FORMULATION, CHARACTERIZATION, IN VITRO, IN VIVO AND...

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Chapter 6 Formulation, characterization, in vitro, in vivo and correlation

study of mucoadhesive sustained release tablets

FORMULATION, CHARACTERIZATION, IN VITRO, IN VIVO

AND CORRELATION STUDY OF MUCOADHESIVE

SUSTAINED RELEASE TABLETS

The extracted Natural Mucoadhesive Materials from the Seeds of Caesalpinia

pulcherrima and Leucaena leucocephala has proved as a promising candidate for the

development of Sustained release drug delivery system.

This part of the work investigates the feasibility of using NMM01 and

NMM02 as the rate retarding polymer for the formulation of sustained release tablets.

Characteristics of the tablets containing NMM01 and NMM02 were compared with

tablets formulated with commercially available polymers like Sodium alginate and

Hydroxy propyl cellulose.

6.1. Construction of Standard Calibration curve for Salbutamol sulphate and

Theophylline

6.1.1. Preparation of acid buffer pH 1.2

250 mL of 0.2 M potassium chloride solution is transferred to a 1000 mL

volumetric flask. 425 mL of 0.2 M HCl was added into it and the volume is made up

to 1000 mL1.

Preparation of 0.2 M Potassium chloride: 14.911 g of KCl is dissolved in water and

diluted with sufficient quantity of water to make up to 1000 mL.

Preparation of 0.2 M HCL: 16.66 mL of concentrated HCl is placed in a 1000 mL

volumetric flask it is diluted with water up to 1000 mL.

a. Preparation of standard solution of Salbutamol sulphate in acidic buffer of pH 1.2

Stock solution: Stock solution of Salbutamol sulphate was prepared by dissolving

100 mg in 100 mL of acid buffer pH1.2 to get 1000 μg/mL concentration.

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Sample solution: The known aliquots of Salbutamol sulphate were diluted to obtain

the different concentrations. The absorbance of each solution was measured at 276 nm

in UV-Visible spectrophotometer against a blank solution of acid buffer (pH 1.2)2,3

. A

standard curve was plotted from the absorbance values at 276 nm. The values for the

calibration curve of Salbutamol sulphate were given in table 6.1 and calibration plot

was shown in fig 6.1.

b. Preparation of standard solution of Theophylline in acidic buffer of pH 1.2

Stock solution: Stock solution of Theophylline was prepared by dissolving 100 mg in

100 mL of acid buffer to get 1000 μg/mL concentration.

Sample solution: The known aliquots of the Theophylline were serially diluted with

acid buffer pH 1.2 to obtain the different concentrations. The absorbance of each

solution was measured at 271 nm in an UV-Visible spectrophotometer against a blank

solution of acid buffer (pH 1.2). A standard calibration curve was plotted with the

absorbance at 271 nm against concentration4. The experimental results for the

calibration curve of Theophylline were given in table 6.1 and calibration plot was

shown in fig 6.2.

Table 6.1: Results for standard calibration curve of pure drugs in pH 1.2

Salbutamol sulphate Theophylline

Concentration in

µg/mL

Absorbance at

276nm in pH1.2

Concentration in

µg/mL

Absorbance at

271nm in pH1.2

0 0 0 0

2 0.054 2 0.070

4 0.109 4 0.126

6 0.163 6 0.190

8 0.214 8 0.250

10 0.277 10 0.304

12 0.327 12 0.362

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Fig. 6.1: Standard calibration plot of

Salbutamol sulphate in pH 1.2

Slope 0.0273

Intercept -0.001

Regression 0.9997


Theophylline in pH 1.2

Slope 0.0299

Intercept 0.0062

Regression 0.9994

6.1.2. Preparation of phosphate buffer pH 7.4

250 mL of 0.2 M potassium dihydrogen phosphate was placed in a 1000 mL

volumetric flask. 195.5 mL of 0.2 M NaOH was added into it and diluted with water

to make up to 1000 mL1.

Preparation of 0.2 M Potassium Dihydrogen Phosphate: 27.218 g of potassium

dihydrogen phosphate was dissolved in distilled water and diluted up to 1000 mL.

Preparation of 0.2M NaOH: It was prepared by dissolving 8g of NaOH in sufficient

quantity of water to make up to 1000 mL3.

a. Preparation of standard Solution of Salbutamol sulphate in alkaline buffer of pH 7.4

Stock solution: 100 mg of Salbutamol sulphate was dissolved in 100 mL of alkaline

buffer (pH 7.4), to get a solution of 1000 μg/mL concentration1.

Sample solution: The known aliquots of Salbutamol sulphate solution were diluted

with phosphate buffer pH 7.4. The absorbance of each solution was measured at

276 nm in an UV-Visible spectrophotometer against a blank solution of phosphate

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buffer pH 7.42. A standard calibration curve was plotted with the absorbance at

276 nm against Salbutamol sulphate concentration. The values for the calibration

curve of Salbutamol sulphate were given in table 6.2 and calibration plot was shown

in fig 6.3.

b. Preparation of standard Solution of Theophylline in alkaline buffer of pH 7.4

Stock solution: 100 mg of Theophylline was dissolved in 100 mL of pH 7.4, to get a

solution of 1000 μg/mL concentration.

Sample solution: The stock solution of the Theophylline was serially diluted with

alkaline phosphate buffer pH 7.4 The absorbance of each solution was measured at

271 nm in an UV-Visible spectrophotometer against a blank solution of phosphate

buffer pH 7.42. A standard calibration curve was plotted with the absorbance at 271

nm against theophylline concentration. The experimental results for the calibration

curve of Theophylline were given in table 6.2 and calibration plot was shown in fig

6.4.

Table 6.2: Results for standard calibration curve of pure drugs in pH 7.4

Salbutamol sulphate Theophylline

Concentration in

µg/mL

Absorbance at

276nm in pH7.4

Concentration in

µg/mL

Absorbance at

271nm in pH 7.4

0 0 0 0

2 0.068 2 0.104

4 0.136 4 0.212

6 0.195 6 0.294

8 0.248 8 0.407

10 0.319 10 0.498

12 0.387 12 0.612

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Salbutamol sulphate in pH 7.4

slope 0.0316

Intercept 0.0031

Regression 0.9993


Theophylline in pH 7.4

slope 0.0503

Intercept 0.0018

Regression 0.9995

6.2. Drug-Polymer Interaction Study

The presence of incompatibility between drug and other excipients were

evaluated by performing drug – polymer interaction studies. The interaction study

done by using Infrared spectroscopy and Differential Scanning Calorimetry. The IR

spectra and DSC spectra of pure drugs (Salbutamol sulphate and Theophylline),

polymer (NMM01, NMM02, HPC and Sodium alginate) and drug polymer blends

were taken and compared for studying the presence of incompatibility between drug

and polymer.

6.2.1. Interaction study by FTIR

IR spectroscopy studies were carried out using Perkin Elmer model 2000 by

KBr pellet method. The IR spectrum was recorded from the range of 4000 to 400 cm-1

and peaks obtained were identified5-6

. FTIR spectrums are shown in fig 6.5 to 6.18

and interpretations of spectral data are presented in table 6.3 to 6.13.

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Fig. 6.5: FTIR Spectrum of NMM 01

Fig. 6.6: FTIR Spectrum of NMM 02

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Table 6.3: IR spectral data of NMM

NMM 01

Wave Number in cm-1

Characteristic bands

3416.88 N – H (S)

2919.05 (Methyl or Methylene)

C – H (S)

2851.57 C – H (S)

(Aldehyde)/COOH

1734.51 C = O (S)

1458.06 C – H (B) (CH2)

1384.21 C – H (B) (CH3)

1028.66 C – O (B)

(Ether/Alcohol/Esters/Anhydrides)

871.88, 812.90 C – H (OOP) for Aromatic Ring

NMM 02

Wave Number in cm-1


3447.51 O – H, N – H (S)

2920.94 (Methyl or Methylene)

C – H (S)

1561.86, 1543.56,

1510.61

N – H (B)

(Amines/Amides)

1460.41 C – H (B) (CH2)

1384.44 C – H (B) (CH3)

872.75 C – H (OOP)

for Aromatic Ring

*(S) – Stretching, (B) – Bending, (OOP) – Out of Plane, X – Halogen

Fig. 6.7: FTIR Spectrum of HPC

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Fig. 6.8: FTIR Spectrum of Sodium alginate

Fig. 6.9: FTIR Spectrum of Salbutamol sulphate

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Table 6.4: IR spectral data of HPC and Sodium alginate

HPC

Wave Number in cm-1


3612.93 O – H (S)

2924.94 C – H (S)

1666.82 C = C (S)

1377.24 C – H (B)

1270.33 C – O (S)

1046.45 C – O (S) Ether

851.36 C – H (OOP)

571.56 C – X (S)

Sodium alginate

Wave Number in cm-1


3592.31 O – H (S)

3190.03 C – H (S) Aromatic

2929.80 C – H (S) (CH3)

1416.62 C – H (B) (CH3, CH2)

1301.06 C – O – C (S)

1028.56 C – O (S)

Ether/ Esters/COOH

947.88, 892.43, 820.01 C – H (OOP)

548.23 C – X (S)


Table 6.5: IR spectral data of Salbutamol sulphate and Theophylline

Salbutamol sulphate

Wave Number in cm-1 Characteristic bands

3551.32 O – H (S)

3402.32 N – H (S)

2931.47 C – H (S)

1622.30 C = C (S)

1455.80 C – H (B)

1142.94 C – O (S) Ether

1018.19 C – O (S)

873.33, 766.75, 669.19, 602.15 C – H (OOP)

For Aromatic rings

465.97, 450.74, 444.88, 419.45 C – X (S)

Theophylline

Wave Number in cm-1


3122.02, 3059.85, 2986.41, 2824.98 C – H (S) Alkenes

2710.75, 2605.13 C – H (B) (COOH)

1717.29 C = O (S)

1565.75 C – H (S) Aromatic

1485.06, 1444.61, 1313.68, 1283.76 C – H (B)

1240.79, 1186.88, 1049.08 C – O (S) Ether

978.70, 925.76, 845.65, 785.52,

763.06, 742.07, 609.83 C – H (OOP)


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Fig. 6.10: FTIR Spectrum of Theophylline

Fig. 6.11: FTIR Spectrum of blend NMM 01 and Salbutamol sulphate

Fig. 6.12: FTIR Spectrum of blend NMM02 and Salbutamol sulphate

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Table 6.6: IR spectral data of blend NMM01 and Salbutamol sulphate

NMM 01 and Salbutamol sulphate

Wave Number in cm-1


3407.02 N – H (S)

2827.45 C – H (S)

2791.77, 2779.23, 2631.89 C – H (S) (COOH)

2367.82, 2329.85 C ≡ C (S)

1752.10, 1714.80, 1697.24 C = O (S)

1615.27 C = C (S)

1575.73, 1557.41, 1508.23,

1446.51, 1406.01, 1381.90 C – H (B)

1265.11, 1194.82, 1138.78, 1031.05 C – O (S) Ether

1000.90, 947.95, 900.70 830.30,

744.47, 725.18, 688.20, 653.82,

620.07

C – H (OOP)

563.58, 533.28, 469.88, 450.99,

432.99 C – X (S)


Table 6.7: IR spectral data of blend NMM 02 and Salbutamol sulphate

NMM 02 and Salbutamol sulphate

Wave Number in cm-1


3554.03 O – H (S)

3407.55 N – H (S)

2925.45 C – H (S)

1622.43 C = O (S)

1455.93 C – H (B)

1115.36, 1019.51 C - O (S)

873.40, 669.17, 602.02 C – H (OOP)

466.02 C – X (S)


Fig. 6.13: FTIR Spectrum of blend HPC and Salbutamol sulphate

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Fig. 6.14: FTIR Spectrum of blend Sodium alginate and Salbutamol sulphate

Fig. 6.15: FTIR Spectrum of blend NMM01 and Theophylline

Fig. 6.16: FTIR Spectrum of blend NMM 02 and Theophylline

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Table 6.8: IR spectral data of blend HPC and Salbutamol sulphate

HPC and Salbutamol sulphate

Wave Number in cm-1


3553.21 O – H (S)

3401.72 N – H (S)

2930.09 C – H (S)

1622.95 C = C (S)

1457.08 C – H (B)

1143.24, 1018.39 C - O (S) Ether

873.17, 669.42, 601.95 C – H (OOP)

465.18 C – X (S)


Table 6.9: IR spectral data of blend Sodium alginate and Salbutamol sulphate

Sodium alginate and Salbutamol sulphate

Wave Number in cm-1


3607.01 O – H (S)

2929.04 C – H (S)

1621.43 C = C (S)

1416.63 C – H (B)

1295.91, 1143.54, 1023.50 C - O (S)

821.57, 669.32 C – H (OOP)

569.92 C - X


Fig. 6.17: FTIR Spectrum of blend HPC and Theophylline

131



Table 6.10: IR spectral data of blend NMM 01 and Theophylline

NMM01 and Theophylline

Wave Number in cm-1


3437.27 N – H (S)

3122.32 C – H (S)

2826.99 C – H (S) (COOH)

2361.49 C ≡ C (S)

1717.81 C = O (S)

1667.15 C = C (S)

1186.70, 1048.91 C – O (S)

978.89, 925.95, 845.57, 785.36,

763.14, 741.91, 609.84 C – H (OOP)

502.86, 445.32, 420.37 C – X (S)


Table 6.11: IR spectral data of blend NMM 02 and Theophylline

NMM 02 and Theophylline

Wave Number in cm-1


3445.59 N – H (S)

3060.48, 3122.15 C – H (S)

2826.98, 2606.27 C – H (S) (COOH)

2363.40 C ≡ C (S)

1717.65 C = O (S)

1667.30 C = C (S)

1563.77, 1485.83, 1444.75, 1313.53,

1283.66, 1240.82 C – H (B)

1186.66 C – O (S)

1049.03, 978.66, 925.75, 845.42,

785.40, 763.07, 741.94, 666.34,

609.71, 502.71

C – H (OOP)

444.71, 419.69 C – X (S)


Table 6.12: IR spectral data of blend HPC and Theophylline

HPC and Theophylline

Wave Number in cm-1


3574.19 N – H (S)

3121.64, 3057.45 C – H (S)

2606.99 C – H (S) (COOH)

1717.57 C = O (S)

1667.23 C = C (S)

1566.85, 1445.24, 1313.75, 1284.50,

1240.84 C – H (B)

1187.76, 1049.16 C – O (S)

979.48, 926.94, 846.63, 785.27,

763.79, 742.71, 666.93, 610.32 C – H (OOP)

503.57, 445.31, 419.72 C – X (S)


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Fig. 6.18: FTIR Spectrum of blend Sodium alginate and Theophylline

Table 6.13: IR spectral data of blend Sodium alginate and Theophylline

Sodium alginate and Theophylline

Wave Number in cm-1


3122.28 C – H (S)

2826.87 C – H (S) (COOH)

1667.28 C = C (S)

1567.57, 1443.90, 1313.57, 1284.68,

1240.73 C – H (B)

1186.97, 1047.24 C – O (S)

979.88, 926.99, 845.00, 785.37,

763.70 C – H (OOP)

742.64, 666.83, 610.21 C – H (B)

503.51, 445.23, 419.39 C – X (S)


6.2.2. Interaction study by DSC

Differential scanning colorimetric analysis were performed to characterize the

drug–polymer compatibility. The DSC thermograms of pure drug (Salbutamol

sulphate, Theophylline), polymer (NMM01, NMM02, HPC, Sodium alginate) and

drug-polymer blends were recorded in a DSC analyzer Model DSC-50 Shimadzu7.

DSC spectrums were shown in fig. 6.19 to 6.32 and interpretations of spectrums are

presented in Table 6.14.

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Fig. 6.19: DSC Spectra of NMM 01

Fig. 6.20: DSC Spectra of NMM 02

Fig. 6.21: DSC Spectra of HPC

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Fig. 6.22: DSC Spectra of Sodium alginate

Fig. 6.23: DSC Spectra of Salbutamol sulphate

Fig. 6.24: DSC Spectra of Theophylline

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Fig. 6.25: DSC Spectra of blend NMM01and Salbutamol sulphate

Fig. 6.26: DSC Spectra of blend NMM02 and Salbutamol sulphate

Fig. 6.27: DSC Spectra of blend HPC and Salbutamol sulphate

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Fig. 6.28: DSC Spectra of blend Sodium alginate and Salbutamol sulphate

Fig. 6.29: DSC Spectra of blend NMM 01 and Theophylline

Fig. 6.30: DSC Spectra of blend NMM 02 and Theophylline

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Fig. 6.31: DSC Spectra of blend HPC and Theophylline

Fig. 6.32: DSC Spectra of blend Sodium alginate and Theophylline

Table 6.14: DSC spectral Data

Drug Substance Temperature °C Heat Flow (J/g)

NMM 01 338.9 -8.83

NMM 02 330.6 -3.24

Hydroxypropyl Cellulose (HPC) 129.6 -2.84

Sodium alginate 302.4 -4.84

Salbutamol sulphate 157.3 -11.65

Theophylline 271.1 -6.30

NMM 01 and Salbutamol sulphate 158.2 -11.8

NMM 02 and Salbutamol sulphate 157.8 -24.22

HPC and Salbutamol sulphate 157.2 -8.74

Sodium alginate and Salbutamol sulphate 156.4 -10.13

NMM 01 and Theophylline 273.4 -14.49

NMM 02 and Theophylline 271.3 -14.63

HPC and Theophylline 271.3 -4.53

Sodium alginate and Theophylline 272.2 -10.34

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6.3. Formulation of mucoadhesive sustained release tablets of Theophylline and

Salbutamol sulphate using NMM01, NMM02, HPC and Sodium alginate

a. For Salbutamol sulphate tablets

The granules were prepared by wet granulation method8-10

. Accurately known

weighed quantities of the ingredients exclusive of lubricants are shown in table 6.15.

were mixed and required quantity of warm water was added to the powder and mixed

thoroughly. The wet mass was then passed through BSS sieve no. 16. The wet

granules were dried in a hot air oven at 60° C for 30minutes.

The dried granules of Salbutamol sulphate were mixed with the magnesium

stearate and talc. The lubricated granules were compressed into tablets containing 100

mg of Salbutamol sulphate using 8/32 biconcave punch in Chamunda rotary tablets

punching machine to a hardness of 6-7 kg/cm2.

b. For Theophylline tablets

The granules were prepared by wet granulation method8-10

. Accurately

weighed quantities of the ingredients exclusive of lubricants were mixed and required

quantity of warm water was added to the powder and mixed thoroughly. The wet mass

was then passed through BSS sieve no. 16. The wet granules were dried in a hot air

oven at 60° C for 30minutes.

The dried granules of Theophylline were mixed with the magnesium stearate

and talc. The lubricated granules were compressed into tablets containing 400 mg of

Theophylline using 12/32 biconcave punch in Chamunda rotary tablets punching

machine to a hardness of 6-7 kg/cm2. The composition of the different formulations of

Salbutamol sulphate tablets and Theophylline tablets are listed respectively in the

table 6.15 and 6.16. The Salbutamol sulphate formulations containing different

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mucoadhesives are coded with SF01 to SF12. The Theophylline formulations

containing different mucoadhesives are coded with TF01 to TF12. The prepared SR

tablets were used for the further evaluation.

6.4. Evaluation of granules

Evaluation of granules is an investigation of physical properties of a drug

along with excipients. It is the first step in the rational development of dosage forms.

The overall objective of the evaluation of granules was to generate useful information

to the formulation in developing stable and bioavailable dosage form11-12

. The

standard limits for various flow and derived properties are given in table 6.17. The

preformulation evaluation values for granules of all formulations are given in table

6.18 and 6.19.

6.4.1. Bulk density and Tapped density

Weighed quantity of granules (W) was carefully poured into the graduated

measuring cylinder and the initial volume (VO) was measured. The measuring

cylinder was then fitted with bulk density apparatus and gently tapped from a height

of 1 inch at interval of 2 seconds for 20 minutes. After tapping the final volume (Vf)

was measured. The tapping was continued till two consecutive readings did not show

any difference. The bulk density and tapped density were calculated using the

following formula,

Bulk density (Db) = W / VO

Tapped density (Dt) = W / Vf

Where, W = weight of the powder, VO = initial volume, Vf = final volume

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Chapter 6 Formulation, characterization, in vitro, in vivo and correlation study of mucoadhesive sustained release tablets

Table 6.15: Formulation of mucoadhesive sustained release tablets of Sulbutamol sulphate

S.No Ingredients Formulation Code

SF01 SF02 SF03 SF04 SF05 SF06 SF07 SF08 SF09 SF10 SF11 SF12

1. NMM-01 25 50 75 - - - - - - - - -

2. NMM-02 - - - 25 50 75 - - - - - -

3. Hydroxy Propyl

Cellulose - - - - - - 25 50 75 - - -

4. Sodium Alginate - - - - - - - - - 25 50 75

5. Salbutamol

sulphate 4 4 4 4 4 4 4 4 4 4 4 4

6. Dibasic Calcium

Phosphate 69 44 19 69 44 19 69 44 19 69 44 19

7. Magnesium

Stearate 1 1 1 1 1 1 1 1 1 1 1 1

8. Talc 1 1 1 1 1 1 1 1 1 1 1 1

All the ingredients are represented in mg.

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Chapter 6 Formulation, characterization, in vitro, in vivo and correlation study of mucoadhesive sustained release tablets

Table 6.16: Formulation of mucoadhesive sustained release tablets of Theophylline

S.No Ingredients Formulation Code

TF01 TF02 TF03 TF04 TF05 TF06 TF07 TF08 TF09 TF10 TF11 TF12

1. NMM-01 25 50 75 - - - - - - - - -

2. NMM-02 - - - 25 50 75 - - - - - -

3. Hydroxy Propyl

Cellulose - - - - - - 25 50 75 - - -

4. Sodium Alginate - - - - - - - - - 25 50 75

5. Theophylline 100 100 100 100 100 100 100 100 100 100 100 100

6. Dibasic Calcium

Phosphate 267 242 217 267 242 217 267 242 217 267 242 217

7. Magnesium

Stearate 4 4 4 4 4 4 4 4 4 4 4 4

8. Talc 4 4 4 4 4 4 4 4 4 4 4 4

All the ingredients are represented in mg.

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Chapter 6 Formulation, characterization, in vitro, in vivo and

correlation study of mucoadhesive sustained release tablets

6.4.2. Angle of repose

Angle of repose is a measurement of flow property of granules. It is the maximum

angle that can be obtained between the free standing surface of granules and the

horizontal plane. Values of are rarely less than 20o, and values up to 40

o indicate

reasonable flow potential. However, above 50o the powder flows only with difficulty.

The sample was taken in a funnel, which is fixed in a holder 5cm above the

surface. A graph sheet was placed on the surface so as to receive the falling granules

from the funnel. The sample was allowed to flow through the limb of the funnel. The

height of the pile and the circumference formed was measured. The experiment was

repeated three times. The angle of repose was calculated by using the following formula,

= tan-1

(h/r)

Where, h = height the pile, r = radius of the pile, = Angle of repose.

6.4.3. Compressibility index (or) Carr’s index

Compressibility index is an important measure that can be obtained from the bulk

and tapped densities. In theory, the less compressible a material the more flow able it is.

A material having values of less than 20 to 30% is defined as the free flowing material,

based on the apparent bulk density and tapped density, the percentage compressibility of

the bulk drug was determined by using the following formula,

CI = [(Dt – Db) / Dt] × 100

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Where, CI = Compressibility index, Dt = tapped density of the granules, Db = bulk density

of the granules.

6.4.4. Hausner’s Ratio

It indicates the flow properties of the powder and is measured by the ratio of

tapped density to the bulk density of the granules.

H = Dt / Db

Where, Dt = tapped density of the granules, Db = bulk density of the granules.

The evaluation studies on granules of all the formulations were proved to be

within limits and were shown good derived and flow properties.

Table 6.17: Standard limits for flow properties of Granules

S.No Type of flow Angle of repose

(degrees)

Carr’s index

(%) Hausner’s ratio

1. Excellent 25-30 10 1-1.11

2. Good 31-35 11-15 1.12-1.18

3. Fair 36-40 (aid not needed) 16-20 1.19-1.25

4. Passable 41-45(may hang up) 21-25 1.26-1.34

5. Poor 46-55(must agitate) 26-31 1.35-1.45

Table 6.18: Data obtained for evaluation of granules containing Salbutamol sulphate

F.Code

Derived Properties Flow Properties

Bulk density

(gm/cc) (mean ± SD)

Tapped

density


Angle of

repose

(degrees) (mean ± SD)

Carr’s index

(%) (mean ± SD)

Hausner’s

ratio (mean ± SD)

SF01 0.43±0.0020 0.47±0.0028 27.79±0.47 7.97±0.33 1.09±0.004

SF02 0.44±0.0020 0.48±0.0018 26.70±0.39 8.56±0.74 1.09±0.009

SF03 0.45±0.0010 0.49±0.0031 25.52±0.46 9.34±0.71 1.10±0.009

SF04 0.42±0.0014 0.44±0.0024 26.04±0.13 3.39±0.22 1.04±0.002

SF05 0.43±0.0014 0.46±0.0032 25.50±0.40 6.20±0.35 1.07±0.004

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SF06 0.44±0.0010 0.47±0.0028 25.13±0.09 7.93±0.75 1.09±0.009

SF07 0.45±0.0016 0.48±0.0018 33.77±0.83 5.59±0.34 1.06±0.004

SF08 0.46±0.0012 0.49±0.0025 33.23±0.17 6.12±0.72 1.07±0.008

SF09 0.47±0.0023 0.50±0.0044 32.39±0.24 5.46±0.96 1.06±0.011

SF10 0.43±0.0018 0.47±0.0056 32.65±0.20 9.01±1.46 1.10±0.018

SF11 0.44±0.0015 0.48±0.0044 32.16±0.06 8.18±1.12 1.09±0.013

SF12 0.45±0.0010 0.49±0.0024 31.69±0.37 8.05±0.58 1.09±0.007

Each value represents the mean ± standard deviation of 3 trails

Table 6.19: Data obtained for evaluation of granules containing Theophylline

F.Code

Derived Properties Flow Properties

Bulk density


Bulk density


Angle of

repose

(degrees) (mean ± SD)

Carr’s index

(%) (mean ± SD)

Hausner’s

ratio (mean ± SD)

TF01 0.44±0.0025 0.47±0.0028 30.49±0.55 6.41±0.43 1.07±0.005

TF02 0.45±0.0012 0.48±0.0042 28.97±0.50 6.18±0.90 1.07±0.010

TF03 0.47±0.0045 0.50±0.0032 26.57±0.50 5.24±1.52 1.06±0.017

TF04 0.44±0.0025 0.47±0.0023 29.93±0.42 6.08±0.98 1.06±0.011

TF05 0.45±0.0027 0.48±0.0011 28.44±0.09 5.19±0.78 1.05±0.009

TF06 0.47±0.0028 0.49±0.0021 26.65±0.48 3.93±0.25 1.04±0.003

TF07 0.45±0.0031 0.48±0.0036 29.62±0.48 6.73±1.15 1.07±0.013

TF08 0.47±0.0017 0.49±0.0043 29.09±0.37 4.86±0.51 1.05±0.006

TF09 0.48±0.0029 0.50±0.0025 27.61±0.64 2.50±0.96 1.03±0.010

TF10 0.44±0.0030 0.47±0.0017 31.60±0.76 7.03±0.54 1.08±0.006

TF11 0.45±0.0021 0.49±0.0030 30.49±0.42 6.87±0.91 1.07±0.011

TF12 0.47±0.0033 0.50±0.0037 28.69±0.15 5.17±1.20 1.05±0.013

Each value represents the mean ± standard deviation of 3 trails

6.5. Physicochemical evaluation of sustained release tablets

6.5.1. Average Thickness

Thickness of the tablets was measured by using digital Vernier Caliper. Three

tablets were used from each batch and results were expressed in millimeter3.

6.5.2. Hardness test

145



Tablet requires a certain amount of strength or hardness and resistance to withstand

mechanical shocks of handling in manufacture, packing and shipping. Monsanto hardness

tester was used to measure the hardness of tablets3. The hardness of three tablets in each

batch was measured and the average hardness was reported in kg/cm2.

6.5.3. Friability test

Friability is a measure of strength of granules. Friability test was done in Roche

Friabilator apparatus where the tablets were subjected to the combined effect of abrasion

and hock by utilizing a plastic chamber that revolves at 25 rpm for dropping the tablets at

a distance of six inches with each revolution. 20 Pre-weighed tablets were placed in the

Friabilator, which is then operated for 100 revolutions. The tablets are then freed from

dust and reweighed3. The prescribed limit for loss on friability is not more than 1%w/w.

The percentage of friability was calculated by the following formula,

Friability = [Weight loss/ Weights of tablets before operations] × 100

6.5.4. Weight variation test

Twenty tablets were selected at random, and their average weight was calculated3.

Each tablet was weighed individually, and the weight was compared with the average

weight. The variation of weight of individual tablet with respect to average weight shall

not be more than 5% for Theophylline SR tablets and 10% for Salbutamol sulphate SR

tablets.

6.5.5. Determination of surface pH

146



In this test, the tablet is placed in small beaker with 4 mL of buffer solution

(pH 7.4 ± 0.50) and the pH was measured at time interval of 2 hr by placing the electrode

in contact with the microenvironment of the swollen tablets15,16

. The average pH of three

determinations was recorded.

6.5.6. Uniformity of drug content

The quantity of active ingredients of different formulations is analyzed as per the

prescribed monograph of Indian pharmacopeia. The determined percentage of the drug

contents are not less than 95% and not more than 105% of the added quantity.

For Salbutamol sulphate tablets

The prepared Salbutamol sulphate SR tablet was crushed in a mortar to fine

powder. Then the powder was dissolved in 100 mL of buffer pH 7.4 in a volumetric

flask8. The flask was shaken for 12 hr using a metabolic shaker. After shaking, the

solution was filtered and from the filtrate, appropriate dilutions were made and the

absorbance was measured at a 276 nm, using UV/Visible spectrophotometer. The amount

of drug was estimated from the absorbance values by using calibration curve.

For Theophylline tablets

Prepared Theophylline tablet was crushed and the powder equivalent to about

0.15 g of Theophylline was weighed accurately and was transferred to a conical flask

containing 100 mL of water, 20 mL of 0.1M silver nitrate was added and shaken4. To the

above solution 1 mL of bromothymol blue solution was added and then titrated against

147



0.1M sodium hydroxide. 1 mL of 0.1 M sodium hydroxide is equivalent to 18.02 mg of

C7H8N4O2.

The amount of drug present in each tablet was calculated. The results obtained are

listed in Table 6.20 and 6.21.

6.5.7. Determination of Swelling Index

Swelling index of all the batches of sustained-release tablets was determined. An

individual tablet was weighed accurately (W1) and placed in a Petri dish containing 4 mL

of buffer solution (pH 7.4). At the end of 30minutes, the tablet was removed from the

Petri dish, and excess surface water was removed carefully using filter paper. The weight

of the swollen tablet was reweighed (W2)16,17

. The same procedure was repeated at

different time intervals (60, 120, 180 and 240minutes). The swelling index was calculated

according to the formula and the mean value is displayed in table 6.22 and 6.23.

Swelling index = [(W2 - W1) / W1]

Table 6.20: Physicochemical parameters of Salbutamol sulphate formulations

F.Code

Thickness

(mean±SD)

(mm)

Hardness

(mean±SD)

(kg/cm2)

Friability

(mean±SD)

(%)

Average

weight

(mean±SD)

(mg)

Drug

content

(mean±SD)

(%)

Surface

pH

SF01 2.2±0.2 5.8±0.1 0.83±0.37 99.85±0.10 99.86±1.30 7.3±0.50

SF02 2.3±0.2 6.2±0.1 0.31±0.34 99.96±0.11 99.47±0.93 7.3±0.38

SF03 2.1±0.0 6.8±0.1 0.49±0.49 100.01±0.15 99.69±0.53 7.1±0.30

SF04 2.1±0.0 5.2±0.2 0.21±0.13 99.98±0.13 99.80±1.35 7.3±0.25

SF05 2.3±0.2 5.9±0.0 0.25±0.14 100.01±0.08 99.88±0.87 7.4±0.35

SF06 2.3±0.1 6.3±0.1 0.63±0.45 99.87±0.04 99.14±1.59 7.2±0.31

SF07 2.2±0.1 5.3±0.2 0.25±0.14 99.85±0.10 100.46±0.77 7.4±0.38

SF08 2.2±0.1 5.6±0.2 0.38±0.30 100.15±0.17 100.38±0.67 7.2±0.31

SF09 2.3±0.1 6.2±0.1 0.56±0.44 99.90±0.24 100.17±1.09 7.0±0.21

SF10 2.2±0.1 5.6±0.2 0.19±0.14 99.91±0.15 99.83±1.35 7.3±0.35

148



SF11 2.4±0.1 6.2±0.1 0.33±0.11 100.11±0.12 99.86±0.87 7.1±0.30

SF12 2.1±0.0 6.6±0.2 0.74±0.51 99.89±0.08 99.13±1.59 7.0±0.12

Each value represents the mean ± standard deviation (n=3)

Table 6.21: Physicochemical parameters of Theophylline formulations

F.Code

Thickness

(mean±SD)

(mm)

Hardness

(mean±SD)

(kg)

Friability

(mean±SD)

(%)

Average

weight

(mean±SD)

(mg)

Drug

content

(mean±SD)

(%)

Surface

pH

TF01 6.1±0.1 5.8±0.03 0.41±0.13 399.28±0.45 99.61±1.41 7.0±0.21

TF02 6.0±0.2 6.1±0.04 0.38±0.13 400.11±0.47 100.00±1.49 7.1±0.15

TF03 5.9±0.0 6.5±0.10 0.32±0.15 401.13±0.77 100.28±0.21 6.9±0.10

TF04 5.9±0.1 5.6±0.08 0.38±0.11 399.67±0.60 101.39±0.40 7.0±0.15

TF05 6.0±0.1 6.0±0.06 0.40±0.10 400.88±0.78 98.99±0.50 7.2±0.15

TF06 6.0±0.2 6.3±0.07 0.33±0.12 399.43±0.26 100.10±0.06 7.0±0.15

TF07 6.1±0.1 5.5±0.09 0.40±0.14 400.92±0.44 100.47±1.32 6.9±0.10

TF08 6.1±0.1 5.9±0.05 0.41±0.09 401.32±0.60 99.34±0.54 7.1±0.10

TF09 6.0±0.0 6.1±0.07 0.32±0.15 399.08±0.38 100.41±0.21 7.0±0.10

TF10 5.9±0.1 5.9±0.06 0.39±0.10 401.35±0.77 101.62±0.40 7.1±0.21

TF11 6.0±0.1 6.3±0.06 0.38±0.13 400.44±0.86 99.46±0.72 7.2±0.17

TF12 6.0±0.1 6.8±0.05 0.32±0.14 400.86±0.71 99.63±0.79 7.1±0.10


Table 6.22: Swelling index of Sustained release tablets of Salbutamol sulphate

F.Code Swelling index in newton

30 min 60 min 120 min 180 min 240 min

SF01 0.14±0.02 0.27±0.02 0.56±0.02 0.61±0.03 0.65±0.03

SF02 0.20±0.02 0.37±0.02 0.72±0.02 0.77±0.01 0.79±0.01

SF03 0.25±0.01 0.41±0.01 0.81±0.01 0.86±0.00 0.89±0.02

SF04 0.10±0.02 0.22±0.02 0.45±0.01 0.52±0.03 0.57±0.03

SF05 0.15±0.01 0.31±0.02 0.61±0.02 0.69±0.01 0.72±0.02

SF06 0.22±0.02 0.49±0.02 0.92±0.03 1.00±0.03 1.04±0.03

SF07 0.07±0.01 0.20±0.01 0.40±0.01 0.49±0.00 0.54±0.02

SF08 0.12±0.01 0.29±0.01 0.62±0.01 0.67±0.01 0.71±0.02

SF09 0.19±0.02 0.35±0.01 0.72±0.02 0.77±0.03 0.81±0.04

SF10 0.20±0.03 0.36±0.02 0.71±0.02 0.80±0.02 0.86±0.02

SF11 0.24±0.02 0.48±0.01 0.96±0.02 0.99±0.00 1.01±0.02

SF12 0.32±0.03 0.60±0.02 1.01±0.03 1.04±0.03 1.06±0.04


Table 6.23: Swelling index of Sustained release tablets of Theophylline

149



F.Code Swelling index in newton

30 min 60 min 120 min 180 min 240 min

TF01 0.031±0.002 0.055±0.002 0.094±0.0011 0.121±0.004 0.159±0.003

TF02 0.037±0.002 0.056±0.003 0.097±0.0018 0.131±0.004 0.165±0.001

TF03 0.037±0.002 0.056±0.003 0.098±0.0034 0.132±0.002 0.164±0.003

TF04 0.031±0.001 0.053±0.001 0.089±0.0017 0.117±0.001 0.153±0.004

TF05 0.031±0.002 0.060±0.004 0.090±0.0043 0.124±0.005 0.162±0.005

TF06 0.041±0.001 0.063±0.006 0.099±0.0014 0.131±0.002 0.163±0.001

TF07 0.029±0.001 0.048±0.001 0.069±0.0024 0.110±0.003 0.150±0.004

TF08 0.033±0.001 0.054±0.001 0.077±0.0034 0.114±0.003 0.152±0.005

TF09 0.040±0.000 0.064±0.002 0.088±0.0027 0.122±0.002 0.164±0.001

TF10 0.042±0.001 0.066±0.002 0.094±0.0040 0.127±0.003 0.157±0.003

TF11 0.046±0.003 0.071±0.003 0.097±0.0035 0.133±0.002 0.163±0.002

TF12 0.047±0.002 0.073±0.002 0.098±0.0019 0.135±0.004 0.167±0.004


6.5.8. Determination of Mucoadhesive strength

The mucoadhesive capacity of all formulations was determined by the method by

Martti Marvola18

. The apparatus used for this study consist of two glass slides, one

modified physical balance, weights, thread, goat intestine, tyrode solution, distilled water

and a beaker to hold the water14

.

The intestine of a goat was removed immediately after slaughter. It is preserved in

tyrode solution until its removal for the experimental use. At the time of an experiment a

suitable portion of the intestine is cutoff and stretched on a glass plate. The glass plate

along with the stretched intestine is placed beneath the right arm of a physical balance.

The right arm of the balance is modified and connected to another glass plate. The

assembly on the right arm is arranged so as both the glass plates super impose each other.

The tablet formulated by using mucoadhesive material that is to be tested is placed on the

stretched intestine. The glass plate hanging on the right arm is allowed to contact the

150



tablet on the skin and is moistened with a drop of water. To the left arm a beaker is

attached and is calibrated to counter the equal weight of the right arm assembly prior to

the placement of tablet. The weight of the left arm is increased successively by dropping

water into the conical flask. The weight of the water required to detach the glass plate

along with tablet from the intestinal skin is determined. The weight of the tablet is

subtracted from the weight of the water added to the left arm, which represents the weight

required to detach the tablet from the intestine. The same procedure was repeated at

different time intervals of 10, 15 and 30minutes by using fresh formulated tablet of the

same mucoadhesive material. The whole experiment was repeated for all the formulations

which were formulated by using different mucoadhesive materials. This procedure is

repeated for all twenty four formulations. The force in Newton‟s is calculated by the

following formula,

F = 0.00981 W/2

Where, W is the amount of water.

The calculated mucoadhesive strength in newton is displayed in table 6.24 and 6.25.

Table 6.24: Mucoadhesive strength of Salbutamol sulphate formulations

F.Code Mucoadhesive strength in newtons

5 min 10 min 15 min 30 min

SF01 0.0432 0.0491 0.0853 0.1001

SF02 0.0441 0.0687 0.1295 0.1432

SF03 0.0500 0.0736 0.1462 0.1535

SF04 0.0407 0.0476 0.0839 0.1010

SF05 0.0461 0.0682 0.1246 0.1457

SF06 0.0486 0.0726 0.1476 0.1476

SF07 0.0378 0.0446 0.0750 0.0907

SF08 0.0402 0.0594 0.1148 0.1182

SF09 0.0441 0.0667 0.1187 0.1319

151



SF10 0.0437 0.0500 0.0858 0.1035

SF11 0.0451 0.0701 0.1300 0.1452

SF12 0.0540 0.0741 0.1467 0.1550

Table 6.25: Mucoadhesive strength of Theophylline formulations

F.Code Mucoadhesive strength in newtons

5 min 10 min 15 min 30 min

TF01 0.0554 0.0657 0.0912 0.1050

TF02 0.0579 0.0682 0.1050 0.1432

TF03 0.0603 0.0726 0.1462 0.1633

TF04 0.0535 0.0594 0.0839 0.1010

TF05 0.0554 0.0684 0.1001 0.1457

TF06 0.0594 0.0716 0.1378 0.1525

TF07 0.0495 0.0594 0.0701 0.0907

TF08 0.0549 0.0618 0.0952 0.1182

TF09 0.0584 0.0667 0.1187 0.1319

TF10 0.0549 0.0662 0.0858 0.1035

TF11 0.0584 0.0692 0.1104 0.1452

TF12 0.0608 0.0741 0.1467 0.1599

6.6. In vitro dissolution study

Dissolution characteristics of the formulated sustained release mucoadhesive

tablets of Salbutamol sulphate and Theophylline were carried out using USP Type II

(paddle) dissolution test apparatus model EDT-08Lx 8 station Electro labs dissolution

tester.

Method

900 mL of acid buffer pH 1.2 was filled in dissolution vessel and temperature of

the medium was set at 37˚C ± 0.5˚C. One tablet of different batch was placed in each

dissolution vessel and rotational speed of paddle was set at 50 rpm. After 2 h the

152



dissolution medium was replaced with phosphate buffer pH 7.4. Samples were withdrawn

at periodic intervals of every hour for 12 hours. The 2.5 mL from withdrawn sample was

diluted to 25 mL in volumetric flask and filtered through 0.45 µ membrane filter. The

resultant samples were analyzed for drug content against acid buffer pH 1.2 as a blank at

276 nm for Salbutamol sulphate and 271 nm for Theophylline sustained release

mucoadhesive tablets using UV-Visible spectrophotometer19,21

. The content of drug was

calculated using the following expression (1). The percentage cumulative drug release

was also calculated using the following expression (2).

----------- (1)

100loaded drug ofAmount

released drug ofAmount release drug percentage Cumulative X ----------- (2)

The in vitro drug release profiles of tablets of each batch are given in table from

6.27 to 6.44. The plot of cumulative percentage release v/s time in hours was plotted for

tablets of each batch and shown from fig. 6.33 to fig. 6.68. Based on the in vitro drug

release the best formulation of Salbutamol sulphate and Theophylline had selected. And

the best formulation of Salbutamol sulphate and Theophylline with natural mucoadhesive

material was comparatively studied with the market formulation product for their drug

release nature. The comparative reports were given in table 6.45.

6.7. Treatment of dissolution data with different kinetic model

The quantity of drug released from the SR tablets was analyzed as a function of

the square root of time, which is typical for systems where drug release is governed by

153



diffusion. However, the use of this relationship in swellable matrix system is not justified

completely as such system can be erodible and the contribution of the relaxation of

polymeric chains to drug transport has to be taken into account. Therefore, analysis of

drug release from the swellable matrix must be performed as proposed by ritger and

peppas20

. To find out the mechanism of drug release from the sustained release tablets,

the dissolution data obtained from the above experiments were applied to the following

different release kinetic models19-22

.

Zero release (Cumulative % drug released Vs time) equation

Q = K0 t ----- (1)

First order release (Log cumulative % of drug remains Vs time) equation

Log Qt = Log Q0 + Kt/2.303 ----- (2)

Higuchi‟s (Cumulative % drug released Vs square root of time) equation

Q = KH t½

----- (3)

Korsmeyer and Peppas (Log cumulative % drug released Vs log time) equation

F = (Mt/M) = Km tn ----- (4)

Where, Q = amount of drug release at time, Q0 = initial amount of drug, Qt = cumulative

amount of drug release at time, Mt = drug release at time, M = total amount of drug in

dosage form, F = fraction of drug release at time, K0 = zero order release rate constant, K

= first order release rate constant, KH = Higuchi‟s square root of time release rate

constant, Km = constant depend on geometry of dosage form, t = time in hours and n =

diffusion exponent value. The different n values and the mechanism of drug release are

represented in table 6.26.

Table 6.26: Diffusion exponent values indicating drug release mechanism

S.No Diffusion exponent value (n) Drug release mechanism

154



1 < 0.45 Fickian release

2 0.45 to 0.89 Non – Fickian transport

3 0.89 Case II transport

4 > 0.89 Super case II transport

Table 6.27: In vitro drug release and Higuchi data for SF01 – SF03

Time (hrs) Square root time Cumulative % drug released

SF01 SF02 SF03

1 1.000 15.40±0.17 12.60±0.32 10.70±0.22

2 1.414 30.20±0.13 25.70±0.22 23.50±0.37

3 1.732 45.80±0.04 41.30±0.20 37.10±0.18

4 2.000 58.20±0.26 51.80±0.15 47.70±0.12

5 2.236 67.70±0.07 60.80±0.15 57.20±0.13

6 2.449 75.30±0.10 68.20±0.58 64.40±0.15

7 2.646 78.40±0.02 72.00±0.77 68.00±0.15

8 2.828 81.50±0.06 76.20±0.24 73.40±0.51

9 3.000 86.70±0.16 82.70±0.26 78.10±0.35

10 3.162 89.50±0.05 85.50±0.20 81.20±0.88

11 3.317 92.00±0.78 87.20±0.53 82.00±0.72

12 3.464 93.27±0.07 88.60±0.42 83.80±0.18


Table 6.28: First order release and Peppa’s data for SF01 – SF03

Time (hrs) Log Time

Log Cumulative % drug

released

Log cumulative % drug

remaining

SF01 SF02 SF03 SF01 SF02 SF03

0 0.00 0.00 0.00 0.00 2.00 2.00 2.00

1 0.00 1.19 1.10 1.03 1.93 1.94 1.95

2 0.30 1.48 1.41 1.37 1.84 1.87 1.88

3 0.48 1.66 1.62 1.57 1.73 1.77 1.80

4 0.60 1.76 1.71 1.68 1.62 1.68 1.72

5 0.70 1.83 1.78 1.76 1.51 1.59 1.63

6 0.78 1.88 1.83 1.81 1.39 1.50 1.55

7 0.85 1.89 1.86 1.83 1.33 1.45 1.51

8 0.90 1.91 1.88 1.87 1.27 1.38 1.42

9 0.95 1.94 1.92 1.89 1.12 1.24 1.34

10 1.00 1.95 1.93 1.91 1.02 1.16 1.27

11 1.04 1.96 1.94 1.91 0.90 1.11 1.26

12 1.08 1.97 1.95 1.92 0.83 1.06 1.21

For all the formulations the results obtained from dissolution evaluation were

applied to the various kinds of kinetic models. For the In vitro drug releases, first order

155



release, Higuchi and Peppa‟s data for all formulations were determined. The results are

shown in table 6.27 to 6.45 and in graphs represented by fig. 6.33 to 6.68.

Fig. 6.33: In vitro drug release plot of

SF01 – SF03

Fig. 6.34: First order release plot of

SF01 – SF03

Fig. 6.35: Higuchi’s plot of SF01 – SF03

Fig. 6.36: Peppa’s plot of SF01 – SF03



SF04 SF05 SF06

1 1.000 29.80±0.48 26.20±0.27 24.60±0.29

2 1.414 59.40±0.23 49.30±0.26 47.50±0.17

3 1.732 79.60±0.28 71.00±0.38 68.00±0.41

4 2.000 88.00±0.31 82.70±0.04 78.40±0.44

5 2.236 93.60±0.41 90.60±0.07 86.20±0.36

6 2.449 96.60±0.50 93.50±0.36 88.90±0.20

7 2.646 98.30±0.29 94.90±0.24 91.70±0.16

8 2.828 - 95.50±0.13 93.70±0.21

9 3.000 - 98.00±0.25 96.20±0.08

10 3.162 - - 98.20±0.54

11 3.317 - - -

156



12 3.464 - - -



Time

(hrs)

Log

Time


released


remaining


0 0.00 0.00 0.00 0.00 2.00 2.00 2.00

1 0.00 1.47 1.42 1.39 1.85 1.87 1.88

2 0.30 1.77 1.69 1.68 1.61 1.71 1.72

3 0.48 1.90 1.85 1.83 1.31 1.46 1.51

4 0.60 1.94 1.92 1.89 1.08 1.24 1.33

5 0.70 1.97 1.96 1.94 0.81 0.97 1.14

6 0.78 1.98 1.97 1.95 0.53 0.81 1.05

7 0.85 1.99 1.98 1.96 0.23 0.71 0.92

8 0.90 - 1.98 1.97 - 0.65 0.80

9 0.95 - 1.99 1.98 - 0.30 0.58

10 1.00 - - 1.99 - - 0.26

11 1.04 - - - - - -

12 1.08 - - - - - -


SF04 – SF06


SF04 – SF06

157







SF07 SF08 SF09

1 1.000 33.70±0.15 32.10±0.38 30.80±0.25

2 1.414 69.10±0.09 63.10±0.43 60.80±0.16

3 1.732 87.40±0.07 84.70±0.15 82.00±0.44

4 2.000 95.00±0.20 92.00±0.29 89.20±0.11

5 2.236 97.20±0.20 96.00±0.19 95.00±0.28

6 2.449 - 98.70±0.14 98.20±0.27

7 2.646 - - 99.20±0.22

8 2.828 - - -

9 3.000 - - -

10 3.162 - - -

11 3.317 - - -

12 3.464 - - -



Time (hrs) Log Time


released


remaining


0 0.00 0.00 0.00 0.00 2.00 2.00 2.00

1 0.00 1.53 1.51 1.49 1.82 1.83 1.84

2 0.30 1.84 1.80 1.78 1.49 1.57 1.59

3 0.48 1.94 1.93 1.91 1.10 1.18 1.26

4 0.60 1.98 1.96 1.95 0.70 0.90 1.03

5 0.70 1.99 1.98 1.98 0.45 0.60 0.70

6 0.78 - 1.99 1.99 - 0.11 0.26

7 0.85 - - 2.00 - - 0.10

8 0.90 - - - - - -

158



9 0.95 - - - - - -

10 1.00 - - - - - -

11 1.04 - - - - - -

12 1.08 - - - - - -


SF07 – SF09


SF07 – SF09





SF10 SF11 SF12

1 1.000 12.20±0.22 11.80±0.46 9.00±0.54

2 1.414 28.00±0.16 24.50±0.19 22.00±0.12

3 1.732 43.30±0.41 39.20±0.37 35.10±0.13

4 2.000 54.50±0.44 49.20±0.32 46.60±0.26

5 2.236 62.50±0.35 59.60±0.18 57.80±0.04

6 2.449 71.00±0.37 68.80±0.14 66.10±0.14

7 2.646 75.30±0.27 72.90±0.15 70.00±0.15

8 2.828 79.00±0.11 77.50±0.20 74.90±0.34

9 3.000 83.70±0.31 81.80±0.22 79.50±0.32

10 3.162 88.80±0.14 86.50±0.35 83.30±0.19

11 3.317 91.20±0.39 88.40±0.29 83.90±0.09

159



12 3.464 94.50±0.23 90.20±0.10 86.00±0.33



Time (hrs) Log Time


released


remaining


0 0.00 0.00 0.00 0.00 2.00 2.00 2.00

1 0.00 1.09 1.07 0.95 1.94 1.95 1.96

2 0.30 1.45 1.39 1.34 1.86 1.88 1.89

3 0.48 1.64 1.59 1.55 1.75 1.78 1.81

4 0.60 1.74 1.69 1.67 1.66 1.71 1.73

5 0.70 1.80 1.78 1.76 1.57 1.61 1.63

6 0.78 1.85 1.84 1.82 1.46 1.49 1.53

7 0.85 1.88 1.86 1.85 1.39 1.43 1.48

8 0.90 1.90 1.89 1.87 1.32 1.35 1.40

9 0.95 1.92 1.91 1.90 1.21 1.26 1.31

10 1.00 1.95 1.94 1.92 1.05 1.13 1.22

11 1.04 1.96 1.95 1.92 0.94 1.06 1.21

12 1.08 1.98 1.96 1.93 0.74 0.99 1.15


SF10 – SF12


SF10 – SF12

160



Fig. 6.47: Higuchi’s plot of SF10 – SF12 Fig. 6.48: Peppa’s plot of SF10 – SF12

Fig. 6.49: Comparison of drug release pattern of all SR tablets of

Salbutamol sulphate with rank order

Table 6.35: In vitro drug release and Higuchi data for TF01 – TF03


TF01 TF02 TF03

1 1.000 19.20±0.31 14.60±0.06 10.90±0.26

2 1.414 26.40±0.20 22.50±0.43 18.50±0.16

3 1.732 36.80±0.16 29.60±0.24 24.50±0.25

4 2.000 44.70±0.34 36.00±0.29 31.30±0.34

5 2.236 55.60±0.33 43.80±0.04 38.70±0.19

6 2.449 64.80±0.32 59.30±0.21 48.80±0.48

7 2.646 77.80±0.35 70.00±0.14 56.50±0.35

8 2.828 85.40±0.20 77.50±0.33 64.80±0.50

9 3.000 93.10±0.09 87.50±0.09 72.20±0.17

10 3.162 99.10±0.15 94.00±0.16 78.70±0.31

11 3.317 - 99.30±0.15 85.40±0.17

12 3.464 - - 90.50±0.07


Table 6.36: First order release and Peppa’s data for TF01 – TF03

Time (hrs) Log Time


released


remaining

TF01 TF02 TF03 TF01 TF02 TF03

0 0.00 0.00 0.00 0.00 2.00 2.00 2.00

161



1 0.00 1.28 1.16 1.04 1.91 1.93 1.95

2 0.30 1.42 1.35 1.27 1.87 1.89 1.91

3 0.48 1.57 1.47 1.39 1.80 1.85 1.88

4 0.60 1.65 1.56 1.50 1.74 1.81 1.84

5 0.70 1.75 1.64 1.59 1.65 1.75 1.79

6 0.78 1.81 1.77 1.69 1.55 1.61 1.71

7 0.85 1.89 1.85 1.75 1.35 1.48 1.64

8 0.90 1.93 1.89 1.81 1.16 1.35 1.55

9 0.95 1.97 1.94 1.86 0.84 1.10 1.44

10 1.00 2.00 1.97 1.90 - 0.78 1.33

11 1.04 - 2.00 1.93 - - 1.16

12 1.08 - - 1.96 - - 0.98


TF01 – TF03


TF01 – TF03

Fig. 6.52: Higuchi’s plot of TF01 – TF03

Fig. 6.53: Peppa’s plot of TF01 – TF03



TF04 TF05 TF06

1 1.000 22.50±0.14 20.60±0.16 17.60±0.18

2 1.414 33.10±0.13 29.20±0.11 26.60±0.30

162



3 1.732 45.60±0.26 40.50±0.11 39.10±0.12

4 2.000 58.00±0.40 53.50±0.24 49.10±0.12

5 2.236 72.00±0.14 67.40±0.19 63.70±0.12

6 2.449 83.60±0.18 80.30±0.24 76.40±0.17

7 2.646 94.00±0.08 90.50±0.18 85.40±0.16

8 2.828 99.21±0.13 96.00±0.14 91.00±0.05

9 3.000 - 99.50±0.34 96.20±0.16

10 3.162 - - 99.50±0.14

11 3.317 - - -

12 3.464 - - -



Time (hrs) Log Time


released


remaining


0 0.00 0.00 0.00 0.00 2.00 2.00 2.00

1 0.00 1.35 1.31 1.25 1.89 1.90 1.92

2 0.30 1.52 1.47 1.42 1.83 1.85 1.87

3 0.48 1.66 1.61 1.59 1.74 1.77 1.78

4 0.60 1.76 1.73 1.69 1.62 1.67 1.71

5 0.70 1.86 1.83 1.80 1.45 1.51 1.56

6 0.78 1.92 1.90 1.88 1.21 1.29 1.37

7 0.85 1.97 1.96 1.93 0.78 0.98 1.16

8 0.90 2.00 1.98 1.96 - 0.60 0.95

9 0.95 - 2.00 1.98 - - 0.58

10 1.00 - - 2.00 - - -

11 1.04 - - - - - -

12 1.08 - - - - - -


TF04 – TF06


TF04 – TF06

163







TF07 TF08 TF09

1 1.000 44.00±0.19 41.00±0.31 39.40±0.30

2 1.414 74.00±0.27 69.00±0.16 66.70±0.38

3 1.732 91.70±0.37 86.80±0.23 84.70±0.22

4 2.000 97.00±0.30 94.70±0.20 93.10±0.11

5 2.236 99.10±0.26 97.20±0.20 96.00±0.16

6 2.449 - 99.30±0.21 98.10±0.16

7 2.646 - - 99.80±0.16

8 2.828 - - -

9 3.000 - - -

10 3.162 - - -

11 3.317 - - -

12 3.464 - - -



Time (hrs) Log Time


released


remaining


0 0.00 0.00 0.00 0.00 2.00 2.00 2.00

1 0.00 1.64 1.61 1.60 1.75 1.77 1.78

2 0.30 1.87 1.84 1.82 1.41 1.49 1.52

3 0.48 1.96 1.94 1.93 0.92 1.12 1.18

4 0.60 1.99 1.98 1.97 0.48 0.72 0.84

5 0.70 2.00 1.99 1.98 - 0.45 0.60

6 0.78 - 2.00 1.99 - - 0.28

164



7 0.85 - - 2.00 - - -

8 0.90 - - - - - -

9 0.95 - - - - - -

10 1.00 - - - - - -

11 1.04 - - - - - -

12 1.08 - - - - - -


TF07 – TF09


TF07 – TF09





TF10 TF11 TF12

1 1.000 16.40±0.25 13.70±0.43 11.10±0.18

2 1.414 23.80±0.14 20.60±0.22 17.40±0.16

3 1.732 36.27±0.37 32.00±0.25 26.90±0.17

4 2.000 39.30±0.69 38.00±0.17 33.10±0.12

5 2.236 52.50±0.19 46.00±0.16 40.70±0.15

165



6 2.449 62.00±0.17 56.30±0.10 51.60±0.10

7 2.646 68.50±0.41 62.70±0.21 57.40±0.30

8 2.828 75.00±0.17 71.80±0.18 65.50±0.21

9 3.000 81.30±0.30 76.70±0.14 69.70±0.24

10 3.162 85.60±0.14 82.60±0.19 78.00±0.12

11 3.317 93.10±0.44 88.70±0.23 82.60±0.23

12 3.464 97.50±0.14 92.60±0.22 89.10±0.15



Time (hrs) Log Time


released


remaining


0 0.00 0.00 0.00 0.00 2.00 2.00 2.00

1 0.00 1.21 1.14 1.05 1.92 1.94 1.95

2 0.30 1.38 1.31 1.24 1.88 1.90 1.92

3 0.48 1.56 1.51 1.43 1.80 1.83 1.86

4 0.60 1.63 1.58 1.52 1.76 1.79 1.83

5 0.70 1.72 1.66 1.61 1.68 1.73 1.77

6 0.78 1.79 1.75 1.71 1.58 1.64 1.68

7 0.85 1.84 1.80 1.76 1.50 1.57 1.63

8 0.90 1.88 1.86 1.82 1.40 1.45 1.54

9 0.95 1.91 1.88 1.84 1.27 1.37 1.48

10 1.00 1.93 1.92 1.89 1.16 1.24 1.34

11 1.04 1.97 1.95 1.92 0.84 1.05 1.24

12 1.08 1.99 1.97 1.95 0.40 0.87 1.04


TF10 – TF12


TF10 – TF12

166





Fig. 6.66: Comparison of drug release pattern of all SR tablets of

Theophylline with rank order

167



Table 6.43: Release kinetics of Salbutamol sulphate formulations of SF01 to SF12

Formula

code

Zero order First order Higuchi's Plot Koresmayer

and Peppa's

K0 r2 K1 r

2 slope r

2 n r

2

SF01 7.4715 0.9440 -0.2298 -0.9987 30.1501 0.9859 0.7070 0.9763

SF02 7.2967 0.9570 -0.1901 -0.9981 29.0698 0.9868 0.7688 0.9796

SF03 7.0071 0.9584 -0.1613 -0.9960 27.8193 0.9849 0.8092 0.9787

SF04 13.4893 0.9173 -0.5922 -0.9978 40.6100 0.9805 0.6025 0.9564

SF05 10.1242 0.9000 -0.4326 -0.9926 35.7676 0.9731 0.5852 0.9545

SF06 8.8536 0.8987 -0.3797 -0.9944 33.3716 0.9747 0.5691 0.9538

SF07 19.6629 0.9404 -0.7581 -0.9947 47.3990 0.9828 0.6683 0.9604

SF08 16.1714 0.9278 -0.7223 -0.9929 43.9866 0.9820 0.6273 0.9598

SF09 13.5857 0.9128 -0.6993 -0.9926 41.0423 0.9791 0.5911 0.9540

SF10 7.6275 0.9598 -0.2311 -0.9930 30.3428 0.9882 0.7810 0.9766

SF11 7.5269 0.9633 -0.2012 -0.9986 29.7625 0.9859 0.8068 0.9827

SF12 7.3500 0.9610 -0.1755 -0.9966 28.9957 0.9812 0.8068 0.9768

Table 6.44: Release kinetics of Theophylline formulations of TF01 to TF12

Formula

code

Zero order First order Higuchi's Plot Koresmayer

and Peppa's

K0 r2 K1 r

2 slope r

2 n r

2

TF01 9.7291 0.9959 -0.3709 -0.8849 33.0879 0.9745 0.7563 0.9929

TF02 9.1570 0.9964 -0.3481 -0.8728 32.4558 0.9612 0.8480 0.9909

TF03 7.6055 0.9987 -0.1825 -0.9646 28.4118 0.9656 0.8839 0.9971

TF04 13.0071 0.9959 -0.3611 -0.9475 35.8990 0.9759 0.7542 0.9953

TF05 11.2915 0.9903 -0.4990 -0.9073 36.3616 0.9760 0.7786 0.9929

TF06 10.2273 0.9872 -0.4430 -0.9138 35.2104 0.9779 0.8081 0.9950

TF07 19.2057 0.9213 -0.9566 -0.9931 47.4704 0.9873 0.5166 0.9664

TF08 15.5714 0.9081 -0.8126 -0.9919 43.3542 0.9838 0.4973 0.9639

TF09 12.9571 0.8896 -0.8093 -0.9728 40.0837 0.9770 0.4713 0.9560

TF10 7.8632 0.9901 -0.2584 -0.9397 30.2686 0.9864 0.7459 0.9976

TF11 7.6742 0.9940 -0.2034 -0.9731 29.2332 0.9800 0.8040 0.9978

TF12 7.3868 0.9972 -0.1706 -0.9740 27.8432 0.9728 0.8040 0.9980

K0 = Zero order rate constant, r2 = Regression, K1 = First order rate constant,

n = Release exponent.

168



Table 6.45: Comparison of dissolution data of formulation SF03, TF03 and the

corresponding Marketed tablets

Time

In hour

% of drug release

Marketed tablet

contains

Salbutamol sulphate

% of drug

release

SF03

% of drug release

Marketed tablet

contains

Theophylline

% of drug

release

TF03

0 0 0.00 0.00 0.00

1 10.13 10.70 11.50 10.90

2 21.99 23.50 19.62 18.50

3 36.43 37.10 23.35 24.50

4 46.29 47.70 31.30 31.30

5 56.30 57.20 40.04 38.70

6 64.35 64.40 48.80 48.80

7 69.33 68.00 57.72 56.50

8 72.70 73.40 65.53 64.80

9 78.01 78.10 74.61 72.20

10 81.04 81.20 80.17 78.70

11 82.38 82.00 86.34 85.40

12 82.62 83.80 92.13 90.50


6.8. Similarity factor for Marketed formulation with best formulations

The similarity factor f2 as defined by FDA and EMEA is a logarithmic reciprocal

square root transformation of one plus the mean squared (the average sum square)

difference of drug percent dissolved between the test and reference products23

. It is given

by following equation:

-------------------------- (1)

Where, n is the number of pull points, Wt is an optional weight factor, Rt is the profile at

time point t and Tt is the reference profile at the same time point.

169



For a dissolution profile to be considered similar, the value of f2 should be

between 50 and 10023, 24

. An f2 value of 100 suggests that the test and reference profiles

are identical and as the value becomes smaller, the dissimilarity between release profiles

increases. The similarity factor (Sd) is given as

------------------- (2)

Where, „n‟ is the number of data points collected during the in vitro dissolution test and

AUCRt and AUCTt are the areas under curves of the reference and test formulation,

respectively, at time „t‟. For the test and reference formulations to be identical, the Sd

value should be zero25-27

. The result of the similarity factor is given in table 6.46.

Table 6.46: Data of Similarity factor

Formulations

contains Parameter Value

Salbutamol sulphate

f1 1.27571019

f2 93.9556407

Sd 0.00854545

Theophylline

f1 1.9980669

f2 90.4727134

Sd 0.00979779

Fig. 6.67: Comparison of drug release

pattern of Best formulation SF03

Fig. 6.68: Comparison of drug release

pattern of Best formulation TF03

170



and Marketed formulation and Marketed formulation

6.9. In vivo study

Based on the results obtained from the in vitro drug release study and mucoadhesion

strength study the formulations SF03 and TF03 were selected as best formulations containing the

natural mucoadhesive material obtained from Caesalpinia pulcherrima. Hence these two

formulations were selected for further in vivo evaluation.

6.9.1. Bio-analytical method development for Salbutamol sulphate

a. Chromatographic conditions

The reverse phase HPLC (RP-HPLC) analysis were carried out using Perkin

Elmer equipped with a LC 200 pump PE nelson 1020S integrator and a variable

wavelength UV-VIS detector, The column used was an Hypersil® (Thermo-scientific,

India), 5 µm, Rp C18 250×4.6 mm. This RP-HPLC method was used for the determination

of Salbutamol sulphate and chloramphenicol (internal standard) in rabbit plasma. A

mixture of water: methanol: acetonitrile: 70:20:10 (v/v), pH of which is adjusted to 2.5 by

10% phosphoric acid was used as the mobile phase. Flow rate and Injection volume were

1.2 mL/min and 20 µL respectively. The running time was 18 min for each sample.

Ambient temperature was maintained throughout running time28

.

b. Standard solutions

Accurately weighed 100 mg of Salbutamol sulphate was dissolved in 100 mL of

methanol as primary stock solution. Working standard solutions were prepared by

diluting the stock solution with methanol to the concentration of 1, 2, 3, 5, 7, 10 and

15 μg/mL. An internal standard stock solution was prepared in methanol 1.00 mg/mL.

171



A working solution was made by dilution of the stock solution with methanol to

10 μg/mL.

c. Extraction

Rabbit blood containing heparin as an anticoagulant was used for the preparation

of calibration standard. Calibration standard solution samples were freshly prepared in

rabbits plasma by adding to 0.5 mL of plasma appropriate aliquots of working standard

solutions to yield concentration of 100, 200, 300, 500, 700, 1000, 1500 ng/ mL. After 2

min of agitation, samples were mixed with 5 mL of 0.1M-bis-(2 ethyl hexyl) phosphate in

chloroform centrifuged at room temperature at 2000 rpm for 10 min. About 2.5 mL of the

supernatant liquid was then transferred into a second tube and 1 mL of 0.5N HCl was

added to it. After 5 min of centrifuge, the aqueous layer was separated and 20 µL was

injected to HPLC. The chromatogram was recorded and response of major peaks was

measured. The experimental result for calibration curve of pure drug is given in Table

6.47 and calibration plot is shown in Fig. 6.69.

Amount of drug in %= AS/AT×WS/100×5/50×100/WT×50/5×P/100×AV×100

Where, AS = average area of drug peak for standard, AT = average area of drug peak for

test sample, WS = weight of drug taken for standard (in gm), WT = weight of drug taken

for test sample (in gm), P = percentage purity of standard, AV = average weight in gm.

Table 6.47: Result of Standard Calibration plot for

Salbutamol sulphate in rabbit plasma

Concentration

ng/mL Peak area

100 9265

200 18564

300 29564

500 48265

700 72153

1000 97856

172



1500 152463

Slope 101.4866

Regression 0.999556

Fig.6.69: Calibration plot for Salbutamol sulphate in rabbit plasma

6.9.2. Bio-analytical method development for Theophylline

a. Chromatographic conditions

The reverse phase HPLC (RP-HPLC) analysis was carried out using shimadzu

equipped with a liquid chromatogram of SCL-10 Avp and SPD-10A detector of variable

wavelength UV-VIS detector at 271 nm, The column used was an Hypersil® BDS

(Thermo-scientific, India), 5 µm, Rp C18 250×4.6mm and with the support of Class-vp®

version 6.1 software. This RP-HPLC method was used the determination of Theophylline

in rabbit plasma. The mobile phase of (acetonitrile) ACN: 0.2 M acetate buffer solution

(pH 4.5) in the ratio of 6.5: 93.5 (%, v/v) was applied at a flow rate of 1.0 ml/min. The

run time was 15 min for each sample. Ambient temperature was maintained throughout

the run time.29

b. Sample solutions

173



Sample was prepared by adding 400 μL of acetonitrile solution to 100 μL of

freshly withdrawn serum. After vortex for 1min and then centrifuged at 4500 x g for 30

min, the clear supernatant liquid was transferred to another microtube and evaporated to

dryness. The residue was reconstituted with 100 μL of mobile phase, and 20 μL of this

solution was subjected to HPLC analysis.

c. Standard solutions

100 mg of Theophylline was accurately weighed and dissolved in 100 mL of

mobile phase as primary stock solution. 20 μL of solution was spiked into 180 μL serum

to afford serum standards consisting of 0.5, 2.5, 5.0, 10.0 and 25.0 µg/mL. To 100 μL of

serum standard, 400 μL of acetonitrile was added. The peak areas of serum standard were

determined. The calibration curve is drawn after linear regression of the peak-area with

concentrations. The experimental result for standard calibration curve of pure drug is

given in Table 6.48 and calibration plot was shown in Fig. 6.70.

Amount of drug in % = AS/AT×WS/100×5/50×100/WT×50/5×P/100×AV×100

Where, AS = average area of drug peak for standard, AT = average area of drug peak for

test sample, WS = weight of drug taken for standard (in gm), WT = weight of drug taken

for test sample (in gm), P = percentage purity of standard, AV = average weight in gm.

Table 6.48: Result of Standard Calibration plot for

Theophylline in rabbit plasma

Concentration

(µg/ml) Peak area

0.50 4125

2.50 12532

5.00 23032

10.00 42153

25.00 99845

Slope 3934.83

174



Regression 0.9995

Fig. 6.70: Calibration plot for Theophylline in rabbit plasma

6.9.3. Pharmacokinetic study

The study was approved by the Institutional Animal Ethical committee (IAEC) of

Santhiram College of Pharmacy, Nandyal, India with the Registration No:

1519/PO/a/11/CPCSEA. Twelve healthy white albino rabbits of either sex weighing 2.5-

3.0 kg were housed individually in standard cages on a 12 h light-dark cycles. The rabbits

were fasted for 24 h before drug administration but were allowed free access to water.

The animals were divided at random into two groups (six animals each), and under

random study design, first group animals were received uncoated marketed tablet of

Salbutamol sulphate and Theophylline respectively. And second group received

mucoadhesive formulation SF03 and TF03 corresponding to a dose of 4 mg and 100 mg.

These formulations were administrated by the oral route with an oral gavage.

About 2 mL of blood sample was collected through peripheral ear vein prior to

drug administration (0 h) and 1, 2, 3, 5, 6, 8, 10, 12, 16, 18, 20, 24 hrs after oral

administration. Samples were transferred immediately into heparin containing test tubes.

175



After centrifugation at 2000 rpm for 10 minutes, plasma samples were harvested and

stored at 20°C until analysis. The concentration of drug in plasma was determined by

high performance liquid chromatographic technique with UV detection at 276 nm for

Salbutamol sulphate and 271 nm for Theophylline. Estimation of drug concentration was

carried out by interpolating the peak area of the best formulation on calibration curve

spiked the blank plasma over the range assayed.

h. Pharmacokinetic Analysis

The pharmacokinetic parameters such as Cmax (ng/mL), Tmax (h), Kel(h-1

),

t1/2(h), Vd(ng/mL), AUC0-24 (ng.h/mL), AUC0-∞(ng.h/mL), AUMC0-24(ng.h2/mL) and

AUMC0-∞ (ng.h2/mL), of drug were determined from plasma concentration time profile.

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

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

area under the plasma concentration time curve up to the last time (t) showing a

measurable concentration (Ct) of the analyte (AUC0-t) was determined by applying the

linear trapezoidal rule. The apparent elimination rate constant (Kel) was calculated by log-

linear regression of the data point describing a terminal log-liner decaying phase. The

AUC0-∞ values were determined by adding the quotient of *Ct and the appropriate Kel to

the corresponding AUC0-t .

AUC0-∞ = AUC0-t + *Ct / Kel

Where *Ct is the last detectable plasma drug concentration.

The apparent elimination half life (t1/2) of drug in plasma was calculated by using the

following equitation,

t1/2 = (ln2)/Kel

176



The ratio of Cmax/AUC0-∞ was also computed and used to to measure the rate of

absorption. All values are expressed as the mean ± standard deviation (SD). The

pharmacokinetic parameters obtained by following a single dose administration of the

reference standard tablets and the formulated tablets to normal rabbits were compared

using paired „t‟ test, considering a probability of P<0.05 to be significant.30

Bioavailability test is performed by using „PK function‟ (Microsoft Excel add In)

programme31

and „The Modern BiopharmaceuticsTM

Version 6‟ software.32

Statistical

analysis was performed using Microsoft Excel (Analysis Tool Park add in). Results of

pharmacokinetic parameters are presented in table 6.49 and 6.50. Observed plasma drug

concentration graphs are shown in Fig 6.71, 6.72 and 6.73. An HPLC spectrum of subject

1, 2, 3, 4, 5 and 6 for Salbutamol sulphate reference tablet was given in fig 6.74 - 6.79.

HPLC spectra of the subject 1, 2, 3, 4, 5 and 6 administered with formulation SF03

containing Salbutamol sulphate was given in fig 6.80 - 6.85.

Observed plasma drug concentration graphs for Theophylline are shown in

fig 6.86, 6.87 and 6.88. HPLC spectra of subject 1, 2, 3, 4, 5 and 6 for Theophylline

reference tablet was given in fig 6.89 - 6.94. HPLC spectra of the subject 1, 2, 3, 4, 5 and

6 administered with formulation TF03 containing Theophylline was given in

fig 6.95-6.100.

177



Fig. 6.71: Observed plasma drug concentration of Reference in six subjects

Fig. 6.72: Observed drug plasma concentration of Test SF03 in six subjects

178



Fig: 6.73: Observed mean drug plasma concentration of

Reference and Test SF03

Table 6.49: Pharmacokinetic parameters of Reference and Test of SF03

Parameters Reference Test

Mean ±SD CV % Mean ±SD CV %

Cmax (ng/mL) 235.89 10.62 4.50 186.63 2.45 1.313

tmax (h) 3.00 0.00 0.00 6.00 0.00 0

Ke (h-1

) 0.41 0.01 2.08 0.12 0.01 3.79

t1/2 el (h) 1.68 0.03 1.99 5.68 0.22 3.86

AUC0-t (ng.h/mL) 1558.45 16.41 1.053 2230.36 30.49 1.37

AUC0-∞ (ng.h/mL) 1558.52 16.42 1.05 2388.14 55.48 2.32

AUMC0-t (ng.h/mL) 9169.85 178.01 1.94 20717.33 460.02 2.22

AUMC0-∞

(ng.h/mL) 9171.64 178.01 1.94 25797.83 1338.92 5.19

MRT0-∞ (h) 5.88 0.08 1.29 10.80 0.32 2.98

Each value represent the mean ± standard deviation (n=6)

179



Fig. 6.74: Chromatogram of Salbutamol sulphate Reference tablet

for subject 1 at Cmax





180







181





Fig. 6.80: Chromatogram of Salbutamol sulphate Test SF03 tablet


182









183







184



Fig. 6.86: Observed plasma drug concentration of

Theophylline Reference tablet in six subjects

Fig. 6.87: Observed plasma drug concentration of

TF03 Test formulation in six subjects

185



Fig. 6.88: Observed mean drug plasma concentration of Reference and Test TF03

Table 6.50: Pharmacokinetic parameters of Reference and Test of TF03

Parameters Reference Test

Mean ±SD CV % Mean ±SD CV %

Cmax (µg/mL) 8.50 0.20 2.32 6.29 0.12 1.89

tmax (h) 4.00 0.00 0.00 10.00 0.00 0.00

Ke (h-1

) 0.16 0.00 1.99 0.08 0.00 3.14

t1/2 el (h) 4.45 0.09 1.98 8.01 0.29 3.66

AUC0-t (µg.h/mL) 93.91 1.67 1.78 86.79 1.81 2.09

AUC0-∞ (µg.h/mL) 99.56 1.84 1.85 111.34 3.09 2.78

AUMC0-t (µg.h/mL) 785.20 17.38 2.21 1027.26 25.40 2.47

AUMC0-∞ (µg.h/mL) 873.40 17.55 2.01 1936.53 85.74 4.43

MRT0-∞ (h) 8.77 0.06 0.67 17.39 0.35 2.01

Each value represent the mean ± standard deviation (n=6)

Fig. 6.89: Chromatogram of Theophylline Reference tablet for subject 1 at Cmax

186





187





188




Fig. 6.95: Chromatogram of Theophylline Test TF03 tablet for subject 1 at Cmax

189





190





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6.10. IVIVC Study

In vitro-in vivo correlation

Correlations between in vitro and in vivo data (IVIVC) are often used during

pharmaceutical dosage development in order to reduce formulation development time and

optimize the formulation. A good correlation is a tool for predicting in vivo results based

on in vitro data. IVIVC allows dosage form optimization with the fewest possible trials in

man, fixes dissolution acceptance criteria, and can be used as a surrogate for further

bioequivalence studies; it is also recommended by regulatory authorities.33-37

Various definitions of in vitro-in vivo correlation have been proposed by the

International Pharmaceutical Federation (FIP), the USP working group,38

and regulatory

authorities such as the FDA or EMEA.34-37

The FDA defines IVIVC as “a predictive

mathematical model describing the relationship between an in vitro property of an

extended release dosage form (usually the rate or extent of drug dissolution or release)

and a relevant in vivo response, e.g. plasma drug concentration or amount of drug

absorbed.”

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Five correlation levels have been defined in the IVIVC- FDA guidelines.39

The

concept of correlation level is based up on the ability of the correlation to reflect the

complete plasma drug level-time profile which result from administration of the given

dosage form. Level A correlation represents a point to point relationship between in vitro

dissolution rate and in vivo input rate of the drug from the dosage form.40

The wagner –

Nelson method was used to determine the fractional oral absorption at each sampling

time.

The fraction of drug absorbed was calculated by using the following formula

Fraction of drug absorbed = F (t)/ Ke*AUC0-∞

F (t) = C (t) +Ke*AUC0-t

Where, C (t) = plasma drug concentrations at 't' time, Ke = Elimination rate constant,

AUC0-t = Area under the curve from '0' time to't', AUC0-∞ = Area under the curve from '0'

time to 't' and '∞' time.

In vitro - In vivo data are given in table 6.51 and 6.52. The corresponding graphs are

shown in fig 6.101 and 6.102 for the formulations SF03 and TF03 respectively.

Table 6.51: In vitro – In vivo data of formulation SF03

Time (h)

Fraction of drug

absorbed (%)

Fraction of drug

dissolved (%)

1 16.65 10.70

2 44.98 23.50

3 61.57 37.10

5 86.03 .57.20

6 97.08 64.40

8 105.77 73.40

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10 98.19 81.20

12 91.79 83.80

Table 6.52: In vitro – In vivo data of formulation TF03

Time (h) Fraction of drug

absorbed (%)

Fraction of drug

dissolved (%)

1 17.02 11.5

2 43.30 19.62

4 77.66 31.30

8 96.99 65.53

10 104.16 80.17

12 107.56 92.13

Fig. 6.101: In vitro - in vivo correlation of

formulation SF03

Fig. 6.102: In vitro - in vivo correlation of

formulation TF03

6.11. Results and discussion

6.11.1. Construction of calibration curves for drug solution

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The standard calibration curves for the estimation of drug in solution were

constructed for Salbutamol sulphate and Theophylline in acid buffer pH 1.2 and

phosphate buffer pH 7.4. The calibration curves showed a straight line with regression

>0.999 making it suitable for estimation.

6.11.2. Drug-polymer interaction studies

a. FTIR spectral study

The presence of incompatibility between drug and polymer was evaluated by

FTIR spectral studies and Differential scanning calorimetric studies. The FTIR spectra of

the pure drug, polymer and blends containing drug with polymers were obtained and

compared. FTIR studies dealt with recording of FTIR spectra of pure drugs

(Theophylline, Salbutamol sulphate) polymers (NMM01, NMM02, HPC, Sodium

alginate) and blends (NMM01 with Salbutamol sulphate, NMM01 with Theophylline,

NMM02 with Salbutamol sulphate, NMM02 with Theophylline, Theophylline with HPC,

Theophylline with sodium alginate, Salbutamol sulphate with HPC and Salbutamol

sulphate with sodium alginate)

The Salbutamol sulphate characteristic peaks due to C-H (stretching) at 2931 cm-1

in the pure spectra was found at 2827 cm-1

in the spectra of the blend, peak at wave

number 1622 cm-1

due to C=C (stretching) and out of plane C-H were shown in pure

spectra of Salbutamol sulphate as well as in the spectra of the blend containing

Salbutamol sulphate and NMM01. The characteristic peaks of NMM01 namely at wave

numbers 3400 cm-1

due to N-H (Stretching) around 1700 cm-1

due to C=O (stretching)

were found in the spectra of pure polymer as well as in the spectra of the blend containing

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Salbutamol sulphate and NMM01. The presence of characteristics peaks in both the

spectra implied that there was no incompatibility between NMM01 and Salbutamol

sulphate.

The Theophylline characteristic peaks at wave numbers 3122 cm-1

due to C-H

(stretching), peak at 1717 cm-1

due to C=O (stretching) and peaks due to C-H out of plane

bending and peaks due to C-X (stretching) were evident in the spectra obtained for pure

drug as well as in the spectra obtained for the blend containing Theophylline and

NMM01. The characteristic peaks of NMM01 namely at wave numbers 3416 cm-1

due to

N-H (Stretching) around 1734 cm-1

due to C=O (stretching) were found in the spectra of

pure polymer as well as in the spectra of the blend containing Theophylline and NMM01.

The FTIR studies revealed that there was no incompatibility between Theophylline and

NMM01.

The characteristic peaks of Salbutamol sulphate at wave number 3551 cm-1

due to

O-H (stretching) at wave number 3402 due to N-H (stretching) at wave number 2931 cm-1

due to C-H (stretching) were retained in the spectra of the pure drug as well as in the

spectra of the blend containing Salbutamol sulphate and NMM01. Evaluation of NMM02

spectra showed peaks at wave numbers 1155 cm-1

due to C-O (stretching) and at around

870 cm-1

due to out of plane bending caused by aromatic ring of NMM02. The above said

were also found in the spectra obtained by the blend containing Salbutamol sulphate and

NMM02 proving the fact that there was no incompatibility between Salbutamol sulphate

and NMM02.

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The Theophylline peaks at wave numbers 3122 cm-1

due to C-H (stretching) peak

at 1717 cm-1

due to C=O (stretching) peak at 1667 cm-1

due to C=C (stretching) and out

of plane peaks at around 970 cm-1

wave number were seen in the spectra of pure

Theophylline as well as in the spectra of the blend containing Theophylline and NMM02.

The characteristic peaks of NMM02 at wave number 3447 cm-1

due to O-H, peak at

2920 cm-1

due to C-H (stretching) at 1460 cm-1

due to C-H (bending) and out of plane

peak at around 870 cm-1

wave number due to the presence of aromatic ring was found in

both spectra of pure polymer as well as in the spectra of the blend containing

Theophylline and NMM02 confirmed the absence of any significant incompatibility

between Theophylline and NMM02.

The FTIR studies revealed the absence of any onward incompatibility between the

drugs and polymers taken for the study.

b. Differential scanning calorimetric study

Based on the data listed out in the table 6.10 it was evident that the pure drugs

Theophylline and Salbutamol sulphate showed endothermic peak at 271o

C and 157o

C

respectively. The natural mucoadhesive polymers NMM01 and NMM02 showed

endothermic peaks at 338o

C and 330o

C respectively. The standardized polymers taken in

the study namely HPC and sodium alginate showed endothermic peaks at 129o C and 302

o

C respectively. DSC spectra of the blend containing NMM01 and Salbutamol sulphate

demonstrated a endothermic peak at 158.2o

C evident to the fact that the Salbutamol

sulphate is still in the crystal form in the formulation. DSC spectra of the blend containing

NMM01 and Theophylline showed a peak at 273.4o

C suggesting that the Theophylline

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might be available as crystals and not in amorphous form in the formulation. The same

trend was witnessed in the case of NMM02 also. The spectra of the blends containing

NMM02 with Salbutamol sulphate showed a peak at 157.8o

C suggesting the presence of

the drug in crystal form and the spectra of the blend containing NMM02 and

Theophylline showed a peak at 271.3o

C due to the crystalline nature of Theophylline.

The DSC spectra of the HPC blends (HPC with Theophylline and HPC with Salbutamol

sulphate) showed peak at 271.3o

C and 157.2o

C which are similar to the pure drug peaks

confirming the status of the pure drug to be intact form without melting or any changes.

The DSC spectra of the sodium alginate blends (sodium alginate with Theophylline and

sodium alginate with Salbutamol sulphate) showed peaks at 272.2o

C and 156.4o

C owing

to the fact that the pure drugs are available in crystals. The presence of the characteristic

peaks and absence of appearance of new peaks suggested that there was no

incompatibility between the drug and the polymer taken for the study. The selection of

the drug and polymer were justified and further taken for preparation of formulations.

c. Evaluation of granules

Table 6.14 listed out the experimental values obtained for the determination of

derived properties such as Bulk density, tapped density, angle of repose, compressibility

index and Hausner ratio for granules containing Salbutamol sulphate. The bulk density

were found to be in the range of 0.42 -0.45 gm/cc. The tapped density was found to be in

the range of 0.44- 0.50 gm/cc. The granules had excellent flow property having angle of

repose within 25o-30

o. The compressibility index were well below 10% and the hausner‟s

ratio values were found between 1.04 -1.10. The overall physical properties of the

granules of Salbutamol sulphate were good and suitable for compression into tablets.

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The physical evaluation of granules of Theophylline were done and tabulated in

table 6.15. The bulk density of the granules was between 0.44 -0.48 gm/cc. The tapped

density of the granules was in the range of 0.47 gm/cc to 0.50 gm/cc. The granules

showed angle of repose below 30o. The compressibility index of the granules was

between 2.50 % - 7.3 % well below the optimum value and the hausner‟s ratio was about

1.05-1.08. The granules of the Theophylline showed optimum derived properties making

it suitable for high speed compression.

d. Physiochemical evaluation of sustained release tablets

The SR tablets containing Salbutamol sulphate and Theophylline were prepared

and evaluated for its thickness, hardness, friability, uniformity of weight, uniformity of

drug content, and surface pH. The tabulation 6.16 listed out the values of various

physicochemical tests done on SR tablets of Salbutamol sulphate.

The average thickness of the tablets was found between 2.1–2.4 mm. The

hardness of the tablets ranges from 5.2 kg to 6.8 kg. The % Friability was 0.1% to 0.8%.

The average weights of the tablets were between 99.85 mg to 100.11 mg. The drug

content in each tablet was found to be within acceptable range 99.13 -100.46%. The

surface pH of the mucoadhesive tablets was in the range of 7.0 -7.4.

The tabulation 6.17 listed out the values of various physicochemical tests done on

SR tablets of Theophylline. The average thickness of the tablets was found between 5.9-

6.1 mm. The hardness of the tablets ranges from 5.5 kg to 6.8 kg. The % Friability was

0.3% to 0.4%. The average weights of the tablets were between 399.08 mg to 401.13 mg.

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The drug content in each tablet was found to be within acceptable range 98.99 -101.62%.

The surface pH of the mucoadhesive tablets was in the range of 6.9 -7.2.

The physicochemical tests of the SR tablets of Salbutamol sulphate &

Theophylline prepared by natural mucoadhesive materials NMM01 and NMM02

possessed optimum strength, hardness, friability to withstand the abrasion and attrition

contributed due to handling and transportation. The drug loading and uniformity of drug

content were satisfactory and sufficient to be processed in a large scale and using high

speed compression machines.

The surface pH of the tablets was close to that of salivary glands thereby

decreased the chances of irritation and discomfort at the point of contact with the mucosa.

The average pH of all formulations was around neutral pH significantly similar to that of

saliva and hence no mucosal irritation was expected thus improving patient compliance.

e. Swelling index

The swelling index values of various SR formulations containing Salbutamol

sulphate was tabulated in table 6.22. At the end of 240 minutes Formulation SF03

(containing 75 mg of NMM01) registered swelling index of 0.89 and Formulation SF06

(containing 75 mg of NMM02) showed swelling index of 1.04 which are similar to the

swelling index produced by SF09 (containing 75 mg of HPC) 0.89 and SF12 (containing

75 mg of sodium alginate) 1.06. These results illustrated the fact that the Natural

mucoadhesive materials showed swelling index equivalent to the standardized

mucoadhesive polymer namely Hydroxy propyl cellulose and sodium alginate.

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The swelling index values of various SR formulations containing Theophylline

was given in table 6.23. After 240 minutes the swelling index of the formulation TF03

containing 75 mg of NMM01 showed 0.164 and formulation TF06 containing 75 mg of

NMM02 showed 0.163. These values are on par with the swelling index values of 0.164

demonstrated by the formulations TF09 (containing 75 mg HPC) and 0.167 showed by

TF12 (containing 75 mg of sodium alginate). The natural polymers NMM01 and NMM02

produced good swelling indices.

f. Mucoadhesive strength

Mucoadhesive strength of SR formulations containing Salbutamol sulphate was

listed in table 6.24. The force of adhesion of the formulations containing NMM01 found

to increase on increasing the concentration of the polymer. At the end of 30 minutes the

formulation SF03 containing highest concentration of the polymer NMM01 (75 mg)

showed mucoadhesive strength of 0.1535 N. The formulation SF06 containing 75 mg of

NMM02 polymer showed 0.146 N. The Mucoadhesive strength or force of adhesion was

comparable to the SR tablets formulated using HPC and sodium alginate.

Mucoadhesive strength of SR formulations containing Theophylline was given in

table 6.25. The concentration of the polymers found to influence the mucoadhesive

strength of the formulations. The formulation TF03 having highest concentration of the

polymer NMM01 illustrated mucoadhesive strength of 0.1633 N. The same trend was

witnessed in case of the polymer NMM02 also. The formulation TF06 containing 75 mg

of NMM02 had mucoadhesive strength of 0.1525N. The SR tablets formulated with

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NMM01 and NMM02 were found to have good force of adhesion when compared with

the formulations with polymers like HPC and sodium alginate. The force of adhesion was

found to be directly proportional to the concentration of the polymer.

g. Invitro drug release kinetics

Drug release data for the formulations containing various compositions of

NMM01 and Salbutamol sulphate was tabulated in table 6.27 and Fig 6.33. At the end of

12 h the formulation SF01 with 25 mg of NMM01 showed drug release of 93%. On

increasing the concentration of NMM01 the drug release in SF03 gradually decreased to

83%. The decrease in the drug may be due to the fact that the polymer NMM01 upon

contact with aqueous medium start absorbs water and as a consequence the polymer

swells forming a gel layer. This layer increases in thickness as time passes creating a

considerable barrier for both penetration of solvent into the tablet and drug release from

it. The same phenomenon was observed in case of formulations containing NMM01 with

Theophylline. From the table 6.35 and Fig 6.50, the formulation TF01( containing 25 mg

of NMM01) showed drug release of 99.10 % in 10 h where as formulation TF02 with 50

mg of NMM01 showed drug release of 99.30% in 11 h. The formulation containing

highest concentration of NMM01 in TF03 demonstrated drug release of 90.50% in 12 h.

Based on the data given in table 6.29, the release of Salbutamol sulphate from the

formulations containing NMM02 slowed on increasing the concentration of the polymer.

The formulation SF04 with 25 mg of NMM02 released 98.3% of its content within 7 h.

On increasing the concentration NMM02 to 50 mg showed release 98% in 9 h where as

the formulation SF06 with 75 mg of NMM02 showed release of 98.2% in 10 h. It was

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observed that NMM02 showed less retardant effect when compared with NMM01. The

drug release data for the formulation containing NMM02 with Theophylline was listed in

table 6.37 and fig 6.54. The same phenomenon with NMM01 was observed with NMM02

also. The increase in concentration of the NMM02 produced increase retardant effect

evident from the fact that formulation TF04 ( 25mg of NMM02) showed 99.2% in 8 h,

formulation TF05 (50 mg of NMM02) 99.5% at 9 h and TF06 (75 mg of NMM02)

showed 99.5% drug release at 10 h.

From the data observed in table 6.31, 6.33, 6.39, 6.41 and Fig 6.41, 6.45, 6.58 and

6.62 it was concluded that the natural mucoadhesive material NMM01 was found to

exhibit better drug retardant characteristics compared with the polymer NMM02, HPC

and sodium alginate.

In order to determine the exact mechanism of drug release from the formulations,

the invitro drug release data was analyzed according to zero order kinetics, first order

kinetics, Higuchi and Korsemeyer Peppas equation. The criterion for selecting the most

appropriate model was on the basis of goodness of best fit. Based on the summary of

results given in table 6.44 and 6.45 the formulations containing NMM01 showed first

order drug release for Salbutamol sulphate with r2 value >0.9. The release exponent value

of 0.8 determined from korsemeyer peppas plot indicate non-fickian drug release that

means drug release occurred by diffusion and erosion of the polymer. The formulations

containing NMM01 with Theophylline followed zero order drug release. The release

exponent value >0.88 suggested case II transport.

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The overall best formulation SF03 and TF03 was compared with existing

marketed formulations containing Salbutamol sulphate and Theophylline respectively.

The in vitro drug release data was given in table 6.45, 6.47 and Fig 6.67, 6.71. The

f2 similarity factor was calculated and observed between >50 and <100 indicates both

formulations are bioequivalent their respective marketed formulations.

h. Similarity factor

Comparison of dissolution data of formulation SF03, TF03 and the corresponding

Marketed tablets were shown in table 6.45 and Fig 6.67 & 6.68. The similarity factor and

Sd were calculated by using MS Excel add In-software. The obtained similarity factor

values where shown in table 6.46. An f2 of 93.95 and 90.47 and Sd value of 0.008 and

0.009 indicates that the release profile of SF03, TF03 and their corresponding marketed

tablets were comparable and in a good agreement with each other.

i. In vivo bioavailability studies

The pharmacokinetic parameter of the formulation containing Salbutamol sulphate

and its reference were listed in table 6.49. The Cmax was found to be 235.89 and 186.63

ng/mL for the reference and formulation SF03, respectively and the corresponding tmax

were 3 h and 6 h. Table 6.50 showed pharmacokinetic values of the formulation

containing Theophylline and it reference. The Cmax was found to be 8.5 and 6.29 µg/mL

for the reference and formulation TF03, respectively and the corresponding tmax were 4 h

and 10 h. It was observed that the Cmax values of the formulation and the reference differ

and significant difference were obtained between them. The formulation SF03 and TF03

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were exhibited delayed tmax. The value of the MRT, which was the non compartmental

analogue of t1/2 was found parallel to those of t1/2 of the reference. The formulation SF03

showed a higher MRT of 10.8 h compared to that of Salbutamol sulphate reference

standard of 5.88 h, TF03 showed higher MRT 17.39 h than the Theophylline reference

standard of 8.77 h.

Statistical differences were observed in the AUC0-∞ for the Salbutamol sulphate

reference standard 1558.52 ng.h/mL and the formulation of SF03 2388.14 ng.h/mL, the

differences were also observed in AUC0-∞ for the Theophylline reference standard 99.56

µg.h/mL and the formulation TF03 111.34 µg.h/mL. It was concluded that the Cmax, tmax

and AUC0-∞ values lie within the acceptable range of FDA guidelines (80-125%)193

.

j. In vitro-In vivo correlation studies

The feasibility of developing a Level A correlation for SF03 and TF03 were

evaluated by plotting the percentage fraction of drug dissolved in vitro with respect to

percentage fraction of drug absorbed in vivo. Consistent correlations (r2>0.9) were

observed between in vitro and in vivo profiles of both the formulations shown in fig 6.101

and 6.102.

6.12. Conclusion

The extracted natural mucoadhesive materials NMM01 and NMM02 were been

incorporated as mucoadhesive material into the formulations of sustained release tablets

of Salbutamol sulphate and Theophylline. The prepared tablets were been evaluated for

various standards such as thickness, friability, Hardness, weight variation and for

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uniformity of the active ingredients. The results of the evaluation reveal that there is no

compliance with the standards set in given in the Indian pharmacopeia.

The formulated tablets were also been subjected to in vitro kinetic studies. The

formulation containing NMM01 as showed more retarded release of the active ingredient

than the formulation made of NMM02. The kinetic study also reveals that the NMM01

exhibit better drug retardant characteristics compared with the polymer NMM02, HPC

and sodium alginate. The in vitro release profile of the formulations with NMM01 was

compared with the commercially marketed formulation of the same drugs. The results

revealed that the formulations of NMM01 and the formulation of the market are having

comparable release profiles.

The in vivo bioavailability studies of the formulations containing the NMM01

were carried out on animal models. The reports reveal that The Cmax was found to be 8.5

and 6.29 µg/mL for the reference and formulation TF03, respectively and the

corresponding tmax were 4 h and 10 h. It was observed that the Cmax values of the

formulation and the reference differ and significant difference were obtained between

them. The formulation SF03 and TF03 were exhibited delayed tmax. The value of the

MRT, which was the non compartmental analogue of t1/2 was found parallel to those of

t1/2 of the reference. The formulation SF03 showed a higher MRT of 10.8 h compared to

that of Salbutamol sulphate reference standard of 5.88 h, TF03 showed higher MRT

17.39 h than the Theophylline reference standard of 8.77 h.

The feasibility of developing a Level A correlation for SF03 and TF03 were

evaluated by plotting the percentage fraction of drug dissolved in vitro with respect to the

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percentage fraction of drug absorbed in vivo. Consistent correlations (r2>0.9) were

observed between in vitro and in vivo profiles of both the formulations.

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