Experimental

4 . 0 E X P E R I M E N T A L

4.1 MATERIAI.S AND EQUIPMENTTable 4.1 and 4.2 list the chemicals/materials (obtained ex gj'aiis* or

pui'chased) and the various instmments used respectively, during die

course of study.

Table 4.1: List of Materials

Chemical/Material Soiii'ce/Manufactiiitei'

Acetone AR S.D. Fine Chem., India

Acetonitrile (HPLC Grade) S.D. Fine Cheni., India

Activated cairbon Norit CN Extra & SX Plus, Norit, Netherlands

Acyclovir BP Ranbaxy Research Labs, Gurgaon, India

Ammonium acetate AR S.D. Fine Chem., India

Butan-2-onc AR S.D. Fine Chem., India

Chloroform AR BDH, IncUa

Citric acid AR CDH, India

Co-processed Microcrystalline cellulose and Lactose

Cellactose, Meggle GnibFI, Germany

Croscarmellose sodium NF Ac-Di-Sol, FMC Corporation, USA

Cross Linked PVP NF Polyplasdone XL, ISP Tech Inc., USA

Diethyl amine AR S.D. Fine Chem., India

Dimethyl formamide AR CDFI, India

Dimethyl sulfoxide AR CDI-I, IndiaEgg-phosphaddylchoUne Lecithin) Lipoid E PC, Lipoid Gmbh., GermanyEthyl acet'dtc AR BDH, India

Fulvic acid (T^aurentian) Fredriks Research Products, Netherlands

Glacial acedc acid AR S.D. Fine Chem., India

Hydroxypropyl'P-cyclodextrin Signaa-Aldrich Chem, Corp. Inc., USA

Hydrochloric acid AR S.D. Fine Chem., IndiaHumic acid Sigma Aldrich, USAIon Exchange Resins Tulsion ADS-400, Thermax Ltd., India

Tulsion T-42 FI, Thermax Ltd,, India Indion 225 FI, Ion Exchange India Ltd

65

Experimental

Chemical/Material Soiii'ce/Masmfacttttef

Isoaniyl alcohol A ll Merck, India

Itraconazole BP Lee Pharma, Chennai, India

Ranbaxy Research Labs., Gurgaon, India

Ketoconazole BP Ranbaxy Research Labs., Gurgaon, India

Lactose LR S.D. Fine Chem., India

Low substituted Hydi:oxy|3ropyl cellulose

L-IIPC LH-II, Shiii-Etsu, Japan

Magnesium steamte LR S.D. Fine Chem., India

Methanol (HPLC Grade) Merck, India

Methanol AR Merck, India

MicrocrystalLine cellulose Avicel PFI102, FMC Corporation, USA

Morpholine propane sulfonic acid buffer AR

Merck, India

ii-heptai\e AR Merck, India

Perchloric acid AR Merck, India

Potassium dihydrogen ortho phosphate AR

CDI-I, IncUa

Potassium hydroxide AR CDH, India

Potato dextrose agar Himedia Labs Pvt. Ltd., India

RPMI 1640 medium Llimedia Labs Pvt. Ltd., India

Shilajit (Rock) Dabur Research Foundation, Sahibabad, India

Shilajit (Shudh) Gurukul Kangri Pharmacy, Haridwar, IndiaShilajit Extract Natural Remedies, Bangalore, India

Pioneer Enterprises, Mumbai, IndiaSodium chloride AR S.D. Fine Chem., India

Sodium hydroxide AR CDH, India

Sodium octane sulfonate AR Merck, India

Sodium taurocholate AR Merck, India

Sulphuric acid AR Merck, India

Tissue culture medium, TCI 99 Himedia Labs Pvt. Ltd., India

Tween 80 Merck, India

Water for HPLC Merck, India

66

Experimental

Table 4.2: L ist of E qu ipm ent

Eqiiipnient Model/Maiiufactwer

D ifferential Scanning Calorimeter Perkin Eim er, USA

Dissolution Apparatus D isso 2000, Labiiidia, India

D ouble beam U.V. spec trop h o torne ter

Shimaclzu, 1601 U V/VS, Shim adju, Japan

Double D istillation Unit Borosil, India

Electronic W eighing Balance B244, M etder Toledo, Sw itzerland

Fourier I 'ransfom i Infrared Spectrophotom eter

W IN 111, PTS 40, B io llad , USA

Freezer N ew Brunswick Scientific, G erm any

FriabiUty T est Apparatus Scientific System , India

Hot ait oven Scientific System s, India

HPLC Systems Shimaclzu, Japan :® System controller: SCLIO-VP ® Pum p: LC-IOATVP ® U V detector; SPD -IO A W• Software; Class VP (5.03)

W aters, USA:• W aters 1525 b inary pum p® W aters 2487 D ual X D etector ® Software: Breeze

Lyophilizer Drywinner, DVV-8-85, Fleto H olten , D enm ark

Magnetic stirrer Rcm i M otors, India

M echanical Stirrer Scientific System s, India

M elting Point Apparatus Scientific System s, India

pFI m eter Flanna M icrocom puters, USA

Powder X -ray diffractometer PW 1710 AN D P W 1729, Panalytical, USA

Refrigerated Centrifuge M egafuge 20B , Fleraeus Sepatech, Sw itzerland

Rotary Evaporator Scientific System s, India

Rotary Tabletting M acliinc Clit Jem akay Eng. Ltd., India

Scanning E lectron M icroscope JS M -8 40 ,Jeo l Scanning, USA

Shaldng W ater Bath N SW 133, N ational Scientific W ork, India

Sonicator Bath Pram a Instruments Pvt. L td ., India

Spray Dryer S M Scientech. India

Stability Chamber M etrex Scientific Instrum ents, India

Vortex M ixer Spinix, India

67

Experimental

4.2 PREPARATION OF STANDARD SOLUTIONS /

BUFFERS

4.2.1 Sirrmlated gastric fluid without pepsin (SGF) (pH 1.2)Sodium chloride (2.0 g), taltcn iii 1000 ml volumetric flask, was dissolved

in 7 ml of concentrated hydrochloric acid and 500 ml of water. The

volume was finally made up to the mark with water. The solution had a pH

of 1.2.

4.2.2 Acetate buffer pH 4.0Glacial acetic acid (28.6 ml) and 50% sodium hydroxide solution (10 ml)

were placed in a 1000 ml volumetiic flask and made up to volume with

water.

4.2.3 Phosphate buffer pH 6.0Monobasic potassium phosphate (6.8 g) was dissolved in 250 nil of water.

To diis was added 70 ml of 0.2N sodium hydroxide and the volume was

made up to 1000 ml with water.

4.2.4 Fed State Simulated Intestinal Fluid (FeSSIF)Composition:

Sodium taurocholate 15 mM

Lecithin 3.75 mM

NaOH Pellets 4.04 g

Glacial acetic acid 8.65 g

NaCl 11.874 g

Distilled Water c|s to 1,000 ml

Pteparation;

FeSSIF was prepared by tlie method described by Prof. Dressman

(Dressman, 2003; Kostewicz, et. al., 2002). Blanlc FeSSIF was first prepared

by dissolving 11.875 g of NaCl, 4.04 g of NaOH pellets and 8.65 g of glacial

acetic acid in sufficient quantity o f distilled water to make tlie volume to 1.0

litre and adjustitig die pH to 5.0 widi IN NaOH or IN ITCL.

68

Experimental

To 250 ml of this solution was added 8.25 g of sodium taurocholate and

stirred to dissolve. About 29.5 ml of a chloroform solution contiiining 100

mg/ml of lecithin was added to this solution resulting in die formation of

an emulsion. The chloroform was then driven off using a Rotary

evaporator under vacuum at about. 40°C. The final solution was a clear,

niicellar solution, with no perceptible odour of chloroform. After cooling

to room temperature, the volume was made up to 1 litre witii blank

FeSSIF,

43 CHAEACTERISATION AND IDENTIFICATION OF

ITRACONAZOLEThe sample of itraconazole was characterized on the basis of its physico­

chemical properties such as colour, odour, taste, hydl'ophilic/hydrophobic

behaviour, solubility in water and other organic solvents, etc. M elting

point, UV and IR spectt'al analysis were carried out on the obtdned sample

and matched \vitii that of a reference worldng standard. Assay of tlie drug

was done titrimetricaUy as per die mediod described in British

Pharmacopoeia (1999).

4.4 ANALYTICAL METHODOLOGY FOR

ITRACONAZOLE

An HPIvC analysis method for the determination of itraconazole was

developed. The mediod is based on the method reported by Badcock,

1990 and Miyake et. a l, 1999 and uses Water : Acetonitrile : Diethyl amine

(40 : 60 ; 0.05, v/v) as the mobile phase and a 250 x 4.6 mm Cig

Spherisorb column having a 5 p n packing as tlie stationaiy phase. This

mediod was used for the routine analysis of itraconazole such as for

determining the assay, solubility, dissolution, etc. For the determination of

itraconazole in plasma a liquid-liquid extraction method using an internal

standard followed by HPLC analysis was used.

69

Experimental

4.4.1 HPLC method for routine Analysis4.4.1.1 M aterials used

Itraconazole

Water

Dictliylamiiie

Acetonitrile

Metlianol

Dimethyl foi'maniide

4.4.1.2 Preparation of M obile Phase

The mobile phase was prepared by mixing water, acetonitrile and

diethylamine in die ratio of 40;60;0.05, v/v. The mobile phase was degassed

by sonication and filtered through 0.45 [.im membrane filter under vacuum

just before die HPLC analysis.

4.4.1.3 Chfomatogmphic conditions

Column ; Waters Spherisorb ODS-2, 250 x 4.6 mm, 5 jam

W ater: Acetonitrile : Diethyl amine (40 : 60 : 0.05, v/v)

20)al

1.5 ml/min

UV at 263 nm

4.4.1.4 Preparation of calibration cuive

Calibration curves for itraconazole were prepared in medianol or

niethanolic HCl in die range of 1 to 100 |a.g/ml for routine analysis.

About 100 mg of itraconazole was accurately weighed and dissolved in 5

ml of dimethyl formamide by sonication. The volume was made up to 100

tnl widi medianol. Serial dilutions from this stock solution were prepared

by diluting die required aliquots with eitiier medianol or an equal volume

of medianol and O.OIN HCl.

Mobile phase

Itij action volume

Flow rate

Detection

70

Experimental

4.4.1.5 Sample prepafation for HPLC

i. A ssay of Com plex/Tablets:

A quantity of complex/tablet eqiiivalent to 100 mg of itraconazole was

taken in a 100 ml volumetric flask. Two ml of dimetliyl formaniicle was

added to the powder and the solution was sonicated for 5 minutes. Fifty

ml of equal volume of medianol and O.OIN hydrochloric acid (Solvent A)

was added to the above and furdier sonicated for 5 minutes. 'I'he final

volume was made up widi die Solvent A and mixed. Five ml of this

solution was again diluted to 100 ml widi Solvent A to give the test

solution. Twent}r ;al of tliis solution was filtered through a 0.22 |am

membrane filter and injected into die HPLC column.

11. Solubility and D issolution Studies:

Solubility and d isso lu tion study samples were filtered th ro u gh a 0.22 jim

membrane filter and injected directly into the HPLC column,

4.4.2 HPLC method for Plasma AnalysisA liquid-lic|uid exttaction method followed by HPLC was developed for

the analysis of itracona2ole in plasma. The method was based on the

methods reported by Woestenborghs et. al. (1987) and Warnock et al.

(1988) with slight niodification.

4.4.2.1 M aterials used

Itraconazole

Ketoconazole (internal standard)

Water

n-heptane

Isoamyl alcohol

Diethylamine

Acetonitrile

Methanol

Dimethyl formamide

71

Experimental

Phosphate buffer 0.05M: Prepared by dissoh'iiig 6.8 g of potassium

dihydrogen ordio pliosphate in 930 ml water. The pH was adjusted to

7.8 with 30% w/v solution of potassium hydroxide. The volume was

finally made up to 1000 rnl widr water.

Sulphuric acid 0.05M: Prepared by ciiluring 2.7 ml of sulphuric acid

with cc|ual volume of water and making up the volume to 1000 ml

with water.

Potassium hydroxide 5M: Prepared by dissolving 28.05 g of

potassium hydroxide in sufficient quantity of water to make up the

volume to 100 ml.

4.4.2.2 Preparation of M obile Phase

The mobile phase was prepared by mixing water, acetotiittile and

diethylamine in the ratio of 40:60:0.05, v/v. Tlie mobile phase was degassed

by sonication and filtered through 0.45 |.Lm membrane filter under vacuum

just before die HPLC analysis.

4.4.2.3 Chfom atographic conditions

Column : Lichrosper 100, RP-18, 250 x 4.0 mm, 5 p,m

Guard Column : Nucleosil C-18, Machery-Nagel, Germany

Mobile phase : W ater: Acetonitiile : Diethyl amine (40: 60 : 0.05, v/v)

Injection volume : 50 )j,l

Flow rate : l,5m l/min

Detection : UV at 263 nm

4.4.Z.4 Preparation of internal standard

About 100 mg of ketoconazole (accurately weighed) was dissolved in 5 ml

of dimethyl formamide and tiie volume was made up to 100 ml with

methanol to give a stock solution of 1000 |J,g/ml, 5 ml of this solution was

diluted to 50 ml with methanol to give a concentration of 100 pg/ml. 10

ml of this solution was again diluted to 50 ml witii methanol to give an

internal standard solution of 20 jag/ml.

72

Experimental

4.4.2.5 Preparation of calibration curve for Itraconazole

i. Preparation of Itraconazole Stock solution:

About 100 rng of itjaconazole was accurately weighed and dissolved in

5 ml of dimethyl formamide by sonication. The volume was made up

to 100 ml widi methanol to give a stock solution of 1000 )^g/ml. Five

ml of diis solution was diluted to 50 ml widi methanol to give a

concenttation of 100 |uig/ml. Ten ml of tliis solution was again diluted

to 50 ml with methanol to give a stock solution of 20 |-ig/ml.

ii. Preparation of dilutions of itraconazole stock solution:

Dilutions as per the following scheme were prepared to give dilutions

of itraconazole ranging from 20 ng/ml to 10000 ng/ml.

S.No.

Cone, of Stock

Solution (ng/ml)

Aliquot of Stock

solution taken (ml)

Volume of methanol

added (ml)

Final concentration of

Itraconazole dilution (iig/ml)

1, 20000 2,5 2.5 10000

2. 10000 3.2 1.8 6400

3. 6400 2.5 2.5 3200

4. 3200 2.5 2.5 1600

5. 1600 2.5 2.5 800

6- 800 2.5 2.5 400

7. 400 2.5 2.5 200

8. 200 2.5 2.5 100

9. 100 1.0 4.0 20

iii. Spildng of Plasma:

a) For calibration curve standards: Aliquots (0.25 ml) of tlie following

stock solutions were taken in 5 ml volumetric flasks and tlie volume

was made up with plasma to achieve the concentrations described

below. The solution was mixed by agitating on a vortex shaker for 30

seconds.

73

Experimental

S.No.

Cone, of Stock

Soliitioii (iig/iiil)

Aliquot of Stock

solution taken (ml)

Voluine of plasm a

added (ml)

Final concentration of Itraconiazole in spiked plasma

(tig/ml)

1. 10000 0.25 4.75 500

2, 6400 0.25 4.75 320

3. 3200 0.25 4.75 160

4. 1600 0.25 4.75 80

5. 800 0.25 4.75 40

6. 400 0.25 4.75 20

7. 200 0.25 4.75 10

8. 100 0.25 4.75 5

9. 20 0.25 4.75 1

b) For quality control sam ples: One ml aliquots of the following stock

solutions were taken in 25 ml volumetric flasks and the volume was

made up witli plasma to achieve the concentrations described below.

The solution was mixed by agitating on a vortex shaker for 30

seconds.

S.No.

Cone, of Stock

Solution :(ng/ml)

AEquotof Stock

solution taken (ml)

Volume of plasm a

added (ml)

Final concenttation of

^: Iti’aconazole iti sp iked plasm a

(ng/inl)

1. 10000 1.0 24.0 400

2. 6400 1.0 24.0 256

3. 1600 1.0 24.0 64

4. 100 1.0 24.0 4

Aliquots (3 ml) of die above quality control samples were transferred into

labelled polyj^ropylene tubes and frozen along with actual samples at

-70°C pending analysis. These samples were processed and analyzed

whenever die actual samples were analyzed.

74

Experimental

4A.2.6 Sample preparatioti

a) The calibration cun^e, quality control and subject samples were

withdrawn from the deep freezer and allowed to thaw to room

temperature in a water bath maintained at room temperature. The

thawed samples were vortexed on a A ortex mixer for 15 sec. to ensure

complete mixing.

b) Two ml alic|uots of each of the samples were taken in stoppered test

tubes. One hundred jxl o f the internal standard was added to all tiie

samples except subject blank and calibration curve blank and vortexed

for 30 seconds to ensure proper mixing.

c) Four ml of 0.05M phosphate buffer was added to each test tube and

mixed followed by addition of 4 ml o f n-heptane~isoamyl alcohol

(98.5:1.5% v/v) to each test txibe.

d) The tubes were vortexed for 2 minutes to allow extraction of

itracona2ole and ketoconazole into the organic layer. The tubes were

centxifuged at 4000 rpm for 5 minutes and die supernatent organic layer

was transferred to anodier tube.

e) The process of extraction was repeated widi 4 ml of n-heptane-isoamyl

alcohol. The tubes were centrifuged and the supernatant organic layer

was transferred to the tube containing the earlier organic extract.

f) The two organic layers were mixed and 3 ml of 0.05M sulphuric acid

was added. The tubes were vortexed for 2 minutes in order to allow

extraction of the drugs into die acidic aqueous layer.

g) The tubes were centiifuged at 4000 rpm for 5 min, and tlie organic

layer was discarded.

h) One hundred |o.l of 5M potassium hydroxide solution was added to each

test tube and mir.ed. The resulting alkaline solution was extracted with 4 ml

of n-heptane-isoamyl alcohol and centrifuged to separate die organic layer.

i) Ihree ml of the organic layer was separated and evaporated to dryness

under nitrogen at 55°C. The residue was redissolved in 50 pi of acetonitrile

and 50 )al of water and 50 pi was injected into the HPLC column.

75

Experimental

4.5 CHARACTERISATION AND IDENTIFICATION OF

ACYCLOVIRThe obtained sample of acyclovir was characterized on the basis of its

physico-chemical properties such as colour, odour, taste,

hydrophilic/hydrophobic behaviour, solubility in water and other organic

solvents, etc. Melting point, UV and IR spectral analysis were also carried

out on the obtained sample. Assay of the drug was determined by the

method described in tlie British Pharmacopoeia (1999).

4.6 ANALYTICAL METHODOLOGY FOR ACYCLOVIRAn HPLC analysis method for tlie determination of acyclovir was

developed. The method is based on the modification of mediods reported

by Jalon et. a l, 2002 and Park et. al., 1992. The method uses 10 mM

ammonium acetate buffer pH 5.0 along with 1 mM sodium octane

sulfonate : acetonitrile (96 : 4) as the mobile phase and a 250 x 4 mm Cis

Hypersil column hrving a 5 \xm packing as the stationary phase.

4.6.1 M aterials used

Acyclovir

Water

Acetonitrile

Sodium octane sulfonate

Ammonium acetate

4.6.2 Preparation of M obile Phase

The mobile phase was prepared by mixing 10 mM ammonium acetate

buffer pH 5.0 con'-aining 1 mM sodium octane sulfonate : acetonitrile in

the ratio of 96 : 4, v/v. The mobile phase was degassed by sonicarion and

filtered through 0.45 jam membrane filter under vacuum just before the

HPLC analysis.

76

Experimental

4.6.3 Chromatographic conditions

Column : Lichrosper C-18, 250 x 4,6 mm, 5 |am pacldng

Mobile phase ; 10 mM ammonium acetate buffer pH 5.0 containing

1 mM sodium octane sulfonate: acetonitrile (96 : 4, v/v)

Injection volume ' : 20 |.il

Flow rate : l.Onil/min

Detection : UV at 254 nm

4.6.4 Preparation of calibration curve

Ciilibration curve for acyclovir was prepared in water in the range of 1 to

100 |J-g/ml. About 100 mg of acyclovir was accurately weighed and dissolved

in 50 ml of water by sonication. The volume was made up to 100 ml with

water. Serial dilutions in die range of 1 to 100 fig/ml were prepared from

diis stock solution by diluting the required jiUquots with water.

4.6.5 Sample preparation for HPLC

i. Assay of Complex:

A quantity of complex equivalent to 100 mg of acyclovir was taken in a

100 ml volumetric flask. Two ml of dimethyl sulfoxide was added to the

powder and the solution was sonicated for 5 minutes. Fifty ml of water

was added to the above and further sonicated for 5 minutes. The final

volume was made up with water to 100 ml. Twenty [xl of this solution was

filtered through a 0.22 |nm membrane filter and injected into the HPLC

column.

ii. Perm eab ility Studies:

The samples obtained during permeability studies were centrifuged at 4000

rpm for 5 min, Sample (0.2 ml) from die supernatant was taken and mixed

on a vortex mixer for 1 min with 0.2 ml of acetonitrile. The obtained

sample was again centrifuged at 4000 rpm for 5 min and 50 )j.l of die

svipernatant was injected into the HPLC column.

77

Experimental

4.7 PHYSICAL CHARACTERIZATION AND

AUTHENTICATION OF SHILAJITAn authentic sample of Rock sliilajit was obtained from Dabur Research

Foundation and evaluated on the basis of its physico-chemical

characteristics such as colour, odour, taste, solubiJity in water and odier

organic solvents, etc.

Spectral analysis such as UV and FTTR were performed. Since huiTiic

substances usually yield uncharacteristic spectra in the UV and visible

regions of die electromagnetic spectmm, E4/E6 ratio is often used for the

characterisation of such substances (Schnitzer, M., 1972). FU/Efi ratio is die

rario of the absorbances of the solution at 465 and 665 nm and is

independent of the concentration of the liumic material.

Scanning electton inicrograph and X-ray diffraction pattern of the sample

were also obtained. An FIPTLC finger print of die sample was obtained by

using chloroform : medianol (90 : 10) as the mobile phase.

4.8 AUTHENTICATION OF SHILAJIT FROM DIFFERENT

SOURCESSHlajit samples were obtiiined from different sources and audienticated by

comparing their FIPTLC fingerprints, FTIR and UV-visible spectra widi

that of the authenticated sample of Rock shilajit obtained from Dabur

Research Foundation. The procured samples included a purified (shodhit)

sample of shilajit in the form of Shudh shilajit marketed by Gurukul

Kangri Pharmacy (GK), Flaridwar and aqueous extracts of shilajit from

commercial suppliers [Natural Remedies (NR), Bangalore and Pioneer

Enteiprises (l^E), Mumbai].

78

E xperim enta l

4 J EXTRACTION OF HUMIC ACIDS AND FULVIC ACIDS

FROM SHBLAJITInitiaUy, die folio \ving method reported by Ghosal et. al. (1989) was

adopted for the extraction of fulvic and humic acids from shilajit:

a) Finely powdered shilajit (200 g.) was extracted with 500 ml of hot

chloroform by soflicarion followed by stirring for 6 hours. I'he

suspension was filtered and washed twice widi 200 ml of hot

chloroform. The remaining marc was dried and powdered again.

b) The entire process of extraction was repeated, on the powder obt'ained

in die above step, with solvents of increasing polarity i.e. ediyl acetate

followed by medianol to furdier remove die interfering substances.

c) 10 g powder from die above Extracted shilajit was taken and

dispersed in 1000 ml of 0.1 N ac}ueous sodium hydroxide solution

widi intermittent shaldng in presence of Nitrogen at room

temperature for 24 hours.

d) The suspension was filtered and the filtrate was acidified with dilute

HCl to a pFI of less dian 3.

e) The solution was allowed to stand at room temperature (25°C)

overnight, humic acid, wliich separated out as coagulate, was filtered,

dried and pulverized.

To die filtrate was added 5 g of activated carbon and die suspension

was stirred for 2 hours and kept overnight.

g) The activated carbon was filtered off and dried at 40°C in an oven.

The dried carbon was then suspended in 50 ml of acetone and the

suspension was stirred for 2 hours in order to dissolve the adsorbed

fulvic acids in acetone.

h) The acetone was filtered off and two more cycles of elution with

acetone were carried out, to completely remove the fulvic acid from

carbon.

i) The acetone was evaporated to dryness to obtain the fulvic acid.

79

Experimental

However, it was observed tliat tlae yield of fulvic acid obtained by this

method was very 13w. Additionally, the fulvic acid obtained by die above

method was not completely soluble in water. Hence, an improved method

for the extraction of fulvic acid was developed and standardized. The

method used indigenously available ion-exchange resins instead of

activated carbon for the separation of fulvic acids. The developed method

gave improved yields of fulvic acid as compared to die earlier mediod.

The developed method for die extraction of humic and fijlvic acid

essentially consisted of die following steps:

a) Finely powdered sliilajit (200 g) was successively extracted with 500 ml

cach of hot organic solvents of increasing polarity, chloroform, ethyl

acetate and methanol to remove the bioactive components specifically,

oxygenated dibenzo-a-pyrones.

b) 50 g of the extracted sliilajit so obtained was taken and dispersed in

500 ml of 0.1 N aqueous sodium hydroxide solution widi intermittent

slialdng in presence of Niti:ogen at room temperature for 24 hours.

c) The suspension was filtered and the filtrate thus obtained was acidified

widi dilute HCl to a pH! of less tiian 3.

d) The solution was allowed to stand at room temperature (25°C)

overnight, humic acid, which separated out as coagulate, was filtered,

dried and pulverized.

e) The filtrate obtained in the above step was shaken w idi 20 g of

macroporous ion-exchange resin, TULSION ADS-400 from M/s

Thermax Ltd., India for 5 minutes in order to adsorb die fulvic acids

on the macroporous resin.

Q Fulvic acid adsorbed on the macroporous resin was then eluted using

100 ml of 0.1 N aqueous sodium hydroxide solution,

g) The process was repeated several times ( 6 - 8 times) using die same

macroporous resin and O.IN aqueous sodium hydroxide solution, tiU

complete adsoirption and elution of fulvic acid took place.

80

___________________ E xperim en ta l

h) The fulvic ac’d solution tlius obtained was shaken with 10 g of

hydrogen saturated cation exchange resin (INDION 225 H from M/s

Ion Exchange Lidia Ltd. or TULSION 1-42 H from M/s Thcrmax

Ltd., India) for 5 minutes in order to exchange die socUum ions witli

hyckogen ions.

i) The final fulvic acid solution was freeze dried to obtain amorphous

fulvic acids.

4.10 PHYSICO-^CHEMICAL CHARACTERIZATION OF

HUMIC AND FULVIC ACIDSThe humic and fulvic acids extracted from Rock sliilajit were characterized

on tlie basis of thei^ physical properties, solubility, E4/E6 ratio, UV spectra,

IR spectra, XRD spectra and DSC scan. Humic and fulvic acids extracted

from odier shilajit sources were characterized on die basis of their physical

properties, E4/Er, ratio, and UV and IR spectra and were compared with

those of humic and fulvic acids obtained from Rock shilajit: Dabur,

4.11 EVALUATION OF SURFACTANT PROPERTIES OF

HUMIC AND FULVIC ACIDSThe surfactant properties of humic and fulvic acids were investigated by

determining tiie effect of increasing concentration of humic and fulvic

acids on the surface tension of water. The surface tension of the solutions

was determined by the drop-weight mediod using a stalagmometer.

Solutions of humic or fulvic acids in the concentration range of 0 to 1.4%

w/v were prepared. Each solution was separately sucked into die

stalagtnometer and allowed to drop slowly from it. ITie drop rate was

adjusted to approximately 2-3 drops/min. and the weight of 10 drops was

measured. The determination was repeated twice. Surface tension of die

solution was calculated using die formula;

Surface tension of Surface tension of water Weight of humic/humic/fulvic acid solution = ------------------------------------------ x fulvic acid solution

Weight of water

81

Experimental

4.12 PHASE SOLUBILITY STUDIESThe effect of increasing concentration of liurnic and fulvic acids on die

ac|ueous solubility of itiraconazole was determined by carrying out phase-

solubility studies in water according to die method of Higuchi & Connors

(1965). Aqueous solutions (10 ml) of humic or fulvic acids were prepared

in the concentration range of 0 to 1.6 % w/v. An excess amount of

itiraconazole (about 20 mg) was added to each sample contained in a

stoppered glass tvtbe. The txibes were shaken on a mechanical shaker,

equipped with a tiiermostarically controlled water batli, for 7 days at

25±2°C. After 7 df.ys, aU die samples were centrifuged and die supernatant

was filtered through a 0.22 |o,m membrane filter and analyzed by HPLC

mediod. The concentration of drug in die solution was determined from

the calibration curve.

4.13 PREPARATION OF ITRACONAZOLE COMPLEXESA number of complexes of itraconazole were prepared witii humic and

fulvic acids extracted from shilajit. Various techniques such as freeze drying,

solvent evaporation and spray drying were used for die preparation of

complexes. Since preliminary results with humic acids were not very

promising, furtiier trials were taken only widi fulvic acids. The complexes

were prepared in die molar ratio of 1 : 0.5 for itraconazole : humic acid.

With fulvic acid, die complexes were prepared in die molar ratios of 1 : 0.5,

1 : 1 and 1 : 2 of dnag ; complexing agent by die solvent evaporation

technique. Since the 1 : 1 molar ratio gave die best results, complexes by the

odier techmques were prepared only in 1 : 1 molar ratio. The quantity of

itraconazole and humic/fulvic acids used for the preparation of complexes

in the different molar ratios is shown in Table 4.3.

82

Experimental

T able 4.3; Q uantity of itraconazole, hum ic ac id and fulvic ac id used for com plexes prcpared in different m olar ratios

Ratio (Drug : Cotnplexiiig

agent)

Quatitity of Itraconaxole

(g)

Qty. of Humic acid*

(g)

Qty. of Fulvic acid**

(g)1 : 0,5 7.05 32.5 -

1 : 0.5 7.05 - 6.0

1 : 1 7.05 - 12.0

1 :2 7.05 - 24.0

* Avci'agc Mol. Wt. : 6500

** Average Mol. Wt.: 1200

4.13.1 Preparation of Itraconazoie-hum ic ac id com plex by Solvent

evaporation in a Rotary Evaporator

Complexes of itraconazole and huinic acid in the molar ratio of 1 : 0.5 was

prepared by suspending the reqiured quantity (shown in Table 4.3) of

itraconazole and humic acid in 250 ml of water and Stirling die mixture for 6

hours followed by sonication in an Liltrasonicator bath for 3 hours. The

resulting mixture was dried in a rotary evaporator under vacuimi to yield die

itraconazole-humic acid complex. Evaporation was carried out at 100°C by

dipping the rotating flask in a boiling water bath. Tlie dried complex was

sieved through sieve no 60 and stored in a vacuum desiccator till use.

4.13.2 Preparation of Itraconazole-hum ic ac id com plex by Freeze drying

Freeze dried complex of itraconazole with humic acid were prepared in a

manner similar to 4.13.1 above, with the difference that the mixture of

itraconazole and humic acid, after stirring and sonication, was frozen to

-70°C and tlien dried for 24 hours in a Heto freeze dryer to obtain die

freeze dried complex. The dried complex was sieved through sieve no 60

and stored in a vacuum desiccator till use.

4.13.3 Preparation of Itrr'.conazole-liiimic ac id com plex by Spray drying

Spray dried complex of itraconazole widi humic acid was prepared in a

manner similar to 4.13.1 above, with die difference that the mixture of

83

Experimental

, ilraconazole and huniic acid, after stifling and soiiication, was spray dried

at an inlet temperature of 260 to 280°C and a flow rate o f 10 ml per

minute to obtain the spray dried complex. The obtained complex was

stored in a vacuum desiccator till use.

4.13.4 Pfeparation of Itraconazole-fulvic ac id com plexes by Solvent

evaporation in a Rotary Evaporator

Complexes of itraconazole and fxilvic acid in the molar ratio of 1 : 0.5, 1 : 1

and 1 : 2 molar ratios were prepared by dissolving the requit:ed quantity

(shown in Table 4.3) of itraconazole in 100 ml of glacial acetic acid and

fulvic acid in 150 ml of water. The fulvic acid solution was then added to

the itraconazole solution witii stirring and the solution was sonicated in an

ultirasonicator badi for 3 hours. The solution thus c^btatned was dried in a

rotary evaporator under vacuum to yield the itraconazole-fulvic acid

complex. Evaporation was carried out at 100°C by dipping the rotating

flask in a boiling water batii. The dried complex was sieved through sieve

no 60 and stored in a vacuum desiccator tiU use.

4.13.5 Preparation of Itraconazole-fulvic ac id com plexes by Freeze drying

Freeze dried complexes of itraconazole widi fulvic acid were prepared in a

manner similar to 4.13.4 above, witii the difference that die solution of

itraconazole-fulvic acid complex was frozen to -70°C and then dried for

24 hours in a Heto freeze dryer to obtain the freeze dried complex. The

dried complex was sieved tlirough sieve no 60 and stored in a vacuum

desiccator till use.

4.13.6 Preparation of Itraconazole-fulvic acid com plex by Spray drying

Complexes of itraconazole widi fulvic acid were prepared in a manner

similar to 4.13T above, with the difference diat the solution of

itraconazole-fulvic acid complex was spray dried at an inlet temperature of

260 to 280°C and a flow rate of 10 ml per minute to obtain the spray dried

complex. The obtained complex was stored in a vacuum desiccator till use.

84

Experimental

4.13.7 Preparation of Itfaconazole-hum ic acid/fulvic acid com plexes by

Physical m ix ing

Physical inixtures of ittaconazole and fulvic acid were ptepared by

intimately mixing the required quantities of itraconazole and fulvic acid in

a pcsdc mortar.

4.13.8 Preparation of IttaconazoIe-fuM c ac id com plex by Freeze drying the

P hysical m ixture

A physical mixture of iti'aconazole and fulvic acid in 1 : 1 molar ratio was

prepared by intimately mixing 7.05 g of itraconazole and 12.0 g of fxilvic acid

in a pestle mortar. The mixture was added to 250 ml of water and sonicated

in an ultrasonicator for 3 hours. The resulting mixture was frozen at -70°C

and dried in a Heto Freeze drier for 24 hours. The dried complex was sieved

dirough sieve no 60 and stored in a vacuum desiccator tiU use.

4.14 CHARACTERIZATION OF COMPLEXESThe prepared itraconazole-fulvic acid complexes were characterized by

means of Differential Scanning Calorimetry (DSC), X-Ray Diffraction

(XRD), Fourier Transform Infrared Spectroscopy (FTIR) and Scanning

Electi'on Microscopy (SEM).

4,14.1 Differential Scanning CaloiimettyDSC thermograms (instrument calibrated by using Idium as a standard

widi melting point at 165°C) were recorded using a Perkin Elmer

Differential Scanning Calorimeter. All samples were treated according to

die following specifications:

Atmosphere : Nitrogen

Heating Rate : 10°C/min,

Temperature range : 40 - 400°C

Sample size ; Itraconazole or itraconazole-fulvic acid complexequivalent to 2 mg of itraconazole

85

Experimental

4.14,2 Powder X-Ray DifffactionPowder ,X-ray diffraction patterns of tlic samples were obtained using a

Paiialytical X-ra)^ diffractometer PW3719. All the samples were treated

according to die following specifications:

Target/Filter (Monochromator)

Voltage/Current

Scan Speed

Smoothing

Cu

40 KV/50 mA

4°/min.

0

4.14.3 Fouiief Transform Infra-Red SpectroscopyFTIR spectra of the samples were recorded on a Perldn lilm er 16 PC

FTIR instrument using the ICBr pellet technique. Two mg of previously

dried sample was mixed with 100 mg KBr and compressed into a pellet on

an IR hydraulic press. These pellets were made immediately prioi: to tlie

recording of die spectLiim. Scanning was done from 4000 to 450 cm'i.

4.14.4 Scanning Electron Microscopy (SEM)Scanning electron micrographs of prepared samples were obtained using a

Joel JSM-840 Scanning Microscope with a lOKV accelerating voltage. The

surface of samples for SEjM were made electrically conductive in a

sputtering apparatus (Fine Coat Ion Sputter JFC-1100) by evaporation of

gold. Magnifications of 1500 and 3000 were used for all samples.

4.15 AQUEOUS SOLUBILITY STUDIESThe solubilit^r of drug is an important physico-chemical property because it

affects die bioavailabiUty of die drug, the rate of drug release into the

dissolution medium and consequendy, die dierapeutic efficacy of the

pharmaceutical product.

In order to determine the effect of compiexation on the solubility of

itraconazole, the saturation solubility of itraconazole, prepared complexes

and physical inixtute was determined in different media (simulated gastric

86

Experimental

fluid without: pepsin, acetate buffer pJ-I 4.0, water and fed state simulated

intestinal fluid) at a temperature of 25°±2°C by shake flask method.

An excess quantity of the drug or complex (about 20 mg) was added to 10

ml of media in stoppered glass tubes which were placed in a

thermostatically controlled water bath and agitated continuously for 7 days.

Preliminary experiments had shown that satxiration solubilit}^ could be

achieved by shaldng for 7 days. After 7 days, the solution was centxifuged

and the supernatant was liltered tlirough a 0.22 |.Lm membrane filter and

analyzed lay HPIvC mediod. The concentration of drug in the solittion was

determined from the calibration curv e.

4.16 FORMULATION OF TABLETS USING COMPLEXESFast disintegrating tablet formulation incorporating the drug alone or

complexes was developed keeping die dispersion time as the critical

parameter. A number of diluents including microcrystalline cellulose,

lactose, co-processed microcrystalline cellulose-Iactose (Cellactose), starch,

dicalcium phosphate and their combinations in different ratios were tried

along witii different super-disintegrants like croscarmellose sodium,

crospovidone, low substituted hydroxyl propyl cellulose, etc. A prototype

formula was selected on die basis of best dispersion time, physical

appearance and dissolution and was used for further evaluation. Table 4.4

gives die composition of tlie prototyjae formulation and Table 4.5 gives

tlie tablet parameters used for the study.

87

Experimental

T ab le 4.4: Com position of Prototype fom iu lation

S.N o.

In gfed ien ts Qt / 1 ah. (miFot Tablet containing Uncomplex

cd dm g

For Tablet containing ITRA-HA complex

1:0.5

Fot Tablet containing ITRA-FA complex

1 : 0.5

For Tablet contain ing ITRA-FA complex

1 :1

Fot Tablet containing ITRA-FAcomplex

1 :2

1. Itraconazole 100 - -.... ■■■ 1

2. ITlliV-HA physical mixture or complex

- 560 - -

3. ITR/V-FA physical inixture or complex

- - 185 270 4401

4. Cellactose 632 350 547 462 292

5. Crospovidone 60 80 60 60 60

6, M agnesium stearate 4 5 4 4 4

7. Purified Talc 4 5 4 4 4

T able 4.5: T ab let Param eters used for the study

SI.No.

Patameters L— . ™ mFor Tablet containing

UncoiBLpIexed drug

For Tablet containing :

Ittaconay.ole- Humic acid complexes

For Tablet cotitaining

Itracoiiaxok- Fulvic acid complexes

1. Tablet weight 800±20 mg 1000±20mg 800±20 mg

2. Tablet ' Dimensions

19.0 X 9.0 mm 19.0 X 9.0 rm-n 19.0 X 9.0 mm

3. Tablet shape Oval, Standard concave

Oval, Standard concave

Oval, Standard concave

4. Hardness 4.0±2.0 kg/cm" 4.0±2.0 kg/cm" 4.0±2.0 kg/cm^

5. DisintegrationTime

NMT 2.0 min NMT 2.0 min NMT 2.0 min

6. Dispersion Time during

dissolution

NMT 2.0 min. NMT 2.0 nm. NMT 2.0 min,

7. Friability NMT1.0%w/w NMT 1.0% w/w NMT 1.0% w/w

Experimental

Tablets containiag eithei: 100 mg of the uiicompicxed drug ot: containing

the physical mixture or complexes equivalent to 100 mg of itraconazole

were prepared as per the formula given in I ’able 4.4. For tlie preparation

of tablets, the ingredients were intimately mixed and compressed on a 16

station rotary tabletdng macliine.

4.17 EV/ILUATION OF TABLETSThe prepared tablets were evaluated for the following parameters:

SI.No.

Parametet Test

1, General Appearance General appearance of tablets was recorded on die basis of colour, surface and overall appearance.

2. Hardness The hardness of tablets was determined using a Monsanto hardness tester. The test was conducted on five tablets.

3. Friability The tablets were tested for friability using a Roche friabilator. Tablets, pre-weighed were subjected to combined effects of shock and abrasion in a plastic chamber of friabilator revolving at 25 r.p.m. for 4 minutes. The tablets were then dusted and reweighed.

4. Dl g Content Ten (or Five) tablets were powdered in a glass pestie and mortar. An accurately weighed portion equivalent to 100 mg of pure drug was taken and the assay was performed using FIPLC method. The amount of drug in die formulation was calculated from die standard calibration curve.

5. Disintegration test One tablet was placed in each of die six tubes of the basket of tablet disintegration test apparatus and the test was performed. The assembly was raised and lowered 30 times per minute. The temperature of bath was maintaitied at 37+1 °C and mediiu-n was water. The time required for complete disintegration of the tablets was noted

89

Experimental

4.18 RELEASE AND IN-VITRO EQUIVALENCE STUDYPi'eliminaij solubility and dissolution studies of the drug alone and

cotnplexcs in water, acetate buffer pH 4.0, phosphate buffer pH 6.0 and

Fed state simulated intestinal fluid had shown tliat the solubility of

itraconazole in these media was not adequate to provide the sink

conditions required to carry out the dissolution study. Hence, aU further

dissolution studies were carried out in simulated gastric fluid widiout

pepsin at 37+l°C by the USP paddle method at 100 ipm. This medium

and condition has earlier been used by a number of investigators (Jung et.

al., 1999; Yoo et. a l, 2000) for determining the dissolution of itraconazole

formulations including that of the innovator’s product, Sporanox capsules.

During the study, samples were withdrawn at 5, 15, 30, 45 and 60 min.,

filtered through a 0.22 |.mi membrane filter and were analyzed for the

amount of itraconazole dissolved by HPLC method. Fresh aliquots of

dissolution medium were added to compensate for the sample withdrawn.

The dissolution profile of die optimized formulation was compared witii

that of the innovator’s product (SPORiVNOX capsule 100 mg of M/s

Janssen Pharmaceutica, USA) using WUcoxon signed rank test as well as

as well as using die Similarity factor (f2) and the Difference factor (fl)

(Moore and Planner, 1996).

4.19 DRUG PERMEATION STUDY ACROSS RAT EVERTED

GUT SAC

4.19.1 Itfaconazole Permeability StudiesIn order to study the effect of complexation on the intestinal permeability

of itraconazole, tlie permeability of itraconazole-fulvic acid complex

prepared by spray drying was compared witii itraconazole alone as well as

witli itxacona'zole-fulvic acid physical mixture by the la t everted gut sac

technique (Barthe, ct a l, 1998a&b; Carreno-Gomez, et. al., 2000)

90

Experimental

For the study, rat everted intestinal sacs of about 5 cm length were

prepared in Tissue culture medium, TCI99 and filled with about 3 ml of

Fed State simulated intestinal fluid. Tlie sacs were placed in t:ubcs

containing 25 m l of Fed State simulated intestinal flmd in which exccss

(about 25 mg equivalent of itraconazole) of eidier itraconazole alone or

itraconazole-fulvic acid (1 ; 1) physical mixture or itraconazole-fulvic acid

(1 : 1) spray dried complex had been suspended/dissolved. The tubes were

maintained in a shaking water bath at 37°C, continuously bubbled with

oxygen and agitated at a speed of 60 rpm.

Samples were withdrawn from the mucosal side at the start and from the

mucosal and seroscil side after 1 hour. T'he samples were centrifuged for 5

min. at 4000 tpm, filtered through 0.22 |j,m membrane filter and analysed

by HPLC method. Table 4.6 summarizes the parameters used for carrying

out die permeability stxidies.

Table 4.6: Param eters for perm eab ility study of Itraconazo le

S.N o .

Pafainetef Condition

1. Samples Itraconazole or complex or physical mbcture equivalent to 25 m g of ittaconazole

2. Liquid used for washing the lumen of intestine "

N orm al saline (0.9% sodium chloride at 37°C)

4. Liquid used for storing the intestine before mounting

Tissue culture m edium TC199 at 37°C

5. Mucosal fluid Fed state sim ulated intestinal fluid at 37°C

6. : Serosal fluid Fed state sim ulated intestinal fluid at 37°C

7. Lengtli o f the Sacs 5 cm

8. Vol. o f Mucosal fluid 25 ml

9. Vol. o f Serosal fluid 3.0 m l

10. Oscillations during tire study

60/iTiin

11. Sampling tiiiie One hour

91

Experimental

4.19.2 Acyclovir Pefmeability StudiesIn order to determine the effect of complexation on the permeability o f

BCS Class III drugs, permeability studies were carried out by the rat

everted gut sac method using acyclovir as a model drug.

The study was carried out by a metliod similar to that described for

itraconazole except that Tissue culture medium TC I99 was used as die

mucosal as well as serosal Uquid. The method is based on the methods

reported by Mizuma et. al. (1999); Barthe et. a l, 1998a&b. In order to

study the effect of complexation, a 1 : 1 molar freeze dried complex of

acyclovir and fulvic acid was prepared and compared with acyclovir alone,

1 : 1 physical mixture, 1 ; 1 complex of acyclovir and ITP-P-cyclodextrin

(HP-P-CD) prepared by freeze drying and the innovator product (Zovirax

tablet).

Since it has been reported diat the acyclovir shows a regional permeability

in the different regions of die intestine (Park et. al., 1992), the rat intestine

was divided into three portions. The first 15 cm length comprising of the

duodenum was considered to be die upper intestine followed by the next

15 cm length which was considered to be the middle intestine. The

remaining portion of tlie intestine comprising of the lower jejunum and

Ueum was considered as the lower intestine. The permeability studies were

carried out separately in the three portions. Since it has been reported in

die literature tiiat the permeability of acyclovir follows a linear pattern in

the concentration range of 5 )iM to 5 mM (Fujioka, et. a l, 1991), a single

concentration of 0.5 mM (112.6 |u.g/ml) of acyclovir or complexes

equivalent to acyclovir were taken on the mucosal side for the permeability

studies. Table 4.7 summarizes die various parameters used for the

permeability study of acyclovir.

92

Experimental

T able 4.7: Param etefs for perm eab ility study of Acyclovir

S.No.

Parameter Condition

1. Samples Acyclovir or complcx or physical mixture equivalent to 0.5 m M o f acyclovic (112.6 (J,g/ml)

3.

Intestinal segments

Liquid used for wasliinF the lumen ofOintestine

liq u id used for storing the intestine before mounting

s Segm ent 1: F irst 15 cm length com prising the duodenum.

® Segment 2: N ext 15 cm length com prising mainly the upper jejunum.

e Segm ent 3: Rem aining portion o f the intestine comprising d ie lower jejunum and Ueum.

Normal saline (0.9% sodium chloride at 37°C)

Tissue culture medium T C I99 at 37°C

5. Mucosal fluid Tissue culture medium TC199 at 37°C

6. Serosal fluid Tissue cultxire medium T C I99 at 37°C

7. Length ot the sacs 5 cm

8. Vol. of mucosal fluid 25 ml

9. Vol. o f serosal fluid 3.0 ml

10. Oscillations during the study

60/ min

11. Sampling time One hour

4.20 ANTI-FUNGAL STUDIESThe minimum inlaibitory concentration of ittaconazole-fulvic acid complex

prepared by spray drying was deterixiined in comparison to the

uncomplexed drug against a non-filamentous flingus, Candida alhkam and a

filamentous fungi A sper0 u s fumigatus by the brodi dilution method. The

studies were carried out according to die macrodilution procedure of die

National Committee for Clinical Laboratory Standards described in

document M27-A2, Reference Method for Broth Dilution Susceptibility

Testing of Yeasts (NCCLS, 2002a) for Candida albicans and according to

93

Experimental

document M38-A, Reference Method for Broth Dilution Susceptibility

Testing of Filamentous Fungi (NCXLS, 2002b) i o t Aspergilhisfiimigaliis.

4.20.1 Culture MediumIIPMI 1640 medium (with L-glutamine and phenol, red, without

bicarbonate) and buffered witir 0.165 M morpholino propane sulfonic acid

(MOPS) buffer at pH 7.0 was used as the culture medium..

For preparation of the medium, 10.4 g of powdered RPMI 1640 medium

was dissolved in 900 ml of distilled water. I'o this was added 34.53 g of

MOPS buffer and the pFI was adjusted to 7.0 using 1 mol/L sodium

hydroxide and the final volume was made up to 1000ml with distilled

water. The medium was filter sterilized using a 0.22 jj, membrane filter and

stored at 4°C till further use.

4.20.2 Antiftmgal agentsItraconazolc alone and Itraconazole-fulvic acid (1:1) complex prepared by

spray drying were used as the anti-fungal agent, Fulvic acid alone was used

as a control.

4.20.3 Organisms and Inoculum Prepatation

4 . 2 0 . 3 . 1 Candida albicansA cHnical isolate of C. albicans (C. albicans NVQl 193) was obtained from

Vallabhai Patel Chest Research Institute, New Delhi and used for tlie

study. Before use in the test, the isolate was subcultured twice on

Sabauraud dextrose agar plates at 35°C for 24 hours each. Five colonies of

about 1 mm diameter from the 24 h growtli plates were picked and

suspended in sterile saline (0.85 % w/v). The resulting suspension was

vortexed for 15 seconds and die cell density was adjusted by adding

sufficient sterile saline in order to match the transmittance to drat

produced by 0.5 McFarland turbidity standard (BaS04 airbidity standard)

at 530 nm wavelengdi. This procedure yields a stock suspension of 1 x

94

Experimental

to 5 X K)''' cells/rnl. A working suspension was prepared from tiiis stoclc

suspension by diluting 0.1 ml of stock suspension to 10 ml with llPM l

1640 medium followed by further dilution of 1ml of die resulting

suspension to 20 ml with RPMI 1640 medium.

4.20.3.2 A sp erg i l lu s fu m ig a tu s

A clinical isolate of A. fumigatus (A. fttmigalus VPCI 68) was obtained from

Valhibhai Patel Chest Research Institute, New Delhi and used for the

study. Before use in the test, the isolate was subcultured on potato

dextrose agar plates at 35°C for 7 days. Seven-day-cJd colonies were

covercd with approximately 1 ml of sterile saline (0.85% w/v) containing 1

% Tween 80 and the conidia were harvested by probing the colonies widi

the tip of the transfer pipette. The resulting mixt-ure was transferred to a

sterile tul)e and the heav)7 particles were allowed to settle for about 5

minutes. The upper homogeneous suspension was transferred to another

sterile tube and vortexed for 15 seconds. The ceU densit} was adjusted by

adding sufficient sterile saline in order to obtain a transmittance of 80 -

82'*/) for the stock suspension. A worldng suspension containing about 0.4

X 10“' to 5 X 10“* CFU/ml was prepared from this stock suspension by

diluting 0.1 ml of stock suspension to 10 ml widi RPMI 1640 medium .

4.20.4 ProcedureThe tests were performed by following the standard additive twofold drug

dilution scheme described in the NCCLS reference method. Stock

solutions of itraconazole or itraconazole-fulvic acid complex were

prepared at a concentration ecjuivalcnt to 1600 )-ig/ml of itraconazole in

DMSC3. Stock solution of fulvic acid was prepared at a concentration of

2725)ag/ml which corresponded to the concentration of fulvic acid present

in the itraconazole-ftilvic acid complex. Dilutions in the range of 160 to

0.3125 i-ig/ml equivalent of itraconazole were prepared by additive dilution

of the stock solutions widi die culture medium, as described in the

NCCLS method. E ach dmg dilution was then pipetted in 0.1-ml volumes

95

Experimental

into i:ound-botl:om, polysLyrcnc, snap-cap, sterile (xibes (12 by 75 mm) and

inoculated by adding 0.9-ml volumes of die coiTespondiiig well-mixed

working culture suspension of C albicans ox A. fumigatus. This step diluted

each drug to the final test concentiations (16 to 0.03125 |j.g/ml ec[uivalent

of itraconazole). I'he growth control tube contained a 0.9-ml volume o f

inoculum suspension and a 0.1-nil volume of drug-free medium. Steiiiity

control was performed by including 1 ml of un-inoculated, drug-free

medium. The whole procedure was repeated thrice with each micro­

organism.

All tubes were incubated at 35°C and observed for growdi after 48 hours.

MIC was visually determined as tlie lowest drug concentration which

prevented any discernible growth.

4.21 STABILITY STUDIES OF OPTIMISED FOEMULATIONStal)iliLy of a dosage form refers to tlie chemical and physical integrity of

the dosage unit and when appropriate, the ability of die dosage unit to

maintain protectiorx against microbiological ct^ntaminatioa. The developed

itraconazole tablets were subjected to stability studies to evaluate any

physical or chemical changes on storage. Tablets were packed in sealed

polythene lined aluminium pouches and stored in stability chambers at

40“C and 75% R li for 6 months. The samples were withdrawn

periodically at predetermined time intervals (1, 2, 3 and 6 mondis) and

evaluated for physical changes, drug content, hardness, disintegration time

and dissolution profile following die procedure outlined earlier. Zero time

samples were used as controls.,

4.22 SAFETY STUDIES OF THE OPTIMIZED

FORMULATIONIn order to confirm the safety of die optimized formulation, sub-acute

toxicity studies were carried out using rat as the model animal.

96

Experimental

4.22.1. Type of animals used:Wistar rats weighing between 150 180 g were used in the study.

4.22.2. Number of animals used:Five animals were used in each group, i.e., control, placebo and test

groups.

4.22.3. Dose g-iven to the animals:l l i e test formulation containing itraconazole-fulvic acid complex

equivalent to 30 mg/kg body weight was given to the test group. This

corresponds to about 10 times the average recommended daily dose for

humans.

4.22.4. Procedure:I ’he animals were divided into diree groups: conti:ol, placebo and test

group. The test group was given the formulation containing itraconazole-

fulvic acid comple'i equivalent to 30 mg/kg body weight, suspended in

water. The placebo group was administered the complexing agent, i.e.,

fulvic acid solution in distilled water, in the dose of 50 mg/kg body weight.

This amount corresponded to die amount of fiilvic acid present in the test

formulation being administered. The control group was given die vehicle

alone.

Before starring the experiment, about 2.0 mi blood was collected from the

tail vein of each rat from all die diree groups. The blood samples were

analysed for haematological parameters, Iddney function test and liver

function test.

The three groups were dien administered the respective products daily for

21 days, witli the help of a moudi feeder, and observed for physical activity

and body weight. After a period of 21.0 days, blood samples were again

witlidrawii from die rats and tested for haematological parameters, liver

function test and Iddney function test.

97

Experimental

After giving tlie test medication for a period of 21 days, the control,

placebo and test animals were sacrificed. Liver, Iddney, heart and spleen

were taken, out o f die rats and stored in 10% formalin solution. The tissues

were washed with normal saline and were seen for:

Gross Changes: Colour changes or die development of any patch was

observed.

H istopathological Changes: Tissues were cut into tliin slices and kept in

10% formalin solution for fixing for a period of 48 hours. This prevented

the post-mortem changes such as putrefaction and autolysis and preserved

the cell constituents in as life-like manner as possible. It protected the

tissues by hardening the naturally soft tissues thereby aUowing easy

manipulation during subsequent processing. Slides were prepared and

observed under the microscope.

4.23 IN-VIVO BIOAVAILABILITY STUDIES IN HEALTHY

HUMAN VOLUNTEERSThe bioavailability of tablets containing itraconazole-fulvic acid complex

prepared by spray drying was determined in comparison to the innovator’s

formulation (Sporanox capsule 100 mg of M/s Janssen Pharrnaceutica,

USA) in 6 healthy human volunteers in die fasting state. Blood samples

were witlidrawn at regular intervals and analyzed by a validated HPLC

analytical method

4.23.1 ObjectiveTo compare die smgle-dose oral bioavailabiHty of itraconazole 100 mg

tablet containing itraconazole-fulvic acid complex widi Sporanox capsules

100 mg of Janssen Pharmaceutica, USA in heaWiy, adult, human subjects

under fasting conditions.

98

Experimental

4.23.2 Study DesignThe study was conducted as open, balanced randornized, two period, cross

over witli a wash out period of 14 days.

4.23.3 SummaryThe study was an open randomized, two treatment, two sequence, two

period, single dose, crossover, comparadve bioavailability study on

itraconazole formuladons comparing itraconazole 100 mg tablet prepared

using itraconazole-fulvic acid complex witli Sporanox 100 mg capsules of

janssen Pharmaceutica, USA in healthy, adult, male, human subjects under

fasdng conditions

4.23.4 Number of Subjects06 healdiy male subjects were recruited for the study.

4.23.5 Admission and StayThe subjects were admitted and housed in the Clinical Pharmacology Umt

from at least 10-12 hours before dose administration and were discharged

24 hours after administration of the test or reference products during each

period. After discharge at 24 hours, subjects made 2 ambulatory visits to

die Clinical Pharmacology Unit for blood sampling at 48 and 72 hours.

4.23.6 DoseA single oral dose of itraconazole 100 mg was administered during each

period of the study under supervision of a trained Medical Officer.

4.23.6.1 Rcfcrcncc - R

A single oral dose of a Sporanox 100 mg capsule (Janssen Pharmaceutica,

USA) was administered with 240 ml of drinldng water at ambient

temperature.

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Experimental

4.23.6.2 T est~ T

A single oral dose of an iti'aconazole 100 mg tablet (containiftg

iri'aconazole-fulvic acid complex) was administeted with 240 ml of

drinking wafer at ambient temperature.

4.23.7 Fasting/MealsAll subjects were required to fast overnight after admission for at least 10

hours before the morning dose and for 4 hours post dose. The subjects

received standard meals, i.e., lunch, snacks and tiinner at approximately 4,

9, and 13 hours respectively, after first dosing. During housing, aU meal

plans were identical for the two periods. In case where tlie meals and

blood sample collection time coincided, sainples were collected before

meals were provided.

Drinking water was not allowed from 1 hour before dosing- and until 2

hours post dose. Thereafter, it was allowed at all times.

4.23.8 Sampling ScheduleA total of thirty six 5-mL blood samples for bodi the ti'eatments were

collected in EDTA vacutainers during die course of die study through an

indwelling cannula placed in the forearm vein, l l i e blood samples were

collected pre-dose and at 0.5, 1,1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6 , 8, 12, 24,

48 and 72 hours after each oral dose in each period.

The pre-dose blood sample was collected widiin a period of 1 hour before

dosing and die post dose samples was generally collected widiin two

minutes of the scheduled time. For each subject, the total number of blood

draws during the study were 36 and the total volume of blood drawn,

including 16 ml for screening and 18 ml ‘discarded’ blood prior to venous

cannula collections, did not exceed 214 ml.

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Experimental

4.23.9 Washout Period

There was a washout period of fourteen days between the administration

of test and reference products.

4.23.10 Restrictions4.23.10.1 M edication

Only subjects who had not received any medication including over the

counter (OTC) medications during die two weeks period prior to the onset

of the study were recruited. They were instixicted during screening not to

take any prescription and OTC medications subsequendy until tiie

complerion of die study.

4.23.10.2 Diet

AH subjects were instructed to abstain from any alcoholic products for 48

hours prior to dosing until completion of die study. They also abstmned

from any xanthine containing food or beverages during in-house stay in

each period

4.23.10.3 Activity

All subjects were dosed whUe seated and were instructed to remain seated

or ambulatory for the first two hours following each drug administration.

Thereafter, subjects were aUowed to engage only in normal activities while

avoiding severe physical exertion.

4.23.11 Selection of SubjectsAdequate number of subjects were selected randomly and were subjected

to a standardized screening procedure. Six healthy male subjects were

selected from the screened ones on die following inclusion and exclusion

criteria:

4.23.11.1 Screening Assessments

Medical histories and demograpliic data, including name, sex, age, body

weight (leg), height (cm) and tobacco use (including number of cigarettes

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Experimental

smoked per day) were recorded. Each subject underwent a physical

examination and die laboratory tests o f haematology, iiepatic and renal

functions as described in Table 4.8. Only medically healthy subjects with

clinically normal laboratory profiles were enrolled in the study.

Table 4,8: Labotatoiy Tests for Screening of Volunteers

HEMATOLOGY URINALYSIS ADDITIONALTESTS

Haemoglobin PHYSICAL I-lIV I & 11

Total leucocyte count Colour HBsAg

Differential leucocyte count Appearance HCV

Platelet count pH VDRL

Specific gravit)^ Urine drug screen

Protein Cannabinoids

Glucose Opioids

BIO-CHEMISTRY MICROSCOPICEXAMINATION

BUN RBC

Creatinine WBC

Total bilirubin E. CeUs

AUcaline phosphatase Crystals

AST Casts

ALT Otliers

Glucose

Cholesterol

102

4.23.11.2 Inclusion Criteria'

a) Be in the age range of 18-45 yrs.

b) Be neither overweight nor underweight for his height as per the Life

hisurance Corporation of India height/weight chart for non-medical

cases.

c) Have voluntarily given written informed consent to participate in this

study.

d) Be of normal health as determined by the medical history and physical

examination of tlie subjects performed widiin 28 days prior to the

commencement of the study.

4.23.11.3 Exclusion Criteria

a) History of allergy to itraconazole and/or related drugs.

b) Any evidence of organ dysfunction or any clinically significant

deviation from the normal, in physical or clinical determinations.

c) Presence of decease markers of HIV 1 and 2 , Hepatitis B and C viruses

and syphilis infection.

d) Presence of values which ate clinically significandy different from

normal reference ranges for haemoglobin, total white blood cells

count, differential WBC count and platelet count.

e) Positive for urinary screen testing of drugs of abuse (opiates and

cannabinoids).

f) Presence of values wliich are significandy different from normal

reference ranges for serum creatinine, blood urea, serum aspartate,

aminotransferase (ASl^, serum alanine aminotransferase (AUf), serum

alkaline phosphatase, seiTim bilirubin, plasma glucose and serum

cholesterol.

____________ ____________________ Experimental

103

E xperim enta l

g) Cliiiically abnormal chciiiical and microscopic examination of uiitie

clefmed as presence of RBQ W13C (>4/HPF), epiriieUa cells

(>4/HPF), glucose (positiTC) and protein (positive).

h) Clinically abnormal ECG and C]hest X-ray.

i) I-Iistory of serious gastrointestinal, hepatic, renal, cardiovascular,

pulmonary, neurological or haematological disease, diabetes or

glaucoma.

j) History of any psychiatric illness, which may impair the ability to

provide, written informed consent.

k) Regidar smokers who smoke more than 10 cigarcttes daily or have

difficulty abstaining from smoking for the duration of each study

period.

1) History of drug dependence or exxessive alcohol intake on a habitual

basis of more than two units of alcohoUc beverages per day (one unit

equivalent to half pint of beer or one glass of wine or one measure of

spirit) or have difficulty in abstaining for the duration of each study

period.

m) Use of any enzyme modifying ckugs within 30 days prior to day one of

this study,

n) Participation in any clinical trial widiin 12 weeks preceding day one of

tiiis smdy.

4.23.12 Safety4.23.12.1 C linical Safety m easurem ents

Vital signs of oral temp, sitting blood pressure and radial pulse were

measured during subject admission, prior to dosing and 2 , 8 and 12 hours

after administration of stxidy drug and before discharge in each period.

Clinical exairrinarion of tiie subjects was conducted by a qualified medical

designate on duty after subject admission, prior to dosing of study drug

and before discharge.

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Experimental

4.23.13 Handling of Safety Parametets

4.23.13.1 Adverse Events

The Clinical Pharmacologist or a Medical Officer was available at the site

of investigation until 24 hours post-dose during each period. Subjects were

monitored throughout tlie study period for adverse events. Subjects were

advised to bring to the notice of the nurse or the doctor any adverse event

diat may occur during dieir stay at die site of investigation. Subjects were

also specifically asked about any adverse events tliroughout the study

period every four hours.

4.23.14 Statistical AnalysisStatistical and pharmacoldnetic parameters were calculated using die

WinNonlin Pharmacoldnetic software, ANOVA and correlation analysis

was applied for pharmacoldnetic parameters.

4.23.15 DeviationsThere was no deviation during the study from the Protocol approved by

the Ediical committee.

4.23.16 Ethical Consideration4.23.16.1 Basic Principles

The study was carried out as per ICH (Step 5), 'Guidance for Good

Clinical Practice’ and the principles enunciated in die Declaration of

Helsinld (South Africa 1996).

4.23.16.2 Institutional Review Board

l l i e protocol and the corresponding informed consent form (IGF) used to

obtain informed consent of the study subjects was reviewed and approved

by the Jamia Hamdard Institutional Review Board (IllB).

4.23.16.3 Informed Consent

The purpose of the study, die procedure to be carried out, potential

hazards and rights of the subjects were described to the subjects in non-

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Experimental

technical terms, through an oral presentation. Subjects were also required

to understand and sign a consent form summarising the discussion prior tc:>

admission for the study in Period-I.

4.23.16.4 Dfop-out/ W ithdrawal of Subjects from study

Subjects were informed that they were free to dropout from die study at

any time without stating any reason. It was also informed that the

investigator may withdraw a subject from the study for any of the

following reasons;

a) The subject suffers from significant intercurrent illness or undergoes

surgery during the course of the study.

b) The subject experiences adverse e\'ent, when withdrawal would be in

die best interest of the subjects.

c) The subject fjuls to comply with the rec[uirements of the protocol.

T’his would include pre-study directions regarding alcohol and drug

use, fasting or if the subject is uncooperative during the study.

106

Results & Discussion

Fig 5.3: U V Spectra o f Itraconazole d issolved in m ethanol

Fig 5.4: U V Spectra o f Itraconazole dissolved in m ethanolic H Cl

1.4 D ifferential Scann ing C alorim etry

DSC] fhcrm ogram s o f irraconitzolc drug sam ple and rcfcrcncc standard were

o ljta incd in the tem perature range o f 40 to 4(K)°C] and arc shown in f ig 5.5 and

5.6, respectively.

Both the sam ples show ed shatp endotherm ic peaks at about 167°C] w liich is

indicative o f their m elting points. The results arc sim ilar to those reported in

tlic literature (VX'ang, et. al., 2004; V'erreck, ct. al., 2003).

KJ9