Faculty of Pharmacy and Alternative Medicine The Islamia...

Development of Self-Microemulsifying DrugDelivery System (SMEDDS) for Poorly Water

Soluble Anticancerous Drug

A Thesis Submitted

in partial fulfillment of the requirement for the degree

of

DOCTOR OF PHILOSOPHY(Pharmaceutics)

by

NAYAB KHALIDB.Pharm., M.Phil.,

Department of PharmacyFaculty of Pharmacy and Alternative Medicine

The Islamia University of BahawalpurPAKISTAN

2017

In the name of Allah, the Most Merciful, the Most Kind

Dedicated to

My Parents,my wife,and little daughter Anabia Nayab

DEDICATION

I

CONTENTS

PART-A S. NO. CONTENTS PAGE NO.

1 Front Page

2 Bismillah

3 Declaration

4 Certificate

5 PhD open defense

6 Dedication

7 Contents I

8 List of Figures IV

9 List of Tables V

10 Acknowledgment VI

11 List of Abbreviations VIII

12 Abstract X

13 Research Publication XII

PART-B

1 INTRODUCTION 1

1.1 Biopharmaceutical Classification System (BCS) 1

1.2 Lipid Drug Delivery System 2

1.3 Self-Microemulsifying Drug Delivery System (SMEDDS) 4

1.4 Self-emulsification process and drug loading in SMEDDS 5

1.5 Formulation of SMEDDS 6

1.5.1 Lipid vehicle 6

1.5.1.1 Triglycerides 7

1.5.1.2 Fatty acids esters of polyacohols 8

1.5.2 Surfactants 9

1.5.2.1 Water insoluble surfactants 9

1.5.2.2 Water soluble surfactants 10

1.5.3 Co-Surfactants/Co-solvents 11

1.5.4 Preparation of SMEDDS 12

1.5.4.1 Selection of Drug 12

1.5.4.2 Excipient Screening and Solubility studies 13

1.5.4.3 Construction of phase-diagrams 13

1.5.5 Mechanism of oral drug absorption by SMEDDS 14

1.6 In vitro Characterization of SMEDDS 16

16.1 Globule size, Zeta potential, PDI 16

1.6.2 Refractive index 17

1.6.3 Percentage Transmittance 18

1.6.4 Rheological studies 18

1.6.5 Transmission Electron Microscope (TEM) 19

1.6.6 Dissolution 19

1.6.6.1 Rotating paddle USP method 20

1.6.6.2 Dialysis bag method 20

1.6.6.3 Modified cylinder method 21

1.6.7 Permeation 21

1.6.7.1 Intestinal rat permeability study 22

II

1.6.7.2 Franz diffusion cell method 22

1.7 In vivo characterization of SMEDDS 23

1.7.1 HPLC analysis of SMEDDS of anticancer drugs 23

1.8 Objective of study 23

2.0 MATERIAL AND METHODS 25

2.1 Materials 25

2.2 Methods 25

2.2.1 Preparation of calibration curve of pure etoposide 25

2.2.2 Drug solubility studies 26

2.2.3 Selection of oil, surfactant, co-surfactant, and solubilizer 26

2.2.4 Compatibility/Miscibility studies 26

2.2.5 Phase-diagram study 26

2.2.6 Preparation of SMEDDS 28

2.3 In vitro Evaluation of SMEDDS 29

2.3.1 Dilution study 29

2.3.2 Globule size, zeta potential, and polydispersity index (PDI) 29

2.3.3 pH, RI, Viscosity and percent transmittance 29

2.3.4 Drug release study 30

2.3.5 Accelerated and thermodynamic stability study 30

2.3.6 Transmission Electron Microscope (T.E.M) 31

2.4 In vivo evaluation of SMEDDS 31

2.4.1 Study design 31

2.4.2 Method of sampling 32

2.4.3 Plasma Etoposide standard curve and extraction procedure 32

2.4.4 Analysis of Etoposide in Plasma 33

2.4.5 Pharmacokinetics Analysis 33

2.5 Statistical Analysis 33

3.0 RESULTS 34

3.1 Preparation of Standard curve of pure Etoposide 34

3.2 Drug solubility studies 35

3.3 Selection of oil, surfactant, co-surfactant, and solubilizer 37

3.4 Development of phase diagram 37

3.5 Drug loading, globule size, zeta potential and polydispersity index

(PDI) 38

3.6 Viscosity, pH, Refractive index(RI) and percent transmittance 39

3.7 Dilution, Accelarted and Thermodynamic stability study 40

3.8 In vitro release study 41

3.9 Transmission Electron Microscope (TEM) 44

3.10 In vivo evalutation of SMEDDS 44

3.10.1 Stanadard curve of ETO in rat plasma 44

3.10.2 Pharmacokinetics analysis 48

4.0 DISCUSSION 60

4.1 Selection of oil, surfactant, co-surfactant, and solubilizer 60

4.2 Development of phase-diagram 60

4.3 Drug loading, Globule size, zeta potential, and Polydispersity index

(PDI) 60

4.4 Viscosity, pH, Refractive index (RI), and percent transmittance 61

4.5 Dilution, Accelarted and thermodynamic stability study 62

III

4.6 In vitro release study 62

4.7 In vivo evaluation of SMEDDS 63

5.0 CONCLUSION 71

6.0 Future Prospects 77

7.0 REFERENCES 73

Pharmacy Research Ethics Committee (PREC) approval letter 89

IV

LIST OF FIGURES S. NO. CONTENTS PAGE NO.

1.1 Biopharmaceutical Classification System 2

1.2 Etoposide (Chemical Structure) 12

1.3 Preparation Steps of SMEDDS/SNEDDS 14

1.4 Schematic process of lipid absorption, digestion and solubilization of

drug in the small intestine 16

2.1 Modified dissolution apparatus used for SMEDDS 30

3.1 Standard curve of ETO in methanol 34

3.2 Solubility of etoposide in different types of oils 35

3.3 Solubility of etoposide in different surfactants 36

3.4 Solubility of etoposide in different co-surfactants 36

3.5

Ternary phase diagram of different %ages of MCT (oily vehicle), PSM

20 (surfactant), PGM type-I (co-surfactant), DGME (Solubilizer)

mixtures in 100 fold water

37

3.6 Effect of percentageof oil concentration on globule size 39

3.7 In vitro release study of different “FD” formulations Vs VePesid 50 mg

capsules in SGF at pH 1.2 42

3.8 In vitro release study of different “FLD” formulations Vs VePesid

® 50

mg capsules in SGF at pH 1.2 42

3.9 In vitro release study of different “FD” formulations Vs VePesid

® 50

mg capsules in SIF at pH 6.8 43

3.10 In vitro release study of various “FLD” formulations Vs VePesid

® 50

mg capsule in SIF at pH 6.8 43

3.11 Representative TEM image of optimized SMEDDS formulation 44

3.12 Mean plasma Standard curve of ETO in Wistar Albino rats 45

3.13 Chromatograms of blank plasma before drug administration (0 hour)

Wistar Albino rat 45

3.14 Chromatogram of plasma at 2 h after dosing of VePesid

® in Wistar

Albino rat. 46

3.15 Chromatogram of plasma at 4 h after dosing of VePesid

® in Wistar

Albino rat 46

3.16

Chromatogram of plasma at 2 h after dosing of SMEDDS in Wistar

Albino rat 47

3.17

Chromatogram of plasma at 4 h after dosing of SMEDDS in Wistar

Albino rat 47

3.18 Plasma ETO concentration versus time profile of Rat-1 49










3.28 Mean plasma ETO concentration Vs time profile of VePesid

® and

SMEDDS in Wistar Albino Rats 56

V

LIST OF TABLES

S. NO. CONTENTS PAGE NO.

1.1 Lipid Formulation Classification System 3

1.2 Commonly used Surfactants with Trade Marks 11

2.1 Formulation mixtures used for phase diagram study 27

2.2 Formulations without co-surfactant 28

2.3 Formulations with co-surfactant 29

2.4 Weights of Rats colored with Black marker and Red marker 32

2.5 Administration of Test and Standard Formulation in crossover design 32

3.1 Known concentrations of etoposide in methanol 34

3.2 Globule size, zeta potential, PDI of F and FD formulations (mean±SD,

n=3) 38

3.3 Globule size, zeta potential, PDI of FL and FLD formulations

(mean±SD, n=3) 38

3.4 pH, Refractive index and Viscosity of “F” formulations (mean±SD,

n=3) 40

3.5 pH, Refractive index and Viscosity of “FL” formulations (mean±SD,

n=3) 40

3.6 Dilution, accelerated and thermodynamic stability study 41

3.7 Plasma concentrations verses time profile of VePesid

® in male Wistar

Albino Rats 54

3.8 Plasma concentrations verses time profile of SMEDDS formulation in

Wistar Albino Rats 55

3.9 Individual Pharmacokinetic values (AUC 0-∞, Cmax and Tmax) of

VePesid®

and SMEDDS in Wistar Albino Rats 57

3.10 Individual Pharmacokinetic values (AUMC 0-∞, and MRT) of VePesid

®


3.11 Individual Pharmacokinetic values (Ke, t1/2 and Vd) of VePesid

®


VI

Thanks to ALMIGHTY ALLAH the most Merciful and Beneficent, who bestowed

me with philosophy, perception and courage to perceive, to pursue and to present this

work by enabling me to overcome so many hardships in this accord. I profoundly

praise the Holy Prophet Muhammad (PBUH) for leading all the humanity &

mankind to the path of eternal success and prosperity. It’s because of his entreaty that

we are living with such a blessed lives.

I have yet find words to express my gratitude & sincere thanks to my supervisor Prof.

Dr. Nisar-ur-Rehman, under whose sagacious supervision, worthwhile suggestions,

persistent inspiration, wise counseling and ablest guidance, it became possible for me

to complete this attempt. I also thank him for continued motivation, always supportive

behavior. I am again thankful to him because he has arranged consent letter from

Professor Dr Raimar Loebenberg.

I am grateful to Professor Dr. Raimar Loebenberg, Director, Drug Development and

Innovation Center (DDIC), Faculty of Pharmacy and Pharmaceutical Sciences,

University of Alberta, Canada for his valuable suggestions, numerous technical

discussions on drug delivery systems particularly on SMEDDS.

I am highly obliged to Professor Dr. Mahmood Ahmad, Dean of Faculty of Pharmacy

and Alternative Medicine who has always been encouraging and supportive whenever

I need his help.

I am thankful to the Higher Education Commission of Pakistan (HEC) who awarded

me schorlarship through a 6 months research programe titled “International Research

Support Initiative Program”(IRSIP). Here I would like to pay special thanks to

Jehanzeb Khan (Project Director, IRSIP).

Thanks are due to Prof. Dr. Naveed Akhtar, Chairman, Department of Pharmacy for

his precious advises he made during the study and always helping and accommodative

attitude. I am thankful to Dr. Muhammad Khan Sarfraz and Dr. Muhammad Waheed

Asghar who provided excellent hospitality and helped me during my stay in

Edmonton, Alberta, Canada.

I am extremely thankful to Dr. Muhammad Akhtar, Assistant Professor who

motivated me and also helped me in organizing of manuscript as well as my thesis

and always supportive behavior throughout my research work. I would like to say him

VII

a special thanks for his continued support to me. I have find no more words to write

more about him.

I also want to say thanks to Dr. Qaiser Jabeen who had always been remaining source

of encouragement and also because of her maintained animal house I become able to

perform sampling on Wistar Albino Rats. I say thanks to Mr. Muhammad Farhan

Rasheed (PhD scholar Pharmacology) who helped me during the sampling procedure

of Rats.

I pay my gratitude to my B.Pharm class fellow Dr. Muhammad Usman Minhas

whose unlimited help not only in my research work but also everywhere. Special

thanks to Mr. Muhammad Arfat, who keep me motivated during HPLC studies.

I wish to express my thanks to all my family members especially my wife. I owe my

success to their proper support; spiritual guidance and special care for me.

There are many others within & outside the department who have been very helpful to

me in many different ways. There is no way to list all of them, however, I am simply

thankful.

May ALLAH bless them all.

Nayab Khalid

VIII

LIST OF ABBREVIATIONS

AUC Area Under the Curve

AUMC Area Under (first) Moment Curve

Cmax Maximum plasma concentration

CoS Co-Surfactant

DGME Diethylene Glycol Monoethyl Ether

ETO Etoposide

F SMEDDS formulations without additional COS and

without drug

FD SMEDDS formulations with additional CoS and with

drug

FL SMEDDS formulations with additional CoS and

without drug

FLD SMEDDS formulations with additional CoS and with

drug

GIT Gastrointestinal tract

LCT Long chain triglycerides

MCT Medium Chain Triglycerides

PGM Type-I Propylene glycol monolaurate type 1(Lauroglycol FCC)

PGM Type-II Propylene glycol monocaprylate Type II

PSM(20) Polyoxyethylene Sorbitan monooleate (20) (Tween 80)

SGF Simulated Gastric Fluid

SIF Simulated Intestinal Fluid

SMEDDS Self-Microemulsifying Drug Delivery System

T.E.M Transmission Electron Microscopy

IX

µg Microgram

Approx. Approximately

Conc. Concentration

g Gram

Inf. Infinity

Ke Elimination rate constant

Kg Kilogram

mg milligram

ml milliliter

r.p.m. Revolutions per minute

t½ Half life

Tmax Time to reach max concentration

Vd Volume of distribution

X

ABSTRACT

Self-Microemulsifying Drug Delivery System (SMEDDS) is one of the promising

technique to enhance the solubility and bioavailability of hydrophobic drugs such as

anticancerous drugs. Very few studies on SMEDDS as drug delivery system to

enhance solubility and bioavailbility of anticancer drugs have been conducted. The

current study was aimed to enhance the solubility and bioavailability of poorly water

soluble anticancer drug etoposide (ETO) by developing its SMEDDS for oral

administration. Two different kinds of SMEDDS formulations were made with and

without using co-surfactant (propylene glycol monolaurate type 1). The excipients

were selected on the basis of ETO solubility in different vehicles. The first

formulation used medium chain triglycerides (MCT), polyoxyethylene sorbitan

monooleate 20 (PSM 20) and diethylene glycol monoethyl ether (DGME) as oily

vehicle, surfactant and solubilizer, respectively. While the second formulation

comprised of the same ingredients, but with the incorporation of propylene glycol

monolaurate type 1 as a co-surfactant. Phase diagram was used to identify

microemulsion area. The formulations were characterized for globule size, zeta

potential, polydispersity index, pH, viscosity, refractive index, percentage

transmittance and transmission electron microscope (TEM). Dissolution studies were

employed using a modified cylinder method. Dilution study was performed to check

transparency using water, SGF (pH=1.2) and SIF (pH=6.8). Accelerated and

thermodynamic stability studies comprised of three phases as heating-cooling cycles,

centrifugation and freeze-thaw cycles were performed on SMEDDS. TEM was

performed to evaluate the surface morphology of the globules. The in vivo studies

were performed on 10 Wistar Albino healthy male rats. The study was an open, single

dose, crossover complete two periods of treatment dosing. The plasma samples were

analyzed using a reversed-phase high-performance liquid chromatographic (RP-

HPLC) method. The most common pharmacokinetic parameters such as total area

under the plasma concentration-time curve (AUC0-), peak plasma concentration

(Cmax) and time to reach maximum plasma concentration (tmax) were estimated from

the plasma concentration-time profiles. The above pharmacokinetic parameters were

calculated as per non-compartmental method of analysis using Kinetica® version

4.2.1. The data was statistically analyzed by Sigmaplot 12.5 and studies were carried

out in triplicates, and the results showed the mean±SD. The statistical study was done

XI

via Student's t-test. The optimized SMEDDS formulation was thermodynamically

stable, having small globule size, improved zeta potential and faster dissolution

profile as compared to commercial VePesid® capsules. The percent drug release of

optimized formulation was 1.6 and 1.4 folds more than VePesid® capsule in SIF and

SGF, respectively. The optimized formulation has globule size 15.84 ± 0.1 nm, zeta

potential -12.0 ± 0.2 mV, (polydispersity index (PDI) 0.094 ± 0.3, pH 5.35 ± 0.04,

Refractive index (RI) 1.42 ± 0.05, Viscosity 52.10 ± 0.45 cP, percent transmittance

99.65 ±1.20% and %drug release was 77.44% in SGF and 88.31% in SIF,

respectively. The pharmacokinetic parameters values (Mean±SEM) were calculated

of both the optimized SMEDDS and VePesid® are as for AUC0-∞ 3.2356 ± 0.1135

µg/ml*h and 1.3217 ± 0.1096 µg/ml*h, for AUMC0-∞, 11.88 ± 0.92 µg.h2/ml and 3.41

± 0.41 µg.h2/ml, for Cmax 1.125±0.020 µg/ml and 0.618 ± 0.029, for tmax 0.925 ± 0.038

h, and 0.875 ± 0.042 h, for t1/2 2.37 ± 0.69 h, and 1.66 ± 0.48 h, for MRT (h) 3.55 ±

0.71, and 2.60 ± 0.41, for Ke (h-1

) 0.32 ± 0.11, and 0.44 ± 0.11, and for Vd 512.71 ±

137.98 L, and 948.24 ± 316.50 L, respectively. The use of oily vehicles, surfactant

and co-surfactants present in SMEDDS has improved the bioavailability by enhancing

the solubility of ETO and permeability in the gastrointestinal tract. The

pharmacokinetic parameters such as AUC0-∞ and Cmax showed that oral bioavailability

was successfully enhanced. The relative bioavailability of optimized SMEDDS and

VePesid®

was also calculated and found 2.4 fold increased in bioavailability of

optimized SMEDDS formulation as compared to standard formulation.

Keywords: Etoposide, SMEDDS, Modified dissolution apparatus, Pharmacokinetic,

Bioavailability, Wistar Albino rats, RP-HPLC

XII

RESEARCH PUBLICATION (One research publication as minimum requirement by HEC-Pakistan for PhD degree

award)

Title:

Design and Evaluation of SMEDDS to Enhance Solubility and Dissolution of

Anticancer Drug Using Modified Cylinder Method. Authors:

Nayab KHALID, Nisar U. RAHMAN, Raimar LÖBENBERG, Muhammad

AKHTAR, Muhammad K. SARFRAZ, & Braa M. HAJJAR

Journal:

Latin American Journal of Pharmacy (formerlyActa Farmacéutica Bonaerense)

36 (4): 647-57 (2017) JCR Impact Factor 0.372

1

1. INTRODUCTION

Oral route remained the most accepted route for drug administration as compared to the

parenteral route because of ease of administration, more patient acceptability (Roger et

al., 2011). This route also prevents hospitalization, sterilization of finished dosage form,

and technical trained staff (Thanki et al., 2013). Pharmacoeconomic evaluation revealed

that oral drug delivery system is cost effective than parenteral route of administration

(Mazzaferro et al., 2013). But for poor water soluble drug entities oral drug

administration is remained the trouble route because of decreased absorption by this

route. After oral administration, initially the drug is first solubilized and then permeation

process led to absorption so the solubility and permeability are rate-limiting steps. Poor

water solubility of these drugs generate problems in free drug diffusion through GIT and

ultimately leads to less availability of drug in blood stream (Burcham et al., 1997;

Stegemann et al., 2007).

1.1. Biopharmaceutical Classification System (BCS)

The active pharmaceutical ingredient is classified as Biopharmaceutical classification

system (BCS). The BCS recognized by Food and Drug Administration is actually

research performed by Amidon and coworkers. It is a technical and scientific guide in

order to classify drug entities on the basis of their aqueous solubility as well as

intestinal permeability (Food and Administration, 2000; Yu et al., 2002). During

formulation development stage BCS provide important guide from the biopharmaceutical

view. In BCS three factors are very important comprising of drug dissolution, solubility

and intestinal permeability. Drug entity is supposed to be more soluble when the

maximum dose of corresponding drug substance is soluble in 250 ml or less in water

based media over a wide pH range of 1 to 7.5 having equilibrium solubility at a

temperature 37°C. Whilst drug entity is believed to be highly or extremely permeable

when the amount of drug absorption in humans is find out to be 90% or more of dose of

drug to be administered which is based on determination of mass balance or in

comparison to an intravenous standard dose (Amidon et al., 1995). P-glycoprotein (P-gp)

is widely dispersed which are present in the intestine where they plays significant role

that it pumped out drug substances back to the intestinal lumen. So P-glycoprotiens

played the main role for low oral systemic availability (bioavailability) of several drug

substances. P-gp inhibitors are searched to overcome the poor or low bioavailability of

2

many drug substrates (Srivalli and Lakshmi, 2012). According to the BCS, drug entities

are categorized into four classes shown in Figure 1.1.

Figure 1.1: Biopharmaceutical Classification System

It was found that 50% of marketed drugs are categorized as having poor aqueous

solubility (Dahan et al., 2013). However, recent data showed that almost 70% of all

chemical entities are classified as having a poor aqueous solubility. This can lead to low

or erratic drug bioavailability, increased intra and inter-subject variability and

subtherapeutic plasma levels due to inadequate dissolution. These problems might be

overcome by formulating drugs with suitable lipid based carrier system.

1.2 Lipid Drug Delivery System

Lipid based drug formulation system is promising approach to increase systemic

availability by improving the oral absorption of drug substances which is involved by

distressing various biological and physiological mechanisms. These formulations

perform by stimulating bile and pancreatic enzymes secretions (Singh and Kim, 2002),

3

increasing gastric emptying times (Welling, 1977), improving the fluidity of membrane,

as well as tight junctions opening (Porter et al., 2007) and gearing up of lymphatic

transport of drugs (Benameur, 2006), also by escaping the first pass effect, and inhibiting

efflux transporters (Martignoni et al., 2004). It was found that poorly soluble drugs when

administered in the presence of diet containing more fats resulted in increase in the

bioavailability of these drugs which further has got much attention for formulation of

these drugs with lipidic vehicles (Grove et al., 2006; Perlman et al., 2008). Pouton

(2000) has categorized lipid based drug delivery system into 3 types dependent upon

their composition and properties. This categorization was restructured after the addition

of Type IV class as summarized below in Table 1.1 (Pouton, 2006).

Table 1.1: Lipid Formulation Classification Systems

Excipients type with HLB range

Formulation Composition (%w/w or %v/v)

Type

I

Type

II

Type

IIIA

Type

IIIB

Type

IV

Oils: Mono glycerides or either mixed

diglycerides and triglycerides 100 40-80 40-80 <20 -

Lipophilic surfactants (HLB*<12) - 20-60 - - 0-20

Hydrophilic surfactants (HLB*>12) - - 20-40 20-50 30-80

Hydrophilic co-solvents/co-surfactants

e.g. Transcutol HP®

- - 0-40 20-50 0-50

*HLB Hydrophilic-lipophilic Balance

A formulation which based on oils are classified as type I and is made up of only one

excipient which either alone a triglyceride or otherwise it‟s a combination of a

triglyceride with a monoglyceride or diglyceride. While self-emulsified drug delivery

systems (SEDDS) are categorized as a type II system which contain insoluble

surfactants, having an HLB value less than 12. Type III systems comprised of Self-

emulsifying Drug Delivery System (SEDDS) and/or Self-microemulsifying drug

delivery systems (SMEDDS) are sub categorized as type IIIA and type IIIB. In type IIIA

formulations, water-soluble components are less while type IIIB formulation contains

more water-soluble excipients. Type IIIB formulations have large quantity of hydrophilic

components, but a small quantity of oil or lipid vehicles. The emulsification or self-

dispersion efficiency is more in case of type IIIB as compared to IIIA. Formulations

without oils are classified as type IV systems. If the drug is only combined with co-

solvent in type IV, then there are chances of precipitation, which leads to small crystals

4

and system destabilization. On the other hand, if drug incorporated only with surfactants

then there are comparatively less chances of precipitation but high surfactants leads to

irritation in the stomach and local mucosal membrane. One common requirement for all

lipid formulation system that they must maintain the drug in solubilization state and if,

for example, drug precipitated out, then it is considered as a failure of lipid formulation

(Pouton, 2006).

1.3 Self-Microemulsifying Drug Delivery System (SMEDDS)

From all lipid based drug delivery systems like liposomes, microemulsions (ME), self-

emulsifying formulations (SEF), self-microemulsifying drug delivery system

(SMEDDS), solid lipid nanoparticles (SLN), SMEDDS are excellent carriers for BCS

class IV drugs (Singh et al., 2009a). They are very promising drug delivery system for

oil-soluble drugs. They are pre-mixtures of oil, surfactants or either co-surfactants (CoS)

or co-solvents which on gentle stirring solubilized the drug to form a microemulsion in

an appropriate solvent. The agitation or motility which produced from stomach and

intestine is needed for self-microemulsification in vivo (Shah et al., 1994). Normally the

microemulsion which formed after dilution of SMEDDS are clear, optically transparent

and thermodynamically stable system with a very small particle size (< 100 nm) (Shah et

al., 1994; Hauss, 2007a). SMEDDS proposed various merits over common emulsions by

assisting the solubility of lipophilic drug substances, long term thermodynamic stability

due to which these could be stored for a longer period as compared with conventional or

coarse emulsions. The basic difference is of globule size that coarse emulsion represents

droplet size is in range from 0.2 to 10 µm approximately whilst microemulsion formed

from SMEDDS has range lied from 20 to 100 nm. As small globule size provide larger

surface area which result in improved absorption and then bioavailability (Lawrence and

Rees, 2000).

SMEDDS have gained numerous advantages over ready to use microemulsions, which

include their physical as well as chemical stability, enhancement in storage time i.e. long

period of time, option of filling of SMEDDS into soft as well as hard gelatin capsules,

which make it commercially good-looking, and further patient compliance was

increased. SMEDDS provide opportunity for improved capacity for drug loading and

improvement in oral bioavailability or therapeutic effect of several lipophilic drugs

because of the drug solubility in formulation excipients. The improvement in loading of

5

drug and bioavailability can lead to a decrease in the dose of drug. Usually a large

quantity of main surfactant (> 40 to 45%) was required during SMEDDS development

process (Mason et al., 2006) as SMEDDS preparation could only produce microemulsion

when in contact with water and small agitation provided and then self-emulsification

process happened when oil and water interfacial tension become nearly zero (Bowcott

and Schulman, 1955).

1.4 Self-emulsification process and drug loading in SMEDDS

Drugs which have poor water solubility create a main challenge when formulator come

to develop drug delivery systems because their hydrophobic nature avoid them as being

dissolved in a variety of solvents. However, it has been observed in literature that

synthetic oils, some oily vehicles, surfactants, co-surfactants and their mixtures

solubilize significantly larger amount of lipophilic drugs when these compared with

vegetable oils. As synthetic oils are amphiphilc in nature which led improved solubility

of hydrophobic drugs (Cuine et al., 2008). For poorly water-soluble drugs having poor

aqueous solubility SMEDDS was frequently selected as developing a dosage form. It has

become ideal if SMEDDS dissolved at least therapeutic dose of drug entities, which was

not possible in all cases. SMEDDS have the potential to dissolve larger amount of the

drug, which is lipophilc in nature. The drug has some effect on self-emulsifying ability.

Sometimes it increased the self-emulsification ability and sometime decrease in self-

emulsifying power and at the same time self-emulsification process was not affected by

drug incorporation (Pouton, 1985; Charman et al., 1992). Self-microemulsifying drug

delivery systems have found to be more responsive on change in composition or ratios of

formulation excipients therefore care was taken before formulation as comprehensive

solubility studies and phase diagram study was performed before choosing optimized

SMEDDS formulations (Meinzer, 1995).

During SMEDDS development, formulation excipients were added in appropriate order

and when added in aqueous media the free energy required for the formation of

formulation is very low this energy either in the form of positive or sometimes negative

and resulting in formation of microemulsion spontaneously and is referred to as a

thermodynamic stable system. The energy required for the formulation of microemulsion

is very less that is why these formulations are called self-microemulsifying formulations.

It has been proposed that liquid crystalline phase formed between oil-surfactant-co-

surfactant interface and water go in this interface slowly through the agitation come from

6

stirring if in glass vial or by stomach agitation if present in vivo. When water enters into

this phase, it breaks it to some extent and form small globules, which is required for

microemulsion. This formed system is highly stable because of liquid crystalline phase

and also prevent the small globules to coalescence with each other (Groves and De

Galindez, 1976; Wakerly et al., 1986). SMEDDS enhance the rate as well as extent of

absorption of lipohilic drugs because dissolution was the rate limiting step for various

lipophilic drugs especially BCS-IV drug (Pouton and Porter, 2008).

1.5 Formulation of SMEDDS

Inert excipients are used in formulating SMEDDS formulations because it has been

revealed that some excipients produce toxic effects if used in increased concentrations.

U.S Federal Drug Regulatory Authority (FDA) provides a comprehensive list of inactive

ingredients. This list is time to time updated and upgraded. By using this formulator can

add these excipients if they tend to formulate a product for market (2007). Once FDA

has approved any inactive ingredient for formulations used through a specific route of

administration so it can be easily selected for developing a new formulation. Various

oils, surfactant and co-surfactants are basic components of SMEDDS and can be selected

based on drug solubility.

1.5.1 Lipid vehicle

Lipidic vehicles in SMEDDS is to solubilize the lipophilic drug entity which is aimed to

enhance drug loading capacity, which in turn enhance dissolution and ultimately

bioavailability of the hydrophobic drug substances (Prajapati et al., 2011). If oily vehicle

contained larger lipohilic portion than hydrophilic so it can solubilize large amount of

hydrophobic drug and vice versa (Gursoy and Benita, 2004). The oils also help in self-

emulsification process, which is necessary step in the formation of microemulsion. These

lipidic vehicles helps some highly hydrophobic drugs to facilitate lymphatic

transportation in order to improve absorption of drugs (Kimura et al., 1994). Some oils

has dual nature i.e. some part is hydrophilic and some part is lipophilic. These types of

lipidic vehicles are called amphiphiles (Nagel, 1989). Oils were classified as:

7

1.5.1.1 Triglycerides

Triglycerides are easily absorbed and digested and can be used safely in lipid based drug

delivery systems i.e. SMEDDS. These are safe to use and pose no risk when given orally

during development of SMEDDS formulations (Pouton and Porter, 2008). Triglycerides

are further categorized as Triglycerides (Long chain) and Medium chain triglycerides

(MCT) and Mixed partial glycerides.

They have fatty acids with chain length 14-20 carbons also called fixed oils (Pouton and

Porter, 2008; Prajapati et al., 2011). Fixed oils are also generally referred to as vegetable

oils and composed of glyceride esters having unsaturated fatty acids containing long

chain carbon length (LCT). Larger lipohilic part in these types of glycerides make them

suitable for highly hydrophobic drugs due to increased solvent capacity (Ellaithy and El-

Shaboury, 2002). As these vegetable oils are safest oily vehicles and more appropriate

oily vehicle for the fabrication and design of SMEDDS but unfortunately not commonly

used because of their failure to solubilize larger quantity of poorly water soluble

lipophilic drug entities (Tolle et al., 2000). The solvent capacity of triglycerides was

based on ester group concentration (Cao et al., 2004). Commercial example is Neoral®

which contained olive oil produced superior bioavailability (Ellaithy and El-Shaboury,

2002).

Lipidic vehicles that have fatty acids with chain length 6-12 carbons are classified as

medium chain triglycerides (Prajapati et al., 2011). MCT have gained much priority as

compared with LCT because they are least resistant to oxidation, excellent solvent or

solubilizing capacity. MCT formed from coconut oil by the process of distillation and

contained fatty acid having C8 and C10 in saturated liquid form. These MCT‟s also

called capric/caprylic acid triglycerides (Pouton and Porter, 2008). The example is

Labrafac CM 10®

it is a MCT which is shown to have good solubilizing capacity for

fenofibrate than Maisine 35® which is long chain triglyceride (Patel and Vavia, 2007).

Various highly lipophilic drugs do not show good solubilizing and self-dispersing

potential in LCT (vegetable oils) than MCT (Pouton, 1997; Stegemann et al., 2007)

which become the reason that MCT have benefit over LCT. MCT are actually the

products obtained after hydrolysis of LCT or vegetable oils. These are mixtures of mono,

di-, tri-glycerides. These mixtures which have unsaturated fatty acids and also medium

carbon chain length are useful for improving systemic availability of poorly water

8

soluble drugs while those have long chains are for sustained release reasons (Jannin et

al., 2008). The hydrolysis of vegetable oils improve polarity and solvency which results

in good self-emulsification efficiency in order to improve solubility and stability of

vegetable oils (Strickley, 2004). The oils go through hydrogenated process and their

some properties become changed as increases in stability characteristics chemically.

Examples is hydrogenated soybean oil available with registered trade name (LIPO®)

(Pouton and Porter, 2008). Partial hydrolysis of triglycerides results in mixed partial

triglycerides which have improved characteristics. These characteristics depend on

which type of fatty acids is present and how much esterification of these fatty acids has

done during hydrolysis. Mixed partial glycerides having medium chain triglycerides

include Capmul MCM® has chemical name glyceryl monocaprylocaprate.

It has observed that higher concentrations of surfactant is required when LCT‟s were

used while less quantity of surfactant is required when MCT‟s/mixed partial glycerides

were used. Latest synthetic and semi-synthetic oily vehicle such as MCT derivatives also

pronounced as amphiphilic lipid vehicles having surfactant like properties are day by day

replacing vegetable oils or conventional medium chain triglycerides in SMEDDS

formulations (Lawrence and Rees, 2000). It has been well established that oils with high

length of carbon chain have high molecular weight such as castor oil and soybean oil are

not easy to self-microemulsify as compared to low molecular weight large hydrocarbons

chain length e.g. Capmul MCM is medium chain triglyceride which has superior self-

emulsification ability compared with LCT (Patravale, 2009). It has reported that increase

in drug solubility in SMEDDS depends not entirely on oily vehicles but also surfactant

and co-surfactant/solubilizers also play major role. Testosterone propionate is an

important example of such improved solubilization enhancement (Malcolmson et al.,

1998).

1.5.1.2 Fatty acids esters of polyalcohols

Some new oily type excipients which actually derivatives of oils which have surfactant

type of properties because of its amphiphilic nature and are effective in replacing

conventionally used vegetable oils of natural origin (Constantinides, 1995). Their nature

is dependent on the type of alcohol was used. Some of these are referred as polyglycerol

(Plurol Oleique CC 497), glycerol based esters such as Maisine®

35-1 , Peceol, Labrafac.

9

1.5.2 Surfactants

Surface-active agent or surfactants are important components of SMEDDS development.

The problem is that only few surfactants are given by orally. They act as emulsifier when

added in aqueous media. These agents were responsible for reducing oil-water interfacial

tension a step necessary to make system stable when SMEDDS added in aqueous media.

Nonionic surfactants were given preference over nonionic surfactants because of their

least toxic effects produced and CMC (critical micelle concentration) required to

microemulsion is less as compared to ionic surfactants (Jiao, 2008). Surfactants are

amphiphilic in their nature containing hydrophobic group (non-polar portion) and

hydrophilic group (polar portion) which by self-emulsification process enhance the

solubilization process. The surfactant selection is purely based on the fact that how

quickly it will self-emulsify (self-microemulsify) the oily vehicle, and solubilizing ability

for drug as well as safety of selected surfactant. In some special case the objective is to

include P-glycoproteins (P-gp) if selected drug have properties to act as P-gp substrate

(Date and Nagarsenker, 2008). In SMEDDS mostly surfactants having a HLB value

greater than 12 were exclusively used. As far as safety is concerned, the surfactant from

natural origin has good safety profile but they have less capacity to self-emulsify as

compared to synthetic surfactants, which are more commonly used now days.

1.5.2.1 Water insoluble surfactants

These, also called polar oils, as they are esters of non-ionic in origin, which do not

polyethoxylated or not even polyglycerated. These are actually intermediates type in

nature and have shorter HLB value (8-12). This group has not commonly used in

SMEDDS formulations due to their lesser self-microemulsifying nature as they are not

much hydrophilic in their property. These surfactants are also referred to as water

dispersible surfactants because they need shear to micro-emulsify. Tween-85 and Tagat

TO having HLB 11 and 11.5 are typical examples of such type of surfactants (Wakerly et

al., 1986; Pouton, 1997, 2000). Other important examples are Propylene glycol

monocaprylate (CapryolTM

90), Propylene glycol monolaurate (LauroglycolTM

90),

Polyglyceryl-3-dioleate (Plurol® Oleique CC 497).

10

1.5.2.2 Water soluble surfactants

These types of surfactants are most extensively used surfactant for the development of

SMEDDS (Pouton, 2006; Hauss, 2007b). They have large HLB value as more than 12,

which is requisite to develop SMEDDS. These have advantage of forming micelles even

at low concentration. They are actually produced by hydrogenation of natural oils with

addition of polyethylene glycols (Schick, 1987). Sorbitan esters when reacted with

ethylene oxide resulted in formation of Polysorbates (Pronounced as ether ethoxylates

(Kalepu et al., 2013). Cremophor RH40 (hydrogenated castor oil and Cremophor EL

(Ethoxylated castor oil) are their examples (Collnot et al., 2006; Grove et al., 2007).

Polyethylene portion produce hydrophilic properties in the surfactants. The optimum

surfactant concentration should between 30-60% in order to develop maximum

SMEDDS stability (Fanun, 2010). Care must be taken while formulating SMEDDS that

minimum concentration should be used in order to protect from GIT irritations as if

surfactant used in higher concentration it will lead to gastric irritation (Charman et al.,

1992). Surfactant has found to have different type of effects that increase in surfactant

concentration would produce mean small globules in size, which lead to improved

absorption from gastric mucosa due to adherence of molecules of surfactant oil-water

interphase (Georgakopoulos et al., 1992; Gursoy and Benita, 2004). While in contrary

increase, surfactant will produce opposite effect on globule size. So it has been

concluded that surfactant at some specific concentration would be beneficial and produce

small droplets while in larger concentrations it will results in larger globules which

destabilize the system (Pouton, 1997; Gursoy and Benita, 2004). The surfactants improve

oral bioavailability by following mechanisms;

Improvement in dissolution of poorly water soluble drugs

Increased intestinal permeability

Opening of tight junctions

By inhibition of P-glycoprotiens

Inhibiting cytochrome enzymes CYP3A

Some commonly used surfactants are presented in table 1.2 (Gurram et al., 2015)

11

Table 1.2: Commonly used Surfactants with Trade Marks

Sr.No Surfactant chemical name Registered Trade name HLB value

1 Polyoxyethylene sorbitan monolaurate Tween®

20 17

2 Polyoxyethylene sorbitan monopalmitate Tween®-40 15.6

3 Polyoxyethylene Sorbitan monostearate Tween®-60 15.0

4 Polyoxyethylene sorbitan monooleate Tween®-80 15.0

5 Polyoxyethylene tristearate Tween®-65 10.5

6 Polyoxythylene sobitan trioleate Tween®-85 11.0

7 Polyoxyethylene glycerol trioleate Tagat TO 11.5

8 Polyoxyethylene-40 hydrogenated castor

oil

Cremophor RH40 (solid

state) 14.0-16.0

9 Polyoxyethylene-35 castor oil Cremophor EL(Liquid

state) 12.0-14

10 Polyoxyethylene-Vitamin E Alpha tocopherol TPGS 13.0

1.5.3 Co-Surfactants/Co-solvents

Co-surfactants or co-solvents actually act to aid in the development process of

SMEDDS. The development of SMEDDS to be optimum needs increased concentration

(more than 30%) of main surfactant. In this way, the quantity of surfactant would be

reduced due to the addition of co-surfactant/co-solvent. They help with surfactant in

reducing the interfacial tension sometimes to become very low as reached to negative.

Hydrophilic co-solvents were most commonly used in SMEDDS such Transcutol-HP,

Ethanol, Polyethylene glycol (PEG-400) (Cole et al., 2008) Propylene glycol

monolaurate (Lauroglycol), they also aid in self-emulsifying process of SMEDDS.

Concentration of co-solvent play an important role in SMEDDS as too high

concentration leads to drug precipitation because of moving of co-surfactant from

SMEDDS when diluted in aqueous media (Lawrence and Rees, 2000). Co-solvent acts as

solubilizer to improve the solvent capacity to dissolve large quantity of drug e.g.

Transcutol-P is good solubilizer for ETO (poorly water-soluble BCS-IV drug). It has

been reported that surfactant alone was not able to reduce oil-water interfacial tension in

order to form microemulsion upon dilution of SMEDDS (Attwood and Kreuter, 1994),

the co-surfactant act as amphiphilic in nature as in reducing interfacial tension.

12

1.5.4 Preparation of SMEDDS

SMEDDS can be prepared in the following steps:

1.5.4.1 Selection of Drug

Different classes of poorly water-soluble drugs, which are lipophilic in nature, could be

used to formulate SMEDDS. Some of the examples of poorly water soluble anticancer

drug entities are Paclitaxel, Docetaxel, Exemestane, Bufalin, 9-Nitrocamptothecin (9-

NC), and Etoposide (ETO). ETO is highly soluble in methyl alcohol and chloroform,

slightly soluble in ethanol but only scarcely soluble in water. The chemical formula of

ETO is C29H32O13 (O'Dwyer et al., 1985; Henwood and Brogden, 1990; Slevin, 1991;

Joel, 1996). The permeability of ETO is less using MDCKII 2.42 ±0.08 *10-6 compared

to 3.19± 0.04 *10-6 cm/s for MDCKII-MRP1 cells (Guo et al., 2002). Its Log P value

is1.16 (Wang et al., 2015) and classified into BCS 4 with lowest solubility and also

lowest Permeability and basic pKa (9.8) (O'Neil, 2001). Water solubility is

approximately 0.08 mg/ml (Pharmacists, 1994). Structural formula is revealed in Figure

1.2.

O

OO

O

HO

HO

O

O

O

O

OH

O

O

Figure 1.2 Etoposide (Chemical Structure)

13

1.5.4.2 Excipient Screening and Solubility studies

This is the important step in order to screen out excipients for development and

preparation of SMEDDS (Pouton, 2000). Solubility studies would be performed for drug

in different oily vehicles (oils), surfactants, and co-surfactants and sometimes

solubilizers also added in order to improve drug solubilization (Borhade et al., 2008;

Wang et al., 2011). Mostly these studies will be done by method called shake flask. In

this method drug is added in the different vehicles (excipient) such as oil, surfactant, co-

surfactant, solubilizer. Mostly drug is added in excess amount in vehicle (2 ml or 2 g)

present in glass vial and then this glass vial shaken for at least 48-72 hours then when

drug dissolved and saturation level reached. Then these samples taken and centrifuged

and final quantity of drug has been determined. The major objective is to find out

vehicles which has highest solubility for drugs and would be selected for further studies

(Singh et al., 2009a)

1.5.4.3 Construction of phase-diagrams

These phase-diagrams are diagrams, which show the phase behavior changes as

composition of system changes. The ternary phase diagrams as the name represent phase

behavior of three components. Each corner in phase diagram shows 100% of one

particular excipient. If one more component added in the addition to three components

then this Phase diagram is called pseudo-ternary phase diagram as one of the corner

represent to mixture of two excipients such as surfactant and co-surfactant or solubilizer.

Such as different surfactant to co-surfactant mixture ratios were made as 2:1, 1:3 etc.

(Lawrence and Rees, 2000). As to construct phase diagram then different SME (self-

microemulsify mixture) by varying the percentages of oily vehicle, surfactant, co-

surfactant, solubilizers are prepared and these mixtures are tested for their self-

emulsification efficiency by diluting in water in order to determine the microemulsion

area (Kommuru et al., 2001; Mou et al., 2008). It is very important to reserve in mind

that total percentage of all excipients used must be 100%. The phase diagrams were

plotted by using appropriate software such as Prosim® software used in preparing phase

diagrams for SMEDDS. Then from ME area optimized formulation is determined by

subjecting the ME area formulation to different tests like droplet (globule size), zeta

potential, dissolution, and stability is checked by thermodynamic stability tests.

14

After careful selection of suitable excipients preparation of SMEDDS is very easy. As

drug is added in the mixture of oil, surfactant, and co-surfactant and then this mixture is

then vortexed properly (Balakrishnan et al., 2009). In some cases, first drug is dissolved

in one or two components then in this drug mixture, the other excipients are added and

then vortex until homogeneous SMEDDS mixture is formed. These mixture are then

characterized for turbidity, dilution studies, and other tests for transparency checking

(Wang et al., 2011). Schematic diagram for preparing SMEDDS is presented in Figure

1.3

Figure 1.3 Preparation Steps of SMEDDS/SNEDDS (Singh et al., 2009b)

1.5.5 Mechanism of oral drug absorption by SMEDDS

SMEDDS have the property to evade dissolution step of drug before its absorption

through gastrointestinal tract, which in turn enhance/improve quantity of drug in

Drug Lipid

Emulsifier Co-emulsifier (Optional)

Lipidic Solution

SEDDS/SMEDDS

Micro/nano-

emulsion

Dilution with Water

15

solubilized form. Therefore, the amount of drug in intestinal secretions has become

increased which lead to improved drug absorption. Additionally systemic availability of

drug entity has enhanced by having lipidic vehicle in the SMEDDS formulation (Porter

et al., 2007). Surfactants have shown to delay gastric emptying time, which increased the

time for the drug availability in GIT to get absorbed. They act by making thick mass in

GIT and intestine. Labrasol® is an example of such activity (Chang and Shojaei, 2004).

When SMEDDS are given orally, they contained lipidic vehicle. These lipids converted

into triglycerides by enzyme lipase and further digested into diglyceride and then small

molecule fatty acids. Lipid also stimulates duodenum, gall bladder, pancreas to release

bile salts (complex of bile salts), cholesterol, and an enzyme pancreatic lipase

respectively. These perform their function in absorption process as these secretions

combined with microemulsion droplet (globules) and give them stability. In addition,

they form micelle structures onto which drug entities has adsorbed these micelle

structures containing drug become storage for drug molecules at different absorption

sites in GIT. So drug becomes easily absorbed through these micelle carriers. This

absorption capacity is further enhanced if surfactants and co-surfactants are present.

Increasing the levels of cholesterol and bile salts further has enhancing effects for drug

entities. Therefore, these carriers (micelles) act to enhance solubilizing capacity of drug

entities. The microemulsion and micelles formed by these processes are penetrated

(absorbed) by the process of pinocytosis or diffusion (Gershanik and Benita, 2000; Porter

et al., 2008). The drug absorption is also enhanced by the lymphatic transport system if

incorporated in SMEDDS as in case of lymphatic transport the drugs are not metabolized

by the liver (first pass effect) which leads to improve the bioavailability of some drugs.

This is only possible for drugs, which have high log-P as more than 5. So highly

hydrophobic drugs only get transported by this mechanism (Hauss et al., 1998). It has

been explored that some endogenous substances for example P-glycoproteins (P-gp) act

as to inhibit the absorptive pathway of poorly water-soluble anticancer drugs. They

inhibit the absorption process by acting as substrate of these drugs. Some surfactants like

Tween-80, Cremophor-RH 40 inhibit these endogenous substances so lead to increased

absorption of poorly permeable drugs like ETO (Porter et al., 2007).

Lipids or lipid type vehicles also shown to improve absorption by increasing membrane

permeability through increasing the fluidity of cell membrane in the intestine and also

acts to open the membrane tight junctions in cell. Examples of surfactants which do these

changes are Polysorbate-80 and Labrasol, Cremphor EL. Surfactants adsorbed into the

16

cellular membranes by disturbing the structure of cells which lead to improve

permeability of drug molecule (Gursoy and Benita, 2004). The process of drug

solubilization and lipid digestion is presented in Figure 1.4.

Figure 1.4 Schematic process of lipid absorption, digestion and solubilization of drug in

the small intestine (Kalepu et al., 2013).

1.6. In vitro Characterization of SMEDDS

1.6.1. Globule size, zeta potential and PDI

The globule size was primarily based on the type and amount of surfactant and co-

surfactant used (Constantinides, 1995). Microemulsion which formed immediately after

dilution of SMEDDS have very small globule size which increase the absorption of drug,

improved dissolution rate and extent as well more system stability would result. Globule

size was determined by zetasizer using technique Dynamic Light Scattering (DLS).

SMEDDS were diluted in water 100 times and droplet size was analyzed by DLS.

Zetasizer also provide PDI (polydispersipity index) which give information about size

distribution of developed system. The lesser value of PDI indicate narrow and uniformity

17

of globules (Borhade et al., 2008; Bali et al., 2011). Ajeet et al., (2008) developed

SMEDDS of anticancer drug exemestane ME1, ME2, ME3, ME4 and found globule

sizes as 12.3, 14.1, 25.6, and 28.5 nm, also zeta potential of these formulations as −2.2,

-7.3, -0.7, -9.7, respectively. They also noted PDI of these formulations and found as

0.11, 0.23, 0.08, and 0.06, respectively. These 4 formulations were found

thermodynamically stable (Singh et al., 2008). Lu (2008) prepared SMEDDS of 9-

nitrocamptothecin (9-NC) a potent anticancer agent and droplet sizes measured as (30.8

± 4.6) nm for T-form and (39.8 ± 8.2) for C-form SMEDDS respectively. They also

studied zeta potential values of these SMEDDS were as -4.3 (T-Form) and -5.7 (C-Form)

(Lu et al., 2008). Chen (2012) developed SMEDDS of induribin, which is a poorly

water-soluble drug. They found droplet sizes of three formulations A, B and C as 93.62,

91.38, and 22.12, respectively. The PDI of these formulations were found as 0.284,

0.524,0.193 (Chen et al., 2012). Yang (2004) developed SMEDDS of anticancer drug

paclitaxel and droplet size and zeta potential values were analyzed as 2.0±0.4 nm and -

45.5±0.5 mV (Yang et al., 2004). Singh (2009) formulated SMEDDS and found different

droplet sizes of SMEDDS as 65.6, 22.9, 28.5, and 57.5 nm and zeta potential values

were as -7.2, -10.9, -9.7, -5.6 mV, respectively (Singh et al., 2009a). Yao (2008)

developed SMEDDS of nobiletin and determined globule size, PDI and zeta potential as

28.6 nm, 0.068 and -22.6 mV, respectively (Yao et al., 2008). Wang (2011) prepared

SMEDDS of tacrolimus and studied its zeta size which was found to be 17.7 nm and also

in other formulations it was found less than 20 nm (Wang et al., 2011). Jyothi and

Sreelakshmi (2011) developed SMEDDS of flutamide and zeta-size and zeta-potential

were measured as 148.7 nm and -28.7 mV respectively (Jyothi and Sreelakshmi, 2011).

Patel (2011) developed SMEDDS of telmisartan. They found zeta size and zeta potential

as 40 ± 4.23 nm and -23.9 ± 0.42, respectively (Patel et al., 2011). Ansari (2014)

developed felodipine SMEDDS and droplet size and zeta potential values were found as

65-85 nm and -13.71 mV, respectively and PDI values of different SMEDDS

formulations were 0.213, 0.292, 0.119, 0.211 (Ansari et al., 2014).

1.6.2. Refractive Index (RI)

Refractive index is used to access the isotropicity of microemulsions formed after

dilution of SMEDDS. A study reported by Karamustafa in 2008 in which they observed

refractive indices of optimized formulations between different temperature and different

time periods i.e. 4 and 25°C, it has been found that there is no change in refractive

18

indices significantly observed which justified the consistency of microemulsion

formulation (Karamustafa and Çelebi, 2008). This stable refractive index also justified

the thermodynamic stable nature of microemulsion. RI is based on surfactant to co-

surfactant ratio (total surfactant content) and droplet size of developed microemulsion.

So it increased with decreased concentration of surfactant and vice versa while if globule

size increased then refractive index also increased (de Campos Araújo et al., 2010; Bali

et al., 2011; Parveen et al., 2011) Yang (2013) developed SMEDDS of oleanolic acid

they evaluated refractive index as 1.396 ±0.004 (Yang et al., 2013). Czajkowska-Ko´snik

(2015) developed SMEDDS and determined Refractive index value as 1.334

(Czajkowska-Kośnik et al., 2015). Deshmukh (2015) developed SMEDDS of

Atorvastatin. They measured the refractive index value as 1.499 (Deshmukh and

Mahajan, 2015).

1.6.3. Percentage Transmittance

This test performed in order to check degree of transparency and homogeneity of

developed microemulsions formed after dilution of SMEDDS in water. This was

measured by using spectrophotometer in visible range i.e. 546 nm. The value of

percentage transmittance close to 100% showed clear microemulsion (Singh et al.,

2009a). Jaiswal (2014) formulated SMEDDS of telmisartan and percent transmittance

was measured by adding weighed 50 mg of SMEDDS in 50 ml of water on

spectrophotometer at wavelength of 638 nm (Jaiswal et al., 2014). The values of percent

transmittance were found as 100, 100.2, 99.52, 99.43, 98.16, 97.76 of stable formulations

(Jaiswal et al., 2014).

1.6.4. Rheological studies

Rheological studies are important in order to determine the flow behavior of

microemulsions, which formed after dilution of SMEDDS in water. When compared

shear stress and shear rate graphically the microemusion showed Newtonian flow (Čilek

et al., 2006). Once Newtonian flow identified it has confirmed that globules formed are

smaller and round (spherical in shape) (Kristis, 1990). The viscosity microemulsions was

mostly determined by rheometers e.g. Brookfield cone rheometers and others are plate

rheometers (Karamustafa and Çelebi, 2008). Jaiswal (2014) formulated SMEDDS of

telmisartan and viscosity was determined by adding 20 g of SMEDDS formulation in

beaker and results of viscosities of different stable undiluted SMEDDS formulations

19

were measured as follows 2042, 2245, 2598, 3014 (mPascal) (Jaiswal et al., 2014). Rao

(2013) formulated SMEDDS of valsartan and diluted 1 ml of SMEDDS in 10 ml and 100

ml of water. The results of determined viscosities were 0.62-0.93 and 0.46-0.62 cP after

dilutions of 10 and 100 times, respectively. While undiluted SMEDDS have viscosities

as 136.7, 118.1, 87.4, 72.9, 65.3 cP (Rao et al., 2013). Patel and Sawant (2007) studied

the viscosity of SMEDDS of acyclovir. The viscosity of undiluted SMEDDS was found

as 1244 cP and its values were 1.92 and 0.72 cP after 10 and 100 times of dilution with

water respectively. Wu (2015) developed SMEDDS of AJS (code of medicative

compound) and viscosity of undiluted SMEDDS were determined by rotational

viscometer and values were found in the range of 264 cP to 350 cP which justified that

these formulations have good fluidity in order to fill in capsules (Wu et al., 2015).

1.6.5. Transmission Electron Microscopy (TEM)

This test was used mostly to determine shape and structure of diluted SMEDDS i.e.

microemulsions. This test was done by using transmission electron microscope at higher

magnification. These can be used for size and morphology of globules formed. It can

also be used for checking the uniformity of globules formed (Shafiq et al., 2007;

Basalious et al., 2010). Singh (2008) formulated SMEDDS of exemestane and performed

TEM in order to get the morphology. The microemulsion droplet were found as dark

and sphere-shaped (Singh et al., 2008). Jaiswal (2014) formulated SMEDDS of

telmisartan and morphological studies by using TEM demonstrated that globules were

spherical in shape and uniformity observed (Jaiswal et al., 2014). Wu (2015) formulated

SMEDDS of AJS (An antidepressant compound) and TEM was performed and it has

revealed that droplets were in round shaped and average globule size was 25 nm (Wu et

al., 2015). Goyal (2012) developed SMEDDS of lovastatin and evaluated TEM images

which revealed that droplets were spherical in shape and has size less than 50 nm (Goyal

et al., 2012).

1.6.6. Dissolution

Dissolution test is important to make sure that the drug release is fast and quick in the

dissolution medium. Moreover, the in vitro drug release studies are performed in order to

ensure the quick release of the drug in the dissolution medium and they act as an

important quality control tool for the dosage forms. Furthermore, it gives an estimation

20

of self-emulsification efficiency of the SMEDDS formulations (Mandawgade et al.,

2008). There are different methods by which dissolution can be performed.

1.6.6.1. Rotating paddle USP method

In this method, paddle USP XXIII apparatus, II was used for in vitro release of drug.

Dissolution was performed separately for pure drug and drug loaded with SMEDDS

(Patel and Sawant, 2007). Dhumal (2015) developed SMEDDS of curcumin and its in

vitro release was much quicker than plain curcumin. SMEDDS release drug in about

97% in 10 minutes while release of drug was 30% from plain curcumin in 60 minutes

(Dhumal et al., 2015). Dissolution was performed in USP apparatus type II. Li (2014)

developed SMEDDS of loratadine and its dissolution was compared with commercially

available tablets. SMEEDS of loratadine showed faster drug release i.e. 90% in 15min

while its only 40% release drug from commercial tablets in 45 minutes (Li et al., 2015).

Mandawgade (2008) formulated SMEDDS of natural lipohile abbreviated as (N-LCT)

named beta artemether. SMEDDS were evaluated using USP apparatus and it has been

observed that all SMEDDS formulations of beta artmether release drug within

15minutes. The drug release was 98% (Mandawgade et al., 2008). Jyothi and

Sreelakshmi (2011) developed SNEDDS of flutamide and it showed higher in vitro

release than pure drug suspension. The percentage drug release was 97% (Jyothi and

Sreelakshmi, 2011).

1.6.6.2. Dialysis bag method

This method was also used to determine the in vitro release profile of drug. Atorvastatin

was loaded in SMEDDS and the release profile compared with commercially available

tablets. SMEDDS released drug faster and also more drug was released (Shen and

Zhong, 2006). Dixit and co-workers (2010) developed SMEDDS of valsartan and

performed dissolution by dialysis the bag method. They observed that SMEDDS release

drug faster than conventionally available tablets and quicker than the standard solution of

valsartan. SMEDDS release more than 90% of drug and its in 1 hour. The release from

conventional tablets and standard valsartan suspension was found as 59% and 19%,

respectively. Very limited drug release was observed from suspension formulation. Shen

and Zhong (2006) developed SMEDDS of atorvastatin and in vitro release profile of

SMEDDS and conventional tablets. It has been observed that SMEDDS releases the drug

faster and more drug release as compared with conventional tablets. Jakki and co-

workers (2013) developed and evaluated SMEDDS of domperidone and in vitro drug

21

release profile was observed by dialysis bag method. The release of SMEDDS B

formulation was higher than that of other SMEDDS formulations because SMEDDS B

has smaller globule size.

1.6.6.3. Modified Cylinder Method

In this current research we have used, modified cylinder method first time for the in vitro

release performance of ETO SMEDDS. This method is simple and easy to handle and

economical as compared to other conventional methods. This method was employed to

check in vitro release of nanoparticles and we have used this apparatus and checked the

SMEDDS in vitro performance and it was observed that modified cylinder method can

be used alternatively to other methods successfully (Gao et al., 2013).

1.6.7. Permeation

It has been determined that membrane permeability and in vitro release (Dissolution) of

drug by the mucosal membrane of the gastrointestinal tract are the most important

parameters during the absorption process. However, a major enhancement in solubility

has been achieved by some solubility enhancing formulations. Utilization of surfactants

as an approach to enhance the aqueous solubility of hydrophobic drugs may lead to

increased membrane permeability sometime it was decreased or in some cases it will not

affect the permeability of membrane. Micellar solubilization approach of drugs allows

highly increases in solubility, but also on the other hand results in a decreased free

concentration of drug available for intestinal membrane permeation. The permeability of

membrane and permeation process slowed if surfactant concentration reached above the

CMC (critical micelle concentration) (Löbenberg and Amidon, 2000; Dahan and Miller,

2012). Therefore, a relationship exist between solubility and permeability which must be

considered when developing solubility enhancing formulations e.g. SMEDDS in order to

enhance the oral absorption of drug (Dahan and Miller, 2012). It has been documented

that many in vitro methods have been designed and developed for measuring the

intestinal absorption (permeability) of drugs. Some of these methods are use of Caco-2

cells and similarly “intestinal like” cells has been used such as A549 cell line,

HT29MTX, MDCK, everted sac, artificial membrane, Follicle-Associated epithelium

model (Gibaud and Attivi, 2012; Shahbazi and A Santos, 2013).

22

As an alternative approach, the artificial membrane has been used as gastro intestinal

membrane in determination of absorption of drug. Primarily many drugs are absorbed by

the process of passive diffusion (transport process). So permeability potential through an

artificial membrane which behaves like passive transport transcellular memebrane give

an excellent sign of drug absorption strength. So during in vitro models these membranes

can used as substitute of GI membrane (Zhu et al., 2002). The most commonly used tests

for the determination of permeation potential which are used for in vitro studies are

culture of cell based i.e. Caco-2 cells and PAMPA assay (parallel artificial membrane

permeability assay) PAMPA is easy and rapid method for determining permeability

when transporters and efflux systems were absent (Shahbazi and A Santos, 2013).

1.6.7.1. Intestinal rat permeability study

This study was successfully used to determine the in vitro intestinal permeability of

hydrophobic drug. The study was reported and comprised male Wistar Albino rats

weighing 250 to 300 g. The rats used in this study were sacrificed to extract duodenum in

order to study the intestinal perfusion (permeability) of drug (Ghosh et al., 2006;

Thakkar et al., 2011). Subudhi and Mandal (2013) developed SMEDDS of ibuprofen and

performed studies in order to determine the permeability. The ibuprofen loaded

SMEDDS has greater and increased permeability than ibuprofen plain drug solution and

marketed formulation. The percent fraction absorption (Permeability) values were 85.5 ±

1.9% for ibuprofen loaded SMEDDS while 59.3 ± 3.1% for plain drug solution of

ibuprofen and 81.2 ± 2.2% for marketed formulation of ibuprofen (Subudhi and Mandal,

2013). Chitneni (2011) developed SMEDDS of sulpiride and permeability studies were

performed on SMEDDS, drug solution of sulpiride, and micelles of sulpiride. The values

were found as 70.70 ± 5.24%, 31.78±6.10, 68.85 ± 4.13%, respectively. These are also

human percent absorption of sulpiride (Chitneni et al., 2011).

1.6.7.2. Franz diffusion cell method

In a reported study in vitro release of oral micro-emulsions were found by using

modified Franz diffusion cell method. The dissolution release profile of oral

microemulsion was faster than commercially available product as well as plain solution

of drug (Solanki et al., 2012).

23

1.7 In vivo characterization of SMEDDS

1.7.1. HPLC analysis of SMEDDS of anticancer drugs

SMEDDS of anticancer 9-nitrocamptothecin (9-NC) and its concentration in mice was

determined by using HPLC. Cmax (ng/ml) and Tmax (h) of SMEDDS C-form, SMEDDS

T-form, suspension of 9-NC was determined as 159.45±15.81, 229.60 ± 12.50 and 62.97

± 8.64 ng/ml and 0.5, 0.5, 0.17, 0.33 (h), respectively. Similarly, AUC0–8h (ng h/ml) of

SMEDDS C-form, SMEDDS T-form and suspension were dertemined as 351.71±33.66,

360.12 ± 19.44, and 161.24 ± 24.31, respectively (Lu et al., 2008). Pharmacokinetic

parameters of another SMEDDS (exemestane) were reported. The Cmax values of

optimized exemestane SMEDDS and suspension were 99.03 ± 13.03 and 64.67 ± 8.01

ng/ml. The Tmax (h) and AUC(0–∞) (ng h/ml) of SMEDDS and suspension were 2 and 1

and 1357.04 ± 191.79 and 473.00 ± 47.97 ng/ml, respectively (Singh et al., 2009a).

Hong and co-workers (2016) developed SMEDDS of antitumor drug bortezomib and

determined its pharmacokinetic parameters. The Cmax of microemulsion of bortezomib

was 3.44 ± 0.81 and its suspension in distilled water was found as 7.92 ± 4.8 and in

labrasol solution it was 15.59 ± 13.67 ng/ml. The AUC0-24 of SMEDDS, drug in distilled

water and labrasol® solution were found as 8.62 ± 4.41, 15.97 ± 2.19, and 27.58 ± 6.68

µg.h/ml, respectively. Liu and co-workers (2011) developed SMEDDS of novel

antitumor drug sorafenib and pharmacokinetic parameters were determined. The Cmax of

sorafenib SMEDDS and sorafenib suspension were 845.4 ± 86.1 and 271.8 ± 31.5 ng/ml,

respectively. The Tmax of SMEDDS and suspension were found as 8.3 ± 2.0 and 10.4 ±

2.2 h, respectively. Similarly AUC0-72h (ng.h/ml) of SMEDDS and suspension were

28118.7 ± 4619.1 and 7358.9 ± 895.0, respectively. In another study, SMEDDS of

anticancer bufalin showed 2.38 fold more bioavailability than bufalin suspension (Liu et

al., 2010). Pharmacokinetic parameters of paclitaxel SMEDDS were also evaluated and

showed improvement in bioavailability when co administered with cyclosporin-A (Yang

et al., 2004).

1.8. Objectives of study

Hydrophobic drug entities have found to have lesser solubility as compared to water-

soluble drugs, which leads to lesser bioavailability. There are many techniques for

improving bioavailability but among all SMEDDS is one of the promising technique to

enhance the solubility and bioavailability. There are few studies on bioavailability of

24

hydrophobic drugs, which are available commercially in various drug delivery systems.

Our main aim of the study was to enhance the solubility and bioavailability of poorly

water-soluble anticancer drug (etoposide) by developing its SMEDDS for oral

administration.

25

2. MATERIALS AND METHODS

2.1. Materials

Etoposide (ETO) was procured from Tecoland Corporation, USA. Oils, surfactants, co-

surfactants, solubilizers were a gift from Gattefosse, Canada. Lauroglycol FCC®

(propylene glycol monolaurate Type I), Lauroglycol 90 (Propylene Glycol monolaurate

type II), Peceol® (Glycerol monoleats Type 40), Labrasol

® (Caprylocaproyl Polyoxyl-8

Glycerides), Labrafac CC® (Medium chain triglycerides MCT or Caprylic/Capric

Triglyceride), Transcutol HP® (Diethylene Glycol Monoethyl Ether). Polysorbate 80

(polyoxyethylene (20) Sorbitan monooleate) (HLB=15), Pluriol R E 400 NF, was from a

BASF company procured from L.V LOMAS Limited Ontario Canada. Ultra-pure water

was made by Maxima (Ultra-pure water system) from ELGA (England). Methyl alcohol

and acetonitrile used were HPLC grade (Merck, Germany). All other chemicals used in

this study were of reagent grade.

All vegetables and natural oils and empty transparent hard gelatin capsule shells were

procured from PCCA Ontario, Canada. Clove oil and Span 85 (Sorbitane trioleate) were

procured from Sigma-Aldrich Co. USA. Dialysis membrane molecular weight cut-off

(MWCO: 12-14 KDa) were from Spectrum Laboratories (Rancho Domi, guez, CA,

USA). VePesid® (etoposide) capsules were purchased from Bristol-Myers Squibb,

Montreal, Canada having lot number 6B04181. Each capsule contains 50 mg ETO.

2.2. Methods

2.2.1. Preparation of calibration curve of pure etoposide

A calibration curve of etoposide was prepared in methyl alcohol and used to determine

the solubility of the drug in various oils, surfactants, co-surfactants/co-solvents. A

standard solution of etoposide was prepared by dissolving 100 mg of etoposide in 50 ml

of methyl alcohol. Then different dilutions were prepared by diluting the stock solution

in methyl alcohol, which range from 2-14 µg/ml. The absorbance was measured at 283

nm wavelength.

26

2.2.2. Drug solubility studies

The saturation level of etoposide was determined in various oils, surfactants and co-

surfactants. An excess quantity of etoposide (approx. 0.5 g) was added to the appropriate

vehicle (2 ml) with constant stirring at 50°C for 40-45 minutes by using hot plate

magnetic stirrer (VELP sceintifica, Germany). If the mixture did not receive the

saturation level then the extra amount of drug was added to achieve the saturation point

under the same condition. At equilibrium all the samples were centrifuged at 3500 rpm

for 15 minutes to separate undissolved drug. Parts of the supernatant were diluted with

methyl alcohol and the quantity of drug was measured using Ultraviolet visible

spectrophotometer (Milton Roy Spectronic Array 3000) (Cho et al., 2013). After

centrifugation the supernatant was taken and properly diluted with methanol. 0.1 ml of

supernatant was diluted and made the volume upto 10 ml with methyl alcohol. The

absorbance was measured at 283 nm wavelength. Blank solutions were prepared by

similarly diluting the respective oil, surfactant, and co-surfactant, Co-solvents in order to

deduct their absorbance from respective samples (Yuan et al., 2006; Zhu et al., 2008).

2.2.3. Selection of oil, surfactant, co-surfactant and solubilizer

Medium chain triglycerides MCT (oily vehicle), polysorbate 80®

(polyoxyethylene (20)

sorbitan monooleate) (surfactant), propylene glycol monolaurate type-I (CoS), and

Diethylene Glycol Monoethyl Ether (solubilizing agent) had the highest solubility for

etoposide. All excipients were miscible with each other.

2.2.4. Compatibility/Miscibility tests

The excipients which had shown high solubility with etoposide were selected and mixed

in capped-vial glass all excipients were miscible with each other therefore they are

finalized for development of phase-diagram study.

2.2.5. Phase-diagram study

A phase-diagram was developed with the aim to investigate the stable area for the

SMEDDS (Singh et al., 2009). Ternary phase-diagrams make a possible assessment of

various surfactants and their potentiating effect with co-surfactant as well as with co-

solvents. These diagrams assisted to establish the optimum concentration ranges of

various excipients used in SMEDDS and aid to determine the self-emulsification regions.

27

In phase diagram boundaries of different phases or region were simply be assessed

visually (Subramanian et al., 2004). Oily vehicle, surfactant, co-surfactant, solubilizer

and their mixtures were used to develop Ternary phase diagram. The oily vehicle,

surfactant, co-surfactant and solubilizer representing each corner of the triangle in

Ternary phase diagram. The different percentages of oily vehicle, 10%- 95% v/v,

surfactant 2.5% to 45% v/v, co-surfactant 1.25% to 22.5% v/v and solubilizer 1.25% to

22.5% v/v were used to prepare variety of mixtures (Gupta et al., 2011). These mixtures

were used for developing phase diagram as shown in Table 2.1.

Table 2.1: Formulation mixtures used for phase diagram study

Formulation codes SMEDDS composition % v/v

MCT PSM (20) PGM (type-I) DGME

FL1 10 45 22.5 22.5

FL2 15 42.5 21.25 21.25

FL3 20 40 20 20

FL4 25 37.5 18.75 18.75

FL5 30 35 17.5 17.5

FL6 35 32.5 16.25 16.25

FL7 40 30 15 15

FL8 45 27.5 13.75 13.75

FL9 50 25 12.5 12.5

FL10 55 22.5 11.25 11.25

FL11 60 20 10 10

FL12 65 17.5 8.75 8.75

FL13 70 15 7.5 7.5

FL14 75 12.5 6.25 6.25

FL15 80 10 5 5

FL16 85 7.5 3.75 3.75

FL17 90 5 2.5 2.5

FL18 95 2.5 1.25 1.25

MCT Medium chaintriglyceride

PSM (20) Polyoxyethylene sorbitan monooleate 20

PGM (type-1) Propylene glycol monolaurate Type-1

DGME Diethylene glycol monoethyle ether

28

2.2.6: Preparation of SMEDDS

Two types of SMEDDS formulations were developed by changing the percentages of

oils, surfactants, and solubilizers as well as with and without addition of co-surfactants.

All formulations were given codes as F1, F2, F3, F4, F5 for unloaded and F1D, F2D,

F3D, F4D, F5D for drug loaded SMEDDS. The other formulations having oils,

surfactants, solubilizer with addition of co-surfactant (PGM type-I) were given codes as

FL1, FL2, FL3, FL4, FL5 for unloaded and FL1D, FL2D,FL3D,FL4D,FL5D for drug

loaded SMEDDS as shown in Table 2.2 and Table 2.3. (Dixit et al., 2010). All „F‟

formulations were prepared by adding MCT, PSM (20) and DGME in desending order in

the galss vial with continous stirring at 50°C for 40 minutes until the homogenous

SMEDDS were formed. In all formulations the proportion of surfactant to solubilizer

was maintained at 2:1. All „FD‟ formulations were prepared by adding a fixed amonut

(1% w/v) of drug in a glass vial before incorporating excipients of formulations.

In the same way „FL‟ formulations were prepared as stated above but with the addition

of co-surfctant. In all formulations the proportion of surfactant to co-surfactant to

solubilizer was maintained at 2:1:1. However, in all types of „FLD‟ formulations the

quantity of drug was fixed at 2% w/v of SMEDDS as shown in table 2.2 and 2.3 below:

Table 2.2: Formulations without co-surfactant

Formulation Codes

‘F’

SMEDDS composition (% v/v)

MCT PSM 20 DGME

F1 10 60 30

F2 15 57.5 28

F3 20 53.5 26.75

F4 25 50 25

F5 30 47 23.5

MCT Medium chain triglyceride


DGME Diethylene glycol mono ethyl ether

29

Table 2.3: Formulations with co-surfactant

Formulation

Codes

SMEDDS composition (% v/v)

MCT PSM 20 DGME PGM (type-I)

FL1 10 45 22.5 22.5

FL2 15 42.5 21.25 21.25

FL3 20 40 20 20

FL4 25 37.5 18.75 18.75

FL5 30 35 17.5 17.5

MCT Mediu m chain triglyceride


PGM (type-1) Propylene glycol monolaurate (type-1)

DGME Diethylene glycol mono ethyl ether

2.3. In vitro evaluation of SMEDDS

2.3.1. Dilution study

Dilution studies were performed by dilution of SMEDDS to 50, 100, 250 and 1000 times

with water and buffers. After dilution, they were kept for 12 h and checked for any signs

of phase separation, turbidity as well as drug precipitation. Turbidity was in general

experienced when the oil concentration was more than 20% in all media (pH 1.2, 4.6,

6.8) (Akula et al., 2014).

2.3.2. Globule size, zeta potential and polydispersity index (PDI)

The globule size, zeta potential and PDI of unloaded and drug loaded SMEDDS were

meaured by Zetasizer Nano-DTS 1060 (Malvern Instruments Ltd., UK) at 25oC and at

fixed angle of 173o. The SMEDDS were diluted in distilled water at 1:100, 1:250 and

1:1000 for measurement purpose.

2.3.3. pH, RI, viscosity and percent transmittance

The pH of the SMEDDS was measured by a pH meter (Accumet XL20) at 25°C

(Junyaprasert et al., 2007). The RI of the SMEDDS was determined by Abbe

refractometer using water as reference. The percent transmittance of SMEDDS was

measured by diluting to 250 ml distilled water and measured at wavelength 546 nm

using UV-visible spectrophotometer (Miltron Roy Spectronic Array 3000) using distilled

water as constant (Akhtar et al., 2013). The viscosity was determined by Rheometer

30

(Brookfield, DV-III Ultra). The 10 ml volume of each type of F, FD and FL, FLD

SMEDDS were analyzed using spindle 21 rotated at 150 rpm for 10 minutes and the

corresponding dial readings on the rheometer were noted (Baboota et al., 2007)

2.3.4. Drug release study

In vitro profile was accessed by using modified dissolution apparatus as shown in Figure

2.1. Weighed SMEDDS containing (50 mg ETO) or VePesid® capsules were placed into

the cylinder with the opening covered using dialysis membrane (MWCO 12-14 kDa).

The rotation speed was adjusted at 100 ± 2 rpm and the temperature maintained at 37 ±

0.5°C using a circulating thermostated bath (Haake D1, Germany). The volume 150 ml

of different media of either SGF pH 1.2 or SIF pH 6.8 was used. At various time

intervals (0, 10, 20, 30, 40, 50, 60 minutes) 1 ml sample was taken from the vessel and

replaced with equal volume of relevant medium. The withdrawn sample was measured

with a UV-spectrophotometer at wavelength 283 nm (Gao et al., 2013)

Figure 2.1: Modified dissolution apparatus used for SMEDDS

2.3.5. Accelerated and thermodynamic stability study

Accelerated and thermodynamic stability studies comprised of three phases as heating-

cooling cycles, centrifugation and freeze-thaw cycles were performed on SMEDDS.

Heating-cooling consist of six cycles comprised heating of diluted SMEDDS

formulations in stability chamber (Sanyo Electric Corporation, Japan) at temperatures

45°C then cooling at refrigerator temperature 4°C. Storage was done at each temperature

for at least 48 hours between cycles. The formulations, which showed no turbidity, were

subjected to centrifugation test. 1-2 ml of the previous formulation was taken in

31

eppendorf tube and was centrifuged at 4000 rpm for 40 min (Heraeus BiofugePico

Instruments, USA) and studied for phase separation and precipitation (Jain et al., 2010).

The formulations which were found transparent after centrifugation were subjected to the

freeze-thaw cycle. Three freeze-thaw cycles which include freezing at -4°C and then

thawing at 40°C were carried out. The formulations were placed for 24 hours for each

temperature between each cycle. Centrifugation was done at 3000 rpm for 30 minutes.

The formulations were then checked for turbidity and precipitation (Balata et al., 2016).

2.3.6. Transmission electron microscopy (TEM)

The formulations were diluted with water, which were directly deposited onto a cooper-

based grid, and the surplus water was removed with a filter paper. Staining was done by

adding a drop of 2% aqueous solution of phosphotungstic acid onto the grid and left for

15-20 seconds and the surplus was removed using a filter paper. The dry slide was

placed under a camera for imaging. The images were taken by Philips/FEI (Morgagni)

Transmission Electron Microscope operated with Gatan Digital Camera (Singh et al.,

2008)..

2.4. In vivo Evaluation of SMEDDS

2.4.1. Study design

The study was an open, single dose, crossover complete two periods of treatment dosing.

Ten (10) Wistar Albino Healthy male rats (210-240 g) were participated in the study.

The rats were divided in two groups 1 and 2 of five rats each. Body weight of rats of

each group is given in Table 2.4. In the first phase of sampling 1st group received

standard formulation (VePesid®) by oral gavage and the second group was given

SMEDDS formulation. A washout period of one week was allowed for the next

sampling. Then in the second phase 2nd

group was given standard formulation and 1st

group was administered Test (SMEDDS) formulation (Table 2.5). Following

administration of dose of either formulation, 3 ml of water was given to rats for the

spontaneous formation of microemulsion in GIT (Zhao et al., 2013). The single dose

drug regimen was administered on an empty stomach and was calculated according to

body weight. Each rat was kept fast overnight prior to the treatment visit.

32

Table 2.4: Weights of rats colored with black marker and red marker

Sr. No 1st group (black colored)

(Weight in grams)

2nd

group (red colored)

(Weight in grams)

1 240 230

2 220 210

3 240 230

4 230 230

5 210 230

Table 2.5: Administration of Test and Standard formulation in crossover design

2.4.2. Method of sampling

The blood samples approximately 400 µl (0.4 ml) was collected from the retro orbital

plexus of rats in a tube containing heparin at different time intervals such as 0,0.25, 0.5,

0.75, 1, 2, 4, 6, 8, hours. The blood samples were mixed completely with heparin in

order to prevent clotting. The supernatant (plasma) was separated from blood samples by

centrifugation using an ultracentrifuge machine at 12000 rpm for a time period of 15

minutes and stored at -21°C until analysis were performed.

2.4.3. Plasma etoposide standard curve and extraction procedure

The standard (calibration) curve was prepared with drug free plasma samples. A known

amount of blank plasma was spiked with methanol etoposide drug solution (1 mg/ml) to

form concentrations of 5.0 to 0.0390625 µg/ml. In 200 µl spiked plasma of each

concentration, 200 µl of 4% perchloric acid was added and mixed by vortex agitation for

1 minute and centrifuged for a time period of 15 minutes at 12000 rpm in ultra-centrifuge

machine. After centrifugation, 20 µl supernatant was injected into the HPLC system

(Ahmad et al., 2010).

Group Treatment

Week 1 Week 2

1

2

Standard (VePesid®)

Test (SMEDDS)

Test (SMEDDS)

Standard (VePesid®)

33

2.4.4. Analysis of Etoposide in plasma

The plasma samples were analyzed using a reversed-phase high-performance liquid

chromatographic (HPLC) method. The HPLC system was comprised of Agilent

Technologies series 1100 (USA) with a pump and variable wavelength detector (VWD)

and a Rheodyne 7125 sample injector fitted with a 20 l sample loop. The detector was

operated using a sensitivity range of 0.005 AUFS and wavelength of 203 nm. Data

Processing Modular was connected with the detector and the signals of detector were

analyzed by HPLC Software. Chromatographic data was processed by computerized

integration software HP ChemStation.

Octadecyl silane (ODS) column (5m, 250-x 4.6 mm ID) fitted with a refillable guard

column was used for separation. Mobile phase was prepared by mixing HPLC grade

methanol with double distilled filtered water (1:1) and contents filtered through 0.45 µm

membrane filter (Sartorius Stedim, Germany). The pH was adjusted to 4.20 with 0.2 N

HCl and was then degassed by passing nitrogen gas for about 2-3 minutes (Munawar

Hayat et al., 2011). Analysis was run at a flow rate of 1.0 ml/min and quantification was

by peak area.

2.4.5. Pharmacokinetic analysis

The most common pharmacokinetic parameters such as total area under the plasma

concentration-time curve (AUC0-), peak plasma concentration (Cmax) and time to reach

maximum plasma concentration (Tmax) were estimated from the plasma concentration-

time profiles of the two preparations. The Cmax and Tmax values were obtained directly

from the plasma-concentration data. The above pharmacokinetic parameters were

calculated as per non-compartmental method of analysis using MS Excel®

(Microsoft

Corporation 2007) and Kinetica® (Thermo Electron Corporation).

2.5. Statistical analysis

Sigmaplot 12.5 statistically analyzed all results and studies were carried out in triplicates

and the results showed the mean ± SD. The statistical study was done via Student's t-test.

A difference which was less than the probability level (P<0.05) was assumed as

statistically significant at alpha 0.5.

34

3. RESULTS

3.1. Preparation of standard curve of pure etoposide

Standard solution of etoposide in methanol with concentrations of 2-16 µg/ml was

prepared. The concentration versus absorbance has been plotted on a graph and shown in

Figure 3.1.

Table 3.1: Known concentrations of etoposide in methanol

S. No. Concentration (µg/ml) Absorbance (nm)

1 2 0.012

2 4 0.025

3 6 0.039

4 8 0.052

5 10 0.065

6 12 0.078

7 14 0.089

8 16 0.101

Figure 3.1: Standard curve of ETO in methanol

y = 0.0064x + 0.0001

R² = 0.999

0

0.02

0.04

0.06

0.08

0.1

0.12

0 5 10 15 20

Ab

sorb

an

ce (

nm

)

Concentration (µg/ml)

35

3.2. Drug solubility studies

Solubility of ETO in a variety of oily vehicles has been carried out which indicate greater

solubility in MCT as exposed in Figure 3.2 while Figure 3.2, Figure 3.4 show solubility

of ETO in different surfactants and co-surfactants, respectively. Maximum and minimum

solubility of ETO were found in MCT as 18.88 ± 0.01 mg/ml and oleic acid as 0.92 ±

0.05 mg/ml, respectively. Similarly, maximum and minimum solubility in surfactants

was found in PSM (20) that is 42.1 ± 0.02 and sorbitan trioleate that is 6.48 ± 0.06

mg/ml, respectively. In co-surfactant it was found highest as 99.18 ± 0.02 in DGME and

lowest as 15.75 ± 0.05 mg/ml in propylene glycol monocaprylate type-II (PGMC type-

II).

Figure 3.2: Solubility of ETO in different types of oils

3.98 2.55

3.75

1.22

5.35

0.92

3.87

8.73 10.72

18.88

2.55

4.96

0

5

10

15

20

25

Co

nce

ntr

atio

n m

g/m

l

36

Figure 3.3: Solubility of ETO in different surfactants

Figure 3.4: Solubility of ETO in different co-surfactants

42.1

26.16

21.64

6.48

0

5

10

15

20

25

30

35

40

45

50

Polyoxyethylene(20) sorbitanmonooleate

Polyoxyethylene(20) sorbitanmonolaurate

Caprylocaproylmacrogol-8glycerides

Sorbitane trioleate

Co

nce

ntr

atio

n(m

g/m

l)

30.56

99.18

15.75

0

20

40

60

80

100

120

Propylene glycolmonolaurate type 1

Diethylene glycolmonoethyl ether

Propylene glycolmonocaprylate (type II)

con

cen

trat

ion

mg/

ml

37

3.3. Selection of oil, surfactant, co-surfactant and solubilizer

As ETO showed the highest solubility in MCT (oily vehicle), PSM (20) (surfactant),

propylene glycol monolaurate type-I (co-surfactant) and DGME (solubilizer) and

therefore were selected for further studies.

3.4. Development of phase-diagram

The mixtures (SMEDDS) were diluted 100 folds with distiled water and were checked

visually for transparency and also evaluated by zetasizer for globule size. The mixture

which become turbid and globule size greater than 100 nm are shown in the shaded area

and are marked as non microemulsion area (non ME area) as shown in Figure 3.5

Figure 3.5: Ternary phase-diagram of different proportion of MCT (oily vehicle), PSM

20 (surfactant), PGM type-I (co-surfactant), DGME (Solubilizer) mixtures

in 100 fold water. (Shaded= non-ME area and non-shaded= ME area)

38

3.5. Drug loading, globule size, zeta potential and polydispersity index (PDI)

The drug loading was 1% w/v of SMEDDS in FD formulations while it was 2% w/v in

FLD. The 20 mg/ml was the maximum payload in the current study. The globule size,

zeta potential and PDI of F, FD and FL, FLD formulations were shown in Table 3.2 and

3.3, respectively.

Table 3.2: Globule size, zeta potential, PDI of F and FD formulations (mean±SD, n=3)

Formulation Codes

Parameters

Globule size (nm) Zeta potential (mV) PDI

F1 15.35 ± 0.05 -2.33 ± 0.02 0.266 ± 0.04

F2 51.07 ± 0.03 -4.13 ± 0.09 0.212 ± 0.01

F3 65.89 ±0.03 -3.44 ± 0.06 0.238 ± 0.01

F1D 15.69 ± 0.01 -2.43 ± 0.02 0.254 ± 0.01

F2D 53.21 ± 0.03 -4.95 ± 0.05 0.224 ± 0.08

F3D 66.80 ± 0.02 -3.98 ± 0.01 0.249 ± 0.01

F denotes unloaded and FD drug loaded SMEDDS formulations, respectively

Table 3.3: Globule size, zeta potential, PDI of FL and FLD formulations (mean±SD,

n=3)

Formulation Codes

Parameters

Globule size (nm) Zeta Potential (mV) PDI

FL1 15.84 ± 0.1 -12.0 ± 0.2 0.094 ± 0.3

FL2 23.99 ± 0.01 -6.13 ± 0.04 0.19 ± 00.002

FL3 38.68 ± 1.88 -5.34 ± 0.12 0.225 ± 00

FL1D 15.89 ± 0.21 -12.9 ± 0.03 0.11 ± 0.01

FL2D 30.99 ± 0.01 -6.6 ± 0.02 0.212 ± 0.02

FL3D 45.68 ± 1.68 -5.82 ± 0.08 0.256 ± 0.01

FL denotes unloaded and FLD drug loaded SMEDDS formulations, respectively

39

Figure 3.6 Effect of percentage of oil concentration on globule size

3.6: Viscosity, pH, Refractive index (RI) and Percent transmittance

The pH Refractive index and viscosity of F, FD and FL, FLD formulations were shown

in table 3.4 and 3.5, respectively. Moreover, percent transmittance of SMEDDS

Formulation FL1 to FL3 and F1 to F3 formulations was found to be 99.65 ± 1.20 %,

99.12 ± 0.45%, 98.95 ± 1.10% and 98.86 ± 0.18 %, 98.42 ± 0.16,98.85 ± 0.20 (mean ±

SD, n=3) respectively. This showed the good transparency of the diluted SMEDDS

(microemulsion).

40

Table 3.4: pH, Refractive index and Viscosity of “F” formulations (mean±SD, n=3)

Table 3.5: pH, Refractive index and Viscosity of “FL” formulations (mean±SD, n=3)

Formulation

code

%

oil

pH

(unloaded

SMEDDS)

pH

(drug

loaded

SMEDDS)

Refractive

index

(unloaded

SMEDDS)

Refractive

index

(drug

loaded

SMEDDS)

Viscosity

(cP)

(unloaded

SMEDDS)

Viscosity (cP)

(drug loaded

SMEDDS)

FL1 10 5.14±0.06 5.35±0.04 1.41±00 1.42±0.05 50.6±0.05 52.10±0.45

FL2 15 5.45±0.04 5.65±0.03 1.40±0.00 1.41±0.05 46.1±0.17 48.16±0.15

FL3 20 5.71±0.03 5.83±0.03 1.42±0.01 1.43±0.01 45.2±0.05 47.40±0.17

FL4 25 5.89±0.03 6.02±0.02 1.41±0.06 1.42±00 42.9±0.17 45.43±0.37

FL5 30 5.95±0.04 6.22±0.06 1.420±00 1.43±00 41.1±0.17 43.56±0.49

3.7 Dilution, Accelerated and thermodynamic Stability study

Table 3.6 shows that the formulation F1-F3 and FL1-FL3 passed the dilution studies, but

formulation F4-F5 and FL4-FL5 failed so these were excluded from further experiments.

The F3 Formulation failed in accelerated and thermodynamic stability studies. However,

formulation FL1, FL2, FL3 remained stable in both types of studies.

Formulation

code

%o

il

pH

(unloaded

SMEDDS)

pH (drug

loaded

SMEDDS)

RI

(unloaded

SMEDDS)

RI (drug

loaded

SMEDDS)

Viscosity

(cP)

(unloaded

SMEDDS)

Viscosity

(cP) (drug

loaded

SMEDDS)

F1 10 6.37±0.025 6.45±0.045 1.43±00 1.42±0.00 48.6±0.057 50.10±0.45

F2 15 6.40±0.011 6.53±0.01 1.43±0.01 1.41±0.02 46.1±0.17 48.16±0.15

F3 20 6.49±0.01 6.58±0.01 1.43±0.01 1.42±0.01 44.2±0.05 46.40±0.17

F4 25 6.60±0.011 6.60±0.01 1.42±0.04 1.41±0.01 42.9±0.17 44.43±0.37

F5 30 6.64±0.011 6.64±0.01 1.42±0.03 1.43±0.20 40.1±0.17 42.56±0.49

41

Table 3.6: Dilution, accelerated and thermodynamic stability study

3.8. In vitro release study

A modified cylinder method was employed to evaluate the drug release from all

SMEDDS. The dissolution studies were performed only with FD and FLD formulations

and marketed product (VePesid® 50 mg) in two types of dissolution media SGF (pH 1.2)

and SIF (pH 6.8). The in vitro drug release of FD formulations (F1D, F2D, F3D) were

found to be 72.68%, 65.66%, 60.95% in pH 1.2, and 79.24%, 70.97%, 62.34% in pH 6.8

(Figure 3.7, Figure 3.9), respectively. While in vitro release of FL formulations (FL1D,

FL2D, FL3D) were found to be 77.44%, 70.64%, 65.89% in pH 1.2, and 88.31%, 79.70,

72.45 at pH 6.8 (Figure 3.8, Figure 3.10), respectively. The drug release of VePesid® was

52.98% and 55.47% in SGF (pH 1.2) and SIF (pH 6.8), respectively.

SMEDDS

formulations

Dilution study results

Transparent (+)

Turbid (-)

Accelerated and

thermodynamic

stability study

results

Interpretation

Stable (+)

Unstable (-)

F1 + Transparent +

F2 + Transparent +

F3 + Turbid -

F4 - Excluded -

F5 - Excluded -

FL1 + Transparent +

FL2 + Transparent +

FL3 + Transparent +

FL4 - Excluded -

FL5 - Excluded -

42

Figure 3.7. In vitro release study of different “FD” formulations Vs VePesid 50 mg

capsules in SGF at pH 1.2

Figure 3.8: In vitro release study of different “FLD” formulations Vs VePesid® 50 mg

capsules in SGF at pH 1.2

0

20

40

60

80

100

0 10 20 30 40 50 60

F1DF2DF3DVepesid

Time (min)

% D

rug

rele

ase

SGF (pH 1.2)

0

20

40

60

80

100

0 10 20 30 40 50 60

FL1DFL2DFL3DVePesid

% D

rug

rele

ase

Time (min)

SGF (1.2)

43

Figure 3.9: In vitro release study of different “FD” formulations Vs VePesid® 50 mg

capsules in SIF at pH 6.8

Figure 3.10: In vitro release study of various “FLD” formulations Vs VePesid® 50 mg

capsule in SIF at pH 6.8

0

20

40

60

80

100

0 10 20 30 40 50 60

F1D

F2D

F3D

Vepesid

SIF (pH=6.8)

Time (min)

% d

rug

rele

ase

0

20

40

60

80

100

0 10 20 30 40 50 60

FL1DFL2DFL3DVepesid

SIF (pH=6.8)

Time (min)

% D

rug

rele

ase

44

3.9. Transmission Electron Microscope (TEM)

TEM pictures of optimized SMEDDS revealed that the microemulsion droplets were

nearly of round shape with a smooth surface. The appearance was black spherical spots

without aggregation. The average globule size of microemulsion dispersed was found in

the range of 15.35 to 65.89nm and 15.69 to 66.8nm for F1 to F3 and FD1 to FD3 for F

formulations and 15.84 to 38.68nm and 15.89 to 45.68nm for FL1 to FL3 and FLD1 to

FLD3 for FL formulations, respectively and was in good relation with the data analyzed

using particle sizing apparatus zeta sizer (Wu et al., 2011).

Figure 3.11. Representative TEM image of optimized SMEDDS formulation


3.10.1 Standard curve of ETO in rat plasma

Standard curve was constructed to encompass anticipated range of ETO concentration

found in Wistar Albino rats. Blank plasma was spiked with standard solution of ETO to

prepare concentrations of 5, 2.5, 1.25, 0.625, 0.3125, 0.1562, 0.0781, and 0.03906 µg/ml.

Mean plasma standard curve was found to be linear over the concentration range

used (Figure 3.12) with correlation coefficient of 0.9997. The chromatogram of

plasma samples taken from Wistar Albino rats at 0, 2.0 and 4.0 hour after dosing of

VePesid® 50 mg capsule is presented in Figure 3.13, 3.14 and 3.15 while Figure 3.16 and

3.17 represent the chromatogram of plasma samples taken at 2.0 and 4.0 hour after

45

dosing of SMEDDS. The retention time of ETO is 7.26. The blank sample was clean and

no interfering peak was observed at the retention times of ETO.

Figure 3.12: Mean plasma standard curve of ETO in Wistar Albino rats

Figure 3.13: Chromatograms of blank plasma before drug administration (0 hour)

Wistar Albino rat.

46

Figure 3.14: Chromatogram of plasma at 2 h after dosing of VePesid

® in Wistar Albino

rat

Figure 3.15: Chromatogram of plasma at 4 h after dosing of VePesid

® in Wistar Albino

rat

47

Figure 3.16: Chromatogram of plasma at 2 h after dosing of SMEDDS in Wistar

Albino rat

Figure 3.17: Chromatogram of plasma at 4 h after dosing of SMEDDS in Wistar Albino

rat

48

3.10.2 Pharmacokinetic analysis

Individual plasma ETO concentration versus time profiles of VePesid®

capsules and

SMEDDS in Wistar Albino rats is shown in Figure 3.18 to 3.27. Significant differences

in the individual as well as in their mean plasma profiles were observed (Table 3.7 and

3.8 and Figure 3.28). The two formulations are seemed to act in a similar fashion. Plasma

concentrations of ETO were detectable during 6-8 hours from the two preparations.

There was rapid increase in the plasma concentration and reaching maximum at

approximately 1.0 hour after dosing, being typical that obtained with conventional

immediate release preparations.

The individual numerical values of AUC0-, Cmax, and Tmax obtained with VePesid®

capsules and SMEDDS are presented in Table 3.9. The values of the pharmacokinetic

parameters, ke, t½ and Vd of the two formulations are given in the Table 3.10. AUC0-,

of SMEDDS are about 2.4 fold greater than VePesid®

capsules while Cmax of SMEDDS

is twice that of VePesid®

capsules in almost similar Tmax. The values of AUC0-, Cmax, ke,

and Vd were in two formulations are different with each other and significantly different

statistically.

49

Figure 3.18: Plasma ETO concentration versus time profile of Rat-1

Figure 3.19: Plasma ETO concentrations versus time profile of Rat-2

0

0.5

1

1.5

0 1 2 3 4 5 6 7 8Pla

sma c

on

cen

tart

ion

(µg/m

l)

Time (h)

VePesid

SMEDDS

0

0.5

1

1.5

0 1 2 3 4 5 6 7 8

Pla

sma c

on

cen

tarti

on

(µg/m

l)

Time (h)

VePesid

SMEDDS

50

Figure 3.20 Plasma ETO concentrations versus time profile of Rat-3

Figure 3.21 Plasma ETO concentration versus time profile of Rat -4

0

0.5

1

1.5

0 1 2 3 4 5 6 7 8

Pla

sma

co

nce

nta

rti

on

(µg

/ml)

Time (h)

VePesid

SMEDDS

0

0.5

1

1.5

0 1 2 3 4 5 6 7 8

Pla

sma c

on

cen

tarti

on

(µg/m

l)

Time (h)

VePesid

SMEDDS

51

Figure 3.22: Plasma ETO concentration versus time profile of Rat Rat-5

Figure 3.23: Plasma ETO concentration versus time profile of Rat -6

0

0.5

1

1.5

0 1 2 3 4 5 6 7 8

Pla

sma

co

nce

nta

rti

on

(µg

/ml)

Time (h)

VePesid

SMEDDS

0

0.5

1

1.5

0 1 2 3 4 5 6 7 8

Pla

sma c

on

cen

tarti

on

(µg/m

l)

Time(h)

VePesid

SMEDDS

52



0

0.5

1

1.5

0 1 2 3 4 5 6 7 8Pla

sma

co

nce

nta

rti

on

(µg

/ml)

Time (h)

VePesid

SMEDDS

0

0.5

1

1.5

0 1 2 3 4 5 6 7 8

Pla

sma c

on

cen

tarti

on

(µg/m

l)

Time(h)

VePesid

SMEDDS

53



0

0.5

1

1.5

0 1 2 3 4 5 6 7 8

Pla

sma

co

nce

nta

rtio

n(µ

g/m

l)

Time(h)

VePesid

SMEDDS

0

0.5

1

1.5

0 1 2 3 4 5 6 7 8Pla

sma c

on

cen

tarti

on

(µg/m

l)

Time(h)

VePesid

SMEDDS

54

Table 3.7: Plasma concentrations verses time profile of VePesid® in male Wistar Albino

Rats.

Albino

Rats

Time (h)

0 0.25 0.5 0.75 1 2 4 6 8

1 0 0.1138 0.2468 0.7254 0.4595 0.2202 0.0873 0.0288 0.0000

2 0 0.0341 0.2202 0.5659 0.4063 0.2202 0.1138 0.0686 0.0101

3 0 0.0418 0.3000 0.5180 0.3912 0.2468 0.1021 0.0535 0.0000

4 0 0.0208 0.1601 0.4808 0.5659 0.3797 0.2104 0.0819 0.0000

5 0 0.0705 0.2468 0.3898 0.7520 0.3266 0.1306 0.0500 0.0000

6 0 0.0413 0.2899 0.3367 0.7225 0.4436 0.1907 0.0572 0.0000

7 0 0.0293 0.0524 0.3000 0.5848 0.3000 0.1622 0.0530 0.0296

8 0 0.0000 0.2120 0.5925 0.3189 0.1699 0.0694 0.0399 0.0000

9 0 0.1107 0.3000 0.4861 0.3189 0.1447 0.0508 0.0133 0.0000

10 0 0.1548 0.3319 0.3797 0.6645 0.1979 0.0774 0.0452 0.0266

Mean 0 0.0617 0.2360 0.4775 0.5184 0.2650 0.1195 0.0491 0.0066

±SD 0 0.0494 0.0822 0.1303 0.1620 0.0957 0.0534 0.0193 0.0118

±SEM 0 0.016 0.026 0.041 0.051 0.030 0.017 0.006 0.004

55

Table 3.8: Plasma concentrations verses time profile of SMEDDS formulation in Wistar

Albino Rats

Albino

Rats

Time (h)

0 0.25 0.5 0.75 1 2 4 6 8

1 0 0.3000 0.8289 1.1680 0.5556 0.4299 0.2976 0.1082 0.0468

2 0 0.3532 0.6426 1.0891 0.8110 0.4764 0.3291 0.2245 0.1270

3 0 0.3149 0.4701 1.0891 0.7906 0.5618 0.4510 0.2845 0.0948

4 0 0.1907 0.4796 0.8455 1.0921 0.4476 0.2976 0.1861 0.0000

5 0 0.2633 0.4248 0.6335 1.0410 0.7035 0.4068 0.2083 0.0728

6 0 0.2875 0.4780 0.7521 1.1943 0.5499 0.3340 0.1184 0.0406

7 0 0.2367 0.5481 0.8311 1.1253 0.4722 0.2854 0.1784 0.0000

8 0 0.2101 0.5201 0.8069 1.0946 0.4485 0.2709 0.1592 0.0223

9 0 0.3000 0.4168 0.5099 1.0742 0.4420 0.2854 0.2083 0.1038

10 0 0.4595 0.7363 0.9834 1.2530 0.5513 0.2612 0.1784 0.0753

Mean 0 0.2916 0.5545 0.8709 1.0032 0.5083 0.3219 0.1854 0.0583

±SD 0 0.0770 0.1381 0.2117 0.2158 0.0848 0.0616 0.0512 0.0437

±SEM 0 0.024 0.044 0.067 0.068 0.027 0.019 0.016 0.014

56

Figure 3.28: Mean plasma ETO concentration Vs time profile of VePesid

® and

SMEDDS in Wistar Albino Rats

0

0.2

0.4

0.6

0.8

1

1.2

0 1 2 3 4 5 6 7 8

Pla

sma C

on

cen

tra

tio

n µ

g/m

l

Time(h)

TEST(SMEDDS)

STD(VePesid®)

57

Table 3.9: Individual Pharmacokinetic values (AUC 0-, Cmax and Tmax) of VePesid® and

SMEDDS in Wistar Albino rats

VePesid

® Capsule

SMEDDS

Rat

AUC 0- Cmax Tmax AUC 0- Cmax Tmax

(µg/ml*h) (µg/ml) (h) (µg/ml*h) (µg/ml) (h)

R1 1.1602 0.73 0.75 2.5516 1.17 0.75

R2 1.1817 0.57 0.75 3.5775 1.09 0.75

R3 1.2044 0.52 0.75 3.6577 1.09 0.75

R4 1.8004 0.57 1.00 3.4077 1.09 1.00

R5 1.5572 0.75 1.00 3.5276 1.04 1.00

R6 1.8401 0.72 1.00 2.9646 1.19 1.00

R7 1.428 0.58 1.00 3.3264 1.13 1.00

R8 0.9456 0.59 0.75 2.8162 1.13 1.00

R9 0.7599 0.49 0.75 3.1558 1.07 1.00

R10 1.3399 0.66 1.00 3.3714 1.25 1.00

Mean 1.32174 0.618 0.875 3.23565 1.125 0.925

±SD 0.3466 0.0913 0.1318 0.3590 0.0631 0.1208

±SEM 0.110 0.029 0.042 0.114 0.020 0.038

58

Table 3.10: Individual Pharmacokinetic values (AUMC 0-, and MRT) of VePesid® and

SMEDDS in Wistar Albino Rats

VePesid

® Capsule SMEDDS

Rat

AUMC0-∞ MRT AUMC0-∞ MRT

(µg.h2/ml) (h) (µg.h

2/ml) (h)

R1 2.50 2.16 10.54 3.13

R2 1.42 2.59 16.16 4.52

R3 2.23 2.70 13.63 3.73

R4 5.57 3.09 15.18 4.45

R5 3.77 2.42 11.33 3.21

R6 4.74 2.58 7.76 2.62

R7 4.18 2.92 13.38 4.02

R8 2.62 2.77 7.39 2.63

R9 1.33 1.75 12.91 4.09

R10 4.09 3.05 10.54 3.13

Mean 3.41 2.60 11.88 3.55

±SD 1.29 0.41 2.92 0.11

±SEM 0.41 0.13 0.92 0.04

59

Table 3.11: Individual Pharmacokinetic values (Ke, t1/2 and Vd) of VePesid®

and SMEDDS in Wistar Albino Rats

VePesid

® Capsule

SMEDDS

Rat

t1/2 Kel Vd t1/2 Kel Vd

(h) (h-1

) (L) (h) (h-1

) (L)

R1 1.39 0.50 865.17 2.25 0.31 482.19

R2 1.42 0.49 865.90 3.21 0.22 646.41

R3 1.67 0.41 1001.60 2.36 0.29 465.51

R4 1.81 0.38 726.04 3.22 0.22 680.69

R5 1.47 0.47 680.59 1.88 0.37 384.95

R6 1.42 0.49 555.33 1.31 0.53 319.70

R7 1.63 0.43 822.75 2.89 0.24 626.44

R8 1.92 0.36 1461.80 1.38 0.50 354.34

R9 1.03 0.67 981.33 2.91 0.24 684.66

R10 2.83 0.25 1521.89 2.25 0.31 482.19

Mean 1.66 0.44 948.24 2.37 0.32 512.71

±SD 0.48 0.11 316.50 0.69 0.11 137.98

±SEM 0.15 0.04 100.09 0.22 0.04 43.63

60

4. DISCUSSION

4.1. Selection of oil, surfactant, co-surfactant and solubilizer

MCT (oily vehicle), PSM (20) (surfactant) Tween 80, propylene glycol monolaurate

type-I (co-surfactant) and DGME (solubilizer) showed the highest solubility with ETO.

MCT possess higher ester concentration per gram over LCT which become the reason of

increases solubility in MCT compared with LCT (Cao et al., 2004). Natural oils have

lesser solubilization capacity for the poorly water soluble drugs which was confirmed

from our solubility studies. Surfactants with HLB value more than 12 have the ability to

provide fine, uniform ME droplets which is necessary for SMEDDS development, such

as nonionic surfactants (e.g. Tweens) and castor oil derivatives (e.g. Ethoxylated castor

oil). These surfactants when combined with lipidic vehicles support self-emulsification

or micro-emulsification (Hauss, 2007). Non-ionic surfactant were preferred because of

their better safety profile, ionic strength and stability than ionic surfactants. DGME

posses phenomenal dissolvable property for ETO and therefore selected as solubilizer.

All components were found to be compatible and completely form a homogeneous

mixture.

4.2. Development of phase-diagram

The SMEDDS mixture which become turbid and globule size greater than 100 nm are

shown in the shaded area and are marked as non microemulsion area (non ME area).

They were considered as non-acceptable SMEDDS. In contrary the SMEDDS which

remained transparent and presented globule size less than 100 nm are highlighted as

Microemulsion area (ME area) and considered acceptable. Formulations FL1-FL3

showed transparency and were included in ME and others in non-ME (Figure 3.5). The

optimum SMEDDS formulation after 100 fold dilution with water highlited in phase-

diagram as FL1. SMEDDS formulations having more than 20% oily vehicle were moved

in non-microemulsion area (non ME area) and vice versa. It was also found that increase

in oil concentration will result in incresed globule size due to decrease in Smix

concentration. Smix was prepared as PSM (20), PGM type-I, DGME in a ratio of (2:1:1)

(Kamboj and Rana, 2016).

61

4.3. Drug loading, globule size, zeta potential and polydispersity index (PDI)

The drug loading was 1% w/v of SMEDDS in FD formulations while it was 2% w/v in

FLD. In a previously reported study the payload was 1% w/w of SMEDDS (Zhao et al.,

2013). The globule size of FD1, FD2 and FD3 increased from 15.69 to 66.80 nm

(FD1<FD2<FD3) (Table 3.2) for F formulations while a lesser increased in globule size

from 15.89 to 45.68 nm (FLD1<FLD2<FLD3) for FLD formulations (Table 3.3). This

lesser increased in globule size might be due to the addition of co-surfactants. Moreover,

as the oil concentration decreased the surfactant concentration was increased which was

the reason for the smaller globule size (Dixit et al., 2010) as depicted in Figure 3.6. The

reported globule size in a previous study was 24.8, 21.3, 20.7 nm in three different

SMEDDS formulations which is in line with our current study (Zhao et al., 2013). The

current study demonstrated that loading of ETO in SMEDDS did not show a substantial

influence to the globule size and zeta potential (Table 3.2 and 3.3). The high zeta

potential signifies the stability of the system and vice versa (Gershanik and Benita,

2000). The zeta potential of F1D was found -2.43 mV. This zeta potential value was

found less and indicator of less stability in the literature. For improvement of formulation

stability and solubility, the PGM type-I was added as co-surfactant in FD formulations

due to its good solubilizing capacity for ETO as observed in our current study. Then after

addition of co-surfactant the zeta potential of FL1 was found as -12.0 mV and -12.9 mV

for FL1D. In previous study the zeta potential value was found -11.2 ± 1.2 and -11.9 ±

0.6 in two different SMEDDS formulations (Zhao et al., 2013) which are also in close

agreement with our current study. The PDI reflected the uniformity of globule size

within each formulation, and it varies from 0 to 1. The globule size measured after

diluting SMEDDS with distilled water the consequential small globule size with the PDI

found in the range of 0.212 to 0.266 nm and 0.249-0.254 nm for “F” and “FD”

formulations and its 0.094 to 0.233 nm and 0.11 to 0.256 nm for FL and FLD

formulations, respectively. All formulations have PDI less than 0.3 which showed

homogeneity and size uniformity (Gershanik and Benita, 2000; Hathout and Nasr, 2013).

4.4. Viscosity, pH, Refractive index (RI) and Percent transmittance

Table 3.4, Table 3.5 shows no statistically significant difference between viscosities of

unloaded and drug loaded SMEDDS (p=0.349; p>0.05). However, a minor increase in

viscosity was found due to drug loading in SMEDDS. As the oil phase increased the

62

viscosity of SMEDDS formulations, decreased while with drug loading in SMEDDS the

viscosity of formulations was slightly increased.

No statistically significant difference (p=0.406) between pH of unloaded and drug loaded

SMEDDS was found. However, a slight increase in pH of drug loaded SMEDDS

formulations was observed due to the drug‟s basic pKa (9.8)(O‟Neil et al., 2001). ETO

was found to have maximum stability at pH of about 3.5-6 (Rezonja et al., 2013) and

therefore, this pH range would be quite favorable for SMEDDS.

The undiluted SMEDDS showed RI values ranged from 1.40 to 1.44 indicating the

clarity of the formulations. The refractive indices of both unloaded and drug loaded

SMEDDS were in close agreement to the RI of constant or water (RI=1.333). The

reported RI values were in the range of 1.40 to 1.47 and therefore, RI values of our study

are close to the reported study (Bandivadeka et al., 2012). Moreover, percent

transmittance of SMEDDS formulations FL1 to FL3 and F1 to F3 formulations was

found to be 99.65 ± 1.20% and 98.86 ± 0.18% (mean±SD, n=3), respectively. This

showed the good transparency of the diluted SMEDDS (microemulsion). The

formulations F4, F5, FL4, and FL5 were omitted from the study because they showed

turbidity when deionized water was added to SMEDDS formulations.

4.5. Dilution, accelerated and thermodynamic stability study

Table 3.6 shows that the formulations F1-F3 and FL1-FL3 passed the dilution studies

test but formulations F4-F5 and FL4-FL5 failed so these were excluded from further

experiments. F3 failed in the accelerated and thermodynamic stability study. However,

formulations FL1-FL3 remained stable in both studies. The turbidity was normally

observed when the oil concentration was more than 20% in all media. The formulations

those were found stable (transparent) were only selected for further studies.

4.6. In vitro release study

The drug release profiles of F and FL formulations were higher as compared to the

VePesid® 50 mg. The drug release was 88% from SMEDDS in 20 minutes at pH 6.8

while 76.9% at pH 1.2 which was the highest among all the formulations. The release

profiles showed improved dissolution in SIF as compared to SGF and same was the case

with VePesid®. This might be owed to the higher solubility of ETO at pH 6.8 due to its

basic pKa (Weylandt et al., 2007) which was confirmed by a previously reported study

63

(Gao et al., 2013). It was observed that irrespective of the dissolution media used, ETO

exhibited rapid release from SMEDDS formulations. This can be attributed to the

microemulsion formed after adding SMEDDS into media (Goldsmith et al., 1978). Here

very tiny globule size resulted in a larger surface area for drug diffusion and high content

of surfactant leads to improve dissolution rate (Borhade et al., 2008). The increase in the

wettability of the lipophilic drug leads to rapid dissolution and, hence increasing the

solubilizing effect (Constantinides et al., 1994). The dissolution data (Zuo et al., 2014)

was fitted in various models using DDSolver®, but the data only fitted well with the

Weibull model. This kinetic model can be successfully applied to almost all kinds of

dissolution curves and the R2 values were found to be in the range of 0.90 to 0.93.


In vivo studies were done in order to determine the pharmacokinetic parameters of

VePesid® capusles and optimized SMEDDS formulation. The plasma concentrations at

different time intervals were used to compute bioavailability and pharmacokinetic

parameters of both formulations. In current study AUC0-∞ of SMEDDS (Test)

formulation was 3.2356 ± 0.1135 µg/ml*h and for standard VePesid® formulation was

1.3217 ± 0.1096 µg/ml*h. The P value is P < 0.0001 indicating significant difference

between AUC0-∞ of VePesid® and SMEDDS. AUC0-∞ values are in remarkable

concurrence with previous study (Akhtar et al., 2013) in which AUC0-∞ for SNEDDS

(Test) was 4855.93 ng.hr/ml and for standard formulation it was 1541.86 ng hr/ml. In

another study, AUC0-∞ values of different SMEDDS formulations such as Cremophor

RH40 based SMEDDS, Crempohor EL based SMEDDS, Tween-80 based SMEDDS are

3.05 ± 0.39, 3.65 ± 0.92, 5.46 ± 1.30 µg.h/ml, respectively (Zhao et al., 2013). These

results are in good agreement with our current study.

In a previous study presented in 2006 in which they have found that AUC0-24h values of

SMEDDS and conventional tablets of a poorly water soluble drug atorvastain as 2612.96

± 367.64 and 1738.04 ± 207.86 ng.h/ml, respectively. So the value of AUC0-24h for

SMEDDS formulation was enhanced (Shen and Zhong, 2006). Kang et al., (2004)

presented in a research in which the values of SMEDDS of simvastatin AUC0-24h and

conventional tablet were found as 123.75 ± 25.40 and 77.88 ± 21.28 ng.h/ml which

clearly showed that AUC0-24h of SMEDDS formulation was enhanced in this reported

study (Kang et al., 2004). In an earlier study reported where it was concluded that AUC0-

∞ of SMEDDS of exemestane (a poorly water-soluble drug) was found to be higher than

64

that of exemestane suspension. The values of AUC0-∞ of SMEDDS and suspension were

found as 1357.04 ng h/ml and 473.00 ng h/ml, respectively (Singh et al., 2009). Likewise

in other research presented in 2015 where AUC0-∞ of SMEDDS of loratadine and

conventional tablet of loratadine were determined and compared and values were as 4.08

± 0.76 and 0.78 ± 0.23 µg.h/L for SMEDDS and tablets, respectively indicating

enhanced AUC0-∞ of SMEDDS formulation (Li et al., 2015). In an earlier research

conducted on silymarin in which values of AUC0-∞ (µg.h/ml) of silymarin SMEDDS and

silymarin PEG-400 solution were found and compared, as 6.23 ± 1.75 and 3.17 ± 1.63,

indicating that AUC0-∞ value was enhanced in case of SMEDDS formulation (Wu et al.,

2006). The AUC0-∞ values of valsartan SMEDDS and valsartan suspension were

determined and compared. The values of AUC0-∞ (ng.h/ml) of SMEDDS and suspension

were found as 1124.57 ± 79.66 and 893.72 ± 116.56, respectively (Dixit et al., 2010). In

another study conducted on sorafenib SMEDDS where it has been reported and

compared AUC0-72h ng.hr/ml values of sorafenib SMEDDS and sorafenib suspension and

values were 28118.7 ± 4619.1 and 7358.9 ± 895.0 respectively. The reported values

showed increased AUC0-72h in case of SMEDDS (Liu et al., 2011).

The solid SMEDDS of clopidogrel napadisilate were developed in a research study

presented in 2014 and the AUC0-∞ values of solid SMEDDS of clopidogrel napadisilate

were determined and compared with AUC0-∞ values of clopidogrel powder. The values

were found as 1521.30 ± 191.50 and 527.26 ± 147.43 (ng.hr/ml), respectively (Kim et

al., 2014) and showed an enhanced AUC0-∞ for solid SMEDDS. The SEDDS of ETO

phospholipid complex (EPC) and SEDDS of ETO without phospholipids were developed

and compared for AUC0-24h. It was found the values of AUC0-24h were 1819.48±173.64

of EPC SEDDS and 1385.97±99.51 (μg/ml min) (Wu et al., 2011). In another research

reported in 2011 in which they calculated and compared the AUC0-12h values of

irbesartan loaded SNEDDS and pure drug suspension. The values were found as

1037±34.6 ng.h/ml and 138.61±23.11, respectively (Patel et al., 2011). Likewise an

earlier research reported the AUC0-∞ values of SMEDDS (cyclopsporin) and

conventional capsules (cyclosporin) were as 5.330±1.514 and 4.443±1.638 (µg*hr/ml),

respectively (Postolache et al., 2002).

The values of AUMC0-∞ in a current study for VePesid and SMEDDS formulations were

found as 3.41±0.41 and 11.88±0.92 (µg.h2/ml). The p value was found to be less than

0.0001 for both formulations and this difference is considered statistically significant. In

65

a previously reported study in 2013 in which values of AUMC0-t (ng.h2/ml) of SNEDDS

(ETO) and Etosid® were found as 14287.62±992.81 and 4186.40±424.08, respectively

(Akhtar et al., 2013). These results are consistent with this current study. The other study

reported in 2012 in which AUMC0-t (µg.h2/ml) values of SNEDDS of cefpodoxime

proxetil and cefpodoxime proxetil (plain capsules) were observed as 2691.84±2.7 and

38.56±1.7, respectively (Bajaj et al., 2013). In another study reported in 2010 where it

was reported AUMC0-∞ (ng.hr/ml) values of SMEDDS (Valsartan) and (Valsartan)

capsule suspension were determined as 37,933.75±1,609.08 and 33,804.48±1,761.19,

respectively (Dixit et al., 2010).

Cmax in current study obtained for VePesid®

(standard) and SMEDDS (Test) formulations

were 0.52±0.05 µg/ml and 1.080 ± 0.051µg/ml respectively. The P value is P < 0.0001

so there is significant difference between Cmax of VePesid® and SMEDDS. Cmax of

SMEDDS (Test) formulation was significantly increased in comparison with VePesid®

formulation; this might be due to presence of surfactants and co-surfactants which

improved the solubilization of ETO which in turn improve peak plasma concentration in

case of SMEDDS. Secondly the Polysorbate-80 has P-glycoprotein and CYP450 enzyme

inhibiting activity (Cornaire et al., 2004; Constantinides and Wasan, 2007; Bansal et al.,

2009; Akhtar et al., 2011) which act as substrate for ETO. By inhibition of P-

glycoproteins the ETO Cmax was increased. In a previous study reported by Akhtar et al.,

(2013), Cmax of Etosid® and SNEDDS formulations were 523.85±9.97 ng/ml and

1297.63 ng/ml, respectively. In another study reported by Zhao et al., (2013) in which

they found Cmax value as 0.39±0.07, 0.50±0.07, 0.52±0.10, 1.37±0.64. These values are

comparable and consistent with the study. In 2011 a research was contucted to develope

SMEDDS of tacrolimus, evaluated and compared its Cmax (ng/ml) with solution of

tacrolimus. These values were found as 1019.173±389.62 and 121.75±74.37,

respectively (Wang et al., 2011). The value of Cmax was increased in case of SMEDDS.

In another study reported in 2008 in which the Cmax values were observed for

nitrocamptothecin (9-NC) SMEDDS, its suspension and solution. The values (ng/ml)

obtained were as 229.60±12.50, 62.97±8.64 and 132.15±41.97, respectively. The results

showed increased Cmax in case of SMEDDS of 9-NC (Lu et al., 2008). In an earlier

reported study the SMEDDS of an anticancer drug i.e. exemestane (a poorly water

soluble drug) and compared the Cmax of SMEDDS (exemestane) and its suspension. The

66

Cmax of SMEDDS was significantly higher 99.03 ng/ml as compared to suspension

which is only 64.67 ng /ml (Singh et al., 2009).

In a study reported in 2011which presented and compared Cmax values of SMEDDS of

sorafenib and suspension of sorafenib and values are as 845.4±86.1 and 271.8±31.5,

respectively (Liu et al., 2011). The enhanced Cmax value of SMEDDS formulation was

observed as compared to drug suspension. In a previously reported study the enhanced

Cmax value was observed for developed SNEDDS of irbesartan as compared to the Cmax

of pure drug suspension which were found as 9.45±0.99 ng/ml and 0.56±0.08 ng/ml,

respectively (Patel et al., 2011). The Cmax (ng/ml) values of SMEDDS of celecoxib were

determined and compared with conventional capsules of celecoxib which were

1287±126.3 and 1065.06 ±38.9 (Subramanian et al., 2004). This showed that Cmax was

improved in case of SMEDDS of celecoxib. The Cmax (ng/ml) values of SMEDDS of

oleanolic acid were determined and compared with conventional tablets of oleanolic acid

and it was found that Cmax values are higher in case of SMEDDS (209.80±47.19) as

compared to conventional tablets (77.60±16.79) (Subramanian et al., 2004). In a similar

study reported in 2004 in which Cmax of paclitaxel in taxol was compared with paclitaxel

in SMEDDS and values obtained were 45±8 ng/ml and 51±8 ng/ml, respectively which

showed improvement in Cmax value when paclitaxel loaded in SMEDDS (Yang et al.,

2004). A likewise study reported in 2004 in which Cmax values of SMEDDS of

simvastatin were obtained and compared with conventional tablets of smivastatin. The

findings were reported as 35.35±8.22 and 18.19±7.01 ng/ml for SMEDDS and

conventional tablets, respectively (Kang et al., 2004). The Cmax (µg/ml) values of

SMEDDS of nevirapine and pure drug suspension were evaluated and values found as

3.9±0.05 and 2.02±0.35, respectively (Kumar et al., 2015). An increase in value of Cmax

for SMEDDS was observed. In a like manner, the SMEDDS of atorvastatin were

developed and Cmax values were comapared with conventional tablet of drug. The values

were found as 512.98±52.60 and 230.88±30.87 ng/ml for SMEDDS and tablets,

respectively (Shen and Zhong, 2006). In another reported study in 2007 in which Cmax

values of SMEDDS and simple drug suspension of silymarin were determined and

compared. The values (µg/ml) were found as 24.79±4.69 and 3.47± 0.20 (Woo et al.,

2007). In an earlier reported study in 2002 in which the reported Cmax values of

SMEDDS of cyclosporine were found higher than that of conventional capsule of

cyclosporine. The values (µg/ml) were 1.025±0.213 and 0.873±0.207 for SMEDDS and

67

capsules, respectively (Postolache et al., 2002). In a previous study presented in 2014 in

which Cmax (ng/ml) values of SMEDDS of leuprorelin acetate were found higher (15.66)

as compared to leuprorelin acetate solution (5.98) (Hintzen et al., 2014). From all above

reported values it has been concluded that SMEDDS enhanced the Cmax. This may be due

to improved solubility of poorly water soluble drugs and avoidance of first pass effect

with SMEDDS formulations (Akhtar et al., 2013) as compared to other conventional

drug delivery systems including suspensions, tablets, and capsules etc.

In current study Tmax values of VePesid® (Standard) and SMEDDS (test) formulations

were 0.875±0.042 h and 0.925±0.038 h, respectively. The P value was found as P =

0.1679 so P>0.01 so there is insignificant difference between Tmax of VePesid® and

SMEDDS and both formulations reached at the peak plasma concentrations at the same

time. Tmax values are in consistent with the reported study (Akhtar et al., 2013). In

reported study, Tmax of Etosid®

(standard) and SNEDDS (Test) formulations as

0.75±0.03 and 1.3±0.05, respectively. In another reported study Tmax values are

1.00±0.52, 0.88±0.14, 1.17±0.68, 0.83±0.13 for ETO suspension, Cremophor RH40

based SMEDDS, Cremophor EL based SMEDDS, Polysorbate-80 based SMEDDS,

respectively (Zhao et al., 2013). These results are in close agreement with our study. In

another reported study in 2013 in which tmax values of SMEDDS (oleanolic acid) and

tablets as 2.00± 1.00 and 2.75±0.50 were observed, respectively (Yang et al., 2013). In a

likewise study reported in 2006 in which tmax (h) values of SMEDDS of atorvastatin and

conventional tablet were 1.17±0.24 and 2.17±0.37, respectively (Shen and Zhong, 2006).

An earlier study where tmax (h) values of developed SMEDDS of nevirapine and pure

drug suspension of nevirapine were found as 1.00±0.05 and 1.50±0.03, respectively

(Kumar et al., 2015). In another study reported in 2011 in which tmax values of irbesartan

loaded SNEDDS and pure drug suspension were determined as 100 (min) for both

formulations (Patel et al., 2011). In a likewise study conducted in 2011 in which tmax (h)

values of SMEDDS of carbamazepine and pure drug suspension were found 2±1.02 and

8.75±2.67, respectively (Kumar et al., 2011). In another study reported in 2008 in which

tmax (h) values of SMEDDS of nitrocamtothecin (9-NC) and its pure drug suspension

were found as 0.5 and 0.33, respectively (Lu et al., 2008). Tmax (h) values of SMEDDS

of tacrolimus and solution of tacrolimus as 2.5±0.55 and 0.29±0.18, respectively (Wang

et al., 2011). In a study presented in 2010 in which tmax (min) values of SMEDDS of

bufalin and bufalin suspension were compared and reported values are 48±6.7 and

60±1.0, respectively (Liu et al., 2010). Singh et al., (2009) presented in his study in

68

which tmax (h) values of SMEDDS of exemestane and exemestane pure drug suspension

were 2 and 1, respectively (Singh et al., 2009). A research reported in 2014 in which tmax

(h) values of SMEDDS of loratadine and commercial tablet of loratadine were found as

0.81±0.21 and 0.81±0.47, respectively. This showed that both the drug delivery systems

have same tmax (Li et al., 2015). In another study reported in 2011 in which tmax (min)

values of SMEDDS of astilbin and pure drug suspension of astilbin were found as

26.66±19.67 and 36.67±28.76, respectively (Mezghrani et al., 2011). In similar study

reported in 2013 in which tmax (h) values of SMEDDS of tacrolimus and marketed tablet

Prograf®

were found as 1.5±0.8 and 2.6±0.7, respectively (von Suesskind-Schwendi et

al., 2013).

In the current study t1/2 (h) (half-life) of SMEDDS formulation and Vepesid® capsule

were found as 2.37±0.69 and 1.66±0.48 respectively. The P value is 0.0633 so P>0.05 so

this difference is considered not statistically significant at 95% confidence interval. In

previously reported study in 2014 in which they t1/2 (h) values of SNEDDS of glyburide

and micronized tablets as 5.8±1.8 and 4.0±1.9 respectively (Liu et al., 2014). In a study

presented in 2011 compared the t1/2(h) values of SMEDDS of sorafenib and sorafenib

suspension as 21.5±5.7 and 10.1±2.0 respectivley (Liu et al., 2011). A research reported

in 2011 in which they have calculated and compared the t1/2 (h) of SMEDDS

(Carbamazepine) and marketed tablet as 69.445±4.83 and 47.95±6.38 respectively

(Kumar et al., 2011). Wang et al., (2015) calculated and compared the t1/2 (h) values of

SMEDDS of 20(S)-Protopanaxadiol and its pure drug Suspension as 1.96±0.15 and

1.45±0.1 respectively (Wang et al., 2015). In presented study in 2010 compeered the

t1/2(h) values of SMEDDS of Silymarin and marketed brand Legalon®

as 2.69±0.12 and

1.71±0.33 (Li et al., 2010). A research conducted in 2014 in which they have reported

and compared t1/2(h) half life values of SMEDDS of 20(S)-25-methoxyl-dammarane-3β,

12β, 20-triol (25-OCH3-PPD) and its Suspension were as 8.77±7.61 and 4.90±1.79 (Cai

et al., 2014). Jakki and co-workers reported t1/2 (h) of SMEDDS of domperidone and

Suspension (Domperidone) and values were as 6.68 ± 1.57 and 6.65 ± 0.61 respectively

(Jakki et al., 2013). Likewise a research conducted in 2002 reported t1/2(h) values of

SMEDDS (cyclosporin) and conventional capsule (cyclosporine) as 4.755 ± 1.050 and

3.483 ± 1.302 respectively (Postolache et al., 2002). In all reported studies, it is apparent

that t1/2 values of SMEDDS is prolonged compared to conventional dosage.

69

In current study mean residence time (MRT) of the SMEDDS (ETO) and VePesid®

were

calculated and values were found as 3.55±0.71 and 2.60±0.41 (h) respectively. The p

value is 0.0049 so P<0.05 so there is statistically significant difference between these

two formulations. In the current study, the increased MRT in SMEDDS (ETO) was due

to increased Cmax, which was due to avoidance of first pass effect and increased aqueous

solubility. In a reported study in 2006 compared MRT values of SMEDDS (Celecoxib)

and Celecoxib (loaded in soyebean oil) as 4.784±0.107 and 3.81 ± 0.230 respectively

(Iwanaga et al., 2006). So MRT value was found increased in case of SMEDDS. These

results are consistent with our current study. MRT (h) values of Supesaturable SMEDDS

(S-SMEDDS) of indirubin and SMEDDS (indirubin) as 8.56 (h) were reported in 2012.

Both formulations have same MRT (Chen et al., 2012). A reaerch conducted by Lu et

al., 2008 in which they have calculated MRT (h) values of SMEDDS (nitrocamptothecin

9-NC) and suspension of 9NC as 2.93 ± 0.26 and 2.81 ± 0.46 respectively (Lu et al.,

2008). In another study conducted in 2016 in which MRT(min) values of SMEDDS

formulaton of cefuroxime Axetil and Tablets of cefuroxime Axetil were found as 120.83

and 124.54 respectively (Satish Puttachari et al., 2016). MRT(h) values of SMEDDS

(Domperidone) and Suspension (Domperidone) were reported as 8.90 ± 1.72 and 8.04 ±

0.85(h) respectively (Jakki et al., 2013). Similarly, MRT(h) values of SMEDDS

(Vinpocetine) and tablets (Vinpocetine) were presented as 2.96±0.82 and 2.45±0.56

respectivley (Chen et al., 2008). In each reported study, MRT was found to be enhanced

in SMEDDS.

In the current study elimination rate constant (Ke) of the optimized SMEDDS (ETO) and

VePesid®

were calculated and values were found as 0.32±0.11 and 0.44±0.11 (h

-1)

respectively. The p value is 0.0480 so P<0.05 so the results are statistically significantly

different between these two formulations. In a study reported in 2011 elimination rate

constant Ke((h-1

) of SMEDDS of sorafenib and sorafenib suspension and values obtained

were 0.034±0.01 and 0.071±0.016 respectively. These results shows decrease in Ke of

SMEDDS (Sorafenib), therefore these results are in close agreement with our current

study. In previously reported study in 2002 in which Ke (h-1

) values of SMEDDS

(cyclosporin) and Capsule (cyclosporin) were compared as 0.153 ± 0.035 and 0.225 ±

0.076 (Postolache et al., 2002). In an earlier study conducted in 2012 in which Kel (h−1

)

values of SNEDDS of cefpodoxime proxetil and plain tablets of cefpodoxime proxetil

were reported as 0.028 ± 0.09 and 0.36 ± 0.08 respectively (Bajaj et al., 2013). The

elimination rate constant of SMEDDS formulation is found to be less due to minor

70

increase in t1/2. This can be explained by the fact that the t1/2 is inversely proportional to

the elimination rate constant, so in this current study elimination rate constant decreased

due to increase in t1/2 of SMEDDS (ETO).

In the current study, volume of distribution (Vd) of the SMEDDS formulation (ETO) and

VePesid®

were calculated and values found as 512.71±137.98 (L) and 948.24±316.50 (L)

respectively. The p value is 0.0040 so p<0.05 indicating statistically significant

difference between Vd of these two formulations. The decreased Vd in case of SMEDDS

formulation might be due to increased Cmax, because inverse relationship is found

between Vd and Cmax. . In a study reported in 2012 in which Vd (L) values of Teniposide

SMEDDS and Teniposide marketed brand (VUMON) were reported as 0.27±0.094 and

0.16±0.052 (L) respectively (He et al., 2012).

71

5.0 CONCLUSION

In current study an optimized Self-microemulsifying drug delivery system (SMEDDS) of

ETO (ETO), a poor water-soluble anticancer drug, was developed successfully for oral

drug delivery and shown to enhance dissolution and in vivo bioavailability in Wistar

Albino Rats. The SMEDDS formulations loaded with ETO were characterized both for

in vitro and in vivo evaluation and optimized formulation was compared with marketed

brand, VePesid®. Based on statistical inferences our current study clearly illustrated that

SMEDDS have the great potential to be used as an ideal oral drug delivery system

instead of other conventional oral available brands of ETO.

72

6.0 FUTURE PROSPECTS

Super saturable SMEDDS of currently developed system can also be prepared.

Solid SMEDDS of the current drug delivery system can be developed by using an

inert carrier.

Some other oils, surfactants, and co-surfactants can also be investigated for

betterment.

The current SMEDDS can also be evaluated as drug carrier for other drugs of

BCS Class-II and IV.

Cell lines studies of this drug delivery system can also be performed.

After conducting appropriate pre-clinical and clinical studies on the current

SMEEDS, it can also be evaluated in real clinical conditions.

73

7. REFERENCES

1. Ahmad, M., Usman, M., Madni, A., Akhtar, N., Khalid, N., Asghar, W.,

Bakhsh, S., 2010. Development and Validation of Reversed-Phase High

Performance Liquid Chromatographic Method for Analysis of Cephradine in

Human Plasma Samples. Journal of the Chemical Society of Pakistan 32, 58-

63.

2. Akhtar, N., Ahad, A., Khar, R.K., Jaggi, M., Aqil, M., Iqbal, Z., Ahmad, F.J.,

Talegaonkar, S., 2011. The emerging role of P-glycoprotein inhibitors in drug

delivery: a patent review. Expert opinion on therapeutic patents 21, 561-576.

3. Akhtar, N., Talegaonkar, S., Khar, R.K., Jaggi, M., 2013. Self-

nanoemulsifying lipid carrier system for enhancement of oral bioavailability

of etoposide by P-glycoprotein modulation: in vitro cell line and in vivo

pharmacokinetic investigation. Journal of Biomedical Nanotechnology 9,

1216-1229.

4. Akula, S., Gurram, A.K., Devireddy, S.R., 2014. Self-Microemulsifying Drug

Delivery Systems: An Attractive Strategy for Enhanced Therapeutic Profile.

International scholarly research notices 2014.

5. Amidon, G.L., Lennernäs, H., Shah, V.P., Crison, J.R., 1995. A theoretical

basis for a biopharmaceutic drug classification: the correlation of in vitro drug

product dissolution and in vivo bioavailability. Pharmaceutical research 12,

413-420.

6. Ansari, K.A., Pagar, K.P., Anwar, S., Vavia, P.R., 2014. Design and

optimization of self-microemulsifying drug delivery system (SMEDDS) of

felodipine for chronotherapeutic application. Brazilian Journal of

Pharmaceutical Sciences 50, 203-212.

7. Attwood, D., Kreuter, J., 1994. Colloidal drug delivery systems. Kreutzer,

J.(Ecls.) Microemulsions. Marcel Dekker; New York, 33-71.

8. Baboota, S., Shakeel, F., Ahuja, A., Ali, J., Shafiq, S., 2007. Design,

development and evaluation of novel nanoemulsion formulations for

transdermal potential of celecoxib. Acta pharmaceutica 57, 315-332.

9. Balakrishnan, P., Lee, B.-J., Oh, D.H., Kim, J.O., Lee, Y.-I., Kim, D.-D., Jee,

J.-P., Lee, Y.-B., Woo, J.S., Yong, C.S., 2009. Enhanced oral bioavailability

74

of Coenzyme Q 10 by self-emulsifying drug delivery systems. International

journal of pharmaceutics 374, 66-72.

10. Balata, G.F., Essa, E.A., Shamardl, H.A., Zaidan, S.H., Abourehab, M.A.,

2016. Self-emulsifying drug delivery systems as a tool to improve solubility

and bioavailability of resveratrol. Drug design, development and therapy 10,

117.

11. Bali, V., Ali, M., Ali, J., 2011. Nanocarrier for the enhanced bioavailability of

a cardiovascular agent: in vitro, pharmacodynamic, pharmacokinetic and

stability assessment. International journal of pharmaceutics 403, 46-56.

12. Bandivadeka, M.M., Pancholi, S.S., Kaul-Ghanekar, R., Choudhari, A.,

Koppikar, S., 2012. Self-microemulsifying smaller molecular volume oil

(Capmul MCM) using non-ionic surfactants: a delivery system for poorly

water-soluble drug. Drug development and industrial pharmacy 38, 883-892.

13. Bansal, T., Akhtar, N., Jaggi, M., Khar, R.K., Talegaonkar, S., 2009. Novel

formulation approaches for optimising delivery of anticancer drugs based on

P-glycoprotein modulation. Drug discovery today 14, 1067-1074.

14. Basalious, E.B., Shawky, N., Badr-Eldin, S.M., 2010. SNEDDS containing

bioenhancers for improvement of dissolution and oral absorption of

lacidipine. I: development and optimization. International journal of

pharmaceutics 391, 203-211.

15. Benameur, H., 2006. Liquid and semi-solid formulations for enhancing oral

absorption. Bulletin Technique Gattefossé 99, 63-75.

16. Boonme, P., Krauel, K., Graf, A., Rades, T., Junyaprasert, V.B., 2006.

Characterization of microemulsion structures in the pseudoternary phase

diagram of isopropyl palmitate/water/Brij 97: 1-butanol. AAPS

PharmSciTech 7, E99-E104.

17. Borhade, V., Nair, H., Hegde, D., 2008. Design and evaluation of self-

microemulsifying drug delivery system (SMEDDS) of tacrolimus. AAPS

PharmSciTech 9, 13-21.

18. Bowcott, J., Schulman, J.H., 1955. Emulsions Control of droplet size and

phase continuity in transparent oil‐water dispersions stabilized with soap and

alcohol. Berichte der Bunsengesellschaft für physikalische Chemie 59, 283-

290.

75

19. Burcham, D., Maurin, M., Hausner, E., Huang, S.M., 1997. Improved oral

bioavailability of the hypocholesterolemic DMP 565 in dogs following oral

dosing in oil and glycol solutions. Biopharmaceutics & drug disposition 18,

737-742.

20. Cao, Y., Marra, M., Anderson, B.D., 2004. Predictive relationships for the

effects of triglyceride ester concentration and water uptake on solubility and

partitioning of small molecules into lipid vehicles. Journal of pharmaceutical

sciences 93, 2768-2779.

21. Chang, R.-K., Shojaei, A.H., 2004. Effect of a lipoidic excipient on the

absorption profile of compound UK 81252 in dogs after oral administration. J

Pharm Pharm Sci 7, 8-12.

22. Charman, S.A., Charman, W.N., Rogge, M.C., Wilson, T.D., Dutko, F.J.,

Pouton, C.W., 1992. Self-emulsifying drug delivery systems: formulation and

biopharmaceutic evaluation of an investigational lipophilic compound.

Pharmaceutical research 9, 87-93.

23. Chen, Z.-Q., Liu, Y., Zhao, J.-H., Wang, L., Feng, N.-P., 2012. Improved oral

bioavailability of poorly water-soluble indirubin by a supersaturatable self-

microemulsifying drug delivery system. Int J Nanomedicine 7, 709.

24. Chitneni, M., Peh, K.K., Darwis, Y., Abdulkarim, M., Abdullah, G.Z.,

Qureshi, M.J., 2011. Intestinal permeability studies of sulpiride incorporated

into self microemulsifying drug delivery system (SMEDDS). Pak. J. Pharm.

Sci 24, 113-121.

25. Cho, W., Kim, M.-S., Kim, J.-S., Park, J., Park, H.J., Cha, K.-H., Park, J.-S.,

Hwang, S.-J., 2013. Optimized formulation of solid self-microemulsifying

sirolimus delivery systems. International journal of nanomedicine 8, 1673.

26. Čilek, A., Čelebi, N., Tirnaksiz, F., 2006. Lecithin-based microemulsion of a

peptide for oral administration: preparation, characterization, and physical

stability of the formulation. Drug Delivery 13, 19-24.

27. Cole, E.T., Cadé, D., Benameur, H., 2008. Challenges and opportunities in

the encapsulation of liquid and semi-solid formulations into capsules for oral

administration. Advanced drug delivery reviews 60, 747-756.

28. Collnot, E.-M., Baldes, C., Wempe, M.F., Hyatt, J., Navarro, L., Edgar, K.J.,

Schaefer, U.F., Lehr, C.-M., 2006. Influence of vitamin E TPGS poly

76

(ethylene glycol) chain length on apical efflux transporters in Caco-2 cell

monolayers. Journal of Controlled Release 111, 35-40.

29. Constantinides, P.P., 1995. Lipid microemulsions for improving drug

dissolution and oral absorption: physical and biopharmaceutical aspects.


30. Constantinides, P.P., Scalart, J.-P., Lancaster, C., Marcello, J., Marks, G.,

Ellens, H., Smith, P.L., 1994. Formulation and intestinal absorption

enhancement evaluation of water-in-oil microemulsions incorporating

medium-chain glycerides. Pharmaceutical research 11, 1385-1390.

31. Constantinides, P.P., Wasan, K.M., 2007. Lipid formulation strategies for

enhancing intestinal transport and absorption of P‐glycoprotein (P‐gp)

substrate drugs: In vitro/In vivo Case studies. Journal of pharmaceutical

sciences 96, 235-248.

32. Cornaire, G., Woodley, J., Hermann, P., Cloarec, A., Arellano, C., Houin, G.,

2004. Impact of excipients on the absorption of P-glycoprotein substrates in

vitro and in vivo. International journal of pharmaceutics 278, 119-131.

33. Cuine, J.F., McEvoy, C.L., Charman, W.N., Pouton, C.W., Edwards, G.A.,

Benameur, H., Porter, C.J., 2008. Evaluation of the impact of surfactant

digestion on the bioavailability of danazol after oral administration of lipidic

self‐emulsifying formulations to dogs. Journal of pharmaceutical sciences 97,

995-1012.

34. Czajkowska-Kośnik, A., Szekalska, M., Amelian, A., Szymańska, E.,

Winnicka, K., 2015. Development and Evaluation of Liquid and Solid Self-

Emulsifying Drug Delivery Systems for Atorvastatin. Molecules 20, 21010-

21022.

35. Dahan, A., Miller, J.M., 2012. The solubility–permeability interplay and its

implications in formulation design and development for poorly soluble drugs.

The AAPS journal 14, 244-251.

36. Dahan, A., Wolk, O., Kim, Y.H., Ramachandran, C., Crippen, G.M., Takagi,

T., Bermejo, M., Amidon, G.L., 2013. Purely in silico BCS classification:

Science based quality standards for the world‟s drugs. Molecular

pharmaceutics 10, 4378-4390.

77

37. Date, A.A., Nagarsenker, M., 2008. Parenteral microemulsions: an overview.

International journal of pharmaceutics 355, 19-30.

38. de Campos Araújo, L.M.P., Thomazine, J.A., Lopez, R.F.V., 2010.

Development of microemulsions to topically deliver 5-aminolevulinic acid in

photodynamic therapy. European journal of pharmaceutics and

biopharmaceutics 75, 48-55.

39. Deshmukh, A.S., Mahajan, V.R., 2015. Advanced Delivery of Poorly Water

Soluble Drug Atorvastatin By Lipid Based Formulation. Asian Journal of

Pharmaceutical Research and Development Vol 3, 21-38.

40. Dhumal, D.M., Kothari, P.R., Kalhapure, R.S., Akamanchi, K.G., 2015. Self-

microemulsifying drug delivery system of curcumin with enhanced solubility

and bioavailability using a new semi-synthetic bicephalous heterolipid: in

vitro and in vivo evaluation. RSC Advances 5, 90295-90306.

41. Dixit, A.R., Rajput, S.J., Patel, S.G., 2010. Preparation and bioavailability

assessment of SMEDDS containing valsartan. AAPS PharmSciTech 11, 314-

321.

42. Ellaithy, H., El-Shaboury, K.M., 2002. The development of Cutina lipogels

and gel microemulsion for topical administration of fluconazole. AAPS


43. Fanun, M., 2010. Colloids in drug delivery. CRC Press.

44. Food, Administration, D., 2000. Guidance for industry: waiver of in vivo

bioavailability and bioequivalence studies for immediate-release solid oral

dosage forms based on a biopharmaceutics classification system. Food and

Drug Administration, Rockville, MD.

45. Gao, Y., Zuo, J., Bou-Chacra, N., Pinto, T.d.J.A., Clas, S.-D., Walker, R.B.,

Löbenberg, R., 2013. In vitro release kinetics of antituberculosis drugs from

nanoparticles assessed using a modified dissolution apparatus. BioMed

research international 2013.

46. Georgakopoulos, E., Farah, N., Vergnault, G., 1992. Oral anhydrous non-

ionic microemulsions administered in softgel capsules. BT Gattefosse 85, 11-

20.

47. Gershanik, T., Benita, S., 2000. Self-dispersing lipid formulations for

improving oral absorption of lipophilic drugs. European journal of

pharmaceutics and biopharmaceutics 50, 179-188.

78

48. Ghosh, P.K., Majithiya, R.J., Umrethia, M.L., Murthy, R.S., 2006. Design

and development of microemulsion drug delivery system of acyclovir for

improvement of oral bioavailability. AAPS PharmSciTech 7, E172-E177.

49. Gibaud, S., Attivi, D., 2012. Microemulsions for oral administration and their

therapeutic applications. Expert opinion on drug delivery 9, 937-951.

50. Goldsmith, J., Randall, N., Ross, S., 1978. On methods of expressing

dissolution rate data. Journal of pharmacy and pharmacology 30, 347-349.

51. Goyal, U., Arora, R., Aggarwal, G., 2012. Formulation design and evaluation

of a self-microemulsifying drug delivery system of lovastatin. Acta

pharmaceutica 62, 357-370.

52. Grove, M., Müllertz, A., Nielsen, J.L., Pedersen, G.P., 2006. Bioavailability

of seocalcitol: II: development and characterisation of self-microemulsifying

drug delivery systems (SMEDDS) for oral administration containing medium

and long chain triglycerides. European Journal of Pharmaceutical Sciences

28, 233-242.

53. Grove, M., Müllertz, A., Pedersen, G.P., Nielsen, J.L., 2007. Bioavailability

of seocalcitol: III. Administration of lipid-based formulations to minipigs in

the fasted and fed state. European Journal of Pharmaceutical Sciences 31, 8-

15.

54. Groves, M., De Galindez, D., 1976. The self-emulsifying action of mixed

surfactants in oil. Acta pharmaceutica Suecica 13, 361.

55. Guo, A., Marinaro, W., Hu, P., Sinko, P.J., 2002. Delineating the contribution

of secretory transporters in the efflux of etoposide using Madin-Darby canine

kidney (MDCK) cells overexpressing P-glycoprotein (Pgp), multidrug

resistance-associated protein (MRP1), and canalicular multispecific organic

anion transporter (cMOAT). Drug metabolism and disposition 30, 457-463.

56. Gupta, S., Chavhan, S., Sawant, K.K., 2011. Self-nanoemulsifying drug

delivery system for adefovir dipivoxil: design, characterization, in vitro and

ex vivo evaluation. Colloids and Surfaces A: Physicochemical and

Engineering Aspects 392, 145-155.

57. Gurram, A., Deshpande, P.B., Kar, S.S., Nayak, U.Y., Udupa, N., Reddy, M.,

2015. Role of components in the formation of self-microemulsifying drug

delivery systems. Indian Journal of Pharmaceutical Sciences 77, 249.

79

58. Gursoy, R.N., Benita, S., 2004. Self-emulsifying drug delivery systems

(SEDDS) for improved oral delivery of lipophilic drugs. Biomedicine &

Pharmacotherapy 58, 173-182.

59. Hathout, R.M., Nasr, M., 2013. Transdermal delivery of betahistine

hydrochloride using microemulsions: physical characterization, biophysical

assessment, confocal imaging and permeation studies. Colloids and Surfaces

B: Biointerfaces 110, 254-260.

60. Hauss, D.J., 2007. Oral lipid-based formulations: enhancing the

bioavailability of poorly water-soluble drugs. CRC Press.

61. Hauss, D.J., 2007a. Oral lipid-based formulations. Advanced drug delivery

reviews 59, 667-676.

62. Hauss, D.J., 2007b. Oral lipid-based formulations: enhancing the

bioavailability of poorly water-soluble drugs. CRC Press.

63. Hauss, D.J., Fogal, S.E., Ficorilli, J.V., Price, C.A., Roy, T., Jayaraj, A.A.,

Keirns, J.J., 1998. Lipid‐based delivery systems for improving the

bioavailability and lymphatic transport of a poorly water‐soluble LTB4

inhibitor. Journal of pharmaceutical sciences 87, 164-169.

64. Henwood, J.M., Brogden, R.N., 1990. Etoposide. Drugs 39, 438-490.

65. Hong, E.-P., Kim, J.-Y., Kim, S.-H., Hwang, K.-M., Park, C.-W., Lee, H.-J.,

Kim, D.-W., Weon, K.-Y., Jeong, S.Y., Park, E.-S., 2016. Formulation and

Evaluation of a Self-microemulsifying Drug Delivery System Containing

Bortezomib. Chemical and pharmaceutical bulletin 64, 1108-1117.

66. Jain, J., Fernandes, C., Patravale, V., 2010. Formulation development of

parenteral phospholipid-based microemulsion of etoposide. AAPS


67. Jaiswal, P., Aggarwal, G., Harikumar, S.L., Singh, K., 2014. Development of

self-microemulsifying drug delivery system and solid-self-microemulsifying

drug delivery system of telmisartan. International journal of pharmaceutical

investigation 4, 195.

68. Jakki, R., Syed, M.A., Kandadi, P., Veerabrahma, K., 2013. Development of

a self-microemulsifying drug delivery system of domperidone: In vitro and in

vivo characterization. Acta pharmaceutica 63, 241-251.

80

69. Jannin, V., Musakhanian, J., Marchaud, D., 2008. Approaches for the

development of solid and semi-solid lipid-based formulations. Advanced drug

delivery reviews 60, 734-746.

70. Jiao, J., 2008. Polyoxyethylated nonionic surfactants and their applications in

topical ocular drug delivery. Advanced drug delivery reviews 60, 1663-1673.

71. Joel, S., 1996. The clinical pharmacology of etoposide: an update. Cancer

treatment reviews 22, 179-221.

72. Junyaprasert, V.B., Boonme, P., Songkro, S., Krauel, K., Rades, T., 2007.

Transdermal delivery of hydrophobic and hydrophilic local anesthetics from

o/w and w/o Brij 97-based microemulsions. J Pharm Pharm Sci 10, 288-298.

73. Jyothi, B.J., Sreelakshmi, K., 2011. Design and evaluation of self-

nanoemulsifying drug delivery system of flutamide. Journal of young

pharmacists 3, 4-8.

74. Kalepu, S., Manthina, M., Padavala, V., 2013. Oral lipid-based drug delivery

systems–an overview. Acta Pharmaceutica Sinica B 3, 361-372.

75. Kamboj, S., Rana, V., 2016. Quality-by-design based development of a self-

microemulsifying drug delivery system to reduce the effect of food on

Nelfinavir mesylate. International journal of pharmaceutics 501, 311-325.

76. Karamustafa, F., Çelebi, N., 2008. Development of an oral microemulsion

formulation of alendronate: Effects of oil and co-surfactant type on phase

behaviour. Journal of microencapsulation 25, 315-323.

77. Kimura, M., Shizuki, M., Miyoshi, K., Sasai, T., Hidaka, H., Tatakamura, H.,

Matoba, T., 1994. Relationship between the molecular structures and

emulsification properties of edible oils. Bioscience, biotechnology, and

biochemistry 58, 1258-1261.

78. Kommuru, T., Gurley, B., Khan, M., Reddy, I., 2001. Self-emulsifying drug

delivery systems (SEDDS) of coenzyme Q 10: formulation development and

bioavailability assessment. International journal of pharmaceutics 212, 233-

246.

79. Kristis, G., 1990. A viscosity study on oil-in water microemulsion. Int J

Pharm 61, 213-218.

80. Lawrence, M.J., Rees, G.D., 2000. Microemulsion-based media as novel drug

delivery systems. Advanced drug delivery reviews 45, 89-121.

81

81. Li, H., Tan, Y., Yang, L., Gao, L., Wang, T., Yang, X., Quan, D., 2015.

Dissolution evaluation in vitro and bioavailability in vivo of self-

microemulsifying drug delivery systems for pH-sensitive drug loratadine.

Journal of microencapsulation 32, 175-180.

82. Liu, Y., Chen, Z.Q., Zhang, X., Feng, N.P., Zhao, J.H., Wu, S., Tan, R., 2010.

An improved formulation screening and optimization method applied to the

development of a self-microemulsifying drug delivery system. Chemical and

pharmaceutical bulletin 58, 16-22.

83. Liu, Y., Fan, J., Wang, X., Zhang, Q., 2011. Preparation of sorafenib

selfmicroemulsifying drug delivery system and its relative bioavailability in

rats. J Chin Pharm Sci 20, 164-170.

84. Löbenberg, R., Amidon, G.L., 2000. Modern bioavailability, bioequivalence

and biopharmaceutics classification system. New scientific approaches to

international regulatory standards. European journal of pharmaceutics and


85. Lu, J.-L., Wang, J.-C., Zhao, S.-X., Liu, X.-Y., Zhao, H., Zhang, X., Zhou,

S.-F., Zhang, Q., 2008. Self-microemulsifying drug delivery system

(SMEDDS) improves anticancer effect of oral 9-nitrocamptothecin on human

cancer xenografts in nude mice. European journal of pharmaceutics and


86. Malcolmson, C., Satra, C., Kantaria, S., Sidhu, A., Lawrence, M.J., 1998.

Effect of oil on the level of solubilization of testosterone propionate into

nonionic oil‐in‐water microemulsions. Journal of pharmaceutical sciences 87,

109-116.

87. Mandawgade, S.D., Sharma, S., Pathak, S., Patravale, V.B., 2008.

Development of SMEDDS using natural lipophile: application to β-

artemether delivery. International journal of pharmaceutics 362, 179-183.

88. Martignoni, M., de Kanter, R., Grossi, P., Mahnke, A., Saturno, G.,

Monshouwer, M., 2004. An in vivo and in vitro comparison of CYP induction

in rat liver and intestine using slices and quantitative RT-PCR. Chemico-

biological interactions 151, 1-11.

82

89. Mason, T., Wilking, J., Meleson, K., Chang, C., Graves, S., 2006.

Nanoemulsions: formation, structure, and physical properties. Journal of

Physics: Condensed Matter 18, R635.

90. Mazzaferro, S., Bouchemal, K., Ponchel, G., 2013. Oral delivery of

anticancer drugs I: general considerations. Drug discovery today 18, 25-34.

91. Meinzer, A., 1995. Microemulsion-A Suitable Galenical Approach for the

Absorption Enhancement of Poorly Soluble Compounds. Bulletin Technique-

Gattefosse, 21-26.

92. Mou, D., Chen, H., Du, D., Mao, C., Wan, J., Xu, H., Yang, X., 2008.

Hydrogel-thickened nanoemulsion system for topical delivery of lipophilic

drugs. International journal of pharmaceutics 353, 270-276.

93. Munawar Hayat, M., Ashraf, M., UR-Rehman, N., UL-Hassan Nasim, F.,

Ahmad, I., Rahman, J., Saleem, M., Zubair Malik, M., 2011. HPLC

determination of etoposide in injectable dosage forms. Journal of the Chilean

Chemical Society 56, 881-883.

94. Nagel, B., 1989. High‐performance liquid chromatography and lipids. A

practical guide. Oxford, New York, Beijing, Frankfurt, Sao Paulo, Sydney,

Tokyo, Toronto: Pergamon Press, 1987. 272 pp., 63 fig., 21 tab., $38.00,

ISBN 0‐08‐034212‐4. Engineering in Life Sciences 9, 440-440.

95. O‟Neil, M.J., Smith, A., Heckelman, P., 2001. The Merck Index, Merck &

Co. Inc., Whitehouse Station, NJ 309, 405.

96. O'Dwyer, P.J., Leyland-Jones, B., Alonso, M.T., Marsoni, S., Wittes, R.E.,

1985. Etoposide (VP-16–213) Current Status of an Active Anticancer Drug.

New England Journal of Medicine 312, 692-700.

97. O'Neil, M., 2001. Editor. The Merck Index. Whitehouse Station, NJ: Merck

& Co. Inc.

98. Parveen, R., Baboota, S., Ali, J., Ahuja, A., Vasudev, S.S., Ahmad, S., 2011.

Oil based nanocarrier for improved oral delivery of silymarin: in vitro and in

vivo studies. International journal of pharmaceutics 413, 245-253.

99. Patel, A.R., Vavia, P.R., 2007. Preparation and in vivo evaluation of

SMEDDS (self-microemulsifying drug delivery system) containing

fenofibrate. The AAPS journal 9, E344-E352.

83

100. Patel, D., Sawant, K.K., 2007. Oral bioavailability enhancement of acyclovir

by self-microemulsifying drug delivery systems (SMEDDS). Drug

development and industrial pharmacy 33, 1318-1326.

101. Patel, J., Kevin, G., Patel, A., Raval, M., Sheth, N., 2011. Design and

development of a self-nanoemulsifying drug delivery system for telmisartan

for oral drug delivery. International journal of pharmaceutical investigation 1,

112.

102. Patravale, V.B., 2009. Microemulsions: pharmaceutical applications.

Microemulsions: Background, New Concepts, Applications, Perspectives,

259-301.

103. Perlman, M., Murdande, S., Gumkowski, M., Shah, T., Rodricks, C.,

Thornton-Manning, J., Freel, D., Erhart, L., 2008. Development of a self-

emulsifying formulation that reduces the food effect for torcetrapib.


104. Pharmacists, A.S.o.H., 1994. American hospital formulary service drug

information. authority of the Board of Directors of the American Society of

Hospital Pharmacists.

105. Porter, C.J., Pouton, C.W., Cuine, J.F., Charman, W.N., 2008. Enhancing

intestinal drug solubilisation using lipid-based delivery systems. Advanced

drug delivery reviews 60, 673-691.

106. Porter, C.J., Trevaskis, N.L., Charman, W.N., 2007. Lipids and lipid-based

formulations: optimizing the oral delivery of lipophilic drugs. Nature

Reviews Drug Discovery 6, 231-248.

107. Pouton, C., 1985. Effects of the inclusion of a model drug on the performance

of self emulsifying formulations. Journal of pharmacy and pharmacology 37.

108. Pouton, C.W., 1997. Formulation of self-emulsifying drug delivery systems.

Advanced drug delivery reviews 25, 47-58.

109. Pouton, C.W., 2000. Lipid formulations for oral administration of drugs: non-

emulsifying, self-emulsifying and „self-microemulsifying‟drug delivery

systems. European Journal of Pharmaceutical Sciences 11, S93-S98.

110. Pouton, C.W., 2006. Formulation of poorly water-soluble drugs for oral

administration: physicochemical and physiological issues and the lipid

formulation classification system. European Journal of Pharmaceutical

Sciences 29, 278-287.

84

111. Pouton, C.W., Porter, C.J., 2008. Formulation of lipid-based delivery systems

for oral administration: materials, methods and strategies. Advanced drug

delivery reviews 60, 625-637.

112. Prajapati, H.N., Patel, D.P., Patel, N.G., Dalrymple, D.M., Serajuddin, A.T.,

2011. Effect of difference in fatty acid chain lengths of medium-chain lipids

on lipid-surfactant-water phase diagrams and drug solubility. J Excipients

Food Chem 2, 73-88.

113. Rao, B.P., Baby, B., Durgaprasad, Y., Ramesh, K., Rajarajan, S., Keerthi, B.,

Sreedhar, C., 2013. Formulation and evaluation of SMEDDS with Capmul

MCM for enhanced dissolution rate of valsartan. RGUHS. J. Pharm. Sci 3,

33.

114. Rezonja, R., Knez, L., Cufer, T., Mrhar, A., 2013. Oral treatment with

etoposide in small cell lung cancer–dilemmas and solutions. Radiology and

Oncology 47, 1-13.

115. Roger, E., Lagarce, F., Benoit, J.-P., 2011. Development and characterization

of a novel lipid nanocapsule formulation of Sn38 for oral administration.

European journal of pharmaceutics and biopharmaceutics 79, 181-188.

116. Schick, M.J., 1987. Nonionic surfactants: physical chemistry. CRC Press.

117. Sermkaew, N., Wiwattanawongsa, K., Ketjinda, W., Wiwattanapatapee, R.,

2013. Development, characterization and permeability assessment based on

Caco-2 monolayers of self-microemulsifying floating tablets of

tetrahydrocurcumin. AAPS PharmSciTech 14, 321-331.

118. Shafiq, S., Shakeel, F., Talegaonkar, S., Ahmad, F.J., Khar, R.K., Ali, M.,

2007. Development and bioavailability assessment of ramipril nanoemulsion

formulation. European journal of pharmaceutics and biopharmaceutics 66,

227-243.

119. Shah, N., Carvajal, M., Patel, C., Infeld, M., Malick, A., 1994. Self-

emulsifying drug delivery systems (SEDDS) with polyglycolyzed glycerides

for improving in vitro dissolution and oral absorption of lipophilic drugs.


120. Shahbazi, M.-A., A Santos, H., 2013. Improving oral absorption via drug-

loaded nanocarriers: absorption mechanisms, intestinal models and rational

fabrication. Current drug metabolism 14, 28-56.

85

121. Shen, H., Zhong, M., 2006. Preparation and evaluation of

self‐microemulsifying drug delivery systems (SMEDDS) containing

atorvastatin. Journal of pharmacy and pharmacology 58, 1183-1191.

122. Singh, A.K., Chaurasiya, A., Awasthi, A., Mishra, G., Asati, D., Khar, R.K.,

Mukherjee, R., 2009a. Oral bioavailability enhancement of exemestane from

self-microemulsifying drug delivery system (SMEDDS). AAPS


123. Singh, A.K., Chaurasiya, A., Singh, M., Upadhyay, S.C., Mukherjee, R.,

Khar, R.K., 2008. Exemestane loaded self-microemulsifying drug delivery

system (SMEDDS): development and optimization. AAPS PharmSciTech 9,

628-634.

124. Singh, B., Bandopadhyay, S., Kapil, R., Singh, R., 2009b. Self-emulsifying

drug delivery systems (SEDDS): formulation development, characterization,

and applications. Critical Reviews™ in Therapeutic Drug Carrier Systems 26.

125. Singh, B.N., Kim, K.H., 2002. Drug delivery-oral route. Encyclopedia of

pharmaceutical technology 1.

126. Slevin, M.L., 1991. The clinical pharmacology of etoposide. Cancer 67, 319-

329.

127. Solanki, S.S., Sarkar, B., Dhanwani, R.K., 2012. Microemulsion drug

delivery system: for bioavailability enhancement of ampelopsin. ISRN

pharmaceutics 2012.

128. Srivalli, K.M.R., Lakshmi, P., 2012. Overview of P-glycoprotein inhibitors: a

rational outlook. Brazilian Journal of Pharmaceutical Sciences 48, 353-367.

129. Stegemann, S., Leveiller, F., Franchi, D., De Jong, H., Lindén, H., 2007.

When poor solubility becomes an issue: from early stage to proof of concept.

European Journal of Pharmaceutical Sciences 31, 249-261.

130. Strickley, R.G., 2004. Solubilizing excipients in oral and injectable

formulations. Pharmaceutical research 21, 201-230.

131. Subramanian, N., Ray, S., Ghosal, S.K., Bhadra, R., Moulik, S.P., 2004.

Formulation design of self-microemulsifying drug delivery systems for

improved oral bioavailability of celecoxib. Biological and Pharmaceutical

Bulletin 27, 1993-1999.

86

132. Subudhi, B.B., Mandal, S., 2013. Self-microemulsifying drug delivery

system: formulation and study intestinal permeability of ibuprofen in rats.

Journal of pharmaceutics 2013.

133. Thakkar, H., Nangesh, J., Parmar, M., Patel, D., 2011. Formulation and

characterization of lipid-based drug delivery system of raloxifene-

microemulsion and self-microemulsifying drug delivery system. Journal of

Pharmacy and Bioallied Sciences 3, 442.

134. Thanki, K., Gangwal, R.P., Sangamwar, A.T., Jain, S., 2013. Oral delivery of

anticancer drugs: challenges and opportunities. Journal of Controlled Release

170, 15-40.

135. Tolle, S., Zuberi, T., Zuberi, S., Warisnoicharoen, W., Lawrence, M., 2000.

Physicochemical and solubilization properties of N,

N‐dimethyl‐N‐(3‐dodecylcarbonyloxypropyl) amineoxide: A biodegradable

nonionic surfactant. Journal of pharmaceutical sciences 89, 798-806.

136. U.S. Food and Drug Administration. Inactive ingredients for approved drug

products, 2007.. .

137. Wakerly, M.G., Pouton, C.W., Meakin, B.J., Morton, F.S., 1986. Self-

emulsification of vegetable oil-nonionic surfactant mixtures. ACS

Publications.

138. Wang, B., Yu, X.-C., Xu, S.-F., Xu, M., 2015. Paclitaxel and etoposide co-

loaded polymeric nanoparticles for the effective combination therapy against

human osteosarcoma. J Nanobiotechnol 13, 1-11.

139. Wang, Y., Sun, J., Zhang, T., Liu, H., He, F., He, Z., 2011. Enhanced oral

bioavailability of tacrolimus in rats by self-microemulsifying drug delivery

systems. Drug development and industrial pharmacy 37, 1225-1230.

140. Welling, P.G., 1977. Influence of food and diet on gastrointestinal drug

absorption: a review. Journal of Pharmacokinetics and Biopharmaceutics 5,

291-334.

141. Weylandt, K.H., Nebrig, M., Jansen-Rosseck, N., Amey, J.S., Carmena, D.,

Wiedenmann, B., Higgins, C.F., Sardini, A., 2007. ClC-3 expression

enhances etoposide resistance by increasing acidification of the late endocytic

compartment. Molecular cancer therapeutics 6, 979-986.

87

142. Wu, L., Qiao, Y., Wang, L., Guo, J., Wang, G., He, W., Yin, L., Zhao, J.,

2015. A self-microemulsifying drug delivery system (SMEDDS) for a novel

medicative compound against depression: a preparation and bioavailability

study in rats. AAPS PharmSciTech 16, 1051-1058.

143. Wu, X., Xu, J., Huang, X., Wen, C., 2011. Self-microemulsifying drug

delivery system improves curcumin dissolution and bioavailability. Drug

development and industrial pharmacy 37, 15-23.

144. Yang, R., Huang, X., Dou, J., Zhai, G., Su, L., 2013. Self-microemulsifying

drug delivery system for improved oral bioavailability of oleanolic acid:

design and evaluation. International journal of nanomedicine 8, 2917.

145. Yang, S., Gursoy, R.N., Lambert, G., Benita, S., 2004. Enhanced oral

absorption of paclitaxel in a novel self-microemulsifying drug delivery

system with or without concomitant use of P-glycoprotein inhibitors.


146. Yao, J., Lu, Y., Zhou, J.P., 2008. Preparation of nobiletin in self-

microemulsifying systems and its intestinal permeability in rats. Journal of

Pharmacy & Pharmaceutical Sciences 11, 22-29.

147. Yu, L.X., Amidon, G.L., Polli, J.E., Zhao, H., Mehta, M.U., Conner, D.P.,

Shah, V.P., Lesko, L.J., Chen, M.-L., Lee, V.H., 2002. Biopharmaceutics

classification system: the scientific basis for biowaiver extensions.


148. Yuan, Y., Li, S.-m., Mo, F.-k., Zhong, D.-f., 2006. Investigation of

microemulsion system for transdermal delivery of meloxicam. International


149. Zhao, G., Huang, J., Xue, K., Si, L., Li, G., 2013. Enhanced intestinal

absorption of etoposide by self-microemulsifying drug delivery systems:

Roles of P-glycoprotein and cytochrome P450 3A inhibition. European

Journal of Pharmaceutical Sciences 50, 429-439.

150. Zhu, C., Jiang, L., Chen, T.-M., Hwang, K.-K., 2002. A comparative study of

artificial membrane permeability assay for high throughput profiling of drug

absorption potential. European journal of medicinal chemistry 37, 399-407.

151. Zhu, W., Yu, A., Wang, W., Dong, R., Wu, J., Zhai, G., 2008. Formulation

design of microemulsion for dermal delivery of penciclovir. International


88

152. Zuo, J., Gao, Y., Bou-Chacra, N., Löbenberg, R., 2014. Evaluation of the

DDSolver software applications. BioMed research international 2014.

Faculty of Pharmacy and Alternative Medicine The Islamia...

Documents

Transcript of Faculty of Pharmacy and Alternative Medicine The Islamia...