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Chapter 5 Formulation and evaluation of self-

emulsifying formulations of itraconazole using pharmaceutically

accepted lipid-based excipients Page no. 157 to 191

Chapter 5 Itraconazole

Page 157

Chapter 5 Formulation and evaluation of self-emulsifying formulations of

itraconazole using pharmaceutically accepted lipid-based excipients Abstract

Poorly water-soluble drug, itraconazole offers a challenge in developing a drug product

with adequate bioavailability. The potential for lipidic self-emulsifying drug delivery

system to improve the oral bioavailability of itraconazole was investigated in fasted rats. A

series of itraconazole SEDDS were prepared in three groups based on oil:S/CoS mixture

ratios (1:9, 1:6 and 1:4) using Capryol 90 (oil), a mixture of Labrasol and Tween 20

(surfactants) and Transcutol P (co-surfactant). The multi-component delivery systems

were optimised by evaluating their ability to self-emulsify when introduced in simulated

gastric fluid, without pepsin, under gentle agitation and by determination of droplet size

and visual observation. Three formulations (F5, F21 and F27), one from each group were

selected based on droplet size and visual observation. Pseudo ternary phase diagram was

constructed to identify the efficient self-emulsification region. The effect of oil and

surfactants content on mean globule size of resulting emulsions was studied. The

optimized formulations were assessed for dissolution, in vitro diffusion, ex vivo stomach

and intestinal permeability along with the marketed capsule. From the data obtained in this

work, it was clear that oil to surfactant and co-surfactant ratio has the main impact on the

droplet size of the emulsion formed after dilution. However, the relation between droplet

size and in vivo absorption of itraconazole was futile. The order of AUC was 1:4 > 1:6 >

1.9 for oil:S/CoS mixture ratios. Following an oral administration of developed SEDDS

and marketed capsule to rats, a 1.7, 2.0 and 2.33-fold increase in bioavailability was

observed for F5, F21 and F27 respectively, compared with marketed capsule. The results

from this study demonstrate the potential use of SEDDS to provide an effective way of

improving oral absorption of lipophilic drugs.


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5.1. Introduction

In recent years, major advances in healthcare have led to an unwelcome increase in the

number of life-threatening infections due to true pathogenic and opportunistic fungi

(Richardson, 2005). Recently, there have been an increasing number of profound fungal

infections caused by fungi such as those belonging to the genus Candida, the genus

Aspergillus and the genus Cryptococcus. This is a particular complication encountered in

transplant patients, those administered a large quantity of antibiotics, anticancer drugs

(carcinostatic) or a steroidal agents over a long period, AIDS patients, or those suffering

from cancer in the terminal stage, and those with hematological malignancies undergoing

intensive chemotherapy and/or bone marrow transplantation (Bodey, 1992). Invasive

fungal infections are a major cause of morbidity and mortality in patients receiving bone

marrow transplants for leukemia as well as in immunocompromised cancer patients

(Anaissie, 1992).

Itraconazole (ITZ) is a broad spectrum antifungal agent and belongs to trizole group

indicated in the treatment of local and systemic fungal infections (Grant and Clissold,

1989).

Itraconazole is a synthetic anti-fungal drug, which is composed of a 1:1:1:1 racemic

mixture of four diastereomers (two enantiomeric pairs). Three chiral centers are present in

each diastereomer which possess a molecular formula of C35H38Cl2N8O4 and molecular

weight of 705.64 g/mol (Grant and Clissol, 1989; Jain and Sehgal, 2001). Itraconazole is

poorly soluble in aqueous media (less than 1 µg/mL in aqueous solutions at pHs of 1-12.7)

with a partition coefficient greater than 5 in octanol/water at pH 6. Thus, in spite of the

high antifungal activity, the bioavailability of unformulated crystalline itraconazole is

extremely low (Jung et al., 1999; Verreck et al., 2003). In order to enhance its solubility

and dissolution rate and thereby bioavailability, various formulations have been developed

and are briefly reviewed in Section II.

Histoplasmosis is a serious opportunistic infection in patients with AIDS and itraconazole

has demonstrated activity against Histoplasmosis capsulatum (Vyas and Bradsher, 2011).

Itraconazole is a weak base (pKa = 3.7) with high lipophilicity (log D > 5) (Peeters et al.,

2002) Its aqueous solubility is estimated at approximately 1 ng/mL at neutral pH and

approximately 4 μg/mL at pH 1, (Peeters et al., 2002) and thus it is ionized and water

soluble only at low pHs, as in gastric juice.


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Itraconazole is available as capsules or an oral solution, and doses of 200 mg daily or 200

mg twice a day are used therapeutically (Barone et al., 1993; Physician Desk Reference,

2000). Because it has relatively low bioavailability after oral administration, especially

when given in capsule form (Barone et al., 1998; Heykant et al., 1989), gastric acidity is

required for drug dissolution and adequate absorption with capsule formulations.

Decreased absorption has been observed in the fasting state and in patients with low

gastric acidity (Barone et al., 1993). Furthermore, in persons with AIDS, the absorption of

itraconazole from capsules was reduced by 50% compared with absorption in healthy

persons (Smith et al., 1992). Hypochlorhydria and thus a relatively high gastric pH, a

frequent complication of human immunodeficiency virus (HIV) infection, may be

responsible for this phenomenon (Welage et al., 1995).

Co-administration of acidic beverage with itraconazole may be an effective approach in

improving the bioavailability of itraconazole in patients with who are hypochlorhydric, as

AIDS patients (Lange et al., 1997a) or who are taking gastric acid suppressant (Lange et

al., 1997b). Higher consumption of sugar-sweetened beverages (like coca cola) is

associated with weight gain and increased risk for the development of type 2 diabetes

mellitus, and caffeine is a psychoactive substance. In such situation, co-administration of

vitamin C beverage and itraconazole may be useful for achieving maximum absorption of

itraconazole in patients with drug- or disease-related gastric hypochlorhydria (pH > 4)

(Bae et al., 2011). In addition, itraconazole has been also found to be a substrate of P-

glycoprotein (P-gp) (Balayssac et al., 2005; Wang et al., 2002). P-gp activity is an

important mechanism that decreases the oral bioavailability of drugs by limiting intestinal

absorption (Greiner et al., 1999; Yamaguchi et al., 2002).

In light of the aforementioned discussion, we explored the potential application of lipid-

based drug delivery system in the development of a self-emulsifying drug delivery system

(SEDDS) of itraconazole. SEDDS formulations containing itraconazole were developed

using different proportions of oil and surfactants (GRAS excipients) for oral

administration. Systems were evaluated for the quality of emulsion produced and mean

droplet size. Optimized formulations were further evaluated for dissolution, ex vivo

stomach and intestinal permeability across rat tissues and in vivo performance in rats.

Physicochemical properties of SEDDS and pharmacokinetic parameters were evaluated in

comparison to Itaspor® capsules


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The objective of the investigation was to develop and characterize the self-emulsifying

drug delivery systems of poorly water soluble drug, itraconazole with a view to improve

its solubility, dissolution rate , ex vivo permeation and in vivo absorption in animal model.

The formulations were developed by employing excipients with established long term

acceptability. The impact of excipients ratios and droplet size on bioavailability was also

studies for all the developed formulations.

5.2. Materials and methods

5.2.1. Materials

Itraconazole (ITZ) was a generous gift from Matrix Laboratories Ltd. (Hyderabad, India);

Isopropyl myristate (IPM), Propylene Glycol Dicaprylate/Dicaprate (Captex® 200) and

Glycerol Caprylate Caprate (Captex® 355) were obtained from Subhash Chemical

Industries (Pune, India); propylene glycol monocaprylate (Capmul MCM®), Capmul

MCM (8) were obtained from Abitec Corp., Janesville, WI. Propylene glycol

monocaprylate (CapryolTM 90), PEG-8 glycol caprylate (Labrasol®) and diethylene glycol

monoethyl ether (Transcutol P®) were provided by Gattefosse, France. PEG-35 castor oil

(Cremopho EL®) was obtained from BASF Corp., Germany. Glyceryl caprylate (Imwitor®

988) was provided by Sasol GmbH, Witten, Germany. PEG-20 sorbitan monolaurate

(Tween 20) and Tocopherol acetate were purchased from Merck (Mumbai, India).

Propylene glycol was purchased from Spectrochem (Mumbai, India). Capsule shells were

provided by Capsugel, Mumbai. Ketoconazole was purchased from HIMEDIA. All other

chemicals and solvents used were of analytical grade.

5.2.2. Solubility studies

The solubility of itraconazole in various oils, surfactants and co-surfactants was

determined. An excess amount of itraconazole was added to 5 ml of each selected oils and

surfactants and was shaken reciprocally at 25oC for 24 hrs. The supernatant portion of the

supersaturated solution was carefully withdrawn and suitably diluted with methanol, and

solubility of itraconazole was determined using HPLC (Jasco, Japan) at 263 nm (Shim et

al., 2006; Yang et al., 2008; Yoo et al., 2000). The HPLC system consisted of RP column

(LCGC Qualisil BDS C18; 5 µm 250 mm x 4.6 mm i.d) and acetonitrile: water (90:10) as

a mobile phase. A 40µL volume was injected into the column at a flow rate of 0.7mL/min


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5.2.3. Pseudo-ternary Phase Diagrams

The pseudo-ternary phase diagrams were constructed using water dilution method

(Djordjevic et al., 2004). Capryol 90 was used as the oil phase, mixture of Labrasol and

Tween 20 as the surfactant and Transcutol P as the co-surfactant. Phase diagrams were

constructed by a technique well described (Kommuru et al., 2001; Nazzal et al., 2002;

Taha et al., 2004) with ratios as mentioned in Table 5 and all preparations were diluted

100 times with 0.1N HCl. For each phase diagram at specific oil:Smix ratios (1:9, 1:6 and

1:4), mixtures of the oil, the surfactant and the co-surfactant were prepared, and the

mixture was diluted with 0.1N HCl, maintained at 37°C. Dilution medium was added drop

by drop while mixing on a cyclomixer, and the samples were marked as being optically

clear or turbid. Phase diagrams were then constructed using Tri plot v1-4 software (David

Graham and Nicholas Midgley, Loughborough, Leicestershire, UK).

5.2.4. Preparation of itraconazole self-emulsifying formulations

A series of itraconazole SEDDS were prepared with fixed concentration of itraconazole

(25 mg) and varying concentrations of Capryol 90, Labrasol, Tween 20 and Transcutol.

Itraconazole was dissolved in Capryol 90 and then mixture of Labrasol, Tween 20 and

Transcutol were accurately weighed and added to itraconazole oily solution. The mixture

was slightly heated on water bath for 5-10 mins at 60°C until a transparent preparation was

obtained.

5.2.5. Characterization of Self emulsifying formulation

5.2.5.1. Visual observation and droplet size determination

0.05 mL formulations were diluted in 5 mL of dilution media (0.1N HCl) maintained at

37°C, 100 times dilution. Visual observation of the samples was recorded as previously

reported (Craig et al., 1995; Khoo et al., 1998). Globule Size of the same diluted samples

was measured by Zetasizer ZS 90 and measurements are shown in Table 5.3.

5.2.5.2. Characterization of selected formulations

Droplet size, polydispersity index and zeta potential were measured on Zetasizer ZS 90

after 100 times dilution in different media viz. Distilled water (DW), 0.1N HCl and

phosphate buffer saline pH 6.8 (PBS) maintained at 37°C. Drug content was measured by

HPLC.


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Formulations were also evaluated for dissolution study, in vitro diffusion study, ex vivo

stomach and ex vivo intestinal permeability study in rats. In vivo study was also carried out

for developed itraconazole SEDDS and extemporaneous suspension of market capsule.

5.2.5.3. Drug content

SEDDS formulation equivalent to 25 mg of itraconazole was taken and diluted in

methanol. Volume was made up to 25 mL with methanol (1mg/mL). 0.2 mL was

withdrawn from the above solution and to it 4.8 mL of methanol was added and mixed

well. 5 mL of 0.1 N HCl was added to above mixture and mixed well (20 µg/mL).

Samples were prepared in triplicate and absorbances were taken at 258 nm on UV visible

spectrophotometer (Shimadzu UV-2450, Japan) keeping equal mixture of methanol and

0.1N HCl (1:1) as a reference. Placebo was also treated in the same way to check

interference, if any.

5.2.5.4. Spectroscopic characterization of optical clarity

The optical clarity of aqueous dispersions of SEDDS formulation was measured

spectrophotometrically. Composition was prepared according to the design and diluted to

100 times with 0.1N HCl (USP 30) (USP 30/NF25). The % transmittance of solution was

measured at 650 nm, using distilled water as a reference (Cirri et al., 2007; Subramanian et

al., 2004; Zhang et al., 2008).

5.2.5.5. Morphological characterization

The morphology of self emulsifying formulation was observed by using a transmission

electron microscope (TEM) (Phillips Tecnai 20, Holland) at an acceleration voltage of 200

kV and typically viewed at a magnification of 43,000×. The size of the colloidal structures

was determined using AnalySIS® software (Soft Imaging Systems, Reutlingen, Germany).

Formulations were diluted with distilled water 1:25 and shaken. Carbon-coated copper

grids were glow-discharged (Edwards E306A Vacuum Coater, England) and 10 µL of

sample adsorbed on to these holey film grid and observed after drying.

5.2.5.6. Determination of droplet size and zeta potential

Droplet size and zeta potential of the formed emulsion were determined by photon

correlation spectroscopy that analyzes the fluctuations in light scattering due to Brownian

motion of the particles, using a Zetasizer ZS 90 (Malvern Instruments, UK). Light

scattering was monitored at 25°C at a 90° angle.


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5.2.5.7. Dissolution study

Dissolution studies of itraconazole capsule and itraconazole SEDDS filled in hard gelatine

capsule (size # “000”) was carried out using USP XXIII Dissolution apparatus I (basket

type) at speed of 50 rpm in 900mL for 100 mg marketed capsule and 225 mL for 25 mg

SEDDS 0.1 N HCl at 37 ± 0.5 °C. Aliquots of 5 mL were removed at 2, 5, 10, 15, 30, 45,

60, 75 and 120 min. Volume of aliquots was replaced with fresh dialyzing medium each

time. These samples were analyzed quantitatively for amount of itraconazole released at

corresponding time by using UV-visible spectrophotometer (Shimadzu UV-2450, Japan)

at 255 nm.

5.2.5.8. In vitro diffusion study

In vitro diffusion studies were performed for the formulations developed (F5, F21 and

F27), using a dialysis technique (Dixit et al., 2010; Kang et al., 2004; Patil et al., 2004).

The dialyzing medium was 0.1N HCl. One end of dialysis tubing (Dialysis membrane 70,

HIMEDIA; MWCO 12,000-14,000 daltons; pore size: 2.4 nm) (7 cm in length) was

clamped and then 1.1 mL (≡25 mg of drug) of self-emulsifying formulation was placed in

it. The other end of the tubing was also secured with dialysis closure clip (HIMEDIA,

Mumbai) and was allowed to rotate freely in 225 mL of dialyzing medium and stirred

continuously with magnetic bead on magnetic plate at 37°C. Aliquots of 5 mL were

removed at different time intervals. Volume of aliquots was replaced with fresh dialyzing

medium each time. These samples were analyzed quantitatively for itraconazole dialyzed

across the membrane at corresponding time by using UV-visible spectrophotometer at 255

nm.

For marketed formulation (Itaspor®-INTAS, pellets in capsule), capsules were crushed in

mortar with pestle. From the crushed pellets, blend equivalent to 25 mg (113.975 mg

blend) was weighed and made suspension by adding 3 mL of DW and was filled in

dialysis membrane. The membrane was sealed from both the end with the help of dialysis

closure clips and was placed in 225 mL of 0.1N HCl

5.2.5.9. Ex vivo stomach permeability study

Male Sprague-Dawley rats (250-300 g) were euthanized in carbon-dioxide vacuum

chamber. All experiments and protocols described in this animal study were approved by

the Institutional Animal Ethics Committee of B V Patel PERD Centre, Ahmedabad, India

and were in accordance with the guidelines of the Committee for Purpose of Control and


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Supervision of Experiments on Animals, Ministry of Social Justice and Empowerment,

Government of India. To check the stomach permeability, the stomach part was isolated

carefully and taken for the ex vivo permeation study. Then this tissue was washed with

physiological acid solution containing 100 mM HCl and 54 mM NaCl to remove the

mucous and gastric contents. Then the tissue was rinsed with 100 mM HCl. The method

employed was modified from experimental procedures well described in the literature

(Gharzouli et al., 1995; Hung and Neu, 1997; Shah and Khan, 2004).

SEDDS formulations, 0.4 mL (equivalent to 10 mg of drug) were injected into the

stomach and tissues were tightly closed with the help of threads. Similarly 2 mL

extemporaneous suspension of market capsule (Itaspor®-INTAS) equivalent to 10 mg of

drug was filled in stomach tissue and was placed in a beaker with constant stirring and

temperature of 37° C on a magnetic stirrer. The receiver compartment was filled with 30

mL of 100 mM HCl. The absorbance was measured at 255 nm, keeping the respective

blank. The percent permeation of drug was calculated against time and plotted on a graph.

5.2.5.10. Ex vivo intestinal permeability study

This was carried out by the method well described in the literature (Araya et al., 2006;

Ghosh et al., 2006). Male Sprague-Dawley rats (250-300 g) were euthanized in carbon-

dioxide vacuum chamber. All experiments and protocols described in this animal study

were approved by the Institutional Animal Ethics Committee of B V Patel PERD Centre,

Ahmedabad, India and were in accordance with the guidelines of the Committee for

Purpose of Control and Supervision of Experiments on Animals, Ministry of Social Justice

and Empowerment, Government of India. To check the intestinal permeability, small

portion of small intestine was isolated and used for the ex vivo permeability study. The

tissue was thoroughly washed with pH 6.8 phosphate-buffered saline (USP 30) (USP

30/NF25) to remove any mucous and lumen contents.

One mL SEDDS formulations were diluted to 5 mL with distilled water (outside mixing

for 1 minute by vortex mixer), the resultant sample (5 mg/mL) was injected into the lumen

of the duodenum using a syringe, and the two sides of the intestine were tightly closed

with the help of threads. Extemporaneously suspension was prepared from market capsule

(Itaspor®-INTAS), and 1 mL equivalent to 5 mg of drug was filled in stomach tissue

similar to that of SEDDS formulations. Then the tissue was placed in a beaker with

constant stirring and temperature of 37°C on magnetic stirrer. The receiver compartment


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was filled with 30 mL of phosphate-buffered saline containing 20% PEG 400. The

absorbance was measured at 258 nm, keeping the respective blank. The percent diffusion

of drug was calculated against time and plotted on a graph.

5.2.6. HPLC analysis

See Chapter 4 Sec. 4.2.6. HPLC method development and validation

5.2.7. Bioavailability studies

Bioavailability studies were performed in male Wistar rats weighing 280 to 350 g. All

experiments and protocols described in this study were approved by the Institutional

Animal Ethics Committee of Sri Dhanvantary Pharmaceutical Analysis and Research

Centre, Surat, India and were in accordance with guidelines of the Committee for Purpose

of Control and Supervision of Experiments on Animals, Ministry of Social Justice and

Empowerment, Government of India. Two groups were made for the study, and four rats

were kept in each group. The animals were kept under standard laboratory conditions,

temperature at 25 ± 2°C and relative humidity (55 ± 5%). The animals were housed in

polypropylene cages, four per cage, with free access to standard laboratory diet (Lipton

feed, Mumbai, India) and water ad libitum. The animals were fasted overnight prior to the

experiment but had free access to water.

The formulations (SEDDS and extemporaneous suspension prepared from marketed

capsule (Itaspor®)) were given orally using feeding snode. Dose for the rats was selected

as reported (Shin et al., 2004; Van Cauteren et al., 1987) and calculated based on the

weight of the rats (30mg/kg body weight) according to the surface area ratio (Akhila et al.,

2007; Paget and Barnes, 1964). The animals were anesthetized using ether and blood

samples (approximately 500 μL) were collected from the retro-orbital vein using a

heparinized needle (18-20 size) at 0 (pre-dose), 1, 2, 4, 8, 12, 16 and 24 hours after oral

administration of formulations. The blood samples were collected into a vacutainer tube

(13 x 75mm, 2mL, Accuvac, BD, NJ), mixed and centrifuged on a laboratory centrifuge at

5000 rpm for 20 min at ambient temperature. The supernatant plasma was carefully

separated and kept at –20°C (Thermo Scientific, USA) until analysis was carried out using

validated HPLC.

5.2.7.1. Plasma analysis

Frozen plasma samples were thawed just prior to extraction. A 100 µL of plasma sample

was transferred in 2 mL centrifuge tube (Tarson, Kolkata, India). To that 300 µL of


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acetonitrile containing Ketoconazole as an internal standard (Vaughn et al., 2006) (200

ng/mL in acetonitrile) was added and vortex-mixed for 1 min. The tube was centrifuged at

15,000 rpm for 20 min at 4° C. The organic layer was carefully decanted and was

transferred to clean tube and dried under nitrogen stream at ambient temperature. The

residue was reconstituted with a 100 µL aliquot of mobile phase, consisted of a mixture of

5 mM sodium phosphate buffer pH 5.7 and ACN (38:62 v/v) and 50 µL was injected

directly onto the HPLC column at a flow rate of 1.0 mL/min.

Plasma concentration versus time data of itraconazole for rats was analyzed using standard

non-compartment analysis. The area under the plasma concentration-time curve (AUC0→t)

from zero to 24 hour was estimated by the linear trapezoidal method (Han and Lee, 1999;

Lee and Ku, 1999). The relative bioavailability (F) of SMEDDS to the suspensions was

calculated using the following equation:

F = (AUCtest /AUCreference) × 100%

5.2.8. Statistical analysis

Statistical analysis for the determination of differences in diffusion, permeability and in

vivo absorption profiles of itraconazole SEDDS and the marketed preparation was

assessed by the use of Student’s t-test. The pharmacokinetic data between different

formulations were compared for statistical significance by Student’s t-test. Statistical

probability (P) values less than 0.05 were considered significantly different.

5.2.9. Stability studies

Chemical and physical stability of itraconazole SEDDS (F5, F21 and F27) were assessed

under various storage conditions namely room temperature (RT), 30±2°C/65±5% RH and

40±2°C/75±5% RH as per ICH guidelines (ICH Q1A(R2)) in ICH certified stability

chambers (Humidity Chamber, EIE Instruments Ltd., Ahmedabad, India).

Itraconazole SEDDS equivalent to 25 mg was filled in a glass vial with a rubber closure

and aluminium-crimped tops. Eight such glass vials of each F5, F21 and F27 were filled

and stored at various aforementioned storage conditions up to 3 months. Samples were

removed at 0, 1, 2 and 3 months of interval and checked for itraconazole content (by

HPLC), droplet size, polydispersity index and zeta potential after diluting formulation to

100 times with three different media viz. Distilled water (DW), 0.1N HCl and phosphate

buffer saline pH 6.8 (PBS) maintained at 37°C.


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5.3. Results and discussion

5.3.1. Screening of oil and surfactants

The solubilities of itraconazole in various oils and surfactants are presented in Table 5.1.

Among the selected oils that were screened, maximum solubility of itraconazole was

found in Capryol 90 followed by Captex 200, IPM and Captex 355. Among the surfactants

(Table 5.1), Transcutol P followed by Tween 20 showed reasonable solubilizing potential

for itraconazole. Transcutol P proved to be the best solublizer for itraconazole.

Before selecting Transcutol P as a co-surfactant, propylene glycol was also screened for its

efficiency in preparation of a good self emulsifying system. During the formulation

development of Nevirapine, it was found out that Labrasol has poor water uptake capacity

which was improved by adding Tween 80. In the case of itraconazole, Tween 20 was tried

for the development of self emulsifying system.

A series of trials were taken with three different ratios of Oil: Smix (1:9, 1:6 and 1:4).

Capryol 90 was selected as an oil phase, mixture of Labrasol and Tween 20 as a surfactant

and Transcutol P as a co-surfactant. The ratios 9:1, 1:1, 2:1and 4:1of surfactant to co-

surfactant were tried with 1:1, 1:2 and 1:3 ratios of Labrasol:Tween 20 as a surfactant

(Table 5.2).

Table 5.1: Solubility of itraconazole in various oils and surfactants at 25°C (n =3)

Oil /Surfactant Solubility (mg/mL) Isopropyl myristate 0.206 ± 2.07 Captex 200 1.008 ± 2.08 Captex 355 2.127 ± 1.97 Tocopherol acetate 5.22 ± 1.87 Capryol 90 22.132 ± 1.58 Tween 20 3.709 ± 1.04 Labrasol 7.147 ± 1.65 Transcutol 4.6 ± 1.48 Capmul MCM 7.4533 ± 1.15 Capmul MCM (8) 5.8483 ± 1.43 Imwitor 988 6.5708 ± 1.25 Cremophor EL 4.0015 ± 1.21 Propylene glycol 1.1804 ± 0.55


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Table 5.2: Formulation development trials

1 2 3 4 5 6 7 8 9 10 11 12 Oil:Smix 1:9 1:9 1:9 1:9 1:9 1:9 1:9 1:9 1:9 1:9 1:9 1:9 S/CoS 9:1 9:1 9:1 1:1 1:1 1:1 2:1 2:1 2:1 4:1 4:1 4:1 Lab+T20 1:1 1:2 1:3 1:1 1:2 1:3 1:1 1:2 1:3 1:1 1:2 1:3 ITZ(mg) 25 25 25 25 25 25 25 25 25 25 25 25 13 14 15 16 17 18 19 20 21 22 23 24 Oil:Smix 1:6 1:6 1:6 1:6 1:6 1:6 1:6 1:6 1:6 1:6 1:6 1:6 S/CoS 9:1 9:1 9:1 1:1 1:1 1:1 2:1 2:1 2:1 4:1 4:1 4:1 Lab+T20 1:1 1:2 1:3 1:1 1:2 1:3 1:1 1:2 1:3 1:1 1:2 1:3 ITZ(mg) 25 25 25 25 25 25 25 25 25 25 25 25 25 26 27 28 29 30 31 32 33 34 35 36 Oil:Smix 1:4 1:4 1:4 1:4 1:4 1:4 1:4 1:4 1:4 1:4 1:4 1:4 S/CoS 9:1 9:1 9:1 1:1 1:1 1:1 2:1 2:1 2:1 4:1 4:1 4:1 Lab+T80 1:1 1:2 1:3 1:1 1:2 1:3 1:1 1:2 1:3 1:1 1:2 1:3 ITZ(mg) 25 25 25 25 25 25 25 25 25 25 25 25 Smix = Mixture of surfactants and co-surfactant; Lab = Labrasol; T20 = Tween 20; ITZ = itraconazole

5.3.2. Characterisation of formulation development trials

5.3.2.1. Visual observation: The prepared self emulsifying systems of itraconazole were

diluted 100 folds with 0.1N HCl and checked for optical clarity. When the ratio of oil:Smix

was 1:2, prepared systems exhibited turbidity and they were dropped from the study.

Obviously, as the proportion of oil phase increases, the optical clarity of the formulations

becomes poor on dilution.

Table 5.3: Visual observation and droplet size of formulation development trials as shown in Table 5.2

Trial No.

Size (nm)

PdI Visual Observation

Trial No.

Size (nm)

PdI Visual Observation

1 149.2 0.159 Bluish Tinge 19 185.0 0.293 Slight Turbid 2 56.87 0.151 Poor quality 20 141.0 0.277 Bluish Tinge 3 98.77 0.309 Poor quality 21 95.39 0.4 Slight Bluish Tinge 4 162.6 0.248 Bluish Tinge 22 182.4 0.264 Bluish Tinge 5 118.2 0.172 Slight Bluish Tinge 23 121.0 0.208 Bluish Tinge 6 38.85 0.639 Poor quality 24 147.1 0.976 Poor quality 7 163.7 0.275 Bluish Tinge 25 200.3 0.401 Turbid 8 56.89 0.56 Poor quality 26 167.5 0.252 Bluish Tinge 9 18.13 0.6 Poor quality 27 130.2 0.156 Bluish Tinge

10 142.5 0.206 Bluish Tinge 28 546.1 0.392 Turbid 11 25.33 0.205 Poor quality 29 320.3 0.116 Turbid 12 53.14 0.403 Poor quality 30 255.0 0.265 Turbid 13 158.8 0.199 Bluish Tinge 31 452.6 0.136 Turbid 14 105.0 0.278 Slight Bluish Tinge 32 189.3 0.278 Bluish Tinge 15 40.68 0.256 Poor quality 33 180.0 0.239 Bluish Tinge 16 251.7 0.142 Turbid 34 360.9 0.206 Turbid 17 159.9 0.264 Bluish Tinge 35 205.5 0.341 Bluish Tinge 18 155.6 0.259 Bluish Tinge 36 203.1 0.331 Bluish Tinge


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When oil:Smix ratio was 1:9, S/CoS ratio of 9:1 with Labrasol to Tween 20 ratio of 1:1

gave bluish tinge. The effect of various ratios of surfactant to co-surfactant along with

different ratios of Labrasol to Tween 20 has been shown in the Table 5.3.

5.3.2.2. Droplet size measurement: The droplet size of formulations developed with

Oil:Smix ratios were in the range of 18.13 nm – 149.2 nm, 40.68 nm – 251.7 nm, 180 nm –

546.1 nm for 1:9, 1:6 and 1:4, respectively.

A comparative study of data obtained from the droplet size measurements and visual

observations were made. Those formulations which gave bluish tinge and good optical

clarity were selected. In the first group (Formulation trials 1-12), formulations 1, 2, 5, 7

and 10 gave bluish tinge with droplet size in the range of 118.2 nm – 149.2 nm. The

formulation 5 with lowest droplet size (118.2 nm) was selected. Similarly, in the second

group 8 formulations were compared for their droplet size and the formulation 21 with

lowest droplet size of 95.39 nm was selected. Formulation 27 with droplet size of 130.2

nm was selected among the 5 formulation with good optical clarity.

5.3.3. Construction of pseudo-ternary phase diagrams

The regions which showed bluish tinge were identified as transparent and isotropic

mixtures. The percentage of three different phases i.e. oil, water and the mixture of

surfactant and co-surfactant were calculated and plotted with the help of Tri plot v1.4

software for fabrication of ternary plot. The points (formulation trials) which showed

bluish tinge were joined with the line and were shaded (Figure 5.1). It can be seen that, the

formulations which showed bluish tinge in 1:9, 1:6 and 1:4 ratios of Oil:Smix were not

more than 203.1 nm. The formulation prepared within these regions (dark shaded) may

give droplet size in micron size.

Figure 5.1: Psuedoternary phase diagram of Oil to Smix ratios of 1:9, 1:6 and 1:4


Page 170

5.3.4. Characterization of selected formulations

The droplet size, polydispersity index and zeta potential of SEDDS formulation after 100

times dilution in different media is shown in Table 5.4. Size distribution by intensity in

various media for F5 is shown in Figure 5.2 (a), (b) and (c). Data shows that there is not

wide variation in size of droplets in distilled water and 0.1N HCl. The droplet size

increases in phosphate buffer. The size of droplets increases as the percentage of oil

increases. The droplet size follows the order of F27 > F21 > F5.

The polydispersity index is most poor in phosphate buffer. Zeta potential was also

measured of the same diluted samples and shown in the Table 5.4. Drug content of

SEDDS formulations was measured and is shown in Table 5.4. Table 5.4: Characterization of developed formulations

Droplet size D (nm) Polydispersity Index (PdI) Zeta potential (mV) Drug content (%) F5 F21 F27 F5 F21 F27 F5 F21 F27 F5 F21 F27

DW 417.3 201.0 194.4 0.147 0.226 0.211 -6.10 3.54 -7.15 101.06 ± 0.72

100.85 ± 0.44

100.54 ± 0.62 0.1NHCl 152.1 175.2 179.0 0.256 0.491 0.486 3.47 4.72 1.16

PBS 501.3 539.6 742.2 1.0 1.0 1.0 -2.11 -7.25 -3.16

Figure 5.2(a): Droplet size distribution of itraconazole SEDDS F5 after dilution in distilled water


Page 171

Figure 5.2(b): Droplet size distribution of itraconazole SEDDS F5 after dilution in 0.1N HCl

Figure 5.2(c): Droplet size distribution of itraconazole SEDDS F5 after dilution in phosphate buffer saline pH 6.8


2

Size distribution by intensity in various media for F21 are shown in Figure 5.3 (a), (b) and

(c).




Page 173


Size distribution by intensity in various media for F27 are shown in Figure 5.4 (a), (b) and

(c).



Page 174



The TEM picture is shown in Figure 5.5. The microemulsion droplets were observed to be

spherical and homogenous with large population of the smaller droplet in the size range of

less than 200 nm. The droplet size of formed emulsion determined by TEM is in

correlation with that of measured by photon correlation spectroscopy (PCS).


Page 175

Figure 5.5: Transmission electron microscopic image of itraconazole self emulsifying systems after dilution showing size of some oil globules. (Scale: 500 nm). (a) SEDDS F5, (b) SEDDS F21 and (c) SEDDS F27

Statistically there was significant difference in the dissolution profiles of all developed

formulations and market formulation at P < 0.05 and can be seen in Figure 5.6.

(a) (b)

(c)


Page 176

Figure 5.6: Dissolution profiles of itraconazole SEDDS and marketed capsule in 0.1N HCl (n=6)

In vitro diffusion profiles (Figure 5.7) shows that there was no significant difference

between the formulations with F5 & F21. For all other formulations there was a significant

difference in their diffusion profiles at P < 0.05

Figure 5.7: In vitro diffusion profile of itraconazole SEDDS and extemporaneous suspension of marketed capsule in 0.1N HCl (n=6)

-20

0

20

40

60

80

100

120

0 20 40 60 80 100 120 140

% C

umul

ativ

e dr

ug re

leas

ed

Time (min)

Dissolution profiles of SEDDS and Market Capsule in 0.1N HCl

SEDDS F5

SEDDS F21

SEDDS F27

MKT Capsule

0

10

20

30

40

50

60

0 1 2 3 4 5 6

% c

umul

ativ

e dr

ug d

iffus

ed

Time (Hours)

In vitro diffusion study of Itraconazole SEDDS and Market Capsule

SEDDS F5

SEDDS F21

SEDDS F27

MKT Capsule


Page 177

There was significant difference in stomach permeability between all other formulations at

P < 0.05, except F5 & F21 and F21 & F27 (Figure 5.8).

Figure 5.8: Ex vivo stomach permeability of itraconazole SEDDS and extemporaneous suspension of marketed capsule in 100 mM HCl (n=3)

Significant difference (P < 0.05) was also seen in ex vivo intestinal permeability of

developed SEDDS and marketed formulation except for formulation F5 and F21 (Figure

5.9).

Figure 5.9: Ex vivo intestinal permeability of itraconazole SEDDS and extemporaneous suspension of marketed capsule in pH 6.8 PBS (n=3)

0

5

10

15

20

25

30

35

0 0.5 1 1.5 2 2.5 3 3.5

% c

umm

ulat

ive

dru

g pe

rmea

ted

Time (Hours)

Stomach permeability study of Itraconazole SEDDS and Marketed Capsule

SEDDS F5

SEDDS F21

SEDDS F27

MKT Capsule

0

10

20

30

40

50

0 1 2 3 4 5 6

% c

umul

ativ

e dr

ug p

erm

eate

d

Time (Hours)

Intestinal permeability study of Itraconazole SEDDS and Market Capsule

SEDDS F5

SEDDS F21

SEDDS F27

MKT Capsule


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5.3.5. Bioavailability Studies

Typical chromatograms obtained showing peaks for ITZ at 1 h after administration of

marketed suspension and SEDDS F5, F21 and F27 are shown in Figure 5.10 (a) 5.10(b),

5.10(c) and 5.10(d), respectively. Table 5.5 summarises mean pharmacokinetic parameters

for various formulations.

Figure 5.10(a): Typical chromatogram obtained while analyzing plasma for ITZ level after administration of the extemporaneous suspension of marketed capsules at 1 h


Page 179

Figure 5.10(b): Typical chromatogram obtained while analyzing plasma for ITZ level after administration of the SEDDS F5 at 1 h

Figure 5.10(c): Typical chromatogram obtained while analyzing plasma for ITZ level after administration of the SEDDS F21 at 1 h


Page 180

Figure 5.10(d): Typical chromatogram obtained while analyzing plasma for ITZ level after administration of the SEDDS F27 at 1 h

Figure 5.11 depicts the plasma concentration profile of itraconazole after oral

administration of SEDDS and extemporaneous suspension of marketed capsule to rats.

Figure 5.11: Plasma concentration profile of itraconazole after oral administration of SEDDS and extemporaneous suspension in rats (n=4 and 30 mg/kg)

0

50

100

150

200

250

300

350

400

450

0 5 10 15 20 25 30Itra

cona

zole

pla

sma

conc

entr

atio

n (n

g/m

ml)

Time (Hours)

Marketed Capsule

SEDDS (F5)

SEDDS (F21)

SEDDS (F27)


Page 181

There was no significant difference in AUC0→t for extemporaneous suspension with F5

and F21, but F27 was statistically significant different from extemporaneous suspension.

AUC0→∞ of F5 and F27 was significantly different from extemporaneous suspension, but

there was no significant difference between extemporaneous suspension and F21. Cmax of

extemporaneous suspension and F5 was not significant different, but there was a

significant difference in Cmax of extemporaneous suspension with F21 and F27.

The relative bioavailabilities of itraconazole SEDDS F5, F21 and F27 were 170.56%,

204.55% and 233.91%, respectively when compared to extemporaneous suspension. It

was found that there was no significant difference between F5, F21 and F27 with regard to

AUC0→t, AUC0→∞ and Cmax.

The three self emulsifying formulations were prepared, containing varying amounts of oil

and surfactants and as mentioned previously the droplet size followed the order F27 > F21

> F5. However, the formulations developed in this study contained varying concentrations

of oil and surfactants. The observed bioavailability indicated that there was little

correlation of AUC and Cmax with respect to droplet size of formed emulsions. The

observed results are in agreement with few researchers groups in different laboratories.

Yap & Yuen (2004) observed that droplet size difference (1.5 and 10.6 µm) between two

SEDDS containing varying amounts of soybean oil, Tween 80 and Labrasol did not affect

the bioavailabilities of tocotrienols.

In several cases avoidance of drug precipitation could be the predominant factor governing

improvement of oral bioavailability from lipid vehicles than the size of the droplet size. It

has been well documented that the influence of droplet size on bioavailability has been

observed for several molecules for e.g. Vitamin (Julianto et al., 2000), cyclosporine (Trull

et al., 1995), and halofantrine (Khoo et al., 1998); while it is limited for some others, e.g.,

atovaquone (Sek et al., 2006), danazol (Porter et al., 2004), and ontazolast (Hauss et al.,

1998). A very limited number of studies have focused on the influence of the

particle/droplet size of comparable formulations on the absorption and bioavailability.

The formulation F5 showed least particle size having maximum amount of surfactant.

Contrary to expectations that smaller particle size increases absorption, this did not lead to

higher bioavailability. Formulation F21 with slightly bigger droplet size compared to F5

showed higher bioavailability (204.55%). The maximum bioavailability (233.91%) was

shown by F27 with least concentration of surfactant and maximum concentration of oil.


Page 182

The study revealed that the bioavailability decreased when concentration of oil was

decreased from 17.27% to 8.77%.

Formulation F5 exhibited faster absorption and higher Cmax but lower bioavailability

(170.56%) compared to F21 (204.55%) and F27 (233.91%). This indicates that having a

highly dispersed system improves absorption rate which is due to higher amount of

surfactants, but is not enough to ensure higher bioavailability (Nielsen et al., 2008).

It has been observed and reported that lipid systems which contain more amount of

surfactant or hydrophilic excipients are more prone to drug precipitation. Using such

excipients may improve solvent capacity, drug loading and in vitro performance but may

decrease in vivo absorption due to precipitation of drug on oral administration (Pouton,

2006).

The role of droplet size in the performance of the formulation in vivo is generally less

important than the assumption. The possible reason for this is that as soon as the dispersed

formulation leaves the stomach it encounters digestive power of the small intestine. The

fate of the drug after the formulation has been digested is more important than the initial

particle size. Esters can be rapidly hydrolyzed in the presence of pancreatic lipase and

most commonly used surfactants are ethoxylated esters that are rapidly hydrolyzed. The

physical state of the degradation products will be changed significantly by contact with the

mixed bile salt micelles and the drug will partition between the various phases in the gut

lumen, or could precipitate out if the total solvent capacity is reduced as a result of

lipolysis (Pouton, 2006).

Table 5.5: Relative bioavailability and pharmacokinetic parameters of itraconazole after oral administration of itraconazole SEDDS and extemporaneous suspension to the rats (n=4)

MKT SEDDS (F5) SEDDS (F21) SEDDS (F27) AUC0→t (ng.h/mL)

3037.268 ± 198.86

4111.651 ± 1567.14

3521.983 ±1030.00

4664.412 ± 228.138

AUC0→∞ (ng.h/mL)

5264.334 ± 1705.79

8978.953 ± 3191.02

10768.64 ± 8767.113

12314.27 ± 5551.154

Cmax (ng/mL) 171.956 ± 5.715 276.80 ± 138.645

243.499 ± 44.90 304.848 ± 78.942

Tmax (h) 1.0 ± 0.0 1.5 ± 0.577 1.5 ± 0.577 1.5 ± 0.577 Relative bioavailability (%)

--- 170.56 204.55 233.91


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5.3.6. Stability studies

No change in the physical parameters such as homogeneity and clarity was observed

during the stability studies. Droplet size, polydispersity index and zeta potential remained

unchanged, as compared to initial characterization at 0 month (Table 5.4), even after

keeping the formulations at different environmental storage conditions for 3 months,

indicating the physical stability of developed formulations (Table 5.6, 5.7 and 5.8). No

decline in the drug content was observed at the end of 3 months indicating that drug

remained chemically stable in SEDDS.

Table 5.6: Stability studies of itraconazole SEDDS F5 at various storage conditions Storage conditions → 25 ± 2°C (RT) 30 ± 2°C/65 ± 5% RH 40 ± 2°C/75 ±

5%RH Parameters ↓ Medium↓ 1 M 2 M 3 M 1 M 2 M 3 M 1 M 2 M 3 M Droplet Size D (nm)

DW 389.21 372.83 380.41 379.19 389.17 386.45 397.5 386.3 388.47

0.1N HCl 134.35 146.49 149.83 144.9 144.39 151.38 146.3 144.3 147.35

PBS 448.29 459.82 451.96 439.33 479.7 455.26 487.4 477.32 472.21

Polydispersity index (PdI)

DW 0.152 0.153 0.162 0.182 0.145 0.167 0.224 0.314 0.287

0.1N HCl 0.189 0.235 0.226 0.211 0.244 0.212 0.263 0.273 0.278

PBS 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Zeta Potential (mV)

DW -8.3 -4.62 -6.43 -6.42 -5.72 -6.85 -7.75 -6.82 -7.05

0.1N HCl 5.32 4.28 6.19 4.75 5.39 6.21 5.73 6.71 6.62

PBS -3.29 -3.81 -4.21 -3.64 -5.39 -6.63 -3.49 -4.62 -5.26

Drug Content (%) 104.54 ± 1.6

102.02 ± 1.82

100.70 ± 0.46

101.21 ± 1.09

100.60 ± 2.77

100.90 ± 0.30

100.3 ± 0.6

100.8 ± 1.55

103.03 ± 0.60

Table 5.7: Stability studies of itraconazole SEDDS F21 at various storage conditions Storage conditions → 25 ± 2°C (RT) 30 ± 2°C/65 ± 5% RH 40 ± 2°C/75 ±

5%RH Parameters ↓ Medium↓ 1 M 2 M 3 M 1 M 2 M 3 M 1 M 2 M 3 M Droplet Size D (nm)

DW 194.46 200.37 201.33 198.28 182.39 187.63 199.4 200.46 199.84

0.1N HCl 181.2 169.25 167.46 159.86 161.3 163.02 170.6 170.5 173.25

PBS 492.52 482.46 478.36 483.52 503.2 491.73 532.6 489.4 491.06


DW 0.232 0.321 0.233 0.321 0.142 0.21 0.253 0.216 0.224

0.1N HCl 0.476 0.472 0.389 0.462 0.392 0.288 0.421 0.472 0.418

PBS 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Zeta Potential (mV)

DW 5.23 4.47 5.46 4.78 3.58 5.21 5.63 4.68 4.82

0.1N HCl 6.43 6.73 7.21 7.53 6.48 6.75 5.89 5.49 6.25

PBS -5.38 -6.37 -6.61 -4.82 -5.38 -5.44 -6.72 -5.94 -5.58

Drug Content (%) 102.42 ± 1.84

101.91 ± 2.61

101.61 ± 0.63

99.79 ± 0.17

100.80 ± 0.46

100.30 ± 1.51

99.79 ± 0.46

100.20 ± 1.49

100.0 ± 0.30


Page 184

Table 5.8: Stability studies of itraconazole SEDDS F27 at various storage conditions Storage conditions → 25 ± 2°C (RT) 30 ± 2°C/65 ± 5% RH 40 ± 2°C/75 ± 5%RH

Parameters ↓ Medium↓ 1 M 2 M 3 M 1 M 2 M 3 M 1 M 2 M 3 M Droplet Size D (nm)

DW 199.3 188.58 180.89 192.3 192.5 190.55 198.7 189.7 199.96

0.1N HCl 183.53 171.36 183.62 179.28 168.3 172.36 181.5 177.8 180.84

PBS 648.27 649.36 662.57 684.29 669.5 671.05 668.5 678.2 675.42


DW 0.283 0.253 0.261 0.197 0.325 0.268 0.184 0.227 0.231

0.1N HCl 0.457 0.453 0.432 0.456 0.462 0.338 0.473 0.428 0.436

PBS 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Zeta Potential (mV)

DW -9.1 -5.82 -7.83 -8.75 -6.11 -7.84 -9.28 -7.92 -6.98

0.1N HCl 2.87 2.08 3.42 3.62 3.72 4.21 4.51 4.88 5.35

PBS -4.42 -5.28 -4.88 -5.38 -5.27 -5.33 -4.62 -6.72 -5.69

Drug Content (%) 102.12 ± 1.21

102.12 ± 0.80

101.31 ± 1.06

100.50 ± 1.14

100.40 ± 0.46

100.10 ± 0.76

100.10 ± 0.46

100.10 ± 0.69

100.3 ± 0.8

D (nm) = diameter in nanometer; DW = distilled water; PBS = phosphate buffer saline pH 6.8; RT = room temperature; M = month; RH = relative humidity

5.4. Conclusion

In the recent years the utilization of lipid excipients for the enhancement of oral

bioavailability of poorly soluble drugs has attracted attention. Self-emulsifying drug

delivery systems containing itraconazole were formulated using various ratios of

oil:S/CoS mixture in an attempt to increase its release rate and bioavailability. Three

formulations containing itraconazole were developed through the construction of pseudo-

ternary diagrams, droplet size determination and visual inspection. F5 consisted of 8.77%

of Capryol 90, 13.31% Labrasol, 32.15% of Tween 20, 43.32% Transcutol; F21 consisted

of 12.4% Capryol 90, 12.5% Labrasol, 45.3% Tween 20 and 27.23% Transcutol; F27

consisted of 17.27% Capryol 90, 15.73% Labrasol, 57% Tween 20 and 7.5% Transcutol.

Itraconazole was 2.4% in all three SEDDS formulations. After oral administration to rats,

the SEDDS F5, F21 and F27 containing itraconazole had a relative bioavailability of

170.56%, 204.55% and 233.91% compared with the extemporaneous suspension prepared

from marketed capsules. The order of droplet size of formed emulsion was 1:4 (F27) > 1:6

(F21) > 1:9 (F5) for ratios of oil: Smix. Although the three self-emulsifying formulations

with different ratios of oil:Smix did show differences in exposures of itraconazole in rats,

the results were not in consistent with strong dependence on emulsion droplet size and oral

absorption. Contrary to expectations, the droplet size of the formed emulsion played very

little role in in vivo absorption of itraconazole from developed self emulsifying

formulations. The possible explanation for this observation can be a very high lipophilicity

of itraconazole (log P 5.66). Such drugs have more affinity toward lipophilic domain of

the formulation and remain in a solubilised state for better absorption. When the drug


Page 185

solubilisation capacity at interface has been increased with the use of surfactants, dilution

with physiological fluid may lead to migration of surfactant form the oil/water interface

and can result in reduction in drug loading capacity and precipitation. This phenomenon

may be responsible for the decrease in bioavailability as the proportion of surfactant

increased. In conclusion, the data overall showed little apparent correlation between

emulsion droplet size and oral absorption in rats, but statistically (P < 0.05) there was no

significant difference between all the three formulations with respect to AUC and Cmax.

The results obtained illustrate the magnitude of bioavailability enhancement that can be

achieved through rational design of lipid-based formulations.


Page 186

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