Will Shilajit Capsules Help To Cure Weakness And Fatigue Problem?
Experimental - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/42502/9/08... · 2018-07-03 ·...
Transcript of Experimental - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/42502/9/08... · 2018-07-03 ·...
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
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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.
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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
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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.
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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.
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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).
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