Material and Method

80

4. MATERIALS AND METHODS

4.1 Materials

Table 4.1: List of materials used

Sr.No. Name of Chemical Source

1. Ondansetron hydrochloride Aurobindo Pharma, Hyderabad

2. Levocetirizine dihydrochloride Cipla Ltd, Ahmedabad

3. Hydroxy-propyl β-cyclodextrin Alkem Laboratories, Mumbai

4. Hydroxy propyl methyl cellulose AR SD Fine Chemicals, Mumbai

5. Carboxy methyl cellulose sodium AR SD Fine Chemicals, Mumbai

6. Hydroxy propyl cellulose AR SD Fine Chemicals, Mumbai

7. Methocel K3 AR Color Cone, Goa

8. Methocel E3 AR Color Cone, Goa

9. Methocel E5 AR Alkem Laboratories, Mumbai

10. Methocel E15 AR Color Cone, Goa

11. Polyox N10 AR Color Cone, Goa

12. Polyox N 80 AR Color Cone, Goa

13. Polyox N 750 AR Color Cone, Goa

14. Pullulan gum AR Gangwal Chemicals, Mumbai

15. Xanthan gum AR Alkem Laboratories, Mumbai

16. Carrageenan gum LR Himedia Lab., Mumbai

17. Locust bean gum AR SD Fine Chemicals, Mumbai

18. Propylene glycol LR SD Fine Chemicals, Mumbai

19. Polyethylene glycol 400 LR SD Fine Chemicals, Mumbai

20. Tween 80 AR Spectrochem, Mumbai

21. Citric Acid AR SD Fine Chemicals, Mumbai

22. Neotam Alkem Laboratories, Mumbai

23. Acetonitril HPLC grade SD Fine Chemicals, Mumbai

24. Methanol HPLC grade Rankem, Mumbai.

25. Potassium bromide Sigma-Aldrich, Mumbai

26. Dichloromethane Sigma-Aldrich, Mumbai

27. Potassium dihydrogen phosphate SD Fine Chemicals, Mumbai

Material and Method

81

4.2 Instruments used

Tables 4.2: List of instruments used

Sr.No. Name of Chemical Source

1. Differential Scanning Calorimetry DSC-PYRIS-1, Perkin Elmer, USA

2. Desiccator Poly lab, India

3. Digital vernier caliper Aerospace, Munbai, India

4. Dissolution Test Apparatus Electro-DBK, DBK Instruments,

India

5. Electronic Balance AX-200, Shimadzu, Japan

6. FTIR-Spectrophotometer Spectrum GX, Perkin-Elmer, USA

7. High Performance Liquid

Chromatography

Perkin Elmer, USA

8. Hot air oven Electroquip, India

9. Humidity Chamber CIC-64,Cintex, India

10. Magnetic Stirrer EIE, India

11. Micro pipette Hamilton, China

12. pH Meter Elico, India

13. Remi Centrifuge R-8C, Laboratory Centrifuge

14. Rotary mixer Lab Line, India

15. Scanning, Electron Microscope ESEM TMP with EDAX, Philips,

Holland

16. U V Spectrophotometer Shimazdu UV-1700, Japan

17. Winsoft Tensile and Compression

Testing

Shimadzu AG-100kNG

18. X- Ray Diffraction (XRD) Studies Xpert MPD, Philips, Holland

Material and Method

82

4.3 Methods

An attempt has been made to prepare fast dissolving film (FDFs) of ondansetron

hydrochloride (Antiemetic) and levocetirizine dihydrochloride (Antiallergic). The

FDFs were prepared by using different polymers and plasticizers in different

concentrations by solvent casting method.

4.3.1 Preparation of fast dissolving films of ondansetron hydrochloride

4.3.1.1 Identification of drug

Infrared spectroscopy

A pellet of ondansetron hydrochloride and KBr was prepared using hydraulic pellet

press at a pressure of 7 to 10 tones. This was scanned from 4000-400 cm-1

by using

FTIR (Spectrum GX; Perkin-Elmer, USA).

4.3.1.2 Estimation of drug

Determination of UV absorption maxima

A stock solution of ondansetron was prepared by dissolving 10 mg of pure drug in

100 ml of milipore double distilled water. One ml aliquot solution was taken in 10 ml

volumetric flask and diluted with distilled water to obtain final concentration 10

µg/ml. the resultant solution was scanned in the U V region of 200 to 400 nm by

UV-VIS spectrophotometer (Shimazdu UV-1700, Japan).

4.3.1.3 Preparation of calibration curve

A stock solution of ondansetron was prepared by dissolving 100 mg of pure drug in

100 ml of millipore water (1 mg/ml), resulted solution was filtered through Whatman

filter paper (42 No., 0.45 µ). Appropriate aliquots were prepared in different 10 ml

volumetric flask and diluted with millipore water to obtain concentration of 2, 4, 6, 8,

10, 12, 14, 16, 18, and 20 µg/ml. Absorbance of resultant solutions were measured at

210 nm (λmax) against blank and calibration curve of absorbance vs. concentration was

plotted (Figure 5.3).

Material and Method

83

4.3.1.4 Preparation of placebo film for ondansetron hydrochloride

To prepare placebo film, a proper selection of film former and plasticizer is required.

In this research work, various film formers like- HPMC (15 cps), SCMC, HPC,

methocel K3, E3, E5, E15, polyox N10, N80 N750, pullulan gum and plasticizers

like- PG, PEG-400, glycerol and tween 80 were used. Film were prepared by solvent

casting method and dried in hot air own at 50 °C.

Selection of film former

Selection of the film former was done by preparing placebo films of film former like:

HPMC (15 cps), SCMC, HPC, methocel K3, E3, E5, E15, polyox N10, N80 N750,

pullulan gum at different concentrations. Various film modifiers like Xanthan gum,

Carrageenan gum and Locust bean gum were used to provide strength to the films at

different concentrations. Films were prepared by using solvent casting method.

Selection of cellulose derivatives

Initially cellulose derivatives like HPMC (15 cps), SCMC, HPC are employed in

different concentration ranging from 1 – 4 % as shown in Table 4.3.

Table 4.3: Selection of the cellulose derivatives as film former for ondansetron

film

Formulation

Code

HPMC

(% w/v)

CMC

(% w/v)

HPC

(% w/v)

OA1 1 - -

OA2 2 - -

OA3 3 - -

OA4 4 - -

OA5 - 1 -

OA6 - 2 -

OA7 - 3 -

OA8 - 4 -

OA9 - - 1

OA10 - - 2

OA11 - - 3

OA12 - - 4

Material and Method

84

Selection of plasticizers for cellulose derivatives

Various plasticizers like propylene glycol, polyethylene glycol - 400, glycerol and

tween 80 were used. Their concentrations were varying from 5-15 % w/w of

plasticizer concentration as mentioned in Table 4.4.

Table 4.4: Selection of plasticizers for cellulose derivatives (HPMC, OA3) for

ondansetron film

Formulation

Code

HPMC

(% w/v)

Propylene

glycol

(% w/w)

PEG 400

(% w/w)

Glycerol

(% w/w)

Tween

80

(% w/w)

OB1 3 5 - - -

OB2 3 10 - - -

OB3 3 15 - - -

OB4 3 - 5 - -

OB5 3 - 10 - -

OB6 3 - 15 - -

OB7 3 - - 5 -

OB8 3 - - 10 -

OB9 3 - - 15 -

OB10 3 - - - 5

OB11 3 - - - 10

OB12 3 - - - 15

Material and Method

85

Selection of methocel grades

Methocel grade film formers like- Methocel K3, E3, E5 and E15 were used in

different concentrations ranging from 1- 4 % w/v as shown in Table 4.5.

Table 4.5: Selection of methocel grades as film former for ondansetron film

Form.

Code

PG

(% w/w)

Methocel K3

(% w/v)

Methocel E3

(% w/v)

Methocel E5

(% w/v)

Methocel E15

(% w/v)

OC1 10 1 - - -

OC2 10 2 - - -

OC3 10 3 - - -

OC4 10 4 - - -

OC5 10 - 1 - -

OC6 10 - 2 - -

OC7 10 - 3 - -

OC8 10 - 4 - -

OC9 10 - - 1 -

OC10 10 - - 2 -

OC11 10 - - 3 -

OC12 10 - - 4 -

OC13 10 - - - 1

OC14 10 - - - 2

OC15 10 - - - 3

OC16 10 - - - 4

Material and Method

86

Selection of polyox grades

Polyox grade film formers like- polyox N10, N80, and N750 were used in different

concentrations ranging from 2- 4 % w/v as shown in Table 4.6.

Table 4.6: Selection of polyox grades as film former for ondansetron film

Form.

Code

PG

(% w/w)

Polyox N10

(% w/v)

Polyox N80

(% w/v)

Polyox N750

(% w/v)

OD1 10 2 - -

OD2 10 3 - -

OD3 10 4 - -

OD4 10 - 2 -

OD5 10 - 3 -

OD6 10 - 4 -

OD7 10 - - 2

OD8 10 - - 3

OD9 10 - - 4

Selection of pullulan gum

Pullulan, a natural gum widely used in film technology. It is used in different

concentrations ranging from 1- 4 % w/v as shown in Table 4.7.

Table 4.7: Selection of concentration of pullulan gum as film former for

ondansetron film

Ingredients Formulations Code

OE1 OE2 OE3 OE4

Pullulan (% w/v) 1 2 3 4

Water (ml) 25 25 25 25

Selection of plasticizers for pullulan films

Various plasticizers like propylene glycol, Polyethylene glycol - 400, Glycerol and

tween 80 were used. Their concentrations were varying from 5-15 % w/w of

plasticizer concentration. Combination of propylene glycol - tween 80 and

Material and Method

87

polyethylene glycol – tween 80 in different concentrations were also used. Their

concentrations were varying as mentioned in Table 4.8.

Table 4.8: Selection of plasticizers for pullulan films (OE3) for ondansetron

Form.

Code

PG

(% w/w)

PEG 400

(% w/w)

Glycerol

(% w/w)

Tween 80

(% w/w)

PG : Tween 80

(% w/w)

PEG : Tween 80

(% w/w)

OF1 5 - - - - -

OF2 10 - - - - -

OF3 15 - - - - -

OF4 - 5 - - - -

OF5 - 10 - - - -

OF6 - 15 - - - -

OF7 - - 5 - - -

OF8 - - 10 - - -

OF9 - - 15 - - -

OF10 - - - 5 - -

OF11 - - - 10 - -

OF12 - - - 15 - -

OF13 - - - - 1:1 -

OF14 - - - - 1:2 -

OF15 - - - - 2:1 -

OF16 - - - - 2:2 -

OF17 - - - - - 1:1

OF18 - - - - - 1:2

OF19 - - - - - 2:1

OF20 - - - - - 2:2

Selection of film modifiers

Various film modifiers like carrageenan gum. Xanthan gum and locust bean gum

were used to provide proper strength for Pullulan films. Their concentration was

varying from 0.2 - 0.6 % w/v as shown Table 4.9.

Material and Method

88

Table 4.9: Selection of film modifier for pullulan film for ondansetron

Form.

Code

Pullulan gum

(% w/v)

Carrageenan gum

(% w/v)

Xanthan gum

(% w/v)

Locust bean

gum (% w/v)

OG1 2 0.2 - -

OG2 2 0.4 - -

OG3 2 0.6 - -

OG4 2 - 0.2 -

OG5 2 - 0.4 -

OG6 2 - 0.6 -

OG7 2 - - 0.2

OG8 2 - - 0.4

OG9 2 - - 0.6

4.3.1.5 Drug – excipients compatibility study

Fourier transform infrared spectroscopy (FTIR)

The FTIR absorption spectra of the pure drug, placebo films and drug loaded films

were recorded in the range of 4000– 400 cm-1

by KBr disc method using FTIR

spectrophotometer (Spectrum GX, Perkin-Elmer, USA).

Differential scanning calorimetry (DSC)

Differential scanning calorimetry (DSC) scans of pure drug, placebo films and drug

loaded films were performed using DSC-PYRIS-1 (Perkin-Elmer, USA). The analysis

was performed with a heating range of 50-480°C and a rate of 10 °C min-1

in an inert

nitrogen atmosphere.

4.3.1.6 Complexation of β-cyclodextrin with ondansetron hydrochloride (ODS)

ODS–HPβ-CD inclusion complex was prepared by co-evaporation method. The molar

ratio of ODS and HPβ-CD was varied from 1:1 to 1:3. The required quantities of ODS

and β-CD were dissolved in the water. The solvent was allowed to evaporate by

heating at 45–50°C. The resultant solid was pulverized and then sieved through 120#.

The complex formation was characterized by using X- Rray Diffraction (XRD)

Material and Method

89

Studies (Xpert MPD, Philips, Holland), DSC Studies, FT-IR Spectroscopy. Here

hydroxyl propyl β-cyclodextrin were used to mask the bitter taste of Ondansetron.

4.3.1.7 Selection of sweetening agent and saliva stimulating agent

Neotam was used as sweetening agent and citric acid was used as saliva stimulating

agents. Their concentration was varied as shown in Table 4.10. Neotam and citric acid

were used to improve palatability.

Table 4.10: Selection of sweetening agent and stimulating agent for FDFs of

ondnasetron

Form.

Code

Inclusion complex

ODS: HPβ-CD

Neotame

(% w/w)

Citric acid

(% w/v)

ONC1 1:1 0.020 -

ONC2 1:1 0.025 -

ONC3 1:1 0.030 -

ONC4 1:1 0.035 -

ONC5 1:1 0.020 0.20

ONC6 1:1 0.025 0.25

ONC7 1:1 0.030 0.30

ONC8 1:1 0.035 0.35

4.3.1.8 Preparation of FDFs of ondansetron hydrochloride

All the selected film formers and modifiers were soaked in half the quantity of water

separately for 8 hours to get uniform dispersion and mixed both the solution with

stirring. ODS – HPβ-CD complex was dissolved in a portion of water. This solution

was added to polymeric solution and mixed well to obtain homogenous solution

followed by addition of plasticizer/s, neotam and citric acid. The solution was mixed

well to get uniform dispersion. Solution was then casted into petridishes having

surface area of 64 cm2

and 1.3 cm wall height. Petridishes were kept in hot air oven

for 8 hours at 50˚ C. After drying films were removed with the help of sharp blade

and kept in desicator for 24 hrs before cutting into small pieces having area of 6 cm2

for each film. Films with air bubbles, cuts or imperfections were excluded from

further study. Selected films were subjected for different evaluation parameters.

Material and Method

90

4.3.1.9 Evaluation parameters

Appearance

All prepared films were checked for their appearances either they are transparent or

opaque.

Weight variation and thickness

Films were evaluated for its weight variation and thickness. Weight variation was

evaluated by using electronic balance (Shimadzu Corp. Japan. Type AX200) and

thickness was measured using Digital Vernier Calipers (Aerospace, Munbai) (Mishra

R et al, 2009).

Mechanical properties

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

folding endurance were evaluated for prepared films (Brindle L et al, 2008. Felton L

et al, 2008).

Tensile strength

It was measured using Shimadzu AG-100kNG (Winsoft tensile and compression

testing). The film of size 5×2 cm2

and free of physical imperfections was placed

between two clamps held 10 mm apart. The film was pulled by clamp at a rate of 5

mm/min. Whole experiment was carried out in triplicate (Mishra R et al, 2009).

Percentage elongation

Percentage elongation was calculated by measuring the increase in length of the film

after tensile strength measurement by using the following formula.

Where,

L = Final length, L0 = Initial length.

Material and Method

91

Elastic modulus

Young's modulus or elastic modulus is the measure of stiffness of strip. It is

represented as the ratio of applied stress over strain in the region of elastic

deformation.

Elastic modulus was calculated by following formula

strainingCorrespondmmareationalCross

strainingcorrespondatForceModulusElastic

1

sec 2

Folding endurance

A film of 6 cm2 was repeatedly folded and unfolded at the same place till it breaks.

The number of times, the film could be folded at same place, without breaking was

recorded as the value of folding endurance. This gives an indication of brittleness of

the film (Kunte S, 2010).

Morphology study of film

Morphology of the prepared films was observed under a scanning electron

microscope (SEM) (ESEM TMP with EDAX, Philips, Holland). The sample was

attached to the slab surface with double sided adhesive tapes and the scanning

electron photomicrograph was taken at 2000 x magnification (Mashru R et al, 2005).

Surface pH

The surface pH of rapid dissolving films was determined in order to investigate the

possibility of any side effects in vivo. As an acidic or alkaline pH may cause irritation

to the oral mucosa, it was determined to keep the surface pH as close to neutral as

possible (Kunte S, 2010). The films were allowed to swell in closed petridish at room

temperature for 30 minutes in 1ml of distilled water. Solution was placed under digital

pH meter (Elico, India) to determine the surface pH.

Content uniformity

The drug content of optimized films were assayed by random sampling of the 5 films

of 6 cm2

from one petridish (64 cm2), each film was dissolved in 50 ml volumetric

flask containing water. Solution was subjected to centrifugation for 15 min at 2500

rpm. The supernatant liquid was diluted to obtain 10 µg/ml solution and passed

through Whatman filter paper. Filtered solution was analyzed by double beam UV

Material and Method

92

Spectrophotometer at 210 nm against 6.4 pH phosphate buffer solution as blank

(Mahesh A et al, 2010).

In-vitro disintegration time

Disintegration time provides an indication about the disintegration characteristics and

dissolution characteristics of the film. The require size of film (6 cm2) of selected

formulations was placed in a glass petridish (9 cm diameter and 1.3 cm wall height)

containing 10 ml of distilled water and left undisturbed. The time was noted down till

film was completely converted into small pieces. Test was performed 3 times on each

formulation (Doaa A et al, 2010).

In-vitro dissolution studies

An in-vitro dissolution study was performed for the films of selected formulations for

3 minutes in USP paddle apparatus using pH 6.4 phosphate buffer solution.

Dissolution medium was kept at 37º C ± 2 ºC and rotated at 500 rpm. The samples

(5 ml) were withdrawn after every 30 sec and replaced with fresh buffer (pH 6.4)

solution. One ml sample was then taken and diluted up to 10 ml in volumetric flask.

The samples were analyzed for the drug content using UV spectrophotometer at

210 nm. Dissolution was performed 3 times for each formulation to calculate drug

release (Dinge A et al, 2008).

Stability study

Short term stability studies was performed for optimized films were placed in plastic

containers and exposed to 40 ± 0.5 ˚C and 75± 5% RH (ICH guidelines) for a period

of 4 months. Different film properties like physical appearance of the film,

mechanical properties and drug content was evaluated at interval of one month

(Francesco C et al, 2008, ICH Steering Committee 2003)

4.3.1.10 Pharmacokinetics studies of FDFs of ondansetron hydrochloride

Selection of mobile phase

Instrumentation

The analysis of ondansetron hydrochloride was carried out on isocratic high pressure

liquid chromatography (HPLC), HPLC system used consist of pump (Perkin Elmer,

Material and Method

93

USA) with universal loop injector (Rheodyne) of injection capacity 20 μl. Detector

consists of U.V. detector; the reversed phase column used was RP-C18 brownlee

(5μm particle size, 250 mm x 4.6 mm i.d.) at ambient temperature (Dedania Z et al,

2009. Venkateshwaran T et al, 1995).

Experimental condition

The HPLC system was operated isocratically at flow rate of 1ml/min. at 25°C ± 0.5°C

for 10 min.

Sample preparation

Stock solution containing 10 ppm ondansetron hydrochloride was prepared in the

millipore water. Sub stock solution was prepared from stock solution by diluting 1 ml

of stock solution up to 10 ml by mobile phase to get 100 µg/ml of drug solution. The

nominal concentrations in range of 2-12 µg/ml were prepared for calibration. All

solutions were stored at room temperature. Each working solution (20 μl) was injected

into the column.

Extraction procedure

In a 10 ml capacity glass tube, 1 ml plasma was mixed with 50 µl of saturated sodium

carbonate solution and 5 ml of dichloromethane and mixture was stirred by rotary

mixer for 15 min at room temperature. The mixture was centrifuged for 5 min at 5000

rpm and 4.5 ml of the organic phase was transferred into another test tube and

evaporated to dryness at 40°C under a stream of nitrogen. The residue was

reconstituted in 100 µl of mobile phase and a volume of 20 µl was injected into the

HPLC for analysis (Colthup P et al, 1989. Depot M et al, 1997).

Preparation of stock solution, calibration standards and quality control samples

Stock solution (100 ng/µl) was prepared by dissolving 10 mg of ondansetron in water

in a 50 ml volumetric flask. The solutions were stored at – 20 °C. Calibrate samples

were prepared with drug free plasma that was spiked with increasing concentrations

of ondansetron (6, 15, 30, 40, 50 and 60 ng/ml). Quality control samples were

prepared with drug free plasma spiked with three different concentrations of

ondansetron. Low, medium and high quality control sample concentrations used were

Material and Method

94

6, 40 and 60 ng/ml, respectively. The solutions were stored at – 20 °C (Bauer S et al,

2002).

Study design

Seven-week-old male Sprague–Dawley rats were used in the present experiment.

Their mean weight was 264.66 ± 8.96 g in the range of 250-275 g. Animals were

housed in a room maintained on a 12 hours light/dark cycle at 23±2 °

C with free

access to food and water. The experimental procedures were approved by the

Committee for the Care and Use of Laboratory Animals at the IICP (Protocol number:

IICP/PH/12-2010/02). For the administration of film preparation, 50 µL aliquot of

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

two halves (1 cm×0.5 cm) of the film preparation were applied to the buccal cavity

bilaterally. For oral administration, rats were orally given with 8 mg of ODS dose

(Equivalent to body weight of rat) solution containing in 1 ml under light ether

anesthesia. Blood specimens were taken (every 0.5 ml) in a centrifuge plastic

capillary tube from the intraorbital route at 0 min, 30 min, 1 h, 2 h, 4 h, and 6 h after

drug administration. Blood was subjected to centrifugation at 10,000 rpm for 15 min

then plasma was taken in a polyethylene tube and stored at −20 ◦

C until assay. The

concentration of ondansetron was determined by HPLC-UV at 220 nm (Shimoda H et

al, 2009).

Pharmacokinetic parameters were derived from the plasma concentration vs. time

plot. The peak plasma concentration (Cmax) and the time to attain peak concentration

(Tmax) were obtained from these plots. The elimination rate constant (Kel) was

determined from the semilogarithmic plot of plasma concentration vs. time.

Elimination half-life (t1/2) was calculated using the formula; t1/2 = 0.693/Kel. AUC was

statistically analyzed applying one-way ANOVA at 0.05 levels in the GraphPad Prism

version 5.01 software.

Material and Method

95

4.3.2 Preparation of fast dissolving films of levocetirizine dihydrochloride

4.3.2.1 Identification of drug

Infrared spectroscopy

A pellet of levocetirizine dihydrochloride and KBr was prepared using hydraulic

pellet press at a pressure of 7 to 10 tones. This was scanned from 4000-400 cm-1

by

using FTIR (Spectrum GX; Perkin-Elmer, USA).

4.3.2.2 Estimation of levocetirizine dihydrochloride

Determination of UV absorption maxima

A stock solution of levocetirizine dihydrochloride was prepared by dissolving 10 mg

of pure drug in 100 ml of millipore water. One ml aliquot solution was taken in 10 ml

volumetric flask and diluted with distilled water to obtain final concentration 10

µg/ml. the resultant solution was scanned in the U V region of 200 to 400 nm by UV-

VIS spectrophotometer.

4.3.2.3 Preparation of calibration curve

A stock solution of levocetirizine dihydrochloride was prepared by dissolving 100 mg

of pure drug in 100 ml of millipore water (1 mg/ml), resulted solution was filtered

though Whatman filter paper (42 No., 0.45 µ). Appropriate aliquots were prepared in

different 10 ml volumetric flask and diluted with distilled water to obtain

concentration of 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 µg/ml. Absorbance of resultant

solutions were measured at 231 nm (λmax) against blank and calibration curve of

absorbance vs. concentration was plotted Figure (5.87).

4.3.2.4 Preparation of placebo films for levocetirizine dihydrochloride

To prepare placebo film, a proper selection of film former and plasticizer is required.

In this research work, various film formers like- HPMC (15 cps), SCMC, HPC,

methocel K3, E3, E5, E15, polyox N10, N80 N750, pullulan gum and plasticizers

like- PG, PEG-400, glycerol and tween 80 were used. Film were prepared by solvent

casting method and dried in hot air own at 50 °C.

Material and Method

96

Selection of film former

Selection of the film former was done by preparing placebo films of film former like:

HPMC (15 cps), SCMC, HPC, Methocel K3, E3, E5, E15, polyox N10, N80 N750,

Pullulan gum at different concentrations. Various film modifiers like Xanthan gum,

Carrageenan gum and Locust bean gum were used to provide strength to the films at

different concentrations. Films were prepared by using solvent casting method.

Selection of cellulose derivatives

Initially cellulose derivatives like HPMC (15 cps), SCMC, HPC are employed in

different concentration ranging from 1-4% as shown in Table 4.11.

Table 4.11: Selection of the cellulose derivatives as film former for levocetirizine

film

Formulation

Code

HPMC

(% w/v)

CMC

(% w/v)

HPC

(% w/v)

LA1 1 - -

LA2 2 - -

LA3 3 - -

LA4 4 - -

LA5 - 1 -

LA6 - 2 -

LA7 - 3 -

LA8 - 4 -

LA9 - - 1

LA10 - - 2

LA11 - - 3

LA12 - - 4

Material and Method

97

Selection of plasticizer

Various plasticizers like propylene glycol, polyethylene glycol - 400, glycerol and

tween80 were used. Their concentrations were varying from 5-15 % w/w of

plasticizer concentration as mentioned in Table 4.12.

Table 4.12: Selection of plasticizers for cellulose derivatives (HPMC, LA3) for

films of levocetirizine

Formulation

Code

HPMC

(% w/v)

Propylene glycol

(% w/w)

PEG 400

(% w/w)

Glycerin

(% w/w)

Tween 80

(% w/w)

LB1 3 5 - - -

LB2 3 10 - - -

LB3 3 15 - - -

LB4 3 - 5 - -

LB5 3 - 10 - -

LB6 3 - 15 - -

LB7 3 - - 5 -

LB8 3 - - 10 -

LB9 3 - - 15 -

LB10 3 - - - 5

LB11 3 - - - 10

LB12 3 - - - 15

Material and Method

98

Selection of methocel grades

Methocel grades film formers like- Methocel K3, E3, E5 and E15 were used in

different concentrations ranging from 1- 4 % w/v as shown in Table 4.13.

Table 4.13: Selection of methocel grades as film former of levocetirizine film

Form.

Code

PG

(% w/w)

Methocel K3

(% w/v)

Methocel E3

(% w/v)

Methocel E5

(% w/v)

Methocel E15

(% w/v)

LC1 10 1 - - -

LC2 10 2 - - -

LC3 10 3 - - -

LC4 10 4 - - -

LC5 10 - 1 - -

LC6 10 - 2 - -

LC7 10 - 3 - -

LC8 10 - 4 - -

LC9 10 - - 1 -

LC10 10 - - 2 -

LC11 10 - - 3 -

LC12 10 - - 4 -

LC13 10 - - - 1

LC14 10 - - - 2

LC15 10 - - - 3

LC16 10 - - - 4

Material and Method

99

Selection of polyox grades

Polyox grades film formers like- polyox N10, N80, and N750 were used in different

concentrations ranging from 2- 4 % w/v as shown in Table 4.14.

Table 4.14: Selection of polyox grades as film former of levocetirizine film

Form.

Code

PG

(% w/w)

Polyox N10

(% w/v)

Polyox N80

(% w/v)

Polyox N750

(% w/v)

LD1 10 2 - -

LD2 10 3 - -

LD3 10 4 - -

LD4 10 - 2 -

LD5 10 - 3 -

LD6 10 - 4 -

LD7 10 - 2

LD8 10 - 3

LD9 10 - 4

Selection of pullulan concentration

Pullulan, a natural gum widely used in film technology. It is used in different

concentrations ranging from 1- 4 % w/v as shown in Table 4.15.

Table 4.15: Selection of concentration of pullulan as film former of levocetirizine

film

Ingredients Formulations Code

LE1 LE2 LE3 LE4

Pullulan (% w/v) 1% 2% 3% 4%

Water (ml) 25 25 25 25

Selection of plasticizer for pullulan

Various plasticizers like propylene glycol, Polyethylene glycol - 400, Glycerol and

tween 80 were used. Their concentrations were varying from 5-15 % w/w of

plasticizer concentration. Combination of propylene glycol - tween80 and

Material and Method

100

polyethylene glycol – tween80 in different concentrations were also used. Their

concentrations were varying as mentioned in Table 4.16.

Table 4.16: Selection of plasticizer for pullulan film (LE2) for levocetirizine film

Form.

Code

PG

(% w/w)

PEG 400

(% w/w)

Glycerol

(% w/w)

Tween 80

(% w/w)

PG : Tween 80

(% w/w)

PEG : Tween 80

(% w/w)

LF1 5 - - - - -

LF2 10 - - - - -

LF3 15 - - - - -

LF4 - 5 - - - -

LF5 - 10 - - - -

LF6 - 15 - - - -

LF7 - - 5 - - -

LF8 - - 10 - - -

LF9 - - 15 - - -

LF10 - - - 5 - -

LF11 - - - 10 - -

LF12 - - - 15 - -

LF13 - - - - 1:1 -

LF14 - - - - 1:2 -

LF15 - - - - 2:1 -

LF16 - - - - 2:2 -

LF17 - - - - - 1:1

LF18 - - - - - 1:2

LF19 - - - - - 2:1

LF20 - - - - - 2:2

Selection of film modifier

Various film modifiers like carrageenan gum. Xanthan gum and locust bean gum

were used to provide proper strength for pullulan films. Their concentration was

varying from 0.2 - 0.6 % w/v as shown Table 4.17.

Material and Method

101

Table 4.17: Selection of film modifier for pullulan films for levocetirizine

Form.

Code

Pullulan

gum (% w/v)

Carrageenan

gum (% w/v)

Xanthan gum

(% w/v)

Locust bean gum

(% w/v)

LG1 2 0.2 - -

LG2 2 0.4 - -

LG3 2 0.6 - -

LG4 2 - 0.2 -

LG5 2 - 0.4 -

LG6 2 - 0.6 -

LG7 2 - - 0.2

LG8 2 - - 0.4

LG9 2 - - 0.6

4.3.2.5 Drug – excipients compatibility study

Fourier transform infrared spectroscopy (FTIR)

The FTIR absorption spectra of the pure drug, placebo formulations and drug loaded

films were recorded in the range of 4000– 400 cm-1

by KBr disc method using FTIR

spectrophotometer (Perkin-Elmer, USA).

Differential scanning calorimetry (DSC)

Differential scanning calorimetry (DSC) scans of pure drug, placebo formulations and

drug loaded films were performed using DSC-PYRIS-1 (Perkin-Elmer, USA). The

analysis was performed with a heating range of 50-480 °C and a rate of 10 °C min-1

in

an inert nitrogen atmosphere.

4.3.2.6 Complexation of β-cyclodextrin with levocetirizine dihydrochloride

(LCZ)

LCZ–HPβ-CD inclusion complex was prepared by co-evaporation method. The molar

ratio of LCZ and HPβ-CD was varied from 1:1 to 1:3. The required quantities of LCZ

and β-CD were dissolved in the water. The solvent was allowed to evaporate by

Material and Method

102

heating at 45–50°C. The resultant solid was pulverized and then sieved through 120 #.

The complex formation was characterized by using X- Rray Diffraction (XRD)

Studies (Xpert MPD, Philips, Holland), DSC Studies, FT-IR Spectroscopy. Here

hydroxyl propyl β-cyclodextrin were used to mask the bitter taste of levocetirizine.

4.3.2.7 Selection of sweetening agent and saliva stimulating agent

Neotam was used as sweetening agent and citric acid is used as saliva stimulating

agents. Their concentration was varied as shown in Table 4.18.

Table 4.18: Selection of sweetening agent and stimulating agent for levocetirizine

film

Formulation

Code

Inclusion Complex

LCZ : HPβ-CD

Neotame

(% w/w)

Citric Acid

(% w/v)

LNC1 1:1 0.020 -

LNC2 1:1 0.025 -

LNC3 1:1 0.030 -

LNC4 1:1 0.035 -

LNC5 1:1 0.020 0.20

LNC6 1:1 0.025 0.25

LNC7 1:1 0.030 0.30

LNC8 1:1 0.035 0.35

4.3.2.8 Preparation of FDFs

All the selected film formers and modifiers were soaked in half the quantity of water

separately for 8 hours to get uniform dispersion and mixed both the solution with

stirring. LCZ– HPβ-CD complex was dissolved in a portion of water. This solution

was added to polymeric solution and mixed well to obtain homogenous solution

followed by addition of plasticizer/s, neotam and citric acid. The solution was mixed

well to get uniform dispersion. Solution was then casted into petridishes having

surface area of 64 cm2

and 1.3 cm wall height. Petridishes were kept in hot air oven

for 8 hours at 50˚ C. After drying films were removed with the help of sharp blade

and kept in desicator for 24 hrs before cutting into small pieces having area of 6 cm2

Material and Method

103

for each film. Films with air bubbles, cuts or imperfections were excluded from

further study. Selected films were subjected for different evaluation parameters.

4.3.2.9 Evaluation parameters

Appearance

All prepared films were checked for their appearances either they are transparent or

opaque.

Weight variation and thickness

Films were evaluated for their weight variation and thickness. Weight variation was

evaluated by using electronic balance (Shimadzu Corp. Japan. Type AX200) and

thickness was measured using Digital Vernier Calipers. (Mishra R et al, 2009)

Mechanical properties

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

folding endurance were evaluated for prepared films (Brindle L et al, 2008. Felton L

et al, 2008).

Tensile strength

It was measured using Shimadzu AG-100kNG (Winsoft tensile and compression

testing). The film of size 5×2 cm2

and free of physical imperfections was placed

between two clamps held 10 mm apart. The film was pulled by clamp at a rate of

5mm/min. Whole experiment was carried out in triplicate.

Percentage elongation

Percentage elongation was calculated by measuring the increase in length of the film

after tensile strength measurement by using the following formula.

Where, L = Final length, L0 = Initial length.

Material and Method

104

Elastic modulus

Young's modulus or elastic modulus is the measure of stiffness of strip. It is

represented as the ratio of applied stress over strain in the region of elastic

deformation.

Elastic modulus was calculated by following formula

strainingCorrespondmmareationalCross

strainingcorrespondatForceModulusElastic

1

sec 2

Folding endurance

A film of 6 cm2 was repeatedly folded and unfolded at the same place till it breaks.

The number of times, the film could be folded at same place, without breaking was

recorded as the value of folding endurance. This gives an indication of brittleness of

the film (Kunte S, 2010).

Morphology study of film

Morphology of the prepared films was observed under a scanning electron

microscope (SEM) (ESEM TMP with EDAX, Philips, Holland) The sample was

attached to the slab surface with double sided adhesive tapes and the scanning

electron photomicrograph was taken at 2000 x magnification (Mashru R et al, 2005).

Surface pH

The surface pH of rapid dissolving films was determined in order to investigate the

possibility of any side effects in vivo. As an acidic or alkaline pH may cause irritation

to the oral mucosa, it was determined to keep the surface pH as close to neutral as

possible. The films were allowed to swell in closed petridish at room temperature for

30 minutes in 1ml of distilled water. Solution was placed under digital pH meter

(Elico, India) to determine the surface pH (Kunte S, 2010).

Content uniformity

The drug content of optimized films were assayed by random sampling of the 5 films

of 6 cm2

from one petridish (64 cm2), each film was dissolved in 50 ml volumetric

flask containing water. Solution was subjected to centrifugation for 15 min at 2500

rpm. The supernatant liquid was diluted to obtain 10 µg/ml solution and passed

Material and Method

105

through whatman filter paper. Filtered solution was analyzed by double beam UV

Spectrophotometer at 231nm against 6.4 buffer solution as blank (Mahesh A et al,

2010).

In-vitro disintegration time

Disintegration time provides an indication about the disintegration characteristics and

dissolution characteristics of the film. The require size of film (6 cm2) of selected

formulations was placed in a glass petri dish (9 cm diameter and 1.3 wall height)

containing 10 ml of distilled water and left undisturbed. The time was noted down till

film was completely converted into small pieces. Test was performed 3 times on each

formulation (Doaa et al, 2010).

In-vitro dissolution studies

An in-vitro dissolution study was performed for the films of selected formulations for

3 minutes in USP paddle apparatus using pH 6.4 buffer solution. Dissolution medium

was kept at 37º C ± 0.5º C and rotated at 500 rpm. The samples (5 ml) were

withdrawn after every 30 sec and replaced with fresh buffer (pH 6.4) solution. One ml

sample was then taken and diluted up to 10 ml in volumetric flask. The samples were

analyzed for the drug content using UV spectrophotometer at 231 nm. Dissolution

was performed 3 times for each formulation to calculate drug release profile (Dinge A

et al, 2008).

Stability study

Short term stability studies was performed for optimized films were placed in plastic

containers and exposed to 40 ± 0.5 ˚C and 75± 5% RH (ICH guidelines) for a period

of 4 months. Different film properties like physical appearance of the film,

mechanical properties and drug content was evaluated at interval of one week.

4.3.2.10 Pharmacokinetics studies of FDFs of levocetirizine dihydrochloride

Selection of mobile phase

Instrumentation

The analysis of levocetirizine dihydrochloride was carried out on isocratic high

pressure liquid chromatography (HPLC), HPLC system used consist of pump (Perkin

Material and Method

106

Elmer, USA) with universal loop injector (Rheodyne) of injection capacity 20 μl.

Detector consists of U.V. detector; the reversed phase column used was RP-C18 (5μm

size, 250 mm 4.6 mm i.d.) at ambient temperature.

Experimental condition

The HPLC system was operated isocratically at flow rate of 1ml/min. at 25°C ± 0.5°C

for 10 min.

Sample preparation

Stock solution containing 10 ppm levocetirizine was prepared in the water. Sub stock

solution was prepared from stock solution by diluting 1 ml of stock solution up to 10

ml by mobile phase to get 100 µg/ml of drug solution. The nominal concentrations in

range of 2-12 µg/ml were prepared for calibration. All solutions were stored at room

temperature. Each working solution (20μl) was injected into the column. (Ambadas R.

Rote A et al, 2010. Dhaneshwar S et al, 2011. Kamarapu S et al, 2010. Basu A et al,

2011, Kumar S et al, 2009)

Extraction procedure

In a 10 mL glass tube 1 ml plasma was mixed with 50 µl of saturated sodium

carbonate solution and 5 ml of dichloromethane and extracted with a rotary mixer for

15 min at room temperature. The mixture was centrifuged for 5 min at 5000 rpm and

4.5 ml of the organic phase were transferred into a new tube and evaporated to

dryness at 40°C under a stream of nitrogen. The residue was reconstituted in 100 µl of

mobile phase and a volume of 20–30 µl was injected into the HPLC for analysis.

(Arayne A et al, 2008. Cranswick N et al, 2005)

Preparation of stock solution, calibration standards and quality control samples

Stock solution (100 ng/µl) was prepared by dissolving 5 mg of levocetirizine in water

in a 50 ml volumetric flask. The solutions were stored at - 20°C. Calibrant samples

were prepared with drug free plasma that was spiked with increasing concentrations

of ondansetron (50, 100, 200, 300 and 400 ng/ml). Quality control samples were

prepared with drug free plasma spiked with three different concentrations of

ondansetron. Low, medium and high QC concentrations used were 50, 200 and 400

ng/ ml, respectively. The solutions were stored at - 20°C.

Material and Method

107

Study design

Seven-week-old male Sprague–Dawley rats were used in the present experiment.

Their mean weight was 264.66 ± 8.96 g in the range of 250-275 g. Animals 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 experimental procedures were approved by the Committee for

the Care and Use of Laboratory Animals at the IICP (Protocol number: IICP/PH/12-

2010/02). For the administration of film preparation, 50 µL aliquot of distilled water

was dropped into the rat oral cavity under light ether anesthesia, then two halves (1

cm×0.5 cm) of the film preparation were applied to the buccal cavity bilaterally. For

oral administration, rats were orally given with 5 mg of LCZ dose (Equivalent to body

weight of rat) solution containing in 1 ml under light ether anesthesia. Blood

specimens were taken (every 0.5 ml) in a centrifuge plastic capillary tube from the

intraorbital route at 0 min, 30 min, 1 h, 2 h, 4 h, 6h and 12 h after drug

administration. Blood was subjected to centrifugation at 10,000 rpm for 15 min than

plasma was taken in a polyethylene tube and stored at −20 ◦

C until assay. The

concentration of levocetirizine was determined by HPLC-UV at 230 nm.

Pharmacokinetic parameters were derived from the plasma concentration vs. time

plot. The peak plasma concentration (Cmax) and the time to attain peak concentration

(Tmax) were obtained from these plots. The elimination rate constant (Kel) was

determined from the semilogarithmic plot of plasma concentration vs. time.

Elimination half-life (t1/2) was calculated using the formula; t1/2 = 0.693/Kel. AUC

was statistically analyzed applying one-way ANOVA at 0.05 levels in the GraphPad

Prism version 5.01 software.

Material and Method

108

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