11_chapter 5.pdf - Shodhganga

Materials and methods

5.1 Materials Materials

Source

Miconazole nitrate

Bhavani Pharmaceuticals, Kanpur

5-Fluouracil

Strides Arcolab, Bangalore

Chitosan

Marine Chemicals, Cochin

Carbopol 71G

Arihant Trading Co, Mumbai.

Carboxymethyl tamarind

Creative polymer industries, Ananthpur

Polycarbophil/Noveon AA-1

Arihant Trading Co, Mumbai.

Sodium alginate

Loba Chemie, Mumbai.

Microcrystalline cellulose


Talc


Sodium deoxycholate

Sigma-Aldrich, Bangalore.

Potassium dihydrogen ortho


Phosphate Sodium hydroxide pellets

Reachem Lab, India.

Methanol


Tween 80

Merck Specialities Pvt Ltd, Mumbai.

Sodium chloride

Merck Specialities Pvt Ltd, Mumbai

Potassium hydroxide

Spectrum reagent and chemical Pvt Ltd, cochin.

Calcium hydroxide

Loba chemie, Mumbai.

Bovine serum albumin

Loba chemie, Mumbai.

Lactic acid

Ranbaxy laboratories limited, Punjab.

Dept of Pharmaceutics JSSCP, Mysore

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Acetic acid


Glycerol


Urea

Loba Chemie, Mumbai

Glucose


Hydrochloric acid

Rankem, New Delhi


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5.2 Instruments and Equipments

Equipments / Instruments

Source

Electronic balance

Shimadzu Corporation, Japan.

FT – IR Spectrophotometer

Shimadzu, 8400S, Japan.

Dissolution apparatus (8 basket)

Model No TDT-08L, Electrolab, Mumbai, India

Orbital shaking incubator

Remi , Mumbai, India

UV-Visible Spectrophotometer

UV1800, Shimadzu, Japan

X-ray diffractometer

Miniflex II Desktop, Rigaku Corporation, Japan

Hardness tester

Erweka .Germany

Micrometer screw gauge

Mitotoyo, Japan.

Friabilator

Electrolab, EF-2, Mumbai, India

Hot air oven

Tempo instruments Pvt Ltd,Mumbai

Magnetic stirrer

Remi, Mumbai.


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METHOD OF STUDY Analytical method 

Analytical method of miconazole nitrate (MN) in buccal pH 6.8 and simulated vaginal pH 4.2



Analytical method of 5-fluorouracil (5-FU) in buccal pH 6.8, simulated vaginal pH 4.2 and rectal pH 7.4

Preformulation studies 

Solubility



Partition co-efficient



Drug excipients compatibility studies

INTERPOLYELECTROLYTE COMPLEX (IPEC) 

Preparation and characterization

PREPARATION OF TABLET FORMULATION 

Formulation of tablets using Chitosan- Carbopol 71G IPEC



Formulation of tablets using Chitosan-carboxymethyltamarind IPEC



Formulation of tablets using Chitosan- Polycarbophil IPEC



Formulation of tablets using chitosan- Sodium alginate IPEC

EVALUATION OF THE TABLET 

Physicochemical properties



Swelling studies



In vitro drug release



In vitro mucoadhesive studies



In vivo studies



Ex vivo permeation studies



Stability studies


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5.3 Preparation of buffer solutions Phosphate buffer (pH 6.8) 50 ml of 0.2M potassium di hydrogen phosphate was taken in 200 ml volumetric flask, to which 22.4 ml of 0.2 M sodium hydroxide solution was added and the volume was made up to the mark with distilled water[154].

Simulated vaginal fluid Simulated vaginal fluid (SVF) was prepared as reported: 3.51 g/l NaCl, 1.40 g/l KOH, 0.222 g/l Ca(OH)2, 0.018 g/l bovine serum albumin (BSA), 2 g/l lactic acid, 1 g/l CH3COOH, 0.16 g/l glycerol, 0.4 g/l urea and 5 g/l glucose. The pH was correct at 4.2 with HCl 0.1N [155].

Phosphate buffer (pH 7.4) Potassium dihydrogen phosphate of 0.2 M (50 ml) and 39.1 ml of 0.2 M NaOH were taken in a 200 ml volumetric flask and made up to the volume with water[154].

0.2M potassium dihydrogen phosphate 27.218 gm of potassium dihydrogen phosphate was added to 1000 ml volumetric flask containing distilled water and the volume was made up to the mark with distilled water.

0.2M sodium hydroxide 8 gm of NaOH was taken in a 1000 ml volumetric flask containing distilled water and volume was made up to the mark with distilled water.


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5.4. ANALYTICAL METHODS 5.1. Estimation of Miconazole nitrate (MN) Determination of max of MN in phosphate buffer pH 6.8 (Buccal pH) Preparation of stock solution Accurately weighed 100 mg of MN was dissolved in small amount of methanol and then 1 % of tween 80 was added. The volume was made up to 100 ml using the phosphate buffer pH 6.8. Scanning From the stock solution, 100-600 mcg / ml solutions were prepared by pipetting 1-6 ml to a series of 10 ml volumetric flasks and the volume was made up to 10 ml with phosphate buffer pH 6.8. The UV scan of these solutions was taken between 400-200 nm. The absorption maximum of MN was found to be 272 nm and this wavelength was used for further studies. The spectrum is shown in Figure 2. The calibration curve data are given in Table 1 and calibration curve is shown in the Figure 11.

Figure 10: UV spectra of MN in phosphate buffer pH 6.8


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Table 1: Calibration curve data of MN in phosphate buffer pH 6.8 Sl. No.

Concentration in μg/ml

Absorbance ± S.D Mean*

1

100

0.1378±0.0005

2

200

0.2724±0.0001

3

300

0.4002±0.0006

4

400

0.5245±0.0003

5

500

0.6460±0.0006

6

600

0.7892±0.0006

* Standard deviation n = 3

Figure 11: Calibration curve of MN in phosphate buffer pH 6.8


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Determination of max of MN in simulated vaginal fluid (SVF) pH 4.2 (Vaginal pH) Preparation of stock solution Accurately weighed 100 mg of MN was dissolved in small amount of methanol and then 1 % of tween 80 was added. The volume was made up to 100 ml using the SVF pH 4.2. Scanning From the stock solution, 25-200 mcg / ml solutions were prepared by pipetting 0.25-2 ml to a series of 10 ml volumetric flasks and the volume was made up to 10 ml with phosphate buffer pH 6.8. The UV scanning of these solutions was taken between 400-200 nm.

The absorption maximum of MN was found to be 271 nm and this

wavelength was used for further studies. The spectrum is shown in Figure 12. The calibration curve data are given in Table 2 and calibration curve is shown in the Figure 13.

Figure 12: UV spectra of MN in SVF pH 4.2


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Table 2: Calibration curve data of MN in SVF pH 4.2 Sl. Concentration No. in μg/ml


1

25

0.1093±0.0002

2

50

0.2273±0.0005

3

75

0.3225±0.0004

4

100

0.4467±0.0002

5

125

0.5499±0.0003

6

150

0.6658±0.0006

7

175

0.7842±0.0006

8

200

0.9153±0.0002


Figure 13: Calibration curve of MN in SVF pH 4.2


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5.4.2 Estimation of 5-fluorouracil (5-FU) Determination of max of 5-Fluorouracil (5-FU) in phosphate buffer pH 6.8. Preparation of stock solution Accurately weighed 100 mg of 5-FU was dissolved in small amount of distilled water. The volume was made up to 100 ml using the phosphate buffer pH 6.8. Scanning From the stock solution, 2-10 mcg / ml solutions were prepared by pipetting 0.2-1 ml to a series of 10 ml volumetric flasks and the volume was made up to 10 ml with phosphate buffer pH 6.8.

The UV scanning of these solutions was taken between

400-200 nm. The absorption maximum of 5-FU was found to be 266 nm and this wavelength was used for further studies. The spectrum is shown in Figure 14. The calibration curve data are given in Table 3 and calibration curve is shown in the Figure 15.

Figure 14: UV spectra of 5-FU in phosphate buffer pH 6.8


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Table 3: Calibration curve data of 5- FU in phosphate buffer pH 6.8

Sl. No.

Concentration (mcg/ml)


1

2

0.1777±0.0001

2

4

0.3221±0.0002

3

6

0.5021±0.0002

4

8

06715±0.0002

5

10

0.8214±0.0001


Figure 15: Calibration curve of 5-FU in phosphate buffer pH 6.8


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Determination of max of 5-FU in simulated vaginal fluid (SVF) pH 4.2. Preparation of stock solution Accurately weighed 100 mg of 5-FU was dissolved in small amount of distilled water. The volume was made up to 100 ml using the SVF pH 4.2. Scanning From the stock solution, 5-25 mcg / ml solutions were prepared by pipetting 0.5-2.5 ml to a series of 10 ml volumetric flasks and the volume was made up to 10 ml with simulated vaginal fluid pH 4.2. The UV scanning of these solutions was taken between 400-200 nm. The absorption maximum of 5-FU was found to be 265 nm and this wavelength was used for further studies. The spectrum is shown in Figure 16. The calibration curve data is given in Table 4 and calibration curve is shown in the Figure 17.

Figure 16: UV spectra of 5-FU in SVF pH 4.2


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Table 4: Calibration curve data of 5-FU in SVF pH 4.2

Sl. No.

Concentration (in mcg/ml)


1

5

0.1831±0.0001

2

10

0.3471±0.0001

3

15

0.5151±0.0002

4

20

0.6723±0.0001

5

25

0.8330±0.0002


Figure 17: Calibration curve of 5-FU in SVF pH 4.2


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Determination of max of 5-FU in phosphate buffer pH 7.4 (Rectal pH) Preparation of stock solution Accurately weighed 100 mg of 5-FU was dissolved in a small amount of distilled water. The volume was made up to 100 ml using the phosphate buffer pH 7.4. Scanning From the stock solution, 100-400 mcg / ml solutions were prepared by pipetting 1-4 ml to a series of 10 ml volumetric flasks and the volume was made up to 10 ml with phosphate buffer pH 7.4.

The UV scanning of these solutions was taken between

400-200 nm. The absorption maximum of 5-FU was found to be 267 nm and this wavelength was used for further studies. The spectrum is shown in Figure 18. The calibration curve data is given in Table 5 and calibration curve is shown in the Figure 19.

Figure 18: UV spectra of 5-FU in phosphate buffer pH 7.4


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Table 5: Calibration curve data of 5-FU in phosphate buffer pH 7.4

Sl. No.

Concentration (in mcg/ml)

Absorbance Mean± S.D*

1

3

0.1591±0.0001

2

6

0.3220±0.0001

3

9

0.4531±0.0003

4

12

0.6121±0.0002

5

15

0.7751±0.0002


Figure 19: Calibration curve of 5-FU in phosphate buffer pH 7.4


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5.5 PREFORMULATION STUDIES Solubility Excess amount of MN was shaken with 2 ml of buffer solution (phosphate buffer pH 6.8 and SVF pH 4.2 separately) at room temperature, until equilibrium was reached. Similarly for 5-FU was shaken in phosphate buffer pH 6.8, SVF 4.2 and pH 7.4 separately. The solution was then filtered and the concentration of drug in solution was determined by U.V spectroscopic method using shimadzu 1800 U.V visible spectrophotometer [156].

Partition coefficient Mutually saturated 1- octanol and phosphate buffer solution (pH 6.8) at 37 °C was used for the study. An aliquot (10 ml) of 1-octanol saturated phosphate buffer solution containing suitable concentration of the 5-FU (100 mcg/m1) was mixed with an equal volume of phosphate buffer (pH 6.8) saturated 1-octanol. Two phases were then allowed to equilibrate at 37 °C for 24 hrs on a magnetic stirrer. Similarly in SVF fluid pH 4.2 and pH 7.4 was carried out [157]. The concentration of the drug in the aqueous phase was determined by U.V Spectroscopic method. The apparent partition coefficient (kp) was calculated as the ratio of drug concentration in each phase by the following equation. Caq-Ceq Kp = Ce


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Where, Caq is initial concentration of drug in aqueous phase and Ceq is the concentration of drug at equilibrium in aqueous phase. Phosphate buffer pH 6.8, dissolved in ocantol phase, is not taken into account in this measurement. Drug–Excipient compatibility In order to ascertain whether or not any interaction occurred between the polymers and drug substances, the characterization of drug, polymer and physical mixture of drug: polymer have been done using differential scanning calorimetry (DSC) and fourier transform infrared spectroscopy (FT-IR). Differential scanning calorimetry: All the dynamic DSC studies were carried out on DSC 50, Shimadzu Scientific Instruments, Japan. Calorimetric measurements were made with empty cell (high purity alpha alumina discs) as the reference. The instrument was calibrated using high purity indium metal as standard. The dynamic scans were taken in nitrogen atmosphere at a heating rate of 10 °C min. The runs were made in triplicate. Fourier Transform Infrared (FT-IR) Spectroscopy: The test sample was dispersed in KBr powder and analyzed. FT-IR spectra were obtained by diffuse reflectance on a FT-IR spectrophotometer type FT-IR 8400S shimadzu, Japan. Compatibility between the drugs and the polymers were compared by FT-IR spectra. The positions of FT-IR bands of important functional groups of drugs were identified and were cross checked with FTIR spectra of drug with excipients in 1:1ratio.


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Formulation design overview Design for oral, cervical and colorectal cancer

Design for oral and vaginal candidiasis

Miconazole nitrate (MN) Part I (14 formulation) Polymers used

Formulation code

5-fluorouracil (5-FU) Part III (12 formulations) Polymers used

Formulation code

Chitosan

MA1,MA2

Polycarbophil

FA1,FA2

Carbopol 71G

MB1,MB2

ChitosanPolycarbophil PM

FB1,FB2

Chitosan-carbopol 71G PM

MC1, MC2

ChitosanPolycarbophil IPEC

FC1,FC2,FC3, FC4 FD1,FD2

Chitosan-carbopol IPEC

MD1,MD2, MD3 MD4

IPEC, chitosan and polycarbophil

FE1,FE2

IPEC, chitosan and carbopol

ME1, ME2,ME3 ME4

IPEC, chitosan, Polycarbophil, SDC

Part IV (12 formulations) Polymers used

Part II (12 formulations) Polymers used

Formulation code

Sodium alginate

FF1,FF2

Chitosan-sodium alginate PM

FG1,FG2

Chitosan-sodium alginate IPEC

FH1,FH2,FH3, FH4

IPEC, chitosan and sodium alginate

FI1,FI2

IPEC, chitosan, Sodium alginate, SDC

FJ1,FJ2

Formulation code

CMT Chitosan- CMT PM

MF1,MF2 MG1,MG2

Chitosan-CMT IPEC

MH1,MH2,MH3, MH4

IPEC, chitosan and CMT

MI1,MI2,MI3, MI4

Figure 20: Schematic representation of formulation design PM: Physical mixture, IPEC: Interpolyelectrolyte complex, CMT: Carboxymethyl tamarind, SDC: Sodium deoxycholate Dept of Pharmaceutics JSSCP, Mysore

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5.6. FORMULATION DESIGN OF MICONAZOLE NITRATE TABLETS Chitosan-carbopol 71G IPEC 5.6.1 Preparation of chitosancarbopol 71G interpolyelectrolyte complex (IPEC) A Carbopol 71G aqueous solution (1 mg/ml) and chitosan aqueous acetic acid solution (5 mg/ml) were mixed. The resulting precipitate (carbopol/chitosan IPEC) was washed with distilled water and filtered under vacuum pump. The filtrate was dried in hot air oven and the dried complex was ground with a grinder. The powder was passed through a 200µm sieve and used for further study.

5.6.2 Turbidity measurement of chitosancarbopol IPEC ratios The carbopol 71G/chitosan ratio in the complex was examined by monitoring the transmittance of the solution at a wavelength of 600 nm using a spectrophotometer (UV-1800, Shimadzu, Japan). An aqueous carbopol 71G solution (0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 mM) and a chitosan aqueous acetic acid solution (0.5, 1, and 2 mM) were used. The concentration was calculated by dividing the weight of chitosan and carbopol by the formula weight of each monomer unit. Each mixture was shaken vigorously. The mixtures were kept aside for 10 min before measuring the transmittance as a function of the various mixing ratios (chitosan/ carbopol 71G) [158].

5.6.3 Preparation of miconazole nitrate tablets Mucoadhesive tablets were fabricated by direct compression method as shown in Table 6. The accurate quantity of miconazole nitrate and excipients was weighed. They were passed through sieve # 100 and thoroughly mixed using mortar and pestle. The blend was


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lubricated and then compressed into compacts by direct compression method using 8-mm flat-faced punches in KBr press (Technosearch, Mumbai, India) at 1 ton pressure with a dwell time of 1 s.

Table 6: Formulation chart for MN matrix tablets

Formulation code

Chitosan (mg)

Carbopol 71G(mg)

Chitosancarbopol 71G physical mixture (1:1) (mg)

MA1

50

---

---

----

45

5

MA2

100

---

---

----

----

---

MB1

---

50

---

---

45

5

MB2

---

100

---

---

---

---

MC1

---

---

50

---

45

5

MC2

---

---

100

--

---

---

MD1

---

---

---

60

35

5

MD2

---

---

---

70

25

5

MD3

---

---

---

80

15

5

MD4

---

---

---

90

5

5

ME1

20

---

---

70

5

5

ME2

25

---

---

65

5

5

ME3

10

10

---

70

5

5

ME4

15

15

---

60

5

5

IPEC (mg)

MCC (mg)

Talc (mg)

Miconazole nitrate is 50mg Total weight of tablet is 150 mg


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Chitosan- carboxymethyl tamarind IPEC 5.6.4 Preparation of chitosan/carboxymethyl tamarind interpolyelectrolyte complex (IPEC) 2 % w/v Chitosan aqueous acetic acid solution and 2 % w/v of Carboxymethyl tamarind solution (CMT) were mixed under homogenization under 1000 rpm. The resulting precipitate (Chitosan/CMT IPEC) was washed with distilled water and filtered under vacuum pump. The filtrate was dried in hot air oven and the dried complex was ground with a grinder. The powder was passed through a 200 µm sieve and used for further study [159].

5.6.5 Viscosity study of chitosan-CMT IPEC ratios Chitosan solution was prepared in 2 % v/v acetic acid. CMT solutions were separately prepared by hydrating them in distilled water. Both the solutions were at 25 °C to obtain different ratios of CH: CMT. The samples were incubated at 37 °C for 24 h. The samples were then centrifuged at 15000 rpm. The viscosity of the supernatant solution was determined using Brookfield RVDV II Pro Viscometer, UK (Spindle 21).

5.6.6 Preparation of miconazole nitrate tablets using chitosan-CMT IPEC Mucoadhesive tablets were fabricated by direct compression method as shown in Table 7. The accurate quantity of miconazole nitrate and excipients was weighed. They were passed through sieve # 100 and thoroughly mixed using mortar and pestle. The blend was lubricated and then compressed into compacts by direct compression method using 8-mm flat-faced punches in KBr press (Technosearch, Mumbai, India) at 1 ton pressure with a dwell time of 1 s.


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Table 7: Formulation chart for MN matrix tablet Ingredients MF1 MF2 MG1 MG2 MH1 MH2 MH3 MH4 MG1 MG2 MG3 MG4 MN (mg)

50

50

50

50

50

50

50

50

50

50

50

50

CMT(mg)

50

100

---

---

----

---

----

---

20

20

30

20

Chitosan– CMT physical mixture (1:1) (mg)

----

-----

50

100

----

---

---

---

---

---

IPEC (mg)

------

----

---

---

60

70

80

90

80

60

60

70

---

---

---

---

---

---

---

---

--

20

10

10

MCC (mg)

45

----

45

---

35

25

15

5

--

---

--

--

Talc (mg)

5

----

5

---

5

5

5

5

--

---

--

---

Chitosan (mg)

Total weight of tablet is 150 mg


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---


5.7. Formulation Design for 5-FU tablets Chitosan-polycarbophil IPEC 5.7.1 Preparation of chitosan-polycarbophil interpolyelectrolyte complex (IPEC) A 3 % w/v Chitosan solution and a 3 % w/v of polycarbophil solution were prepared separately in solutions of 2 % v/v acetic acid. The chitosan solution was added slowly to the polycarbophil solution under homogenization at 1000 rpm over a period of 20 min. the mixture was then stirred for a period of 1 h at a speed of 900 rpm with digital mechanical stirrer. The resulting precipitate (Chitosan- polycarbophil IPEC) was washed several times with 2 % v/v acetic acid solution to remove any noncomplexed polymeric material and filtered under vacuum pump. The product was dried in hot air oven and the dried complex was powdered with a grinder. The powder was passed through a 200 µm sieve and used for further study.

5.7.2 Transmittance measurement of chitosan-polycarbophil IPEC ratios Transmittance measurements were carried out with a UV spectrophotometer (Shimadzu UV visible 1800, Shimadzu Scientific Instruments, Japan) at the wavelength λ= 420 nm for supernatant liquids of various concentration of chitosan-polycarbophil IPEC [160,161].Solutions of 3 % (w/v) of chitosan and various concentration of polycarbophil solution ranging from 0.5 to 5 % (w/v) in 2 % acetic acid solution (v/v) were prepared separately. Then the chitosan solution was slowly added to polycarbophil solution and kept aside. The resultant solution was filtered and the supernatant liquid of the solution was subjected for transmittance.


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5.7.3 Preparation of 5-fluorouracil tablets using chitosan-polycarbophil IPEC Mucoadhesive tablets were fabricated by direct compression method as shown in Table 8. The accurate quantity of 5-fluorouracil and excipients was weighed. They were passed through sieve # 100 and thoroughly mixed using mortar and pestle. The blend was lubricated and then compressed into compacts by direct compression method using 8-mm flat-faced punches in KBr press (Technosearch, Mumbai, India) at 1 ton pressure with a dwell time of 1 s.

Table 8: Formulation chart for 5-FU matrix tablet Ingredients

FA1

FA2

FB1

FB2

FC1

FC2

FC3

FC4

FD1

FD2 FE1 FE2

5-Fluorouracil (mg)

20

20

20

20

20

20

20

20

20

20

20

20

Polycarbophil (mg) Chitosanpolycarbophil physical mixture(1:1) (mg) IPEC (mg)

60

80

---

-----

----

----- -----

-----

10

20

20

20

----

-----

80

100

---

----

-----

-----

-----

----

---

---

-----

-----

----

---

40

60

80

100

80

80

80

80

Chitosan (mg)

----

----

----

-----

----

----

----

---

10

20

20

20

SDC (mg)

----

----

-----

-----

---

----

----

----

----

----

3

4.5

MCC (mg)

65

45

45

25

85

65

45

25

25

5

2

0.5

Talc (mg)

5

5

5

5

5

5

5

5

5

5

5

5



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Chitosan- sodium alginate IPEC 5.7.4 Preparation of chitosan/sodium alginate interpolyelectrolyte complex (IPEC) The chitosan–alginate polyelectrolyte complex was prepared from chitosan solution at 4.0 % w/v in 2 % w/w acetic acid solution and sodium alginate solution at 4.0 % w/v in water. Both solutions were heated separately at 70–80 °C. Both solutions were mixed with agitation until the mixture reached room temperature. Then it was kept aside for 2 h. The interpolyelectrolyte complex (IPEC) was thoroughly washed with distilled water and then separated from water by centrifugation for 30 min at 10000 rpm. Thereafter IPEC was dried in hot air oven and the dried complex was powdered with a grinder. The powder was passed through a 200 µm sieve and used for further study [162].

5.7.5 Viscosity study of chitosan-alginate IPEC ratios Chitosan solution was prepared in 2 % v/v acetic acid and sodium alginate solution was prepared in water. Both the solutions were mixed together to obtain different ratios of IPEC. The samples were incubated at 37 °C for 24 h. The samples were then centrifuged at 15000 rpm. The viscosity of the supernatant solution was determined using Brookfield RVDV II Pro Viscometer, UK (Spindle 21).

5.7.6 Preparation of 5-fluorouracil tablets using chitosan-sodium alginate IPEC Mucoadhesive tablets were fabricated by direct compression method as shown in Table 9. The accurate quantity of 5-fluorouracil and excipients was weighed. They were passed through sieve # 100 and thoroughly mixed using mortar and pestle. The blend was lubricated and then compressed into compacts by direct compression method using 8-mm Dept of Pharmaceutics JSSCP, Mysore

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flat-faced punches in KBr press (Technosearch, Mumbai, India) at 1 ton pressure with a dwell time of 1 s.

Table 9: Formulation chart for 5-FU matrix tablet Ingredients

FF1

FF2 FG1 FG2 FH1 FH2 FH3 FH4 FI1

FI2 FJ1 FJ2

5-Fluorouracil (mg)

20

20

20

20

20

20

20

20

20

20

20

20

Sodium alginate 40 80 --(mg) Chitosan-------- 80 sodium alginate physical mixture(1:1) (mg) IPEC (mg) ------ ----- ----

-----

----

-----

-----

-----

10

20

20

20

100

---

----

-----

-----

----- ----

---

---

---

40

60

80

100

80

80

80

80

Chitosan (mg)

----

----

----

-----

----

----

----

---

10

20

20

20

SDC (mg)

----

----

-----

-----

---

----

----

----

----

----

3

4.5

MCC (mg)

85

45

45

25

85

65

45

25

25

5

2

0.5

Talc (mg)

5

5

5

5

5

5

5

5

5

5

5

5



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5.8 Evaluation 5.8.1 Characterization of IPEC Fourier transform infrared (FT-IR) spectroscopy study The infrared absorption spectra of polymers alone and their IPEC were analyzed using a FT-IR spectrophotometer (shimadzu 8400S). The pellets were prepared by pressing the sample with potassium bromide in the ratio of 1:100. Differential scanning calorimetry (DSC) Thermal analysis of only the polymers and there IPEC was carried out using a differential scanning calorimeter (DSC 50, Shimadzu Scientific Instruments, Japan). The samples were placed in an aluminum-sealed pan and preheated to 200 °C. The sample was cooled to room temperature and then reheated from 40 to 400 °C at a scanning rate of 10 °C/min. Powder X-ray Diffraction Powder X-ray diffraction patterns on polymers alone and their IPEC were obtained by using an X-ray Diffractometer (Miniflex II Desktop X-ray Diffractometer, Rigaku Corporation, Tokyo, Japan). The samples were scanned from 6° to 40° (2θ) with an increment of 0.02° and measurement time of 10 s/increment.

5.8.2 Evaluation of tablets 5.8.2.1 Physicochemical properties Determination of drug content The prepared formulations were analyzed for miconazole nitrate content, by taking 150 mg of tablet powder in 100 ml volumetric flask, to which methanol was added and shaken well. Further, the volume was made up to the mark with methanol. The drug


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content was determined by measuring the absorbance at 272 nm using UVspectrophotometer. Similarly 5-fluorouracil was determined in distilled water at 265 nm using UV spectrophotometer. Determination of weight variation Twenty tablets were randomly selected from each batch and individually weighed. The average weight and standard deviation of 20 tablets was calculated. The batch passes the test for weight variation test, if not more than two of the individual tablet weight deviate from the average weight by more than the percentage shown in table 10. Table 10: Maximum % deviation allowed as per IP Average weight 130 or less 130-324 More than 324

Maximum % deviation allowed 10 7.5 5

Determination of thickness Twenty tablets were randomly selected from each batch and their thickness was measured by using vernier calipers. It is expressed in millimeter. Determination of hardness The hardness of the tablet was determined using Inweka hardness tester. For each batch three tablets were tested. It is expressed in Newton. Determination of friability Twenty tablets were weighed and placed in the friabilator. The apparatus was rotated at 25 rpm for 4 minutes. After revolutions the tablets were dedusted and weighed again. The percentage friability was measured using the formula.


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F

Winitial  WFinal  100 WInitial

5.8.2.2 Swelling studies The swelling index of the prepared matrix tablets was determined by weighing five tablets and recording their weights before placing them separately in weighed beakers. The total weight was recorded (W1). Ten milliliters of phosphate buffer pH 6.8 (similarly with simulated vaginal fluid of pH 4.2 and phosphate buffer pH 7.4) was added to each beaker and then placed in an incubator at 37±0.5 °C. At time intervals of 2, 4, 6 and 8 h excess water was carefully removed, and the swollen tablets were weighed (W2). The experiment was repeated three times, and the difference of W1 and W2 was reported. The percentage swelling index was determined using the formula. Swelling index= W2-W1/W1*100

5.8.2.3 In vitro dissolution studies The drug release rate from buccal tablets was studied using the orbital shaking incubator using (Remi CIS 24, India) 30 mL of phosphate buffer pH 6.8. The temperature was maintained at 37±0.5 °C and 50 rpm (rotation per min). For every one hour of time interval, 3 mL sample was withdrawn and filtered through a Millipore filter of 0.45 µm pore size and assayed spectrophotometrically at 272 nm for miconazole and 266 nm for 5-fluorouracil. Immediately after each sample withdrawal, a similar volume of phosphate buffer pH 6.8 was added to the dissolution medium [163]. The drug release rates from vaginal tablets were studied in 500 ml of simulated vaginal fluid pH 4.2 in type II dissolution apparatus. The temperature was maintained at 37±0.5 °C and 50 rpm. 10 mL sample was withdrawn at hourly interval, filtered through Dept of Pharmaceutics JSSCP, Mysore

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a Millipore filter of 0.45 µm pore size and assayed spectrophotometrically at 271 nm for miconazole and 265 nm for 5-fluourouracil. Immediately after each sample withdrawal, a similar volume of simulated vaginal fluid pH 4.2 was added to the dissolution medium [164]. In vitro drug release for rectal tablets was performed using the dissolution apparatus I; 500 mL phosphate buffer pH 7.4 maintained at 37± 0.5 °C was used as a dissolution medium. Basket was rotated at 50 rpm. 10 mL aliquots were taken at periodic time intervals and replaced by equal volume of phosphate buffer pH 7.4. The solution was suitably diluted and the absorbance was taken at 267 nm for 5-fluorouracil using UV visible spectrophotometer [147].

5.8.2.4 In vitro mucoadhesive studies Mucoadhesive strength of the tablets was measured using modified physical balance. In vitro bioadhesion studies were carried out using sheep buccal mucosa and modified twoarmed balance. The phosphate buffer pH 6.8 was used as the moistening fluid. A glass stopper was suspended by a fixed length of thread on one side of the balance and was counter balanced with the weights on the other side. Fresh sheep buccal mucosa was collected from the slaughter house. It was scrapped off from the connective tissues and a thin layer of buccal mucosa was separated and used for the bioadhesion study. A circular piece of sheep buccal mucosa was cut and fixed to the tissue holder and immersed in phosphate buffer pH 6.8 and the temperature was maintained at 37± 1 °C. Then the tablet was fixed to a glass stopper with the help of cyanoacrylate adhesive and placed on the buccal mucosa by using a preload of 50 gm and kept aside for 1 min to facilitate adhesion


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bonding. After preloading time, the preload was removed and the weights were added on the other side of the balance until tablet detaches from the sheep buccal mucosa. The weight required to detach tablet from buccal mucosa was noted.

Figure 21: Modified physical balance for mucoadhesive studies 5.8.2.5 Ex vivo permeation study Permeation study was carried out for the optimized 5-fluorouracil tablets using Franz diffusion cell. The tablet was placed in the donor compartment on the sheep mucosa. The mucosal layer is on donor compartment. The receptor compartment was filled with phosphate buffer pH 6.8. The temperature was maintained at 37± 0.5 °C and 50 rpm. The amount of 5-fluorouracil permeated through sheep mucosa was determined by withdrawing 3 ml of aliquots from the receptor compartment using a syringe and immediately replacing the same volume of solution.

5.8.2.6 Mathematical model fitting The release data was fitted into various mathematical models using PCP DissoV2.08 software. The parameters like ‘n’ the time exponent, ‘k’ the release rate constant Dept of Pharmaceutics JSSCP, Mysore

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and ’R’ the regression co-efficient were determined to know the release mechanisms. The various models studied were: 

First order



Zero order



Matrix model



Hixon-crowell model



Higuchi model

Peppas model fitting The data obtained from in vitro release studies was put into Peppas model. The various parameters viz., the intercept A, the release constant K and regression coefficient R2 were calculated. Koresmeyer-Peppas equation: Mt/M∞ = 1- A (exp -Kt) Log (1 - Mt/M∞) = log A – kt/2.303 –

Amount of drugs released at time t

M∞

–

Total amount of drug released after an infinite time

K

–

Diffusion constant

A

–

The Intercept

Where, Mt

5.9 In vivo X-ray studies The animal experiment project was cleared and approved by Institutional animal ethical committee, J.S.S. College of Pharmacy, Mysore (Code: 106/2011) The study was performed on a healthy female rabbit, weighing between 1 and 1.5 kg. The optimized formulation was selected in order to study in vivo performance of the preparation. Optimized formulation was modified by adding 50 mg of x-ray grade barium sulfate for miconazole tablets and 20 mg of X-ray grade barium sulfate for 5-fluorouracil tablets. The prepared tablet was placed in the buccal mucosa of a healthy rabbit. During Dept of Pharmaceutics JSSCP, Mysore

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the study, the rabbit was not allowed to eat or drink. The rabbit was exposed to X-ray examinations and photographs were taken at 1st and 8th h after administration of the tablet. Similar procedure was followed for vaginal drug delivery for miconazole and 5-fluorouracil tablets. The rabbit was exposed to X-ray examinations and photographs were taken at 1st and 8th h after administration of the tablet. Similar procedure was followed for rectal drug delivery for 5-fluorouracil tablets. The rabbit was exposed to X-ray examinations and photographs were taken at 1st and 8th h after administration of the tablet.

5.10 Stability Studies Stability is defined as the ability of particular drug or dosage form in a specific container to remain within its physical, chemical, therapeutic and toxicological specification. Drug decomposition or degradation occurs during stability, because of chemical alteration of the active ingredients or due to product instability, lowering the concentration of the drug in the dosage form. The stability of pharmaceutical preparation should be evaluated by accelerated stability studies. The objective of accelerated stability studies is to predict the shelf life of a product by accelerating the rate of decomposition, preferably by increasing the temperature. The optimized formulation of buccal miconazole nitrate tablets was selected for the stability studies. The accelerated stability studies was carried out according to ICH guidelines by storing the samples at 25±2 ºC and 60±5 %RH, 30±2 ºC and 65±5 % RH and 40±2 ºC and 75±5 % RH for 6 months. Samples were withdrawn on 0 day, 3 months, and 6 months and were analyzed for physical stability and drug content.


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