* ^

Ie

rvr*

I Chapter Four

4. MATERIALS AN!) METHODS

4.L MATERIALS

4J.1. Clieinicab:

Rifampicin and Isoniazid (A Gift Sample from Cipla Ltd., Mumbai, India).

Sodium alginate (Loba Chemie Pvt. Ltd., Mumbai, India).

Ispaghula husk (Baidyanath, Jhansi, India).

Eudragit RSPO (Degussa India, Mumbai, India).

Ethylceilulose (Central Drug House, Mumbai, India).

Talc (Central Drug House, Mumbai, India).

Barium carbonate (S.D. Fine Chemicals Ltd., New Delhi, India).

Sodium dodecyl sulfate (Central Drug House, Mumbai, India).

Light liquid paraffin (Central Drug House, New Delhi, India).

Monobasic potassium phosphate (S.D.Fine Chemicals Ltd., New Delhi, India).

Span 80 (Central Dug House, New Delhi, India).

Chloroform (Ranbaxy Fine Chemicals, Chandigarh, India).

Sodium hydroxide pellets (S.D.Fine Chemicals Ltd., New Delhi, India).

Potassium dihydrogen orthophosphate (Merck Ltd., Mumbai, India).

Potassium bromide (Merck Ltd., Mumbai, India).

Stannous chloride (Merck Ltd., Mumbai, India).

Trichloro acetic acid (Merck Ltd., Mumbai, India).

Hydrochloric acid (Ranbaxy Fine Chemicals, Chandigarh, India).

Glacial acetic acid (Merck Ltd., Mumbai, India).

Methanol (Merck Ltd., Mumbai, India).

Sodium bicarbonate (S.D.Fine Chemicals Ltd., New Delhi, India).

Instant thin layer chromatography-silica gel strip (Gellman, Germany).

Methanol HPTLC (S.D.Fine Chemicals Ltd., New Delhi, India).

Acetone HPTLC (S.D.Fine Chemicals Ltd., New Delhi, India).

Water (Ultrapurified) (Millipore, Molsheim, France).

Dichloromethane (S.D.Fine Chemicals Ltd., New Delhi, India).

Ammonia solution (S.D.Fine Chemicals Ltd., New Delhi, India).

All the solvents and chemicals used were of analytical grade satisfying pharmacopoeial

standards.

Chapter 4 ________ Materials and Methods

Jamia Hamdard 77

4.1,2, Imtmmeats!

« Electronic balance (Mettler Toledo Inc., OH, USA).

• Mechanical stiiter (Rerai Motors, Mumbai, India).

® Magnetic stirrer with hot plate (Macro Scientific Works Ltd., Delhi, India).

• Six stage dissolution test apparatus, IP/BP/USP (USP XXVI, VEEGO, VDA-8DR

US? Standards),

• Melting point apparatus (Scientific MP-1, Hyderabad, India).

® Scanning electron microscope (.lEOL, JSM-6360 Scanning Electron Microscope,

Japan).

« U V - Visible spectrophotometer (UV-1601, Shimadzu, Kyoto, Japan).

Differential scanning calorimeter (Pyris 6.0, Perkin Elmer, Shelton, CT, USA),

® Fourier transform- infrared spectrophotometer (Spectrum BX, FTIR system, Perkin

Elmer, Italy).

® X-Ray diffractometer (PW 3710, Philips Analytical X-Ray B.V).

® Freeze dryer (Heto Drywimier, Birkerod, Denmark).

• Gamma camera (Millenium VG, USA).

8 Gamma counter dose calibrator (Capintec, NJ, USA).

® HPTLC applicator (Camag Linomet V, Switzerland),

e HPTLC scanner (Camag Linomet V, Switzerland).

• HPTLC pre-coated plates (E. Merck Ltd. Germany).

® HPLC (Shimadzu, Japan).

® Cooling centrifuge (C 24, Remi, Mumbai, India),

• Homogenizer (Heidolph, DIAX 900, Germany).

® Humidity cabinet (Scientific Equipment, Delhi, India).

9 Hot Air Oven (Scientific Equipment, Delhi, India).

® pH meter (Microprocessor pH System, Punjab, India),

® Stability oven (Nirmal International, Delhi, India).

® Water bath shaker (Nirmal International, Delhi, India).

« Ultrasonicator (PRAMA Ultrasonicator, Mumbai, India).

Chapter 4 _^ _________________ Materials and Methods

Jamia Hamdard 78

Chapter 4 Materials and Methods

4.2. METHODS

4.2.1. PREPARATION OF STANDARD BUFFERS/SOLUTIONS/CULTURE MEDIA

4.2.1.1„ Siniiilated gastric fluid (pH 1.2; without pepsin)

Simulated gastric fluid was prepared as per the composition given below and the

pH of the solution was adjusted to 1.2.

Ingredients Quantity (per L)

Sodium chloride 0.2 g

Hydrochloric acid 0.7 mL

Deionised water 1000 mL

4.2.I.2. Simulated intestinal fluid (pH 7.4; without pancreatine)

Simulated intestinal fluid was prepared as per the composition given below and

the pH of the solution was adjusted to 7.4.

Ingredients Quantity (per L)

Potassium dihydrogen phosphate 6.805 g

Sodium hydroxide 0.896 g

Deionised water 1000 mL

4.2.1.3. 0.01 M disodlum hydrogen phosphate (pH 4,6)

Disodium hydrogen phosphate (3.5815 g) was dissolved in water and diluted with

water to 1000 niL.

4.2.1.4. Culture media

In vitro antimicrobial study was performed to assess the antibacterial (for prolong

release effect) as well as antimycobacterial efficacy (for intestinal antitubercular effect) of the

optimized formulations, For this purpose various types of culture media were used. Nutrient

agar and Mueller Hinton agar medium were used to assess antibacterial efficacy of the

developed formulation. Modified Middlebrook 7H11 agar medium (simulated to gastric as

well as intestinal environment) was used to assess antimycobacterial efficacy (for intestinal

antitubercular effect) of the developed formulation. Lowenstein-Jensen medium was used to

maintain the mycobacterium strain.

Jamia Hamdard 79

Chapter 4 Materials and Methods

Nutrient agar medium.

Nutrient agar (20 g) was suspended in 1000 mL of purified water and dissolved by

slight warming. The media was sterilized by moist heat sterilization (autoclaving) at 15 lbs

pressure (121 °C) for 15 min. and pH after sterilization was adjusted in the range of 7.0.

Ingredients

Peptone from meat

Meat extract

Agar

Purified water (q.s.)

Quantity (per L)

5.0 g

3.0 g

12.0 g

lOOOmL

Mueller Hinton agar medium

Mueller Hinton Agar (38 g) was suspend in 1000 mL o f purified water and

dissolved by slight warming. The media was sterilized by moist heat sterilization

(autoclaving) at 15 lbs pressure (121 °C) for 15 min. Then the media was cooled to 45-50 °C

and pH after sterilization was adjusted in the range of 7.0. Then the media was poured into

sterile petri dishes on a level, horizontal surface to give a uniform depth of about 4 mm.

Ingredients Quantity (per L)

Beef extract powder 2.0 g

Acid digest of casein 17.5 g

Agar 17.0 g

Starch 1.5 g

Purified water (q.s.) 1000 mL

Modified Middlebrook 7HU agar medium (for the assessment o f in vitro intestinal

antitubercular activity o f the optimized formulations)

Modified Middlebrook 7H11 agar medium (simulated to gastric as well as

intestinal environment) was used to assess the in vitro intestinal antitubercular activity of the

optimized formulations. Middlebrook 7H11 agar (19 g) was suspended in 900 mL of

simulated gastric (pH 1.2) as well as simulated intestinal fluid (pH 7.4) containing 5 mL of

glycerol and boiled to dissolve completely. The media was sterilized by moist heat

sterilization (autoclaving) at 15 lbs pressure (121 °C) for 15 min. then the media was cooled

Jamia Hamdard 80

Chapter 4 Materials and Methods

to 45-50 °C and then aseptically added 100 mL of OADC (Oleic acid, Albumin fraction V,

Dextrose and Catalase powder) enrichment.

Ingredients Quantity (per L)

Enzymatic digest of casein 1 g

Disodium phosphate 1.5 g

Monopotassium phosphate 1.5 g

Ammonium sulfate 0.5 g

Monosodium glutamate 0.5 g

Sodium citrate 0.4 g

Ferric ammonium citrate 0.04 g

Magnesium sulfate 0.05 g

Copper sulfate 0.001 g

Pyridoxine 0.001 g

Zinc sulfate 0.001 g

Biotin 0.0005 g

Malachite green 0.00025 g

Agar 13.5 g

Supplement

Glycerol 5 mL

OADC enrichment 100 mL

Lowenstein-Jensen medium.

M ineral salt solution

Dissolve the ingredients in order in the distilled water by heating. The solution

was then sterilized by moist heat sterilization (autoclaving) at 15 lbs pressure (121 °C) for 15

min. Then the solution was cooled to room temperature.

Malachite green solution (2 %)

Using aseptic techniques dissolved the dye in sterile distilled water by placing the

solution in the incubator for 1-2 h.

Jamia Hamdard 81

Chapter 4 Materials and Methods

Homogenised whole eggs

Fresh hens’ eggs, not more than seven days old, were cleaned by scrubbing

thoroughly with a hand brush in warm water and a plain alkaline soap. Then the eggs were

soaked for 30 min. in the soap solution. Rinsed eggs thoroughly in running water and soaked

them in 70 % ethanol for 15 min. then cracked the eggs with a sterile knife into a sterile flask

and beat them with a sterile egg whisk or in a sterile blender.

Preparation of complete iiiediuni

Mineral salt solution (600 niL), malachite green solution (20 mL) and

homogenised eggs (1000 mL) were aseptically pooled in a large, sterile flask and mixed well.

Ingredients Quantity (per L)

Mineral salt solution

Potassium dihydrogen phosphate

anhydrous (KH2P04)

2.4 g

Magnesium sulphate (MgS04.

7H20) 0.24 g

Magnesium citrate 0.6 g

Asparagine 3.6 g

Glycerol (reagent grade) 12 mL

Distilled water 600 mL

Malachite green solution, 2%

Malachite green dye 2.0 g

Sterile distilled water 100 mL

Jamia Hamdard 82

4.2.2. PHYSICOCHEMICAL CHARACTERIZATION AND IDENTIFICATION OF

DRUGS

4.2.2.1. Identification of Isonkzkl

4.2.2.1.1. Orgaiiokiitic properties

The following organoleptic properties have been determined

• Nature

® Color

* Odor

4.2.2.1.2. Solubility

Solubility of the isoniazid in various solvents was estimated at 25 °C. An excess

amount of drug was added to the constant volume of each medium. The mixture was then

shaken at the desired temperature for 72 h. Then the samples were centrifuged at 2000 rpm

for 5 min. The supernatant liquid was suitably diluted and the absorbance was measured

spectrophotometrically (UV-1601, Shimadzu, Japan).

4.2.2.1.3. Melting point

The melting point of isoniazid was determined by melting point apparatus

(Scientific MP-1, Hyderabad, India) and compared with the value from Indian Pharmacopoeia

(2007a).

4.2.2.L4. pH of isoniazid

The pH of a 1 % aq. solution of isoniazid was determined with the help of a

digital pH meter (Microprocessor pH System, Punjab, India). ,

4.2.2.I.5. Identification by fourier-transform infrared spectroscopy (FTIR)

Drug (1-2 rag) was weighed and mixed perfectly with potassium bromide (250-

400 mg) to make a uniform mixture. A small quantity of the mixture was compressed into a

thin semi-transparent pellet by applying pressure. Then the IR spectrum of the pellet was

recorded from 4400 c m t o 400 cm (Spectrum BX, FTIR system, Perkin Elmer, Italy) and

compared with reference spectrum obtained from Indian Pharmacopoeia (2007b).

Chapter 4 _ __ _ ____________ Materials and Methods

Jamia Hamdard 83

4.2.2.1.6. Identification by differential scanning calorimetry (DSC)

The DSC analysis of pure drug (5 mg) was carried out in the heating range of 30-

300 °C at the rate of 10 °C/ min. under the inert atmosphere flushed with nitrogen (20

niL/min.) by using differential scanning calorimeter (Pyris 6 DSC, Perkin Elmer, CT, USA).

4.2.2.1.7. Identification by ultraviolet (UV) spectra! analysis

Approximatly 10 mg of the isoniazid was dissolved in 1 mL methanol. The

methanolic solution was transferred to a 100 mL volumetric flask and the volume was made

up to mark with distilled water, simulated gastric fluid (pH 1.2) and simulated intestinal fluid

(pH 7.4). 10 mL of the resulting solution was transferred to 100 mL volumetric flask and

again the volume was made up to mark with respective fluids and then the samples were

scanned spectrophotometrically (UV-1601, Shimadzu, Japan).

4 2 2 2 . Identification of Rifampiciii

4 2 2 2 .1 . Organoleptic properties

The following organoleptic properties have been determined

« Nature

® Color

• Odor

42.2.2.2. Solubility

Solubility of the rifampicin in various solvents was estimated at 25 °C. An excess

amount o f drug was added to the constant volume of each medium. The mixture was then

shaken at the desired temperature for 72 h. Then the samples were centrifuged at 2000 rpm for

5 min. The supernatant liquid was suitably diluted and the absorbance was measured

spectrophotometrically (UV-1601, Shimadzu, Japan),

4.2.2.23. Melting point

The melting point of rifampicin was determined by melting point apparatus

(Scientific MP-I, Hyderabad, India) and compared with the value from Indian Pharmacopoeia

(2007c).

Chapter 4 _____________ Materials and Methods

Jamia Hamdard' 84

4.2.2.2.4. pH of isonfazici

The pH of a ] % aq. solution of rifampicin was determined with the help of a

digital pH meter (Microprocessor pH System, Punjab, India).

4.2.2.2.5. Identification by foiirier-transforin infrared spectroscopy (FTIR)

Drug (1-2 mg) was weighed and mixed perfectly with potassium bromide (250-

400 mg) to make a uniform mixture. A small quantity of the mixture was compressed into a

thin semi-transparent pellet by applying pressure. Then the IR spectrum of the pellet was

recorded from 4400 cm '' to 400 cm '' (Spectrum BX, FTIR system, Perkin Elmer, Italy) and

compared with reference spectrum obtained from Indian Pharmacopoeia (2007d).

4.1.2.2.6. Identification by differential scanning calorimetry (DSC)

The DSC analysis of pure drug (5 mg) was canied out in the heating range of 30-

300 °C at the rate of 10 °Cf min. under the inert atmosphere flushed with nitrogen (20

mL/min.) by using differential scanning calorimeter (Pyris 6 DSC, Perkin Elmer, CT, USA).

4 . 2 . 2 2 J . Identification by ultraviolet (IJV) spectral analysis

Approximatly 10 mg of the rifampicin was dissolved in 1 mL methanol. The

methanolic solution was transferred to a 100 mL volumetric flask and the volume was made

up to mark with distilled water, simulated gastrointestinal fluid (pH 1.2) and simulated

intestinal fluid (pH 7.4). 10 mL of the resulting solution was transferred to 100 mL volumetric

flask and again the volume was made up to mark with respective fluids and then the samples

were scanned spectrophotometrically (UV-1601, Shimadzu, Japan).

4.2.3. ANALYTICAL METHODOLOGY

4.2.3.I. Ultraviolet (UV) Spectrophotometry

4.2.3.L1. Ultraviolet (UV) spectral analysis of single drug

Preparation o f calibration curve o f isoniazid

10 mg isoniazid was dissolved in 100 mL of simulated gastric fluid (pH L2) and

simulated intestinal fluid (pH 7.4) separately and from these stock solution different dilutions

(2-20 |j.g/mL) were made. Then the absorbance was determined at respective Xmax by using

spectrophotometer (UV-1601, Shimadzu, Japan).

Chapter 4 ______ Materials and Methods

Jamia Hamdard 85

Preparation o f calibration curve ofrifam pidn

10 mg rifampicin was dissolved in small amount of methanol and then volume

was made up to 100 mL by simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH

7.4) separately and from these stock solutions different dilutions (2-20 f.ig/mL) were made.

Then the absorbance was determined at respective ?ini„x by using spectrophotometer (UV-

1601, Shimadzu, Japan).

4.2.3.1.2. Ultraviolet (U V) spectral analysis of drug combination

Preparation o f calibration curve

10 mg o f isoniazid was dissolved in 100 mL (100 f,ig/mL) distilled water and 15

mg rifampicin was dissolved in small amount of methanol and then volume was made up to

100 mL by distilled water. The dilution was prepared and the absorbance was measured at

respective Imas by using spectrophotometer (UV-1601, Shimadzu, Japan).

4.23.2. High Performance Thin Layer Chromatographic (HPTLC) Method

Development for Isoniazid and Rifampicin

The aim was to develop an accurate, specific, repeatable and stability-indicating

HPTLC method for the determination of isoniazid and rifampicin in bulk drug and

pharmaceutical dosage form. The proposed method was validated as per ICH guidelines and

its updated international convention. Acid and base induced degradation, hydrogen peroxide

(H2O2) degradation, photo degradation studies were also carried out to qualify it as a stability-

indicating HPTLC method.

(A) Chromatography

Chromatography was performed on 20 cm x 10 cm aluminium plates coated with

200 (xm layers of silica gel 60?^^ (E. Merck Ltd., Germany), Samples were applied to the

plates as 5 mm wide bands, 5 mm apart, by means of a Camag sample applicator (Camag

Linomet V, Switzerland) fitted with a 100 j.iL syringe. A constant rate of application o f 150

nL/sec, was used. Linear ascending development of the plates to a distance of 80 mm was

performed with dichloromethane and methanol (9:1, v/v), as mobile phase in a 20 cm x 10 cm

twin-trough glass chamber, previously saturated with mobile phase vapour for 15 min at room

temperature (25 ± 2 °C) and relative humidity 60 ± 5 %. After development the dried plates

were scarmed at 254 nm by means of Camag TLC Scanner III in absorbance mode operated

Chapter 4 ^ _ ^ __ ______ Materials and Methods^

Jamia Hamdard 86

by WinCATS software (Version 1.2.0). The source of radiation was a deuterium lamp

emitting a continuous UV spectrum in the range 190-400 nm. The slit dimensions were 5 mm

X 0.45 mm and the scanning speed was 20 mm/sec.

(B) Preparation of Standard Solutions

The 1.0 mg/mL stock solution of rifampicin and isoniazid was prepared in

methanol. An appropriate volume of stock solution was further diluted with methanol to

obtain a standard solution of rifampicin and isoniazid having a final concentration of 100

Mg/mL.

(C) Calibration plots for rifampicin and isoniazid

A stock solution of rifampicin and isoniazid (100 p,g/mL) was prepared in

methanol. Different concentrations of the stock solution (100, 200, 300, 400, 500, 600, 700,

800, 900, and 1000 ng rifampicin and isoniazid) were applied, in duplicate, to a TLC plate.

After development a plot of peak area versus corresponding drug concentration was

constructed.

(D) Method validation

(I) Linearity

The linearity of response for rifampicin and isoniazid was assessed in the range of

50 - 1000 ng/spot for standard drug.

(li) Precision

According to ICH guidelines, precision was determined at two levels, i.e.

repeatability and intermediate precision. Repeatability of sample application was determined

by intraday variation whereas intermediate precision was determined by measuring interday

variation at three different concentrations (300, 600, and 900 ng per spot) for triplicate

determination of rifampicin and isoniazid.

(iii) Robustness

The effect on the result by small changes in chromatographic condition was

examined.

Chapter 4 M o t e r / a / s a » d M g t / i o r f s ’

Jamia Hamdard 87

(iv) Limit o f detection (LOD) and limit q f quantification (L O 0

The limits of detection and limit of quantification concentrations of the rifampicin

and isoniazid were determined on the basis of signal to noise ratio method.

(v) Recovery

Previously analyzed samples were added with 50, 100, and 150 % extra

rifampicin and isoniazid standards and then analysed (in triplicate) for recovery from the

formulations of the drug.

(E) Analysis of rifampicin and isoniazid in formulations

To determine the rifampicin and isoniazid content in the formulation,

microparticles were powdered and powder equivalent to 10 mg drug was weighed. The drugs

were extracted from the powder with methanol. To ensure complete extraction of the drug it

was sonicated for 20 min. The volume was then diluted to 100 mL and the sample (200 ng per

spot) was applied to the TLC plate ibllowed by development and scanning. The analysis

process was repeated in triplicate.

(F) Forced degradation of rifampicin and isoniazid

(i) Acid and base induced degradation

Isoniazid (50 mg) and rifampicin (50 mg) were separately dissolved in 50 mL of

0.1 M HCl and in 0.1 M NaOH. These solutions were heated under reflux for 1 h at 75 °C and

then applied to a TLC plate (200 ng per spot). Chromatography was performed as described

above.

(ii) Hydrogen peroxide ( H p ^ induced degradation

Hydrogen peroxide (30 % v/v, 25 mL) was added to separate solutions of

rifampicin and isoniazid (2 mg/ mL, 25 mL). The solutions obtained (200 ng per spot) were

applied to TLC plates in triplicate and chromatography was performed as described above,

(lii) Photochemical degradation

Rifampicin and isoniazid (50 mg of each) were separately dissolved in methanol

(50 mL) and exposed to direct sunlight for 24 h. The solutions obtained, were applied (200 ng

per spot) to TLC plates and chromatography was performed as described above.

Chapter 4 _______ Materials and Methods

Jamia Hamdard 88

4.2,3.3. High Perforitiiance Liquid Chromatograpliy (HPLC)

A high perforniEince liquid chromatography method was employed (Jindal et a l,

1994) for the determination of drugs in plasma samples.

(A) Iistriim entation

The analysis was performed on an HPLC system consisting o f a Shimadzu Model

(Japan) LC 10 AD pump, a Shimadzu Model SPD 10 A. An ultraviolet detector, a

chromatopac C-R6A integrator (Shimadzu, Japan). Chromatographic separation was achieved

at room temperature on a LiChrocart® 100 RP-18 column (125 mmx4 mm), 5 |o.m particle size

(Merck, Damstadt, Germany).

(B) Chromatographic conditions

The mobile phase consisted of methanol-0.01 M disodium hydrogen phosphate

(70:30, v/v; pH 4.6). The components of the mobile phase were filtered before use through

0.45 |.im membrane filter and degassed for 15 min. The mobile phase was operated at a flow

rate of 1.2 mL/min at ambient temperature. The volume of the injection loop was 20 \iL. The

column was equilibrated for at least 30 min. with the mobile phase.

(C) Preparation of standard solution of isoniazid

Primary stock solution of Img/mL of isoniazid was prepared in distilled water.

Appropriate dilutions of isoniazid from stock solution were made in mobile phase to produce

working stock solutions of 25, 50, 75, 100, 125, 150 ixg/mL. These dilutions were used to

spike plasma in the preparation of a calibration curve.

(i) Calibration curve fo r isoniazid in plasma

Calibration curve was prepared by spiking different 450 p,L samples of blank

plasma with 50 (xL of appropriate working solution, to produce the calibration standards

equivalent to 2.5, 5.0, 7,5, 10.0, 12.5, 15.0 |ig/mL of isoniazid. A blank was also prepared

containing 450 \xL blank plasma. The samples were estimated for isoniazid by HPLC at 263

nm.

(11) Extraction o f isoniazid from the plasma

Isoniazid spiked plasma (50 |aL) was deproteinised with 50 |.iL of 10 % v/v

trichloroacetic acid, vortexed for 5 min. and centrifuged at 5000xg for 20 min. at 4-8 °C. The

Chapter 4 Materials and Methods

Jamia Hamdard 89

supernatant was used for analysis of drug by HPLC method. All the samples were filtered

through 0.25 jj.m membrane filter (Millipore).

(D) Preparation of standard solution of rifainpiciii

Primary stock solution of 1 mg/mL of rifampicin was prepared in methanol.

Appropriate dilutions of rifempicin from stock solution were made in mobile phase to

produce working stock solutions of 25, 50, 75, 100, 125, 150 {.tg/niL. These dilutions were

used to spike plasma in the preparation of a calibration curve.

(I) Calibration curve fo r rifampicin in plasma

Calibration curve was prepared by spiking different 450 i-iL samples of bla.nk

plasma with 50 |.iL of appropriate working solution, to produce the calibration standards

equivalent to 2.5, 5.0, 7.5, 10.0, 12.5, 15.0 |j.g/niL of rifampicin. A blank was also prepared

containing 450 fj,L blank plasma. The samples were estimated for rifampicin by HPLC at 475

nm.

(ii) Extraction o f rifampicin from the plasma

Rifampicin spiked plasma (100 |j.L) was deproteinised with 100 jiL acetonitrile,

vortexed for 5 min. and centrifuged at 5000xg for 20 min. at 4-8 °C. The supernatant was

used for analysis of drug by HPLC method. All the samples were filtered through 0.25 pm

membrane filter (Millipore).

4.2.4. DRU G P O L Y M E R IN T E R FE R E N C E STUDY

To eliminate the possibility of interference between drugs and polymers, a

preliminary interference study was carried out. An aq, polymeric solution (0.05 % w/v), drug

solution (20 |ug/mL) and mixture of drug and polymeric solution (1:9, v/v) were scanned for

the entire UV-Visible range,

4.2.5. DRUG POLYMER IN T E R A C T IO N STU DY

4.2.5.I. Fourier-transform infrared spectoscopy (FT IR )

The drug polymer interactions were investigated by FTIR spectroscopy study. The

spectrum of drug-polymer combination ( 1:1) was recorded by fourier-transform infrared

spectrophotometer (Spectrum BX, FTIR system, Perkin Elmer, Italy) using KBr pellet

Chapter 4 _ Matenals and Methods

Jamia Hamdard 90

technique. The scanning range was from 4400 cm"' to 400 cm‘‘. Then the spectrum was

compared with the pure drug spectra.

4.2.5.2. Differentia! scanning calorimetiy (.DSC)

The DSC analysis of drug-polymer combination (1:1) was carried out at the

heating range of 30-300 ”C at the rate of 10 °C/ min. under the inert atmosphere flushed with

nitrogen (20 mL/min.) by using differential scanning calorimeter (Pyris 6 DSC, Perkin Elmer,

CT, USA). The DSC endotherm was then compared with the pure drug endotherm.

4.2.6. FORMULATION AND OPTIMIZATION OF MICROPARTICLES

4.2.6.I. Formulation and Optiinlzatioii of Isoniazid Microparticles

4.2.6.L1, Alkaline extraction of ispaghiila husk (AEISP)

Ispaghula husk was extracted by using previously reported method (Guo et al.,

2008). Accurately weighed 2.5 g husk was dispersed in 500 mL distilled water at 80 °C for 1

h under constant stirring. The dispersion became a homogenous gel. The dispersion was then

centrifuged (R 23 research centrifuge, Remi, Mumbai) at 18,000x g for 1 h, to separate the gel

and the solution. The gel phase was dissolved in 500 mL 0.5 M NaOH solution at room

temperature while stimng magnetically for 2 h, and the solution and a small amount of

residue was separated by centrifuging at ISOOQxg for 1 h. The alkaline extract (solution) was

neutralized with 2 iVi HCI. During the neutralization process, a large amount of gel like

precipitate was observed and separated by centrifugation from the soluble fraction. The gel

fraction was homogenized (Heidolph, DIAX 900, Germany) for 1 h and washed twice with

distilled water, and dialyzed at room temperature for 24 h, and then freeze dried,

4.2.6.1.2. Preparation of microparticles

The emulsification-intemal ionic gelation method was utilized for the preparation

of microparticles by using barium carbonate as a cross-linking agent (Sultana et al., 2009;

Poncelet et al., 1999; Poncelet et al., 1992; Liu et al., 2002a; Liu et al., 2002b). Solutions of

AEISP (2-4 % w/v) (5 mL) and sodium alginate (2-4 % w/v) (5 mL) in double distilled water

were prepared separately using gentle heat, being stirred magnetically and then both the

solution was mixed together. Then a uniform dispersion of isoniazid (100 mg) and cross-

linking agent (4-10 % w/v) in polymer solution was prepared. The drug dispersion with cross-

linking agent was then emulsified in light liquid paraffin (containing 1.5 % w/v Span 80 and

0.2 % v/v glacial acetic acid ) in the ratio of 1:10, by mechanical stirring (Remi Motors,

Chapter 4 ■ . Materials and Methods

Jamia Hamdard 91

Mumbai, India) at different speeds (1000-2000 rpm). After 1 h, the microparticles were

collected by filtration and traces of the oil were removed by washing them with isopropyl

alcohol. The microparticles were dried at 40 "C in a vacuum oven for 12 h,

4 .2 /k13. Experimental design

A four-factor, three-level Box-Behnken design was used for the optimization

procedure. This design is suitable for exploration of quadratic response surfaces and

constructs a second order polynomial model, thus helping in optimizing a process using a

small number of experimental runs. Sodium alginate concentration (Xj), AEISP concentration

(X2 ), concentration o f cross-linking agent (X3) and stirring speed (X4) were four independent

variables considered in the preparation of microparticles, while the particle size (Yi) and

entrapment efficiency (Y2) were dependent variables. The range of each factor was decided

on the basis of results obtained by preliminary experiment. Then process parameters were

studied by conducting the runs at different levels of all factors. Data collected for responses in

each run were analyzed using the software DESIGN EXPERT 7.1 (Stat-Ease, Minneapolis,

USA) and fitted into a multiple linear regression model.

42.6.1,4. Evaluation of optimized microparticles . .

4,2.6,1.4.1. Determination o f entrapment efficiency

The drug content of the microparticles was determined spectrophotometrically at

Xmax 263 nm (UV-1601, Shimadzu, Japan). The isoniazid-AEISP microparticles (50 mg) were

dissolved in simulated intestinal fluid, pH 7.4 (10 mL) under sonication for 20 min. The

solutions were filtered through 0.22 ^m Millipore filters and the absorbance of isoniazid was

measured. The total amount of isoniazid was calculated by using regression equation of the

calibration curve.

The encapsulation efficiency was calculated by the following formula:

Chapter 4 ________ _ Materials and Methods

Experimental drug contentEncapsulation Efficiency (%) = —— - —r : --------- — ~ x 100

^ Theoretical drug content

4.2.6.L4.2. Surface morphology and particle size analysis

The shape and surface morphology of the drug-loaded as well as blank

microparticles were investigated by using scanning electron microscope (JEOL JSM-6360

Philips, Japan) at 20 kV. Prior to testing, samples were fixed on stubs and sputter coated with

Jamia Hamdard 92

gold in a vacuum evaporator to render them electrically conductive. The sizes of the

microparticles were determined by an optical microscope (Olympus, Japan) fitted with an

ocular micrometer. The ocular micrometer was calibrated with a stage micrometer. The mean

diameter reported was obtained from a total of more than 100 microparticles.

4.2.6,1.43. Drug polymer interaction study in the formulation

The drug polymer interactions study of microparticles was carried out by

differential scanning calorimetry (DSC), fourier transform-infrared spectroscopy (FTIR) and

X-ray diffraction analysis (XRD).

Differential scanning calorimetry (DSC) analysis

The physical state of the drug in the samples was determined by DSC (Pyris 6

DSC, Perkin Elmer, CT, USA). Five mg sample (pure drug, physical mixture o f the excipients

and pure drug and drug loaded microparticles) was placed in aluminium pans and heated from

30 °C to 300 °C at a heating rate of 10 °C/min. under inert atmosphere flushed with nitrogen

at the rate of 20 mL/min.

Fourier transform-infra red (FTIR) spectroscopic analysis

Five mg sample (pure drug, physical mixture of the excipients and pure drug and

drug loaded microparticles) was weighed and mixed perfectly with potassium bromide to

form a uniform mixture. A small quantity of the powder was compressed into a thin

semitransparent pellet by applying pressure. The IR spectrum of the pellet from 4400 cm~' to

400 cm"' was recorded (Spectrum BX, FTIR system, Perkin Elmer, Italy) taking air as the

reference and compared to study any interference.

X-ray diffraction analysis (XRD)

The qualitative X-ray powder diffraction studies were performed using an X-ray

diffractometer (PW 3710, Philips Analytical X-Ray B.V). Pure drug, physical mixture o f the

excipients and pure drug and drug loaded microparticles were scanned from 10-60°

diffraction angle (20) range under the following measurement conditions: Source, Nickel

filtered Cu-Ka radiation; voltage 35 kv; current 30 mA; scan speed 0.05/min. Microparticles

were triturated to get fine powder before taking the scan. X-ray diffractometiy was carried out

to investigate the effect of microencapsulation process on crystallinity of the drug.

Chapter 4 Materials and Methods

Jamia Hamdard 93

4,2,6.L4.4. Study o f mucoadhesive property o f optimized microparticles

The in vitro mucoadhesive property of the microparticles was evaluated by

employing the previously reported method (Lehr et a l, 1990). The mucoadhesivness of

microparticles formulation was compared with non-bioadhesive ethyl cellulose

microparticles. The test was performed with simulated gastric fluid (pH 1.2) as well as in

simulated intestinal fluid (pH 7.4). Freshly excised mucosa from goat intestine was fixed onto

a small glass slide with cyanoacrylate glue. Fifty microparticles were spread onto wet tissue

and immediately the glass slide was hung onto the arm of a tablet disintegrating test machine.

Then the disintegrating test apparatus was operated, the tissue specimen was subjected to a

slow, regular up and down movement in the test fluid at 37±0.5 °C temperature, contained in a

1 L vessel up to 10 h. At the end of the stipulated time, number of microparticles attached to

and removed from the tissue was counted. The percent of mucoadhesion was calculated by the

following formula:

Chapter 4 r,. .. Materials and^Methods^

No. of microparticles adheredMucoadhesion (%) = ~ — — ---------------------------------- —-------- x 100

No. of microparticles applied

4.2.6.1.4.5. Swelling behaviour o f microparticles

Swelling study for optimized microparticles was conducted using two media,

namely, simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 7.4). An accurately

weighed (50 mg) microparticles was immersed in 20 mL of medium contained in a 100 mL

glass beaker. Increase in weight of the microparticles was determined at preset time interval

until a constant weight was observed. All the mass measurement of the swollen microparticles

was taken on a Mettler Toledo single pan electronic balance.

Swelling index was then calculated as-

Weieht of swollen microparticlesSwelling index (%) = . —— . , ■:— :-------------- —r— x 100

Initial dry weight of microparticles

4.2.6.1.4.6. In vitro drug release study

The release profiles of isoniazid from microparticles were examined in simulated

gastric fluid (pH 1.2) and simulated intestinal fluid (pH 7,4) using USP XXVI six stage,

basket type dissolution rate test apparatus (VEEGO, VDA-8DR USP Standards), The drug-

loaded microparticles, equivalent to 10 mg of isoniazid, filled in empty gelatin capsule shells

Jamia Hamdard 94

were put into the basket and stirred in 900 mL of the dissolution medium at 50 rpm

therraostated at 37±0,5 ”C. At preset time intervals, 5 niL sample was withdrawn and replaced

immediately with an equal volume of fresh dissolution medium. The samples were suitably

diluted, filtered and the drug content determined spectrophotometrically at 263 nm (UV-1601,

Shimadzu, Japan). This procedure was done in triplicate,

4J. 6.1.4.7. Release kinetics and Statistical analysis

The data obtained from in vitro release studies were fitted to various kinetic

equations to find out the mechanism of drug release from microparticles. Statistical analysis

of the results was carried out using one way analysis of variance (ANOVA) with post test

(Dunnett’s multiple comparison tests). Statistically significant differences between in vitro

drug release of formulations were defined as p < 0,05. Calculations were performed with the

GraphPad-Instat Software Program (GraphPad-Instat Software Inc., San Diego).

4.2.6.I.5. In vivo studies

Permission for use of experimental animals was obtained from institutional animal

ethical committee of INMAS, Delhi.

4.2.6.1.5,1. Uptake o f sodium pertechnetate f^'"Tc)

Microparticles were labelled using 99m-Technetium (^^"'Tc). One millilitre of

distilled water was added to 3.5 mg of microparticles in a sterile glass vial, to this suspension

50 |ig of stannous chloride dihydrate (1 mg/mL in 10 % acetic acid) was added. The pH was

adjusted to 7.5 with 0.5 M sodium bicarbonate. The content was filtered through a Whatmann

filter paper (No. 41). After that approximately 18,5 mBq ^ "’Tc-pertechnetate was added to it,

mixed and incubated for 10 min. The radiolabeiing efficiency was evaluated with instant thin

layer chromatography-siiica gel (ITLC-SG) strips as stationary phase and acetone 100 % as

the mobile phase,

.... „ .......... . ,, Materials and Methods

Radioactivity (counts) retained in the lower half of the strip ^ % Radiolabeiing = {j itigi radioactivity associated (total count present) with the strip"

4.2.6.1,5.2. Quality controlRadiochemical impurity that is likely to exist in the form of unconjugated

technetium was determined by ascending instant thin layer chromatography (Throll et a l,

Jamia Hamdard yo

1976). Amount of reduced/hydrolysed (R/FI) ^ '”Tc was determined using pyridine: acetic

acid: water [PAW] in the volume ratio of 3:5:1.5 (v/v) as mobile phase and ITLC-SG strip as

the stationary phase. The mobile phase may normally be found involving ethanol and

ammonia as the first two components of the mobile phase, however, in the same volume ratio.

Their replacement, respectively with pyridine and acetic acid while maintaining the volume

ratio and volume fraction contribution was invariably noticed to produced better separation

and therefore, reproducible results, and hence employed as the constituent of the mobile

phase for this part of the experiment, The reduced/hydrolysed technetium remained at the

point of application whereas free pertechnetate and labelled complex moved with the solvent

front.

Chapter 4 Ma terials and Methods

Counts present in the lower part of strip% R/H technetium = - — ------------— -----------------------------------------L x 100

Total count present in the strip

4.2.6. L 5.3. In vitro stability o f radiolabeled microparticles

The in vitro stability of the complex was estimated up to 12 h, Aliquots at

different time intervals were applied on ITLC-SG strips and allowed to run in 100 % acetone

to check for any dissociation/degradation of the labelled complex. The dissociation was

estimated as the percent radiolabeled complex remaining after the incubation time intervals of

Oto 12 h,

Radiolabeled stability (%) = x 100initial (Total) count of strip

4.2.6.I.5.4. Organ distribution study

Organ distribution study of radiolabeled isoniazid microparticles was carried out

in Wistar rats weighing about 300-350 g. 37 mBq of activity in 1 mL of the preparation was

administered orally using a catheter. The animals were sacrificed at 1, 4, and 12 h after

administration of the complex. Various organs were removed and weighed. The radioactivity

was measured in each organ and expressed as percent administered dose per gram of organs.

4.2.6.LS.5. Gamma scintigraphy study

In order to determine the extent of localization of isoniazid microparticles in the

GIT, imaging studies were performed on three Wistar rats weighing about 300-350 g. The

Jamio Hamdard 96

microparticies were administered orally, a dose of 37 niBq of activity in 1 mL preparation,

after overnight fasting for 8-10 h. The animals were freed and allowed to move and carry out

normal activities but were not allowed to take any food or water until the formulation had

emptied the stomach completely. The scintigraphic examination was performed at 0.5, 1, 4

and 12 h to assess the mobilization of the microparticles in the GIT, using a large field view

gamma camera equipped with a high-resolution, parallel-hole collimator and interfaced to a

dedicated computer. Images were recorded for a preset time of 5 min./view with a 15 %

window centered to include the 140 keV photopeak of ‘ '"Tc.

4.2.6.L 5.6, Pharmacohinetic study

The pharmacokinetic studies were conducted in healthy Wistar rats of either sex

weighing 200-225 g. Rats were grouped as follows, with 3 animals per group: Group 1, free

drug (2 mg); Group 2, microparticles loaded with drug (equivalent to 2 mg drug); Group 3,

drug free microparticles (a positive control to explore the influence of microparticles on drug

estimation). All the dosage was administered with an oral cannula. The animals were bled at

several time points and the plasma (50 |aL) obtained from each rat was deproteinised with 50

p,L of 10 % v/v trichloroacetic acid, vortexed for 5 min. and centrifuged at SOOOxg for 20 min.

at 4-8 °C. The supernatant was used for analysis of drug by HPLC method at 263 nm. All the

samples were filtered through 0.25 |am membrane filter (Millipore).

4 2 , 6 . 2 . Formulation and Optimization of Rifampicin Microparticles

4.2.6.2.1, Preparation of rifampicin microparticles

Drug (100 mg) and polymers [Eudragit RSPO (EuRSPO) and ethylcellulose] (1-2

% w/v) were dissolved in a mixture of acetone (5 mL) and dichloromethane (3 mL). After

that, drug dispersing agent (talc, 0,25-0.75 % w/v) was uniformly mixed in the drug-polymer

solution under vigorous shaking. Then the resultant suspension was poured into 100 mL

distilled water containing sodium dodecyl sulfate (SDS) in different concentrations (0.05-

0.15 % w/v) under mechanical stirring (1000-2000) at 40 °C. After agitating the system for

25 min,, another 100 mL of SDS was added slowly and stirring was continued for another 60

min. till the quasi-emulsion droplets turned into opaque microparticles. After one hour

microparticles were collected by filtration and washed with double distilled water. The

microparticles were then dried in an oven at 40 °C for 8 h.

Chapter 4^^ _ _ _ ____ Materials and Methods

Jamia Hamdard 97

4.2,6,2.2. Experimental design

A four-factor, three-level Box-Behnken design was used for the optimization

procedure. This design is suitable for exploration of quadratic response surfaces and

constructs a second order polynomial model, thus helping in optimizing a process using a

small number of experimental runs. Polymer concentration (Xi), drug dispersing agent

concentration (X2), concentration of surfactant (X3) and stirring speed (X4) were four

independent variables considered in the preparation of microparticles, while the pailicle size

(Yi) and entrapment efficiency (Y2) were dependent variables. Then process parameters were

studied by conducting the runs at different levels of all factors. Data collected for responses in

each run were analyzed using the software DESIGN EXPERT 7,1 (Stat-Ease, Minneapolis,

USA) and fitted into a multiple linear regression model.

4,2.6.23. Evaluation of optimized microparticles

4.2,6,23,1. Determination o f incorporation efficiency o f microparticles

The drug content of the microparticles was detennined spectrophotometrically at

niax 475 nm (UV-1601, Shimadzu, Japan). The rifampicin microparticles (10 mg) were

keeping in contact with 10 niL of methanol, the mixture was sonicated for 20 min, to break

down the microparticles completely. The solution was filtered through 0.22 pm Millipore

filter and the absorbance of rifampicin was measured. The total amount of rifampicin was

calculated by using regression equation of the calibration curve. This procedure was done in

triplicate. The incorporation efficiency (%) was calculated by using following equation-

Chapter 4 _ Materials and Methods

, Experimental drug contentEncapsulation Efficiency (%) = — - — — —---------------x 100

Theoretical drug content

4.2.6.23.2, Surface morphology and measurement o f micromeritlc properties o f

microparticles

The shape and surface morphology of the drug-loaded as well as blank

microparticles were investigated by using scanning electron microscope (JEOL JSM-6360

Philips, Japan) at 20 kV. Prior to testing, samples were fixed on stubs and sputter coated with

gold in a vacuum evaporator to render them electrically conductive. Particle size of the

microparticles was determined by the optical microscope (Olympus, Japan) fitted with an

ocular and stage micrometer. In all measurements at least 100 particles in five different fields

were examined. This procedure was done in triplicate. The optimized fonnulation was also

Jamia Hamdard 98

characterized by their micromeritic properties, such as true density, tapped density,

compressibility index and flow properties. The tapped density and percent compressibiHty

index of the microparticles was measured by a tapping method (Martin, 1991) as follows:

Chapter 4 _______ Materials and Methods

Mass of microparticles Tapped density =

Volume of microparticles after tapping

, , volume of the sample after standard tapping ^Compressibility index (%) = [1 ----- j----- ~7 t —■— —j —— :— ] x 100volume of the sample before the standard tappmg

True density was determined using a benzene displacement method. Hausner’s

ratio was also calculated using the equation:

tapped densityHausner’s ratio = —----- — r —

true density

Angle of repose 0 of the microparticles, which measures the resistance to particle

flow, was determined by a fixed funnel method (Ziyaur et ah, 2006) and calculated as;

0 = tan"' h/r

where, 6 is the angle of repose, r is the radius and h is the height of the microparticles heap

that is formed on a graph paper after making the microparticles flow from the glass funnel.

4.2.6,2.3.3. In vitro buoyancy studies (Lee et ai, 1999; Jain et a l, 2005)

Rifampicin microparticles (0.05 g) were spread over the surface o f 900 mL

simulated gastric fluid containing 0.02 % Tween 80 in a USP XXIV dissolution apparatus

(type II).The medium was agitated with a paddle at 100 rpm for 12 h. The floating and settled

portions of microparticles were recovered separately. The microparticles were dried and

weighed. Buoyancy percentage was calculated as the ratio of the mass of the microparticles

that remain floating and the total mass of the microparticles.

Buoyancy percentage (%) - Wf / (Wf + Ws) ><100

where Wf and Ws are the weights of the floating and settled microparticles, respectively. All

the determinations were made in triplicate.

jamia Hamdard 99

4.2.6.2.3.4. Drug polymer interaction study in the formulation

The drug polymer interaction study of microparticles was carried out by

differential scanning ceilorimetry (DSC), fourier transform-infrared spectroscopy (FTIR) and

X-ray diffraction analysis (XRD).

Differential scmming caiarimetry (DSC) analysis

The physical state of the drug in the samples was determined by DSC (Pyris 6

DSC, Perkin Elmer, CT, USA). Five mg Samples (pure drug, physical mixture of the

excipients and pure drug and drug loaded microparticles) was placed in aluminium pans and

heated fi‘om 50 °C to 300 °C at a heating rate of 10 °C/min. under inert atmosphere flushed

with nitrogen at the rate of 20 mL/min.

Fourier tmnsform-infm red (FTIR) spectroscopic analysis

Five mg sample (drug, physical mixture of the excipients and pure dmg and drug

loaded microparticles) was weighed and mixed perfectly with potassium bromide to form a

uniform mixture. A small quantity of the powder was compressed into a thin semitransparent

pellet by applying pressure. The IR spectrum, of the pellet from 4400 cm"' to 400 cm"' was

recorded (Spectrum BX, FTIR system, Perkin Elmer, Italy) taking air as the reference and

compared to study any interference.

X-ray diffraction analysis (XRD)

The qualitative X-ray powder diffraction studies were performed using an X-ray

diffractometer (PW 3710, Philips Analytical X-Ray B.V). Pure drug, physical mixture o f the

excipients and pure drug and drug loaded microparticles were scanned from 10-60°

diffraction angle (20) range under the following measurement conditions: Source, Nickel

filtered Cu-Ka radiation; voltage 35 kv; current 30 mA; scan speed 0.05/min. Microparticles

were triturated to get fine powder before taking the scan. X-ray diffractometry was carried out

to investigate the effect of microencapsulation process on crystallinity of the drug.

4.2.6.2.3.5. In vitro drug release study

An in vitro dissolution test of rifampicin microparticles was carried out using USP

XXVI six stage, paddle type dissolution rate test apparatus (VEEGO, VDA-8DR USP

Standards) at a stirring rate of 50 rpm at 37±0,5 °C in 900 mL of simulated intestinal fluid

(pH 7.4) and simulated gastric fluid (pH 1.2), containing 1.0 % w/v of SDS, Dried

Chapter 4 _ _ . „ ^ , . , » . ______ Materials and Methods^

Jamia Hamdard iqq

microparticles (equivalent to 25 mg rifampicin) were placed into the medium and the paddle

was rotated. Five milliliters of the dissolution medium was withdrawn at certain intervals, and

fresh dissolution medium was immediately replaced in the apparatus to keep the volume

constant, The samples were suitably diluted, filtered and the drug content determined

spectrophotometrically at 475 nm (UV-1601, Shimadzu, Japan). This procedure was done in

triplicate.

4.2,6.2.3,6. Release kinetics and Statistical analysis

The data obtained from in vitro release studies were fitted to various kinetic

equations to faid out the mechanism of drug release from microparticles. Statistical analysis

of the results was carried out using one way analysis of variance (ANOVA) with post test

(Dunnett’s multiple comparison tests). Statistically significant differences between in vitro

drug release of formulations were defined as p < 0.05. Calculations were performed with the

GraphPad-Instat vSoftware Program (GraphPad-Instat Software Inc., San Diego).

4.2.6.2.4. In vivo studies

Permission for use of experimental animals was obtained from institutional animal

ethical committee oflNMAS, Delhi.

4.2.6.2.4.L Uptake o f sodium pertechnetate f'^'"Tc)

Microparticles were labelled using 99m"Technetium ( ^™Tc). One millilitre of

distilled water was added to 3.5 mg of microparticles in a sterile glass vial, to this suspension

50 (,ig of stannous chloride dihydrate (1 mg/mL in 10 % acedc acid) was added, The pH was

adjusted to 7,5 with 0.5 M sodium bicarbonate. The content was filtered through a Whatmann

filter paper (No, 41), After that approximately 18.5 mBq ' "’Tc-pertechnetate was added to it,

mixed and incubated for 10 min. The radiolabeling efficiency was evaluated with instant thin

layer chromatography»silica gel (ITLC-SG) strips as stationary phase and acetone 100 % as

the mobile phase.

Chapter 4 _ ...... _ _ __ _ _ _ _ Materials and M e th q ^

Radioactivity (counts) retained m the lower half of the strip% Radwlabelmg = — — — ----- —---------------------------------------------------------------------;--—-------—------- --------— — x innInitial radioactivity associated (total count present) v\/ith the strip

Jamia Hamdard 2OI

4,2,6.2.4.2. Quality control

Radiochemica! impurity tliat is iiicely to exist in tiie form o f unconjugated

teciinetiuin was determined by ascending ITLC (ThroU et a l, 1976). Amount of

reduced/hydrolysed (R/H) was determined using pyridine: acetic acid: water [PAW] in

the volume ratio of 3:5:1.5 (v/v) as mobile phase and ITLC-SG strip as the stationary phase.

The mobile phase may normally be found involving ethanol and ammonia as the first two

components of the mobile phase, however, in the same volume ratio. Their replacement,

respectively with pyridine and acetic acid while maintaining the volume ratio and volume

fraction contribution was invariably noticed to produced better separation and therefore,

reproducible results, and hence employed as the constituent of the mobile phase for this part

of the experiment. The reduced/hydrolysed technetium remained at the point of application

whereas free pertechnetate and labelled complex moved with the solvent front.

Chapter 4 _ __________ __________Materials and Methods

Counts present in the lower part of stripR/H technetium ~ ------ —— :— -—-— — -— — r—---- — ~ x 100

lo tal count present m the strip

4,2.6.2.4.3. In vitro stability o f radiolabeled microparticles

The in vitro stability of the complex was estimated up to 12 h. Aliquots at

difl'erent time intervals were applied on FfLC-SG strips and allowed to run in 100 % acetone

to check for any dissociation/degradation of the labelled complex. The dissociation was

estimated as the percent radiolabeled complex remaining after the incubation time intervals of

0 to 12 h.

Radiolabeled stability (%)initial (Total) count of strip

4.2.6J.4J. Organ distribution study

Organ distribution study of radiolabeled isoniazid microparticles was carried out

in Wistar rats weighing about 300-350 g. 37 mBq of activity in 1 mL of the preparation was

administered orally using a catheter. The animals were sacrificed at 1, 4, and 12 h after

administration of the complex. Various organs were removed and weighed. The radioactivity

was measured in each organ and expressed as percent administered dose per gram of organs.

Jamia Hamdard iq 2

4,2.6.2.4.5, Gamma scintigraphy

[n oi'der to determine the extent of localization ot isoniazid microparticles in the

GIT, imaging studies were performed on three Wistar rats weighing about 300-350 g. The

microparticles were administered orally, a dose of 37 niBq of activity in 1 niL preparation,

after overnight fasting for 8--10 h. The animals were freed and allowed to move and carry out

normal activities but were not allowed to take any food or water until the formulation had

emptied the stomach completely. The scintigraphic examination was done at 0.5, 1, 4, 6 and

12 h to assess the mobilization of the microparticles in the GIT, using a large field view

gamma camera (Siemens) equipped with a high-resolution, parallel-hole collimator and

interfaced to a dedicated computer. Images were recorded for a preset time of 5 min./view

with a 15 % window centered to include the 140 IceV photopeak o f ‘' ' "’Tc.

4J,6J,4.6. Pharmacokinetic study

The pharmacokinetic studies were conducted in healthy Wistar rats of either sex

weighing 200-225 g. Rats were grouped as follows, with 3 animals per group; Group I, free

drug (2.4 mg); Group 2, microparticles loaded with drug (equivalent to 2.4 mg drug); Group

3, drug free microparticles (a positive control to explore the influence of microparticles on

drug estimation). All the dosage was administered with an oral cannula. The animals were

bled at several time points and the plasma (100 jiL) obtained from each rat was deproteinised

with 100 pL of acetonitrile, vortexed for 5 min. and centrifuged at SOOOxg for 20 min, at 4-8

°C. The supernatant was used for analysis of drug by HPLC method at 475 nm. All the

samples were filtered through 0.25 pm membrane filter (Millipore).

4.2.7. ASSESSMENT OF ANTIBACTERIAL AND ANTIMYCOBACTERIAL

EFFICACY OF OPTIMIZED FORMULATION

Evaluation of in vitro antibacterial efficacy (for prolong release effect) of

optimized formulation was carried out by a procedure, as previously reported method

(Mariappan et al., 2004; Dutta et al., 2000). Evaluation of in vitro antimycobacterial efficacy

(for intestinal antitubercular effect) of optimized formulation was carried out according to a

previously reported method with some modifications (Zahoor et a l, 2005; Banfi et a l, 2003).

The antibacterial as well as antimycobacterial activity was evaluated by measuring zone of

inhibition in millimeters (mm), produced by samples and comparing it with that produced by

the standard drug under similar conditions.

Chapter 4 ..... _ Materials and Methods

JamiaHamdard iq 2

Chapter 4 Materials and Methods

4.2.7.I. Inociiliinis

(A) Mueller Hinton agar niedium was used to culture the test bacteria B. siibtilis

(MTCC 441) whereas S. aureus (NCTC 65710) was grown on nutrient agar media. The

bacterial cultures were grown at 37 °C for 24 h. The microbial cultures were appropriately

diluted with sterile 0.9 % saline solution to obtain the final cell suspension counts of 10 cfu/

m,L. All the microbial cultures were incubated under aerobic conditions.

(B) Modified Middlebrook 7H11 agar medium was used to culture the Mycobacterium

tuberculosis H37RV, The mycobacterial cultures were grown at 37 °C for 30 days. The

microbial cultures were appropriately diluted with Middlebrook 7H11 agar broth to obtain the

final cell suspension counts of lO'** cfu/ml.

4.2.72. Saiiipie preparation for antimicrobial study

(A) An in v i tro dissolution test of isoniazid microparticles was carried out using

simulated intestinal fluid (pH 7,4) using USP XXVI six stage, basket type dissolution rate test

apparatus (VEEGO, VDA-8DR USP Standards).The drug-loaded microparticles filled in

empty capsule shells were put into the basket and stirred in 900 mL of the dissolution medium

at 50 rprn thermostated at 37±0.5 ®C. At preset time intervals, 5 mL sample was withdrawn

and replaced immediately with an equal volume of fresh dissolution medium. This sample

was used to assess the antimicrobial efficacy of the formulation.

(B) An in vitro dissolution test of rifampicin microparticles was carried out using USP

XXVI six stage, paddle type dissolution rate test apparatus (VEEGO, VDA-8DR USP

Standards) at a stinnng rate of 50 rprn at 37±0,5 °C in 900 mL of simulated gastric fluid (pH

1.2), containing 1,0 % w/v of SDS. Dried microparticles were placed into the medium and

the paddle was rotated. Five milliliters of the dissolution medium was withdrawn at certain

intervals, and fresh dissolution medium was immediately replaced in the apparatus to keep the

volume constant. This sample was used to assess the antimicrobial efficacy o f the

formulation.

4.2.73. In vitro antibacterial screening using agar-well diffusion method

A small quantity of B. subtilis culture was transferred to sterile nutrient broth. The

same was shaken for 24 h at 250 rpm at 37 °C . The turbidity was matched with MacFarlane’s

standard (3x10®* cells/mL) and 100 pL of this culture was transferred to fresh 10 mL nutrient

broth. From this, 500 pL was inoculated into each autoclaved petri plates containing Mueller

Hinton agar medium and spreaded uniformly. Micro wells were made on culture media of 6 mm

JamiaHamdard 104

ill diameter with the help of flamed cork borer and 50 f-iL ot the test solution was dropped on

tl'iese micro wells. Fifty jixL of standard solution was also dropped in one micro well o f each

plates. One set of plates was run as negative controls, in which 50 |.tL blank solvent was

applied on the micro wells, instead of the test solution. The plates were incubated tor 24 h and

zone of inhibition was measured as the diameter.

Sterile liquid nutrient agar (20 niL) was poured into presterilized disposable petri

dishes and set aside for solidification for 5 h. Cotton swabs charged with 0.2 mL of diluted

inoculum (10 ' cfti/rnL) of test microorganisms (S. aureus) were inoculated on plates and

spread evenly over the surface of agar plates. Micro wells were made on culture media o f 6

mm in diameter with the help of flamed cork borer and 50 pL o f the test solution was dropped

on these micro wells. Fifty pL of standard solution was also dropped in one micro well o f

each plates. One set of plates was run as negative controls, in which 50 pL blank solvent was

applied on the micro wells, instead of the test solution. The plates were incubated for 24 h and

zone of inhibition was measured as the diameter.

4.2JA , In vitro antiniycobacterial screening for intestiiia! tubercuiosis using agar-well

diffusion in e fh o d

Mycobacterium tuberculosis H37RV strain was maintained on Lowenstin-Jensen

medium and grown for 30 days in modified Middlebrook 7H11 agar medium (simulated to

gastric environment as well as simulated to intestinal environment) supplemented with 10 %

OADC, 0.2 % glycerol and 0.1 % Bacto Casitone (Difeo), Cultures were vortexed and the

supernatant further diluted in complete modified Middlebrook 7H11 agar broth to obtain

viable bacilli count about lO^cfu/mL. This strain dilution was used as inoculum. Fifty pL of

diluted culture was inoculated into each autociaved petri plates containing Middlebrook 7H11

agar medium and spreaded uniformly. Micro wells were made on culture media of 6 mm in

diameter with the help of flamed cork borer and 50 p,L of the test solution was dropped on

these micro wells. Fifty pL of standard solution was also dropped in one micro well o f each

plates. One set of plates was run as negative controls, in which 50 pL blank solvent was

applied on the micro wells, instead of the test solution. The plates were incubated for 30 days

and zone of inhibition was measured as the diameter.

Chapter ..... .. ^ ^ Methods

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4.2.8. STAIMLITY STUDY OF DRUG LOADED MICROPARTICLES O F ISOM AZID

AND MFAIVIPICIN

Stability study for optimized formulation was carried out as per ICH and WFIO

guidelines'. For the estimation of drug content in the microparticles stability indicatiiig

HPTLC method was used.

4.2.8. L Acceleratetl stability study according to ICH guidelines

Microparticles were packed in a laminated aluminium foil and kept in the stability

chamber maintained at temperature of 40±2 °C and 75±5 % RH for 90 days. Samples were

withdrawn at intervals of 0, 30, 60, 90 days. The samples were analyzed for their drug content

by HPTLC analysis using stfindard curve (for isoniazid AUC=6.136><Concentration+417.3

and for rifampicin AUC- 6.425xconcentration+262.7).

4,2.8.2. Accelerated stability study according to WHO guidelines for shelf life

(leterinination

Microparticles were packed in a laminated aluminium foil and kept in the stability

chamber maintained at temperature of 40±0.5 °C, 50±0.5 °C, 60±Q.5 “C for 90 days. Samples

were withdrawn at intervals of 0, 30, 60, 90 days. The samples were analyzed for their drug

content by HPTLC analysis using standard curve (for isoniazid

AUC=6.136xConcentration+4l7.3 and for rilampicin AUC= 6.425xconcentration+262.7).

4.2.9. FORMULATION AND EVALUATION OF ORAL PROLONGED RELEASE

DRUG DELIVERY SYSTEM

4.2.9.1. Formulation of Drug Delivery System

The oral prolonged release dosage form was prepared by incorporating an

equivalent amount of isoniazid and rifampicin microparticles in a hard gelatin capsule shell as

in the marketed FDC product,

4.2.9.2. Evaluation of Drug Delivery System

The in vitro dissolution studies were carried out in simulated gastric fluid (pH 1.2)

and simulated intestinal fluid (pH 7.4) to study any interference and efficacy of the

combination product. Samples were withdrawn at regular intervals and replaced with fresh

-,, , ,-- -. , Materials and Methods

dissolution medium. The absorbance was measured and the concentration was determined by

UV method of drug combination. The dissohition profile of the formulation was compared

with that of the marketed preparations.

Chapter 4 Materials and Methods

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