation of the ASTREE electronic tongue system

Dissolution studies

Dissolution studies of the optimized film (unit dose) and plain drug (equivalent

dose) were performed using USP dissolution paddle apparatus (Electrolab, Mumbai,

India). The dissolution studies were carried out at 37 ± 0.5 ºC using three different buffer

systems (pH 1.2, pH 4.5 acetate buffer and pH 6.8 phosphate buffer). Two milliliters

aliquots of dissolution media were collected at predetermined time intervals and replaced

with equal volumes of respective buffer. The collected samples were filtered through 0.45

μm membrane filter, samples were suitably diluted if necessary and the concentration of

the dissolved drug was determined using the HPLC. The results were the average of six

determinations. Dissolution profile of film formulation was compared with that of the

plain drug. The stirring speed and volume of dissolution media varied based on the drug

used (Table 3) 110, 111

. The data obtained were statistically analyzed using one way

analysis of variance (p < 0.05) to acesses the pH effect on dissolution of film and student

t-test to acesses the difference in release between control and test .



Table 3. Dissolution conditions for various FDFs

Drug Stirring speed Volume of dissolution media (ml)

Methotrexate 50 rpm 900

Cyclophosphamide 100 rpm 900

Anastrozole 50 rpm 500

Imainib mesylate 50 rpm 900

In vivo evaluation

Animals

The study was conducted in accordance with the principles of laboratory animal

care and was approved by institutional animal ethics committee of the JSS College of

Pharmacy, JSS University, Mysore (076/2011). Male Wistar rats, (weighed 240 – 260 g)

used in the present experiment, were housed in a room maintained on a 12 h light/dark

cycle at 23 ± 2 °C with free access to food and water.

Inclusion criteria for animals

i. The animals which have normal behavioral parameters.

ii. The animals which have healthy food consumption and excretory

activities.

iii. The animals with healthy body weight, temperature and heart rate.

iv. The adult animals aging between 3 to 4 months.

Exclusion Criteria

i. The animals which do not meet inclusion criteria.

ii. The animals exposed to insecticides (such as mosquito repellants used for

maintenance of animal house).



Study design

The rats were anaesthetized with an intraperitoneal injection of urethane, 1 g kg -1

,

and the jugular vein was cannulated to facilitate removal of blood sample 112

.

The rats were divided into two groups namely control and test. The rats were

fasted/feeded overnight before administration of film which depends on drugs used

(Table 4). For film application, 50 µl of distilled water was dropped into the rat oral

cavity under light ether anesthesia and then film preparation (test sample; converting

human dose to animal dose in mg/kg) was placed on the tongue. After ensuring

disintegration of the film, anesthesia was discontinued. Drug (equivalent dose) was

dissolved in water (control) and was administered orally using a stomach sonde needle

under light ether anesthesia. The anesthesia was continued for same time as done for

FDFs (test) 55

.

Blood samples (0.25 ml) were obtained immediately before and after dosing at

pre determined time intervals from jugular vein. Samples were collected in heparinized

tubes and were centrifuged for 5 min at 10,000 rpm at 4 °C. Plasma was separated and

stored at – 50 °C until further analysis. Number of animals, administration state and dose

utilized for the studies based on drugs are shown in Table 4.

Table 4. Number of animals, administration state (fed/fasted) and dose employed

for various FDFs

Drug Administration state Human Dose

Animal dose

(mg/kg)

Number of animals

Fast feed Sample control

ANS X 1 mg 0.087 6 6

MTX * X 45 mg

(25

mg/m2)

90 mg

(50

mg/m2)

3.92 7.84 6 6 6 6

IM * X 468 mg 600 mg 40.78 52.28 6 6 6 6

CYP X 5 mg/kg 5 6 6

*MTX and IM FDF had two dose strengths administered separately; hence number of animals in sample group and control group was

divided equally.



Bioanalysis of drug in rat plasma

Drug concentration in plasma samples (test and control) withdrawn at various

time intervals was determined using the procedures given under bio-analytical methods.

Recovery of drug from rat plasma

Plasma sample: The stock solution of drug was added to rat plasma to yield three

different concentrations in the range of standard plot carried out for each drugs.

Diluted sample: Drug stock solution was diluted using suitable solvent to obtain

same three different concentrations.

Both plasma samples and the diluted solutions were processed and analyzed using

the procedures given under bio-analytical methods. The ratio of peak area (plasma

sample/diluted solution) for drug was used to calculate the % recovery in rat plasma 86

.

Pharmacokinetic and statistical analysis

The maximum plasma concentration (Cmax) and the time to reach peak plasma

concentration (Tmax) were obtained directly from the concentration–time data. The

elimination rate constant (KE) was obtained from the slope of the linear part of the

elimination phase. The elimination half-life (t1/2) was calculated from 0.693/KE, while the

area under the curve to the last measurable concentration (AUC0–t) was calculated by the

linear trapezoidal rule. The area under the curve extrapolated to infinity (AUC0–∞) was

obtained as AUC0–t + Ct/KE. The significance of the differences observed for the mean

pharmacokinetic parameters of test (film) and solution (control) was evaluated using

student’s t-test at a significance level of P < 0.05 113

.



Stability studies

Each film (placebo and drug loaded) was wrapped in a butter paper followed by

aluminum foil and placed in an aluminum pouch and was heat-sealed (Dinge and Mangal,

2008). The packed film formulations were subjected to stability studies at storage

conditions such as 25 ± 2 °C / 60 ± 5 % RH, 40 ± 2 °C/75 ± 5 % RH and at refrigerated

temperature (2 - 8 °C). Samples were withdrawn at an interval of 4 weeks up to 6 months

and tested for various parameters such as appearance, pH, mechanical property,

disintegration time, drug content (P < 0.05) and drug release studies (P < 0.05). The

results were the average of six determinations 106

.



FAST DISINTEGRATING TABLETS

Method of preparation of fast disintegrating tablets

1. Direct compression

Fast disintegrating tablets were prepared by direct compression technique using

superdisintegrants (croscarmellose Sodium, crospovidone and sodium starch glycolate),

neusilin and lubricants. All the ingredients were weighed and sieved through a 40-mesh

screen, except for lubricant (magnesium stearate) which was sieved through a 60-mesh

screen. The other excipients used were spray dried mannitol, sweetening and flavoring

agents. The blend was prepared by mixing the ingredients (except lubricant) manually for

10 min by tumbling action in a poly-ethylene bag of suitable size. Finally, lubricant was

added to this blend and mixing was continued for 2 - 4 min to obtain homogenous

powder mixture 114

. The tablets were produced by compressing the powder mixture on a

rotary press using round convex punches at an average hardness of 3.6 - 3.7 kg.

Optimized Anastrozole, cyclophosphamide and methotrexate FDT’s prepared by direct

compression technique are shown in Table 5.

2. Wet granulation

Fast disintegrating tablets were prepared by wet granulation technique using

superdisintegrants (croscarmellose Sodium, crospovidone and sodium starch glycolate),

neusilin and lubricants. The raw materials were passed through sieve no. 100 prior to

mixing. Drug, intragranular fraction (50 %) of superdisintegrants, mannitol and neusilin

were weighed and mixed thoroughly by geometric dilution. Solution of PVP (w/v) was

added to the mixture in a quantity enough to prepare the wet mass. The wet mass was

granulated using sieve no. 44/100, dried in a tray dryer at 35 °C. The moisture content of

the dried granules was determined by an infrared moisture balance with a limit of NMT

2.0 %. The dried granules were mixed with extragranular fraction (50 %) of



superdisintegrants, mannitol, neusilin and required proportion of fines (10 %). Sweetener

and flavor were added to the granules with mixing. Finally the granules were lubricated

with magnesium stearate and punched into tablets using round convex punches at an

average hardness of 3.6 - 3.7 kg on rotary tablet punching machine 115

. Optimized

imatinib and capecitabine FDT’s prepared by wet granulation technique was tabulated in

Table 6.

Table 5. Composition of optimized fast disintegrating tablet prepared by direct

compression technique

Ingredients Methotrexate

Tablet

Quantity (mg)

Cyclophosphamide Tablet

Quantity (mg)

Anastrozole

Tablet

Quantity

(mg)

Drug 45 90 25 50 1

Crospovidone 12 17.5 12 12 5

Neusilin 30 35 30 30 10

Sucralose 5.7 5.7 5.7 5.7 5.7

Strawberry

flavor

12 12 12 12 12

Magnesium

stearate

9 10.5 9 9 3

Spray dried

mannitol

186.3 179.3 206.3 181.3 63.3

Tooling 8 mm 10 mm 8 mm 8 mm 6 mm

Tablet weight 300 mg 350 mg 300 mg 300 mg 100 mg



Table 6. Composition of optimized capecitabine and imatinib mesylate fast

disintegrating tablet prepared by wet granulation technique

Ingredients Capecitabine Imatinib

150 mg

tablet

500 mg tablet 100 mg tablet 400 mg tablet

Quantity

(mg)

Quantity

(mg)

Quantity

(mg)

Quantity

(mg)

Drug 150 500 100 400

Crospovidone Intragranular 8.75 23.75 8.75 20

Extragranular 8.75 23.75 8.75 20

Neusilin 28 28.5 24.50 40

PVP K 30 14 38 14 32

Sucralose 5.7 5.7 5.7 5.7

Strawberry flavor 12 12 12 12

Magnesium stearate 7 9.5 3.5 8

Mannitol

(50:50)

Intragranular 57.9 154.4 86.4 131.15

Extragranular 57.9 154.4 86.4 131.15

Tooling 10 mm 14 mm 10 mm 13 mm

Tablet weight 350 mg 950 mg 350 mg 800 mg



EVALUATION OF FDT’S

Pre and post compression evaluations

Angle of Repose

Bulk Density

Bulk Density

Tapped Density

True Density

Hausner Ratio

Porosity

Compressibility or Carr’s Index

Hardness

Friability

Uniformity of Content

Wetting time and water absorption ratio

In vitro disintegration time

Thermogravimetric and differential thermal analysis (TG-DTA/DSC)

X-Ray Diffraction (XRD) studies


Dissolution studies

In vivo evaluation

Stability studies



Pre and post compression evaluations 91, 116

Angle of Repose

The static angle of repose (q) was measured according to the fixed funnel and free

standing cone method. A funnel was clamped with its tip 2 cm above a graph paper

placed on a flat horizontal surface. The blend/granule was carefully poured through the

funnel until the apex of the cone thus formed just reached the tip of the funnel. To

increase the reliability of the observations, the angle of repose were performed in

triplicate. The mean diameters of the base of the powder cones were determined and the

tangent of the angle of repose calculated using the equation:

Tan q = 2h / D

Where h is the height of the heap of blend/granule and D is the diameter of the

base of the heap of powder.

Bulk Density

The bulk density of blend/granules at zero pressure (loose density) was

determined by pouring a known quantity of the powder (W) into a 250 ml measuring

cylinder and the volume (V0). The bulk density was calculated as Bd = W / V0. The

results presented are the mean of three replicates determinations.

Tapped Density

Tapped density was determined by placing a graduated cylinder containing a

known mass of blend/granule on a mechanical tapper apparatus. Samples were tapped

until no further reduction in volume of the sample was observed. To increase the

reliability of the observations, the tapped density was performed in triplicate.



The degree of volume reduction (C) is calculated from the initial volume ‘V0’ and

tapped volume ‘V’ as:

C = (V0-V)/V

True Density

The true densities (Dt) of blend/granule were determined by the liquid

displacement method using xylene as the immersion fluid

and computed according to the

following equation

Dt = w / [(a + w) - b] x SG

Where ‘w’ is the weight of powder, ‘SG’ is specific gravity of solvent, ‘a’ is

weight of bottle & solvent, ‘b’ is weight of bottle with solvent & powder. To increase the

reliability of the observations, the true density were performed in triplicate

Hausner Ratio

The Hausner ratio was determined as the ratio of tap and bulk density of the

samples.

Porosity

Based on the apparent bulk density and true density, the percentage porosity of

the blend/granule was calculated in triplicate using the eq.

Porosity (%) = (True density – Bulk density / True density) X 100

Compressibility or Carr’s Index

Based on the apparent bulk density and the tapped density, the percentage

compressibility was determined by using the eq.



Carr’s index = [(Tapped density – Bulk density) / Tapped density] X 100

Hardness

Hardness or tablet crushing strength, the force required to break a tablet in

diametric compression was measured using a digital Inweka hardness tester. The results

are reported as average of six determinations.

Friability

Twenty tablets were weighed and placed in a Roche friabilator and the equipment

was rotated at 25 rpm for 4 min. The tablets were taken out, dedusted, and reweighed.

The percentage friability of the tablets was calculated using below Eq.

Percentage friability = [(Initial weight - Final weight)/ Initial weight] X 100

Uniformity of Content

Content uniformity of the tablets (10 no’s) were determined similarly as given

under FDF’s. The content of drug was measured individually as per standard procedures

described under analytical methods.

Wetting time and water absorption ratio

A piece of tissue paper (12 × 10.75 cm) folded twice was placed in a Petri dish

(internal diameter of 9 cm) containing 10 ml of buffer solution (pH 6.8) simulated saliva

and amaranth (dye). The dye solution was used to enable suitable visual end-point

detection. A tablet was carefully placed on the surface of the tissue paper with the help of

forceps and the time required for the dye to reach the upper surface of the tablet was

recorded as wetting time. The wetted tablet was then weighed. Water absorption ratio ‘R’

was calculated using the equation:



R=100 × (Wb–Wa)/Wa

Where Wa is weight of tablet before water absorption and Wb is weight of tablet after

water absorption 116,117

.

In vitro disintegration time

In vitro disintegration time of the tablets was evaluated using 3 different methods 59

.

A. Conventional disintegration apparatus-basket rack assembly (as mentioned in Indian

pharmacopeia) 118

was used to determine the DT of the tablets. Distilled water was

used as dissolution medium. The volume of dissolution medium was 900 ml

maintained at 37 ± 0.5 °C.

B. Modified dissolution apparatus was used to carry out disintegration test. 900 ml of

distilled water maintained at 37 °C as the disintegration fluid and a paddle at 100 rpm

as stirring element were used. Disintegration time was noted when the tablet

disintegrated and passed completely through the screen of the sinker (3 – 3.5 mm in

height and 3.5 – 4 mm in width, immersed at a depth of 8.5 cm from the top with the

help of a hook).

C. Disintegration test was conducted by placing the fast dissolving tablet in a glass

cylinder fitted with 10 mesh at its base. This set up was further placed in a shaking

water bath operated at 150 rpm. 1 ml of purified water maintained at 37 °C

temperature was used as medium. The critical parameters of this method were the

operational speed of shaking water bath and volume of the medium.



Thermogravimetric and differential thermal analysis (TG-DTA/DSC)

The thermal analyses of the samples were carried out using thermogravimetry

(SDT Q600, TA Instruments, USA). The method was carried out similarly as done for

FDF’s 20,

119

.

X-Ray Diffraction (XRD) studies

XRD analysis of optimized tablet in comparison with pure drug and placebo was

performed using Rigaku Miniflex II desktop X-Ray diffractometer (Japan). The method

employed is as described for FDF’s 99

.


The method employed is as described for FDF’s. The analysis was carried out for

sucralose (5.7 mg), pure drug and tablet containing all excipients without drug &

sucralose and lastly final optimized tablet.

Dissolution studies

Dissolution studies of the optimized tablet and plain drug (equivalent dose) were

performed using USP dissolution paddle apparatus. The method employed is as described

for FDF’s. Dissolution profile of tablet formulation was compared with that of the plain

drug. The stirring speed and volume of dissolution media varied based on the drug used

(Table 7). The data obtained were statistically analyzed using one way analysis of

variance (p < 0.05) to acesses the pH effect on dissolution of tablet and student t-test to

acesses the difference in release between control and test .



Table 7. Dissolution conditions for various FDT’s

Drug Stirring speed Volume of dissolution media (ml)

Methotrexate 50 rpm 900

Cyclophosphamide 100 rpm 900

Anastrozole 50 rpm 500

Imainib mesylate 50 rpm 900

Capecitabine 50 rpm 900

In vivo evaluation

The study protocol, ethical clearance, inclusion and exclusion criteria are same as

mentioned for fast disintegrating films.

Study design

Male wistar rats (weighed 240 - 260 g) were housed in a room maintained on a 12

h light/dark cycle at 23 ± 2 °C with free access to food and water. The rats were

anaesthetized with an intraperitoneal injection of urethane, 1 g kg -1

, and the jugular vein

was cannulated to facilitate removal of blood sample.

The rats were divided into two groups namely control and test. The rats were

fasted/feeded overnight before administration of tablet which depends on drugs used

(Table 8). The test group received FDT formulated specially for animals (animal dose =

mg/kg) by reducing the human dose while keeping the excipient ratio constant whereas

the control group received drug solution (equivalent dose). For tablet application, 50 µl of

distilled water was dropped into the rat oral cavity under light ether anesthesia and then

FDT tablet preparation was placed on the tongue. After ensuring disintegration of the

tablet, anesthesia was discontinued. For oral administration of drug solution (control),

rats were orally given with drug (equivalent dose dissolved in distilled water

administered using a stomach sonde needle) under light ether anesthesia and the

anesthesia was continued for same time as done for test.



Blood samples (0.25 ml) were obtained immediately before and after dosing from

jugular vein. Blood samples were collected in heparinized tubes and were centrifuged for

5 min at 10000 rpm at - 4 °C. Separated plasma was stored at - 50 °C until further

analysis. Number of animals, administration state (fed/fasted) and dose utilized for the

studies based on drugs are shown in Table 8.

Table 8. Number of animals, administration state (fed/fasted) and dose employed

for various FDTs

Drug ADM state Human Dose

Animal dose (mg/kg) Number of animals

Fast feed Sample control

ANS X 1 mg 0.087 6 6

MTX* X 45 mg 90 mg 3.92 7.842 6 6 6 6

IM** X 400

mg

600

mg

800

mg

34.85 52.28 69.71 6 6 6 6 6 6

CYP X 5 mg/kg 5 6 6

CAP 1250 mg/m2 196.071 6 6

*MTX FDT had two dose strengths administered separately; hence number of animals in sample group and control group was divided

equally.

**IM FDT had three dose strengths administered separately; hence number of animals in sample group and control group was divided

equally.


Drug concentration in plasma samples (test and control) withdrawn at various

time intervals was determined using the procedures described under analytical methods.

Recovery of the drug from the plasma was carried out similarly as done for FDF’s.


The plasma drug concentration was analyzed for various pharmacokinetic

parameters similarly as done for FDF’s. The significance of the differences observed for



the mean pharmacokinetic parameters of test (tablet) and solution (control) was evaluated

using student’s t-test at a significance level of P < 0.05.

Stability studies

Stability studies were carried out on optimized tablets by storing them at 25 ± 2

°C / 60 ± 5 % RH and 40 ± 2 °C / 75 ± 5 % RH in amber colored bottles for 6 months in

stability chambers. Samples were analyzed at the intervals of one month for various

parameters such as hardness, drug content (P < 0.05), disintegration time and in vitro

drug release (P < 0.05). The results were the average of six determinations.



EATABLE GELS

Method of preparation

Gels were prepared according to the viscosity guidelines provided by National

Dysphagia Diet (NDD) task force (Table 3)

39. Methotrexate was dissolved in sufficient

quantity of distilled water by constant stirring using a magnetic stirrer. pH of the solution

was adjusted to 7.4 using 1N NaOH/1N HCl for solubilization of the drug. Other

excipients like sweetening agent, flavoring agent, preservatives were added and stirred

using magnetic stirrer to form a clear solution.

Polymer was added to distilled water in a separate beaker and stirred well until it

forms a clear polymeric gel. The drug solution was added to polymeric gel solution and

stirred until it forms a clear gel. After complete gelation, the gel was kept until no air

bubbles were visible. Gel was stored at refrigerated temperature until further use 76, 79

.

Gels were prepared using various polymers such as HPMC K4M, HPC, Na Alginate, Na

CMC and Xanthum gum. Eatable gels were also prepared using a silk fibroin polymer

extracted from silk cocoons as described by Dixit and Kulkarni 1. Composition of

optimized gel formulations containing 45 mg of drug in 5 ml (1 teaspoon) are shown in

Table 9.

Table 9. Composition of optimized methotrexate gel formulations

FC

Polymer &

Quantity

(mg)

Drug

(mg)

Sucralose

(mg)

Strawberry

Flavour

(mg)

Methyl

paraben

(mg)

Propyl

paraben

(mg)

1 N sodium

Hydroxide/

1 N Hcl

Distilled

water

(ml)

A HPMC

K4M

40

90

11.4

24

0.5

0.5

Q.S

Make up to

10 ml

B HPMC

K4M

100

C HPMC

K4M

160

D HPMC

K4M

230

FC: Formulation code; Q.S: Quantity Sufficient



EVALUATION OF GELS 1, 2

Appearance and Texture evaluation

pH

Syneresis

Rheological measurements

Drug content and uniformity


In vitro drug release studies

In vivo evaluation

Stability studies

Appearance and Texture evaluation

Appearance of the gels in terms of color, transparency and semitransparency was

observed through naked eyes. The prepared gels were evaluated for the presence of any

particles using microscope (Trinocular microscope, Coslab, Model HL-10). Texture of

the gel was evaluated in terms of stickiness and grittiness by mildly rubbing the gel

between two fingers.

pH

The pH of eatable gel was measured using digital pH meter by dipping the

electrodes into the gel at room temperature. Results are expressed as average of three

determinations.



Syneresis

Syneresis is contraction of the gel upon standing and separation of the water from

the gel. Gels were kept under scrutiny for sign of syneresis (25° C, 55 % RH and 4° C for

six months). The eatable gels showing syneresis were excluded from further studies.

Results are expressed as average of three determinations.

Rheological measurements

The influence of polymer and its concentration on gel strength was measured

using stress controlled rheometer. Cone-plate geometry (cone diameter 50 mm, cone

angle 2°) was used and the experiment was carried out using a shear rate of 50 s-1

at room

temperature (25 °C) for 20 s 39

. Results are expressed as average of three determinations.

Desired gels, as per the NDD guidelines, were evaluated for further studies.

Uniformity of drug content

Content uniformity of the gel [1 Tea spoon full (5 ml)] was determined similarly

as described under FDF’s. The content of MTX (10 no’s) was measured individually as

described under analytical methods.

In vitro drug release studies

In vitro drug release studies of the gels and plain drug (equivalent doses) were

performed using USP dissolution paddle apparatus Type II. Fixed amount of the gel (5

ml) was pressed out through the syringe into the dissolution medium. The studies were

carried out at 37 ± 0.5 ºC using three different buffer systems (pH 1.2, pH 4.5 acetate

buffer and pH 6.8 phosphate buffer) of 900 ml and paddle was stirred at 50 rpm. Two

milliliters aliquots of dissolution media were collected at predetermined time intervals

and replaced with equal volumes of respective buffer. The collected samples were filtered

(0.45 μm membrane filter), diluted if necessary and the concentration of the dissolved

drug was determined using the HPLC analytical technique. The results were the average

of six determinations. Release profile of gel formulation was compared with that of the



plain drug. The data obtained were statistically analyzed using one way analysis of

variance (p < 0.05) to assess the pH effect on release of gel and student t-test to assess the

difference in release between control and test. Gel formulations exhibiting faster In vitro

drug release and qualifying all other evaluation parameters were considered for sensory

and In vivo pharmacokinetic studies.


The method employed is as described for FDF’s. The analysis was carried out for

sucralose (5.7 mg), pure drug, placebo gel (excipients without drug and sucralose) and

optimized gel.

In vivo evaluation

The study protocol, ethical clearance, inclusion and exclusion criteria are same as

mentioned in fast disintegrating films.

Study design

The rats were anaesthetized with an intraperitoneal injection of urethane, 1 g kg -1

,

and the jugular vein was cannulated to facilitate removal of blood sample. Rats were

divided into two sets each containing two groups, control and test, with each group

containing 6 animals.

First set of the rats were administered with the animal dose of 3.92 mg/Kg

(equivalent to human dose of 25 mg/m2). Second set of the rats were administered with

the animal dose of 7.842 mg/Kg (equivalent to human dose of 50 mg/m2). Test group

received MTX gels whereas the standard group received MTX solution (equivalent dose),

administered orally using a stomach sonde needle.

Blood samples (0.25 ml) were withdrawn immediately before dosing and after of

dosing, from jugular vein. After withdrawal, samples were collected in heparinized tubes



and were centrifuged for 5 min at 10000 rpm at - 4 °C to separate the blood plasma.

Separated plasma was stored at – 50 °C until further analysis.


Plasma samples (test and control) withdrawn at various time intervals was

extracted and analyzed using the procedures given under bio-analytical methods. Using

calibration curve drug concentration in the plasma was determined. Recovery of the drug

from the plasma was carried out similarly as done for FDF’s.


The generated data was analyzed for various pharmacokinetic parameters

similarly as done for FDF’s. The significance of the differences observed for the mean

pharmacokinetic parameters of gel (test) and solution (control) was evaluated using

student’s t-test at a significance level of P < 0.05.

Stability studies

The prepared formulations were stored away from light in high-density

polyethylene bottles at 40 ± 2 °C / 75 ± 5 % RH, 25 ± 2 °C / 60 ± 5 % RH and

refrigerated temperature (2 - 8 °C) for 6 months. After an interval of one month; samples

were withdrawn, equilibrated at 25 °C for 2 h and tested for various parameters such as

their physical appearance, pH, syneresis, rheological behavior, drug content (P < 0.05)

and drug release (P < 0.05). The results were average of six determinations.

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Materials and methods - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/28261/12/12_chapter...

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