1

FORMULATION AND EVALUATION OF SOME IN VITRO AND IN VIVO

PROPERTIES OF ARTEMETHER-LOADED SELF EMULSIFYING

DRUG DELIVERY SYSTEM

BY

UGWU CALISTER ELOCHUKWU

PG/M.PHARM/11/59496

DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY AND

INDUSTRIAL

PHARMACY

UNIVERSITY OF NIGERIA, NSUKKA

FEBRUARY, 2014

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TITLE PAGE

FORMULATION AND EVALUATION OF SOME IN VITRO AND

IN VIVO PROPERTIES OF ARTEMETHER-LOADED SELF

EMULSIFYING DRUG DELIVERY SYSTEM

APPROVAL PAGE

3

THIS DISSERTATION WAS APPROVED BY THE DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY AND INDUSTRIAL PHARMARCY, FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA, NSUKKA

…………………………… ...... …………………. PROF. G. C ONUNKWO DR. N. C OBITTE (SUPERVISOR) (SUPERVISOR)

……………………. DR. I. V ONYISHI (HEAD OF DEPARTMENT)

……………………..

……………………. EXTERNAL EXAMINER DEAN OF FACULTY

DEDICATION

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This work is dedicated to my darling, Gabriel and lovely daughters, Ugwu Gabriela and Sonia. 5

DECLARATION

We certify that Ugwu, Calister Elochukwu, a postgraduate student in the Department of

Pharmaceutical Technology and Industrial Pharmacy, University of Nigeria,

Nsukka, has completed the requirements for the award of the degree of Master

of Pharmacy in Pharmaceutical Technology and Industrial Pharmacy.

The research work reported in this dissertation is original and has not been submitted in

support of an application for another degree or qualification of this or any other

university.

…………………………… ...... …………………. PROF. G. C ONUNKWO DR. N. C OBITTE (SUPERVISOR) (SUPERVISOR)

…………………….

DR. I. V ONYISHI (HEAD OF DEPARTMENT)

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ACKNOWLEDGEMENTS

I will start by thanking the Almighty God who has been by my side throughout the period of this work. To Him be the glory. My thanks to Step B management for their support in this research work, may God bless you. I wish to thank my first supervisor, Professor G. C. Onunkwo, who has always given me his support and assistance. I would like to express my warmest gratitude to my second supervisor Dr N. C. Obitte for his constant patience and practical support. You were always present when needed. I enjoyed working under your expert supervision and wished everybody would have a supervisor like you! My gratitude to our honourable HOD, Dr. I. V. Onyishi, who has provided excellent working facilities and environment for research work to progress, may the good Lord continue to shower more wisdom on you, thank you for the endless support. I appreciate our able Professors; Prof A. Ckukwu, Prof S. I Ofoefule and Dr. J. Onyechi, for providing professorial advice and even accommodation to facilitate this work. My thanks to the staff of our sister department such as Prof A. A. Attama, Prof E. Ibezim, Prof K. C Ofokansi, Dr.E. Onuigbo, Dr. P. Nnamani, Dr. M. Mommoh, Pharm. John and Pharm Kene. My thanks to Pharm. Agubata, Pharm. S. A Chime, and Pharm. N. O. Umeh. To my classmates and co- researchers; Pharm. Valentine, Reginald, Kenneth, and Nduka. To our staff Mr Nwodo, Mrs. Ezugwu, Anti Mau-Mau, and labouratory workers Mr. Odoh, Mrs okeh and Mrs. Oledinma, G.O.D, Emeka and KC, they were all helpful. I appreciate my friends and well-wishers; Pharm. (Mrs) Uwaoma Nwanneka, Pharm. (Mrs) Ezeani Chiamaka, Pharm. (Mrs) Peace (Nee Ibezim), Rev. Fr. Charles Ozioko and Mr Clement Okwor for their prayers. They are wonderful too. I cannot thank enough my darling husband, Gabriel, for his financial and moral support. To my dear lovely daughter Anulinna, I appreciate her for her patience always tolerating mummy’s absence. Many thanks to my dear brother, Engr. Emeka, and wife, all my sisters, my niece-Obinwa and my 7

beloved mother, Roseline (always available to assist) for their prayers, financial support and assistance. Thank you all and God bless.

TABLE OF CONTENTS TITLE PAGES i APPROVAL ii DEDICATION iii DECLARATION iv ACKNOWLEDGEMENT v TABLE OF CONTENTS vi LIST OF TABLES ix LIST OF FIGURES x ABSTRACT xi CHAPTER ONE 1.0 GENERAL INTRODUCTION 1 1.1 BIOPHARMACEUTICAL CLASSIFICATION SYSTEM (BCS) OF DRUGS 3 1.2 DISPERSED SYSTEM 6 1.2.1 COLLOIDAL DISPERSION 6 1.2.2 MACROEMULSION OR CONVENTIONAL EMULSION 7 1.2.3 GEL 8 1.2.4 SUSPENSION 9 1.2.5 MICROEMULSION 10 1.2.6 FOAMS 11 1.3 FORMULATION TECHNIQUES OF HYDROPHOBIC DRUGS 12 1.3.1 SALT FORMATION 13 1.3.2 SOLID DISPERSION 13 1.3.3 COMPLEXATION WITH CYCLODEXTRIN 14 1.3.4 SOLID LIPID NANOPARTICLES 15 1.3.5 LIPOSOMES 16 1.3.6 NIOSOMES 17 1.3.7 DENDRIMERS 17 1.3.8 ANTISOLVENT 19 1.3.9 CO-GRINDING 20 1.3.10 SELF EMULSIFYING OIL FORMULATION 22 1.3.10.1 LIQUID SELF EMULSIFYING DRUG DELIVERY SYSTEM 34 8

1.3.10.2 SOLID SELF EMULSIFYING DRUG DELIVERY SYSTEM 34 1.3.10.3 SUPERSATURABLE /GELLED SELF EMULSIFYING DRUG DELIVERY SYSTEM 44 1.4 LIPID FORMULATION CLASSIFICATION SYSTEM (LFCS) 50 1.5 COMPOSITION OF SELF EMULSIFYING OIL FORMULATION 52 1.5.1 LIPOPHILIC BASE 53 1.5.2 SURFACTANT 56 1.5.3 CO-SURFACTANT/CO-SOLVENT 58 1.6 LITERATURE REVIEW OF EXCIPIENTS 60 1.6.1 LABRASOL® 60

1.6.2 TRIACETIN ® 60

1.6.3 TRANSCUTOL P® 61 1.6.4 HYDROXYPROPYLMETHYLCELLULOSE 62 1.7 MALARIA 63 1.8 ARTEMETHER (ARM) 67 1.8.1ASSAY OF ARTEMETHER 71 1.8.2 PHARMACOLOGICAL USES OF ARTEMETHER 73 1.8.2.1 ANTIMALARIAL EFFECT OF ARTEMETHER 73 1.8.2.2 ANTISCHITOSOMIASIS EFFECT OF ARTEMETHER 74 1.8.2.3 ANTITUMOR EFFECT OF ARTEMETHER 75 1.9 SELF EMULSIFYING DRUG DELIVERY SYSTEM (SEDDS) 77 1.9.1 PREPARATION OF SEDDS 77 1.9.2 MECHANISMS OF SEDDS AND ITS APPLICATIONS 78 1.9.3 CHARACTERIZATION OF SEDDS 81 1.9.3.1 DROPLET SIZE 81 1.9.3.2 EQUILIBRIUM PHASE DIAGRAM 81 1.9.3.3 ZETA POTENTIAL MEASUREMENT 82 1.9.3.4 DETERMINATION OF EMULSIFICATION TIME 82 1.9.3.5 LIQUEFACTION TIME 82 1.9.3.6 RELEASE STUDIES 83 1.9.3.7 THERMODYNAMIC STABILITY STUDIES 83 1.10 OBJECTIVES OF THE STUDY 84

CHAPTER TWO 2.0 MATERIAL AND METHODS 85 2.1 MATERIALS AND APPARATUS 85 2.2 METHODS 86 2.2.1 MELTING POINT DETERMINATION 86 2.2.2 STANDARD BEER-LAMBERT PLOT 86 2.2.3 SOLUBILITY OF DRUG IN DIFFERENT VEHICLES 87 2.2.4 CONSTRUCTION OF PHASE DIAGRAMS 87 9

2.2.6 SOLUBILITY STUDIES OF THE OPTIMIZED SEDDS BATCHES 88 2.2.7 PREFORMULATION ISOTROPICITY TESTS 88 2.2.8 FORMULATION OF ARM-LOADED SEDDS 88 2.2.9 FORMULATION OF ARM-LOADED SUPERSATURABLE SEDDS (S-SEDDS) 89 2.3 EVALUATION OF THE SEDDS AND S-SEDDS 89 2.3.1 DROPLET SIZE, POLYDISPERSITY INDEX AND ZETA POTENTIAL 90 2.3.2 POSTFORMULATION ISOTROPICITY TESTS/STABILITY TEST 90 2.3.3 EMULSIFICATION TIME TESTS 90 2.3.4 REFRIGERATION CYCLE TEST 90 2.3.5 CENTRIFUGATION 90 2.3.6 AQUEOUS DILUTION TEST 90 2.3.7 VISCOSITY OF ARM-LOADED SEDDS 91 2.3.8 DETERMINATION OF PH 91 2.3.9 LOADING EFFICIENCY 91 2.3.10 CRYSTALLIZATION/PRECIPITATION STUDIES 91 2.3.10.1 PHOTOMICR. OF STAND. DISPERSION OF ARM-SEDDS AND S-SEDDS 91 2.3.11 DRUG RELEASE STUDIES 92 2.3.12 IN VIVO STUDIES 92

CHAPTER THREE 3.0 RESULTS AND DISCUSSIONS 94 3.1 MELTING POINT 94 3.2 SOLUBILITY OF ARM IN DIFFERENT VEHICLES 94 3.3 PSEUDOTERNARY PHASE DIAGRAM 96 3.4 SOLUBILITY STUDIES OF OPTIMIZED BATCHES 105 3.5 PREFORMULATION ISOTROPICITY TESTS 106 3.6 DROPLET SIZES, POLYDISPERSITY INDEX AND ZETA POTENTIAL 108 3.7 POSTFORMULATION ISOTROPICITY TESTS 110 3.8 EMULSIFICATION TIME 110 3.9 REFRIGERATION CYCLE TEST 111 3.10 CENTRIFUGATION 111 3.11 AQUEOUS DILUTION TEST 112 3.12 VISCOSITY STUDIES 112 3.13 PH STUDIES 119 3.14 LOADING EFFICIENCY OF ARM-LOADED SEDDS AND S-SEDDS 126 3.15 CRYSTALLIZATION/PRECIPITATION STUDIES 132 3.16 RELEASE STUDIES ARM-LOADED SEDDS AND S-SEDDS 135 3.17 RELEASE MECHANISMS OF HPMC BATCHES IN SGF AND SIF 141 3.18 THE RESULTS OF IN VIVO STUDIES OF ARM-LOADED SEDDS 150

CHAPTER FOUR 10

CONCLUSION 153 RECOMMENDATION 154 REFERENCES 155 APPENDICES 177 LIST OF TABLES

Table 1: Lipid formulation classification system 50 Table 2: The percent composition ratios of the SEDDS and S-SEDDS 89 Table 3: Droplet size, polydispersity and zeta potential of the SEDDS 108 Table 4: The loading efficiency of 40 mg ARM-loaded SEDDS of non-HPMC 126 Table 5: The loading efficiency of 50 mg ARM-loaded SEDDS of non-HPMC 127 Table 6: The loading efficiency of 55 mg ARM-loaded SEDDS of non-HPMC 127 Table 7: The loading efficiency of 40 mg ARM–loaded S-SEDDS of HPMC 128 Table 8: The loading efficiency of 50 mg ARM-loaded S-SEDDS of HPMC 128 Table 9: The loading efficiency of 55 mg ARM-loaded S-SEDDS of HPMC 129

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LIST OF FIGURES

Fig 1: Formulation techniques of hydrophobic drugs 12 Fig 2: The role of HPMC as PPI to inhibit the formation of crystalline materials 47 Fig 3: The structural formular of artemether 68 Fig 4: Synthesis of artemisinin derivative 69 Fig 5: Structure of α-β unsaturated decalone 72 Fig 6: Solubility (mg/ml) profile of ARM in different vehicles 94 Fig 7: Pseudoternary phase diagram plot of Smix(1:0) 96 Fig 8: The phase diagram of the Smix ratio 1:0.5 97 Fig 9: The phase diagram of the Smix ratio 1:1 98 Fig 10: The phase diagram of the Smix ratio 1:2 99 Fig 11: The phase diagram of the Smix ratio 1:3 100 Fig 12: The phase diagram of the Smix ratio 3: 1 101 Fig 13: The phase diagram of the Smix ratio 3:2 102 Fig 14: A typical aqueous of SEDDS dispersion 104 Fig 15: Solubility of ARM in the optimized SEDDS batches 105 Fig 16: Capsules of ARM-loaded SEDDS and S-SEDDS 107 Fig 17: Viscosity of non-HPMC batches of 40 mg ARM SEDDS 113 Fig 18: Viscosity of non-HPMC batches of 50 mg ARM SEDDS 114 Fig 19: Viscosity of non-HPMC batches of 55 mg ARM SEDDS 115 Fig 20: Viscosity of HPMC batches of 40 mg ARM SEDDS 116 Fig 21: Viscosity of HPMC batches of 50 mg ARM SEDDS 117 Fig 22: Viscosity of HPMC batches of 55 mg ARM SEDDS 118 Fig 23: pH versus time (month) for 40 mg ARM-loaded SEDDS of non-HPMC batches 120 Fig 24: pH versus time (month) for 50 mg ARM-loaded SEDDS of non-HPMC batches 121 Fig 25: pH versus time (month) for 55 mg ARM-loaded SEDDS of non-HPMC batches 122 Fig 26: pH versus time (month) for 40 mg ARM-loaded SEDDS of HPMC batches 123 Fig 27: pH versus time (month) for 50 mg ARM-loaded SEDDS of HPMC batches 124 Fig 28: pH versus time (month) for 55 mg ARM-loaded SEDDS of HPMC batches 125 Fig 29: Photomicrograph of ARM crystals 132 Fig 30: Photomicrograph of ARM SEDDS of non-HPMC batches after 3 h 133 Fig 31: Photomicrograph of ARM SEDDS of HPMC batches after 3 h 134 Fig 32: Release profile of HPMC batches in SIF 137 Fig 33: Release profile of HPMC batches in SGF 138 12

Fig 34: Release profile of non-HPMC batches in SIF 139 Fig 35: Release profile of non-HPMC batches in SGF 140 Fig 36: Zero order plot of HPMC batches in SGF 142 Fig 37: First order plot of HPMC batches in SGF 143 Fig 38: Higuchi plot of HPMC batches in SGF 144 Fig 39: Ritger–Peppas plot of HPMC batches in SGF 145 Fig 40: Zero order plot of HPMC batches in SIF 146 Fig 41: First order plot of HPMC batches in SIF 147 Fig 42: Higuchi plot of HPMC batches in SIF 148 Fig 43: Ritger–Peppas plot of HPMC batches in SIF 149 Fig 44: The plot of percent antimalarial activity with 40 mg ARM-loaded SEDDS 150

ABSTRACT The aims of this research work were to formulate artemether-loaded self emulsifying drug delivery system (SEDDS), evaluate some of its in vitro and in vivo properties, and prevent drug precipitation through supersaturation approach. The solubility of artemether (ARM) in various oils, surfactants and cosurfactants was determined using the equilibrium solubility method. Vehicles such as Triacetin®, Labrasol® and Transcutol P® were selected as components of the formulation due to their high solubilising capacities. Pseudoternary phase diagrams were constructed in order to select the optimized batches. SEDDS containing 40 mg, 50 mg and 55 mg of ARM respectively were formulated and called non-hydroxypropylmethylcellulose batches (non-HPMC batches) while those containing 5 % hydroxypropylmethylcellulose (HPMC), a precipitation inhibitor, were called HPMC batches. The melting point of artemether was determined. The following evaluation tests: droplet size, preformulation and postformulation visual isotropicity, emulsification time, refrigeration cycle, centrifugation, aqueous dilution, viscosity, pH, drug content, and crystallization/precipitation studies were carried out. In vitro release studies of the formulations were carried out in simulated gastric fluid (SGF) without pepsin (pH, 1.2) and stimulated intestinal fluid (SIF) without pancreatin (pH, 6.8) respectively. Finally, the antimalarial activity of the ARM was evaluated using 25 mice grouped into five groups: SEDDS-treated group, chloroquine-treated group, placebo-treated group, untreated group (negative control) and aqueous ARM dispersion-treated group (positive control). The melting point range was obtained as 86-88 oC. Results showed that ARM has high solubility in Triacetin® (136.00 ± 0.09 mg/ml), Labrasol® (156.00 ± 0.01 mg/ml), and Transcutol P® (166.00 ± 0.02 mg/ml). From the pseudoternary phase diagrams, the optimized batches of Smix (1:0.5, and 3:1) were selected. The preformulation visual isotropicity test carried out on the optimized batches showed that the oil: Smix ratios; 1:2.0, 1:2.5, 1:3.5 and 1:4 were isotropically stable, while the postformulation visual isotropicity test showed that the SEDDS remained thermodynamically stable after 72 h. There was no significant difference in the emulsification times of the HPMC and non- HPMC batches. The result of refrigeration cycle and centrifugation test 13 showed no variation in the physical properties such as colour, odour change or phase separation. Phase separation was not noticed after dilution of the SEDDS to 1 L with 0.1N HCl. There was no significant difference in the loading efficiency (%) between HPMC and non-HPMC batches. Also, there was no significant change in the pH of the HPMC and non-HPMC batches. There was, however, significant difference (p < 0.05) in the viscosity of the HPMC and non-HPMC batches. After three hours of the SEDDS formulation in aqueous phase, the photomicrographs revealed the presence of some drug crystals in the non-HPMC batches, while no crystal was observed in the HPMC batches. The drug release profiles of non-HPMC batches showed T50 (time to release 50 % of the drug) and T85 (time to release 85 % of the drug) values that ranged between 3-8 min and 14-22 min respectively in SIF, and 3- 4 min and 5-18 min respectively in SGF. On the other hand, the drug release profiles of HPMC batches showed T50 and T85 values that ranged between 8- 35 min, and 53-153 min in SGF and T50 and T85 values that ranged between 4- 13 min and 92-213 min respectively in SIF. Results of antimalarial studies showed the following percent activities: SEDDS-treated group (94.00 ± 0.57 %), chloroquine-treated group (59.00 ± 0.85 %), placebo-treated group (16.0 ± 4.1 %), and aqueous ARM-treated group (47.0 ± 1.8 %). The SEDDS- treated group had a significantly (p < 0.05) higher antimalarial activity than the rest.

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CHAPTER ONE

1.0 GENERAL INTRODUCTION

Dosage form selection is as important as the active drug selection itself, because different bioavalabilities can be obtained for the same drug if it was formulated in different dosage forms. Drugs cannot be administered in their pure forms rather they must be developed into convenient dosage forms for their therapeutic effects and successful commercialization. Drugs should have sufficient aqueous solubility to dissolve in a fluid at the absorption site and lipid solubility high enough to facilitate the partitioning of the drug in the lipoidal biological membrane and into the systemic circulation. The therapeutic effectiveness of a drug depends on the ability of the dosage form to deliver the medicament to its site of action at a rate and amount sufficient to elicit the desired pharmacologic response. The poor dissolution characteristics of relatively poorly soluble drugs have been a problem to the pharmaceutical industry. As oral drug delivery remains the most popular route of administration, the oral delivery of hydrophobic drugs presents a major challenge because of the low aqueous solubility.

When lipophilic drugs are administered orally the dissolution rate in the gastrointestinal tract (GIT) becomes the rate limiting step in their absorption from the gut (Hiroshi et al., 2005 and Patel and Patel, 2008).

Dissolution is a process in which a solid substance solubilizes in a given solvent (mass transfer from the solid phase to the liquid phase). Dissolution rate is the most important physicochemical property of orally absorbed drugs with poor solubility. Therapeutic efficacy of a drug with low dissolution rate is assessed by determination of its bioavailability which must be done when a new chemical moiety (NCM) is to be introduced into market. This is best done by using a reliable dissolution test.

15

Transferring oral solid dosage form into systemic circulation involves three steps:

• Delivery of drug to the absorption site or

• movement of drug through the membrane of the GIT and

• movement of active ingredient from site of absorption into systemic circulation.

The rate at which the drug reaches systemic circulation is determined by the slowest of the various steps. This step is known as rate limiting or rate determining step. Some factors influence the rate and extent of absorption of the steps; such factors include physicochemical properties of the drug, the design and production of the dosage form, chemical nature of the drug, the solubility, type and quantity of excipient and compression pressure. These are called pharmaceutical variables. Factors coming from the anatomic and physiological characteristics of the patient are called patient variables; they include: time of administration relative to meal, co-administered drugs, compliance, age, disease state, abnormal genetic characteristics and/ or gastrointestinal physiology.

Dissolution rate is the amount of solid substance that goes into solution per unit time under standard conditions of temperature, pH, solvent composition and constant solid surface area. It is a dynamic process. The process of dissolution is basically dependent on variable parameters with the possible exception of pH dependency which may be patient variable. A quantitative description of dissolution rate is given by the Noyes-

Whitney equation (Noyes and Whitney, 1897) based on diffusion layer model:

= … … … … … … … … … … … … … … … … … . … 1

Where dc/dt is the rate of diffusion

D = diffusion coefficient

S = surface area 16 h = thickness of the diffusion layer

Cs = saturation solubility

C = concentration of drug in the solvent at time ‘t’

In dissolution rate limited absorption ‘C’ is negligible when compared to Cs. Under this condition ‘D’ and ‘h’ remains constant and cannot be altered to any degree by the product formulation. Thus,

=
.
.
……………………………………………………………..………….. (2)

Some researches had made concrete efforts to improve the solubility, dissolution rate and oral bioavailability of the hydrophobic drugs in order to enhance their therapeutic efficacy (Gursory and Benita 2004; Pouton, 2000; Amidon et al., 1995). The techniques include salt formation, solid dispersions (suspension), complexation with cyclodextrins, co-precipitations, colloidal vesicles like liposome, emulsion, micronisation, use of micelles, antisolvent and cogrinding and self emulsifying oil formulations (York, 1988; Sugimoto et al., 1998; Karali, 1992; Trapani et al., 2004;

Weuts et al., 2004; Ammar et al., 2006; Muhrer et al., 2006; Odeberg et al., 2003;

Vikas and Akhtar, 2008).

1.1 BIOPHARMACEUTICS CLASSIFICATION SYSTEM (BCS) OF DRUGS

Drugs are classified or grouped using Biopharmaceutical Classification System (BCS).

BCS was introduced in 1995, and has created a remarkable impact on the global pharmaceutical sciences arena, drug discovery, development and regulation. BCS is a basic guideline classifying drugs based on the solubility and permeability of the drug.

Since only dissolved drug can cross the gastrointestinal membrane, dissolution is one of those factors that limit bioavailability. In general, it can be stated that the rate of absorption, i.e the onset and extent of the clinical effect is determined by the dissolution 17 of the drug and the subsequent transport over the intestinal membrane and passage into the liver. BCS classified drugs into one of four biopharmaceutical classes according to their water solubility and membrane permeability properties which allow the prediction of the rate limiting step in the intestinal absorption process following oral administration. Class 1 BCS are drugs that are highly aqueous soluble and permeable to gastrointestinal tract. They have no associated absorption or permeation problems which may affect oral drug bioavailability. Examples include acetomenaphen, propranolol, metoprolol, etc. Class II drugs are class of drugs with poor aqueous solubility but highly permeable to the membrane. Class II examples include artemether, folic acid, ibuprofen, etc. The formulation strategies that could be used to improve the bioavailability of class II drugs is either by increasing the dissolution rate or by presenting the drug in solution and maintaining the drug in solution in the intestinal lumen. In class III drugs, the permeation over the membrane is a rate limiting step. The strategy for class III drugs is to increase the permeability of the absorbing membrane.

The strategy depends on the transport mechanism over the absorbing membrane, e.g. transcellular, paracellular or matrix mediated. Numerous studies deal with increasing membrane permeability in the gastrointestinal tract. The effect of the contents of the gut lumen or the effect of molecular properties of the drug on permeability is considered.

However, drug metabolism in the intestinal lumen, the intestinal wall and the liver may also reduce its bioavailability.

Efflux-transporters like p-glycoprotein can reduce the uptake and increase the duration of exposure to enzymatic metabolism of CYP3A4. Examples of class III include acetylsalicylic acid, captopril, cimetidine, etc. For a class IV drug, both dissolution and permeability must be increased. However, increasing dissolution is more effective than increasing the permeability because in practice the amount of dissolved drug at the 18 absorption site varies over six orders of magnitude (0.1-100 mg/l) whereas permeability varies over only a 50-fold range.

Therefore, the potential to increase absorption by increasing the drug concentration is larger and it is more practical to increase the solubility even if permeability is further compromised. Class IV includes furosemide, albendazole, methotrexate etc. Class II and IV form approximately 80% of the available drugs in the market with poor solubility and permeability. The candidate drugs suitable for SEDDS carrier systems are poorly water soluble drugs (especially classes 11 and IV biopharmaceutics classification system drugs).

The rate and extent of drug absorption from the gastrointestinal (GI) tract are very complex and affected by many factors. The factors include physicochemical factors such as solubility, stability, pKa, diffusivity, lipophilicity, polar and non-polar, surface area, particle size, crystal form, and presence of hydrogen bonding functionalities.

Physiological factors affecting rate and extent of drug absorption include GI pH, GI blood flow, gastric emptying small intestine transit time, colonic transit time, and absorption mechanism. Dosage factors include tablet, capsule, solution, suspension, emulsion, and gel. Amidon et al (1995) on their work showed that basic factors controlling oral drug absorption are the permeability of drug through the GI membrane and the solubility/dissolution of the drug dose in the GI milieu. The physiological and physicochemical parameters are the fundamentals to the oral absorption process

(Lobenberg and Amidol, 2000; Martinez and Amidol, 2002). Solubility and intestinal membrane permeability characteristics were used to classify drugs into I-IV. According to BCS, ß-Artemether belongs to class II with poor solubility problem that may predispose the drug to plasmodia organisms’ resistance. Therefore, the dissolution rate of a poorly soluble drug can be increased by increasing either solubility or surface area 19 or both. Survey conducted by FDA between 1995 and 2002 showed that only 9% of

New Drug Moieties belonged to class 1 BCS while approximately more than 40% have poor aqueous solubility (Lipinski, 2001; Jing-Ling et al., 2007).

1.2 DISPERSED SYSTEM

Dispersion means suspension of a substance (dispersed phase) in another (dispersion medium or continuous phase). Dispersion is a process by which (in the case of solids becoming dispersed in a liquid) agglomerated particles are separated from each other and a new interface, between an inner surface of the liquid dispersion medium and the surface of the particles to be dispersed, is generated. The dispersion of one phase as small particles into another produces an area of contact between the two phases. Since any system will tend to react spontaneously to decrease its free energy to a minimum, it implies that dispersed system is usually unstable. Dispersion could be lyophobic or lyophilic dispersion. Lyophobic (solvent hating) dispersion is a dispersion that exhibit positive interfacial free energy which makes the particles to aggregate leading to its instability (Carter, 2009). While lyophilic dispersion (solvent loving) is one that has an affinity between dispersed particle and dispersion medium making the system stable.

The terms hydrophobic or hydrophilic is used when the dispersion medium is water

(Aulton, 1999).

1.2.1 COLLOIDAL DISPERSION

A colloid is a substance that is microscopically dispersed evenly throughout another substance. A colloidal system consists of two separate phases: a dispersed phase (or internal phase) and a continuous phase (or dispersion medium). A colloidal system may be solid, liquid, or gaseous. It has at least a particle size dimension within the range of 20

10-9 m (1 nm) to 10-6 m (1 μm). The dispersed-phase particles have a diameter between approximately 5 and 200 nm (Levine, 2001). The size of the dispersed phase may be difficult to measure as colloids may have the appearance of solution. Such particles are normally invisible in an optical microscope, though their presence can be confirmed with the use of an ultra microscope or an electron microscope. Sometimes, a colloid can be considered as a homogeneous mixture. Homogeneous mixtures with a dispersed phase in this size range may be called colloidal aerosol, colloidal emulsions, colloidal foams, colloidal dispersions, or hydrosols.The dispersed particles could be in various shapes such as disc, sphere, cubes, ellipsoids and random coils. Some colloids are translucent because of the Tyndall effect, which is the scattering of light by particles in the colloid. Other colloids may be opaque or have a slight color. Colloids could be characterized by their physicochemical and transport properties. The size exclusion means that the colloidal particles are unable to pass through the pores of an ultrafiltration membrane with a size smaller than their own dimension. There are two classes of colloids, the lyophobic and lyophilic colloids. In lyophobic colloids, particles tend to aggregate in order to lower the free energy of the system. They are thermodynamically unstable and not easy to recover after undergoing phase separation, while lyophilic sols or true solutions are thermodynamically stable.

1.2.2 MACROEMULSION OR CONVENTIONAL EMULSION

An emulsion is a dispersion of two or more immiscible liquids stabilized by a surfactant or emulsifier coating the droplets and preventing coalescence by reducing interfacial tension or creating a physical repulsion between the droplets. It is a system containing two immiscible liquids in which one is dispersed in the form of small globules (internal phase) throughout the other (external phase) (Stedman, 2006). It is a 21 liquid preparation of two immersible liquids, one of which is dispersed throughout the other but which does not completely mix with the molecules of the other. An example of an emulsion is a mixture of water and oil as well as oil and vinegar. Most cosmetics and paints are emulsions. Emulsions are part of a more general class of two-phase systems of matter called colloids. Although the terms colloid and emulsion are sometimes used interchangeably, emulsion tends to imply that both the dispersed and the continuous phase are liquid unlike in the former.

1.2.3 GEL

A gel (from the latin word gelu—freezing, cold, ice or gelatus—frozen, immobile) is a solid, jelly-like material that can have properties ranging from soft and weak to hard and tough. A gel is a soft, solid or solid-like material which consists of at least two components, one of which is a liquid present in abundance (Almdal et al., 1993).

Hydrogel (also called aquagel) is a network of polymer chains that are hydrophilic, sometimes found as a colloidal gel in which water is the dispersion medium. Hydrogels are highly absorbent (they can contain over 99% water) natural or synthetic polymers.

Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content. An organogel is a non-crystalline, non-glassy thermoreversible (thermoplastic) solid material composed of a liquid organic phase entrapped in a three-dimensionally cross-linked network. The liquid can be, for example, an organic solvent, mineral oil, or vegetable oil. The solubility and particle dimensions of the structurant are important characteristics for the elastic properties and firmness of the organogel. Often, these systems are based on self-assembly of the structurant molecules (Terech, 1997; Van Esch et al., 1999). Organogels have potential for use in a number of applications, such as in pharmaceuticals (Kumar and Katare, 22

2005), cosmetics, art conservation (Carretti et al., 2005) and food (Pernetti et al.,

2007). An example of formation of an undesired thermoreversible network is the occurrence of wax crystallization in petroleum (Visintin et al., 2005).

1.2.4 SUSPENSION

Pharmaceutical suspensions are uniform dispersions of solid drug particles (internal phase) in a vehicle (external phase) in which the drug has minimum solubility. Particle size of the drugs may vary from one formulation to the other depending on the physicochemical characteristics of the drug and the rheological properties of the formulation. The internal phase constitutes insoluble solid particles with specific ranges of size which should be uniform throughout the suspending vehicle with the aid of one or more suspending agents. The external phase or suspending medium is generally aqueous in nature but may be organic or oily liquid for non-oral use. A suspension containing particles between 1 nm to 0.5 µm in size is called colloidal suspension.

When the particle size is between 1 to 100 µm, the suspension is called coarse suspension. Most of the pharmaceutical suspensions are coarse suspension formulations.

Properties of ideal suspension

v The dispersed particles should not settle readily and the settled particles should

redisperse immediately on shaking. Ideally, the particles in a suspension should

not sediment at any time during the storage period.

v The particle should not form a cake on settling.

v The viscosity should be such that the preparation can be easily poured.

v It should be chemically and physically stable. 23

v It should be palatable (orally).

v It should be free from gritty particles (external use).

Particle size:

Ø Particle size of any suspension is critical and must be reduced within the range

as determined during the preformulation study.

Ø Too large or too small particles should be avoided as larger particles will settle

faster at the bottom of the container and too fine particles will easily form hard

cake at the bottom of the container.

Ø The particle size can be reduced by using mortar and pestel but in large-scale

preparation different milling and pulverization equipments are used.

Ø Limitation in particle size reduction (after reaching a certain particle size).

Ø Expensive and time consuming; and movement of small particles due to

brownian motion cause particles to aggregate, settle, form hard cake that it is

difficult to redisperse.

1.2.5 MICROEMULSION

In the colloid and surfactant science literature the term ‘microemulsion’ means a thermodynamically stable system. Two immiscible liquids (e.g. water and oil) can be brought into a single phase (macroscopically homogeneous but microscopically heterogeneous) by addition of an appropriate surfactant or a surfactant mixture. This unique class of optically clear, thermodynamically stable and usually low viscous solutions, called microemulsions (Hoar and Schulman, 1943), have been the subject of extensive research over the last two decades primarily because of their scientific and technological importance. Microemulsion is a system of water, oil, and amphiphilic 24 compounds (surfactant and co- surfactant) which is a transparent, single optically isotropic, and thermodynamically stable liquid (Attwood, 1994). The concept of microemulsion was first introduced by Hoar and Schulman (1943); they prepared the first microemulsions by dispersing oil in an aqueous surfactant solution and adding an alcohol as a co-surfactant, leading to a transparent, stable formulation. The main difference between emulsions and microemulsions lies in the size and shape of the particles dispersed in the continuous phase: these are at least an order of magnitude smaller in the case of microemulsions (10–200 nm) than those of conventional emulsions (1–20 μm). Whereas emulsions consist of roughly spherical droplets of one phase dispersed in the other, microemulsions constantly evolve between various structures ranging from droplet-like swollen micelles to bicontinuous structures, making the usual oil in water and water in oil distinction sometimes irrelevant

(Kreilgaard, 2002).

1.2.6 FOAMS

Pharmaceutical foams are pressurized dosage forms, containing one or more active ingredients that, upon valve actuation, emit a fine dispersion of liquid and/or solid materials in a gaseous medium. They differ from most other dosage forms in their dependence on the function of the container, its valve assembly and the pressurized propellant; for the physical delivery of the drug in proper vehicle form (Sciarra and

Sciarra, 2000). Foam formulations are generally easier to apply, are less dense, and spread more easily compared with other topical dosage forms. When assessed, particularly in terms of ointments or even creams and lotions, foams require negligible mechanical shearing force in order to spread the formulation on the skin. This is a major advantage when applying a medicament to highly inflamed skin; for example, in 25 cases of sunburn where rubbing the formulation on to the skin to effect spreading may be painful or cause further inflammation. The maximum drug transfer into the skin takes place when the drug is in saturated solution at the vehicle skin interface (Surber and Smith., 2000; Schwarb et al., 1997). If this is not the case, the rate of drug transfer across the interface is proportional to its degree of saturation (concentration/solubility)

(Surber et al., 2000; Schwarb et al., 1997). Rapid evaporation of the volatile components of foam vehicles results in an appreciable increase in drug concentration in the vehicle. Drugs applied to the skin for their local action include antiseptics, antifungal agents, anti-inflammatory drugs, local anesthetics, skin emollients, and protectants (Sciarra and Sciarra, 2000). As a drug molecule dissolves and enters into the matrix of solvent molecules in the foam delivery vehicle, there are a number of weak bonding interactions that take place between substituent groups on the solute and solvent species. This attractive interaction stabilizes the dissolved molecule in solution and prevents its precipitation.

1.3 FORMULATION TECHNIQUES OF HYDROPHOBIC DRUGS

Figure 1: Showing the formulation techniques of hydrophobic drugs. 26

The oral route of administration is preferred, but for many drugs it can be a problematic and inefficient mode of delivery for a number of reasons. Limited drug absorption resulting in poor bioavailability is paramount amongst the potential problems that can be encountered when delivering an active agent via the oral route. Drug absorption from the gastrointestinal (GI) tract can be limited by a variety of factors with the most significant contributors being poor aqueous solubility and/or poor membrane permeability of the drug molecule. When delivering an active agent orally, it must first dissolve in gastric and/or intestinal fluids before it can then permeate the membranes of the GI tract to reach systemic circulation. A drug with poor aqueous solubility will typically exhibit dissolution rate limited absorption, and a drug with poor membrane permeability will typically exhibit permeation rate limited absorption.

1.3.1 SALT FORMATION

Salt formation is the most common and effective method of increasing solubility and dissolution rates of acidic and basic drugs. Crystalline salt forms of weak bases or acids often provide faster dissolution and a higher apparent solubility as compared to their unionized counterparts. Therefore, dissolving a salt form of a drug may induce supersaturation. An advantage of using a crystalline salt form instead of the amorphous phase is their better stability during storage. An interesting example of the potential use of crystalline salt forms to induce supersaturation and improve intestinal absorption has been reported by Guzma´n et al (2007).

1.3.2 SOLID DISPERSION

Solid dispersions refer to the dispersion of one or more active ingredients in an inert carrier in a solid state, frequently prepared by the melting (fusion) method, solvent 27 method, or fusion-solvent method (Warwick et al., 1971; Sekiguchi and Obi, 1961).

Solid dispersion is a group of solid products consisting of at least two different components, generally a hydrophilic matrix and a hydrophobic drug. The matrix can be either crystalline or amorphous. The drug can be dispersed molecularly, in amorphous particles (clusters) or in crystalline particles (Chiou and Riegelman, 1971). Solid dispersion technologies are particularly important for improving the oral absorption and bioavailability of BCS Class II drugs. Solid dispersion technologies involve drugs that are poorly water-soluble and highly permeable to biological membranes of these drugs since dissolution rate is the rate limiting step to absorption. In the Biopharmaceutical

Classification System (BCS), drugs with low aqueous solubility and high membrane permeability are categorized as Class II drugs (Amidon et al., 1995). Formulation of drugs as solid dispersions offers a variety of processing and excipient options that allow for flexibility when formulating oral drug delivery systems for poorly water soluble drugs.

1.3.3 COMPLEXATION WITH CYCLODEXTRIN

Cyclodextrins are functional excipients that have gained widespread use and attention due to their ability to solubilize, and in some instances stabilize, poorly water-soluble drug candidates enabling both oral and parenteral formulation (Davis and Brewster,

2004; Loftsson et al., 2004; Loftsson and Brewster, 1996; Thompson, 1997; Rajewski and Stella, 1996; Arun et al., 2008). In addition, these materials can convert oils and viscous liquids into free flowing powders and can suppress vapor pressure and therefore can reduce unaesthetic smells and tastes (Brewster et al., 1989). In the case of solubilization and for many of the other property improvements observed, the mechanism suggested is the formation of dynamic inclusion complexes in solution 28 between the cyclodextrin and the compound of interest (Szejtli and Osa, 1996). This interaction occurs because of the specialized architecture of the cyclodextrin molecule wherein the material takes the form of a truncated cone or torus with the primary hydroxyl functions oriented to the narrower end of the torus and the secondary hydroxyl function oriented towards the wider end (Loftsson and Brewster, 1996;

Brewster et al., 1989). This makes the exterior of the molecule relatively hydrophilic while the cavity interior is relatively hydrophobic so that the starch derivative has some degree of water solubility. At the same time, the interior lipophilic cavity allows appropriately sized lipophiles to interact resulting in solubilization while complexation describes many of the pharmaceutically and biopharmaceutically relevant attributes of cyclodextrins.

1.3.4 SOLID LIPID NANOPARTICLES (SLN)

Colloidal particles ranging in size between 10 and 1000 nm are known as nanoparticles.

They are manufactured from synthetic/natural polymers and ideally suited to optimize drug delivery and reduce toxicity. Solid lipid nanoparticles are one of the novel potential colloidal carrier systems in the range of 100-150 nm as alternative materials to polymers which is identical to oil in water for parenteral nutrition, but the liquid lipid of the emulsion has been replaced by a solid lipid (Jenning et al., 2002). They have so many advantages such as good biocompatibility, low toxicity, etc., and lipophilic drugs are better delivered by SLN as the system is physically stable (Cavalli et al., 2002).

SLN may be a promising sustained release and drug targeting system for lipophilic

CNS antitumor drugs (Muller et al., 1997; Karanth et al., 2008).

The drug loading capacity of conventional SLN is limited by the solubility of drug in the lipid melt, the structure of the lipid matrix and the polymeric state of the lipid 29 matrix. If the lipid matrix consists of especially similar molecules (i.e. tristearin or tripalmitin), a perfect crystal with few imperfections is formed. Since incorporated drugs are located between fatty acid chains, between the lipid layers and also in crystal imperfections, a highly ordered crystal lattice can not accommodate large amounts of drug. Therefore, the use of more complex lipids is more sensible for higher drug loading.

1.3.5 LIPOSOMES

Liposomes are spherical vesicles whose walls consist of hydrated bilayers of phospholipids. They are classified as unilamellar and multilamellar. The unilamellar are vesicles enclosed by a 6 to 7 nm thick single phospholipid bilayer. They range between

20-1000 nm (1 µm) which depends on the method of preparation.

Liposomes have different methods of preparations which include subjecting aqueous lipid dispersions to high shear (including ultrasonication); forming their lipid films by solvent evaporation, followed by their hydration and dispersal in water; injecting solutions of lipids as water miscible (alcohol) solvent into water vesicle formation is not restricted to lipids. Liposomes are used as vehicle to deliver synthetic drugs, polypeptides, proteins, including enzymes and antibodies, and nucleic acids as well as recombinant DNA. Oil-soluble drugs are added to solution of the lipid inorganic solvents. Immediately the liposomes are formed, the drugs are solubilized by the hydrocarbon chains of the lipid bilayers. Aqueous soluble drugs can be incorporated into the aqueous phase in which the liposomes are formed. The unencapsulated drug remaining in the continuous aqueous phase is then removed by dialysis, centrifugation or ion exchange. Drugs have also been trapped within the inner, aqueous core of liposomes to protect them from enzymatic degradation as they circulate in the 30 bloodstream. Liposomes have more advantage with their ability to target drugs to specific tissues in the body such as tumors.

1.3.6 NIOSOMES

Niosomes are novel drug delivery system, in which the medication is encapsulated in a vesicle. The vesicle is composed of a bilayer of non-ionic surface active agents and hence the name niosomes. The niosomes are very small, and microscopic in size. Their size lies in the nanometric scale. Although structurally similar to liposomes, they offer several advantages over them. Niosomes have recently been shown to greatly increase transdermal drug delivery and also can be used in targeted drug delivery.

Structure of Niosomes:

Structurally, niosomes are similar to liposomes, in that they are also made up of a bilayer. However, the bilayer in the case of niosomes is made up of non-ionic surface active agents rather than phospholipids as seen in the case of liposomes. Most surface active agents when immersed in water yield micellar structures; however some surfactants can yield bilayer vesicles which are niosomes. Niosomes may be unilamellar or multilamellar depending on the method used to prepare them. The niosome is made of a surfactant bilayer with its hydrophilic ends exposed on the outside and inside of the vesicle, while the hydrophobic chains face each other within the bilayer. Hence, the vesicle holds hydrophilic drugs within the space enclosed in the vesicle, while hydrophobic drugs are embedded within the bilayer itself.

1.3.7 DENDRIMERS

Dendrimers are a new class of polymeric materials. They are nanometer-sized, highly branched and monodisperse macromolecules with symmetrical architecture or in 31 another way are spherical polymeric molecules series of chemical cells built on small core molecules. They are highly branched, monodisperse macromolecules. They consist of a central core, branching units and terminal functional groups. The core together with the internal units, determine the environment of the nanocavities and consequently their solubilizing properties, whereas the external groups determine the solubility and chemical behaviour of these polymers. The structure of these materials has a great impact on their physical and chemical properties. Targeting effectiveness is affected by attaching targeting ligands at the external surface of dendrimers, while their stability and protection from the Mononuclear Phagocyte System (MPS) is being achieved by functionalization of the dendrimers with polyethylene glycol chains (PEG). As a result of their unique behaviour dendrimers are suitable for a wide range of biomedical and industrial applications. Dendrimers were first discovered in the early 1980’s by Donald

Tomalia and co-workers (Tomalia et al., 1985; Klajnert and Bryszewska, 2001) these hyperbranched molecules were called dendrimers. The term originates from ‘dendron’ meaning a tree in Greek. At the same time, Newkome’s group (Newkome, 1985) independently reported synthesis of similar macromolecules. They called them arborols from the Latin word ‘arbor’ also meaning a tree. The term cascade molecule is also used, but ‘dendrimer’ is the best established one.

Properties

Dendrimers are monodispersed macromolecules, unlike linear polymers. The classical polymerization process which results in linear polymers is usually random in nature and produces molecules of different sizes, whereas size and molecular mass of dendrimers can be specifically controlled during synthesis. Because of their molecular architecture, dendrimers show some significantly improved physical and chemical properties when compared to traditional linear polymers. In solution, linear chains exist 32 as flexible coils; in contrast, dendrimers form a tightly packed ball. This has a great impact on their rheological properties. Dendrimer solutions have significantly lower viscosity than linear polymers (Fréchet, 1994). When the molecular mass of dendrimers increases, their intrinsic viscosity goes through a maximum at the fourth generation and then begins to decline (Mourey et al., 1996).

1.3.8 ANTISOLVENT

The supercritical anti-solvent process (SAS), uses the anti-solvent effect of supercritical CO2 to precipitate the substrate(s) initially dissolved in a liquid solvent. It is generally considered more attractive for particle design because it permits to monitor the properties and composition of the particles with a great flexibility and for almost any kind of compounds (Jung and Perrut, 2001; Reverchon, 1999). As a result, this process was widely studied with hundreds of molecules processed at lab-scale, but translating it to large scale still remains a challenge. However, it is now necessary to show to the pharmaceutical industry that particles can be produced at an industrial scale while keeping their characteristics in order to make the technology more attractive than a simple lab tool. The Supercritical Fluid Technology is presently considered as a promising tool by the pharmaceutical industry for formulation and particle design

(Perrut, 2003 and Jung and Perrut, 2001), leading to tailor-made particles of chemical entities and bio-molecules. Moreover, the non-denaturing properties of supercritical carbon dioxide combined with its intrinsic sterility (Fages et al., 1997; Castor et al.,

1999; and Splilimbergo et al., 2002) are of particular interest for the stabilization and formulation of the coming therapeutic proteins. Several processes were developed both for formulating poorly soluble and hydrophilic molecules, including fragile bio- molecules, to come up to the pharmaceutical industry expectations. In particular, an 33 important R and D work is on-going in the following domains : bio-molecules drying and stabilization, increasing bio-availability of poorly-soluble molecules, designing sustained-release formulations and preparing substances for drug delivery less invasive than parenteral (oral, pulmonary, transdermal) (Jung and Perrut, 2001; Reverchon,

1999). The CO2 anti-solvent technique is one of the techniques commonly used to prepare new drug delivery systems. The technique takes advantage of the anti-solvent effect of supercritical CO2 to precipitate the substrate(s) initially dissolved in a liquid solvent.

1.3.9 CO-GRINDING

Particle size reduction generally could enhance dissolution thereby increasing the rate of absorption and then the bioavailability of a drug. However, fine particles may not always produce the expected faster dissolution and absorption thus primarily results from the possible aggregation and agglomeration of fine particles due to their increased surface free energy and subsequently Vander Waal’ s attraction between the weak polar molecules. Fine powders also have poor wettability by aqueous body fluid. Wetting of powder is the primary condition for them to disperse and dissolve in the body fluid

(Lachman et al., 1976). However, all these problems were addressed using a better technology called Co-grinding. Co-grinding is one of the techniques used to improve solubility of hydrophobic drugs. It is simple, scalable grinding with suitable materials that prevent recrystallization. It improves wettability, solubility and dissolution processes. It has an improved stability. The common excipients used for co-grinding are polyvinylpyrrolidone, microcrystalline cellulose, cyclodextrins and various silicates including Neusilin®. Some drugs which have been worked on have showed improvement in dissolution and or solubility of drugs after co-grinding with excipients. 34

The drugs include sulfathiazole, indomethacin, aspirin, ketoprofen, naproxen, progesterone, glibenclamide and NCE. In co-grinding particle size is decreased thereby increasing the surface area which enhances the dissolution rate of the poorly soluble drug. In co-grinding techniques, selection of excipients or adjuvant is of profound significance in the preparation of solvent deposition system because it is one of the controlling features in the enhancement of dissolution rate. There are some methods used in increasing dissolution rate of poorly soluble drug as Noyes-Whitney equation stated that variables to be controlled by formulation of poor soluble drugs are simply the surface area and solubility. The solubility of a weak acid or base can be controlled by buffering the entire dissolution medium by the use of buffers or salts, or through the choice of the physical state such as crystal forms, its hydrates, its amorphous form, etc.

Also, we can control the surface area of the drug by controlling of the particle size.

Other methods of increasing solubility of poorly soluble drug include:

• Buffering of the pH of microenvironment.

• Use of salts of weak acids and weak bases eg. Sodium and potassium salts of

penicillin.

• Use of solvates and hydrates, eg. Ampicillin trihydrate (Poole and Ownen,

1968).

• Prodrug approach, eg. 2, 1- disodium phosphate ester of betamethasone

(Lachman et al., 1976)

• Complexation, eg. Digoxin (Higuchi and Ikeda, 1974) and hydroquinone.

• Use of surfactants, eg. Griseofulvin hexestrol (Lachman et al., 1976)

• Use of polymorphic form, eg. Novobiocin (Mullins and Macek, 1960) and

various steroids

35

Some methods are also used in increasing the surface area. These include: v Micronisation (eg. Griseofulvin, digoxin (Atkinson et al., 1962) v Use of surfactants (eg. Phanecetin (Finhottp et al., 1974)) v Solid dispersion (eg. Griseofulvin-succinic acid, (Goldberg et al., 1966) and

Griseofulvin Griseofulvin-PVP, (Chowdary and Ramamurthy, 1998)

1.3.10 SELF EMULSIFYING OIL FORMULATION

Self emulsifying oil formulation (SEOF) is an isotropic mixture of natural or synthetic oil(s), solid or liquid nonionic surfactant(s) with or without cosurfactants and the hydropobic drug, which when in contact with aqueous medium and under gentle agitation mechanically provided or through gastric motility impelled, results to the formation of oil-in-water emulsion (O/W) or water-in-oil (w/o) emulsion ( Obitte et al.,

2009; Gursoy and Benita, 2004; Enas et al., 2009; Suman et al., 2009). It is a promising strategy to improve the rate and extent of oral absorption of poorly water soluble drugs (PWSD). According to research, there is a great emphasis on SEOF to improve the oral bioavailability of lipophilic drugs (Humberstone and Charman, 1997;

Pouton, 1997). SEOF are either anhydrous or low in water content which allows encapsulation of a unit dose (soft or hard gelatin). SEOF have been formulated using medium chain triglyceride oils and non-ionic surfactants, the latter being less toxic

(Patil and Joshi, 2004). Typical ingredients are triglycerides, diglycerides, monoglycerides, other polar oils, cosurfactants, nonionic surfactants, aqueous cosolvents (e.g. PEG, propylene glycol). The principal characteristic of the system is the ability to form fine oil‐in‐water (o/w) emulsions or microemulsions upon mild agitation following dilution by an aqueous phase through the gastrointestinal tract. Self emulsification is a term used to describe emulsification which occurs with little or no 36 input of energy. The process may be spontaneous or may require low levels of shear but will contrast with conventional emulsification which requires high shear. Self emulsifying formulations spread readily in the gastrointestinal (GI) tract, and the digestive motility of the stomach and the intestine provides the agitation necessary for self emulsification. Self microemulsifying drug delivery system (SMEDDS) was patented by Gattefossé in the 90’s as “latent” microemulsions in the form of a stable, water-free combination of surfactants, co-surfactants and lipophilic phase, which creates a microemulsion when diluted in water or body fluids. Such systems combine the advantages of microemulsions with a water-free formulation protecting sensitive active pharmaceutical ingredient (API) from the chemical degradation they would undergo in an aqueous medium. When the lipid droplet size ranges from 100 nm up to

5 µm, it is called SEDDS and SMEDDS when the lipid droplet size is < 100 nm (Obitte et al., 2009, Rajesh et al., 2010). Rajesh et al., (2010) gave comparative reports on

SEDDS and SMEDDS. SEDDS and SMEDDS form fine droplet size or globules with high interfacial area facilitating/ improving activity and decreasing the irritation due to contact of drug in the gastrointestinal wall with uniform drug absorption which enhance the bioavailability (Pouton, 1985; O’ Dniscol, 2002; Charman et al., 1992; Shah et al.,1994 ; Khoo et al., 1998).

Some SEDDS are transparent/ translucent depending on the extent of dilution with aqueous phase. However, they both have high solubilizing capacity and high dispersity capacity. They can both be prepared as liquids and semi-solid for capsule dosage forms and solid forms for tabletting. Both systems are associated with the generation of large surface area dispersions that provide optimum conditions for the increased absorption of poorly soluble drugs (Wei et al., 2007). Therefore, the fine O/W emulsions produce small droplet of oil dispersed in the gastrointestinal fluids that provide a large 37 interfacial area. This will increase the activity of pancreatic lipase to hydrolyse triglyceride and thereby promoting a faster release of the drug and/ or formation of mixed micelles of the bile salts containing the drug.

These formulations have attracted interest because they can improve the bioavailability of compounds that fall into Class II of the biopharmaceutical classification system

(BCS). Class II compounds are poorly water soluble and highly permeable. The choice of whether a SEDDS or a SMEDDS is the preferred formulation option often depends on the interplay between the intrinsic properties of the drug compound and its solubility and dissolution profile during in vitro screening with a number of excipients.

Effect of Food on SEDDS

The effect of food on the bioavailability of PWSD is determined by multiple factors, including the physicochemical properties of the drug substance, the dose, the nature of the formulation and the amount and composition of the ingested food. Fatty food delays the gastric emptying rate and induces secretion of bile and pancreatic juices, while the passage time of the small intestine remains virtually unchanged. Hence food intake can increase the solubilization time and increase the solubility of a poorly soluble drug, which will affect the pharmacokinetic parameters. Postprandial changes in the GIT that can increase drug absorption, relative to the fasted state (Porter and Charman, 2001), include:

• increased drug solubilization by bile salt mixed micelles

• increased intestinal membrane permeability secondary to the presence of bile

and lipid digestion products.

Furthermore, the food-drug interactions are important to drug absorption. Concomitant food intake has been demonstrated to lead to an increase in drug bioavailability. 38

However, administration of microemulsion reduced the effect of food on bioavailability

(Mueller et al., 1994b).

Efflux effects and Lymphatic effects on SEDDS

Permeability glycoprotein (P-gp) is an efflux pump of multi-drug resistant genes

(MDR) subfamily. P-gp prevents the entry of some drugs into the systemic circulation, thus hampering the oral bioavailability of the drugs. P-gp is energy dependent transporter protein involved in effluxing a number of drugs and impeding their absorption intracellularly. P-gp is highly expressed over Blood Brain Barrier; as a result

P-gp inhibits the entry of various drugs that are used for the treatment of various CNS disorder and chemotherapy. P-gp is also present over the GIT, located in the apical membranes of intestinal absorptive cells, which impedes pharmacokinetic parameter of many drugs that are substrate to this membrane transporter and therefore affects bioavailability and cause therapeutic failures of orally taken drugs. P-gp, a relevant efflux pump is expressed in high amount in biliary tract. Certain lipids and surfactants may reduce the activity of intestinal efflux transporters, as indicated by the P-gp efflux pump and consequently reduce the extent of enterocyte-based metabolism (Benet,

2001; Dintaman and Silverman, 1999; Nerurkar et al., 1996). Inhibition of the efflux effect and/or enterocyte-based metabolism will increase the concentration and residence time of the intact drug in the cell. This may result in increased drug available for partitioning into the lymphatics (O’Driscoll, 2002). It has been shown that

Cremophor EL®, Labrasol®, Miglyol polyethoxylated, vitamin E TPGS, Solutol® HS

15, Gelucire® 44/14 (lauroyl macrogol glycerides) and Polysorbate 80 (Tween 80), and oily phases, including Imwitor® 742 and Akoline MCM® (mono-, and di-glyceride of caprylic acid) and Peceol® (glyceryl monooleate), have the potential to inhibit the P-gp efflux transporter (Hugger et al., 2002; Shono et al., 2004; Cornaire et al., 2004) and 39 thereby potentially improve bioavailability of drug molecules that are P-gp substrates.

Some researches have also shown that bile salts, fatty acids, phospholipids, and surfactants are potent absorption enhancers and efflux-reducing agents in Caco-2 cells and the rat intestine (Lo et al., 1998; Lo and Huang, 2000; Lo, 2000). Stimulation of intestinal lymphatic transport for highly lipophilic drugs may enhance the extent of lymphatic transport and increase bioavailability directly or indirectly via a reduction in first pass metabolism (Porter and Charman, 2001; Muranishi, 1991).

Changes in the physical barrier function of the GI tract: Various combinations of lipids, lipid digestion products and surfactants have been shown to have permeability enhancing properties (Aungst, 2000; Muranishi, 1990). For the most part, however, passive intestinal permeability is not thought to be a major barrier to the bioavailability of the majority of PWSD, and in particular, lipophilic drugs. A number of excipients have shown to influence the lymphatic transport in rats, and have an impact to the chylomicron secretion in caco-2 cells, which is also believed to be an indicator of lymphatic transport (Rege et al., 2002; Seeballuck et al., 2003; Karpf et al., 2004). On the whole, the possibility of lymphatic transport of SEOF reduces first pass metabolism since portal blood transport of drug may be reduced equally. In addition improved drug solubilization predisposes to a more consistent mucosal absorption and bioavailability profile.

Role of Lipolysis

Digestion of dietary triglyceride in the small intestine is very rapid, and many other non-ionic esters, such as mixed glycerides and surfactants, will be substrates of pancreatic lipase (Embleton and Pouton, 1997). Digestion of formulations will inevitably have a profound effect on the state of dispersion of the lipid formulation, and the fate of the drug (MacGregor et al., 1997). The inclusion of highly lipophilic 40 compounds in SEDDS is often reported to result in strongly enhanced oral absorption although it is still controversial whether further lipolysis of the dispersed lipid material is required for final transfer to the enterocyte membranes.

Factors Affecting SEDDS

The Nature and Dose of the Drug: Drugs which are administered at very high dose are not suitable for SEDDS, unless they exhibit extremely good solubility in at least one of the components of SEDDS, preferably lipophillic phase. The drugs that exhibit limited solubility in water and lipids are most difficult to deliver by SEDDS. The ability of SEDDS to maintain the drug in solubilized form is greatly influenced by the solubility of the drug in oily phase. If the surfactant or co-surfactant is contributing to a greater extent for drug solubilization, then there could be a risk of precipitation, as dilution of SEDDS will lead to lowering of solvent capacity of surfactant or co- surfactant. Equilibrium solubility measurement can be carried out to anticipate potential cases of precipitation in the gut. However, crystallization could be slow in solubilizing and colloidal stabilizing environment of the gut. Studies revealled that such formulation can take up to 5 days to reach equilibrium and that the drug can remain in a super saturated state up to 24 hours after the initial emulsification event (Tang et al., 2007).

The Polarity of the Oily Phase: The polarity of lipid phase is one of the factors that govern the release from the micro-emulsion. HLB, chain length and degree or unsaturation of the fatty acid, molecular weight of the hydrophilic portion and concentration of the emulsifier govern polarity of the oil droplets. Polarity reflects the affinity of the drug for oil and/or water, and the type of forces involved. The high polarity will promote rapid rate of release of the drug into the aqueous phase. Example the rate of release of idebenone from SEDDS is dependent upon the polarity of the oil 41 phase used. The highest release was obtained with the formulation that had oily phase with highest polarity (Kim et al., 2000).

Advantages of SEDDS

Potential advantages of these systems (SEDDS) include (Patel et al., 2008):

Enhanced oral bioavailability enabling reduction in dose: The ability of the SEDDS in improving maximium concentration (Cmax)) and oral bioavailability or therapeutic effect has been established for various hydrophobic drugs. Dissolution rate dependant absorption is a major factor that limits the bioavailability of numerous PWSDS.

SEDDS present hydrophobic drug to GIT in solubilised and microemulsified form

(globule size between 1- 100 nm) and subsequent increase in specific surface area enable more efficient drug transport through the intestinal aqueous boundary layer and through the absorptive brush border membrane leading to improved bioavailability.

Reduction in the drug dose leading to reduced dose-related side effects of many hydrophobic drugs, such as antihypertensive and antidiabetic drugs could improve their bioavailability, etc.

More Protection of drug(s) from the hostile environment in gut: SEDDS protect drugs against hydrolysis by enzymes in the GI tract and reduce the presystemic clearance in the GI mucosa and hepatic first-pass metabolism. Drug entrapment creates a healthy barrier between the GIT mucosal surface and the gastric-erosion-potent drug, thus providing an avenue for the delivery of NSAIDS.

Reduced variability including food effects: There are several drugs, such as cyclosporine and ezetimibe that show large inter- and intra-subject variation in absorption leading to decreased performance of the drug and patient noncompliance. It has been demonstrated that fed and fasted state dissolution media have negligible effects on the droplet size of the SEDDS. Hence, it can be anticipated that SEDDS 42

(fabricated with proper optimization) can offer a reduction in ratio of bioavailability between fed and fasted state and can offer reproducibility in plasma profiles of drugs in fed and fasted conditions.

Ease of Manufacture and Scale-up: This is one of the most important advantages that make SEDDS unique when compared with other novel drug delivery systems, such as solid dispersions, liposomes etc. SEDDS require very simple and economical manufacturing facilities, such as simple mixer with an agitator and volumetric liquid filling equipment for large-scale manufacturing. Nanoemulsions can be easily fabricated by low-energy emulsification methods, such as the phase inversion temperature method and phase inversion composition method (spontaneous nanoemulsification method).

Quick Onset of Action: Quick onset of action is required in many disease conditions, such as inflammation, hypertension, angina etc. SEDDS can facilitate oral absorption of the drug, which would result in quick onset of action. The comparative pharmacokinetic analysis of SEDDS to conventional formulation has demonstrated that there is considerable reduction observed in tmax.

Protection of sensitive drug substances: SEDDS formulations create entrapment that protects drugs susceptibility to hydrolytic and oxidative degradation from the hostile environmental conditions.

Liquid or Solid dosage forms: Depending on the choice of dosage form or the type of research beign carried out, SEDDS formulation could be in liquid or solid form.

Consistent temporal profiles of drug absorption: The absorption of SEDDS is temporarily consistent and uniform without inter or intra- interruption.

Selective targeting of drug(s) toward specific absorption Window in GIT: SEDDS may be targeted towards a specific area in the GIT with increased activity. 43

Control of delivery profiles: SEDDS controls the release profiles of the drug by uniformly releasing its active ingredients; it does not release erratically.

High drug payloads: Each dose of SEDDS has high activity as the drug is highly soluble and more bioavailable to the system. SEDDS also provides the advantage of increased drug loading capacity when compared with conventional lipid solution as the solubility of poorly water soluble drugs with intermediate partition coefficient (2 < log

P > 4) are typically low in natural lipids and much greater in amphiphilic surfactants, co surfactants and co-solvents.

Limitation of SEDDS

One of the limitations in the development of SEDDS and other lipid-based formulations is the lack of good predicative in vitro models for assessment of the formulations. Traditional dissolution methods do not work, because these formulations potentially are dependent on digestion prior to release of the drug. To mimic this, an in vitro model simulating the digestive processes of the duodenum has been developed.

This in vitro model needs further development and validation before its strength can be evaluated. Further development will be based on in vitro - in vivo correlations and therefore different prototype lipid based formulations need to be developed and tested in vivo in a suitable animal model.

The following should be considered in the formulation of a SEDDS:

The self‐emulsifying process depends on:

• the nature of the oil–surfactant pair, the surfactant concentration, and the

temperature at which self‐emulsification occurs,

• the solubility of the drug in different oil, surfactants and co surfactant/

co‐solvents, 44

• the selection of oil, surfactant and co‐solvent based on the solubility of the drug

and the preparation of the phase diagram, and

• the preparation of SEDDS formulation by dissolving the drug in a mix of oil,

surfactant and co‐surfactant/co‐solvents.

The addition of a drug to a SEDDS is critical because the drug interferes with the self emulsification process to a certain extent, which leads to a change in the optimal oil: surfactant ratio. So, the design of an optimal SEDDS requires preformulation solubility and phase‐diagram studies. In the case of prolonged SEDDS, formulation is made by adding the polymer or gelling agent. The ideal excipients to use should have all or most of the following properties (Rajesh et al., 2010):

Ø Be safe, inert and available at a purity level suitable for human use.

Ø Not degrade during manufacturing or storage.

Ø Be capable of solubilizing the drug dose in a volume not exceeding that of an

oral capsule.

Ø Preferably possess surface active properties to enable self emulsification or

complete dissolution of the drug dose.

Ø Reliably and reproducibly enhance the oral bioavailability of the drug relative to

a conventional formulation,

Ø Be physically and chemically stable and compatible with a wide range of drugs

and other excipiients,

Ø Be nonhygroscopic and inert to the capsule shell or other packaging

components.

The release of SEDDS loaded drug in vivo has two pathways:

The release of compound from SEDDS based formulation is thought to take place by two major pathways: Interfacial transfer and degradation of vehicle (De Smidt et al., 45

2004; Porter et al., 2004). Interfacial transfer is noted to be a concentration gradient driven process in which the compound diffuses from the formulation into the bulk or directly across the intestinal membrane. The rate and extent of interfacial transfer is thought to be governed by partition coefficient and solubility in the donor (formulation) and recipient phase particle size and hence surface area of formulation (Armstrong and

James, 1980). The second pathway is degradation of the vehicle inducing the release of the compound out of the vehicle. As mentioned above, for lipid based formulations, the most important degradation is the lipolysis catalyzed by pancreatic lipase. The release rate is thought to be dependent on the solubility of the compound in the formulation and rate and extent of the degradation of the vehicle. Lipolysis of triacylglycerols (TG) by the pancreatic lipase–colipase complex releases monoacylglycerols, diacylglycerols and free fatty acids. These lipolysis products are amphiphiles that will further assist the solubilization of poorly soluble compounds in the GI fluids.

Applications/ Uses of SEDDS in Pharmacy

Improvement in solubility and bioavailability

When SEDDS is loaded with a drug, it improves the solubility because it circumvents the dissolution step in case of Class II drug (Low solubility/high permeability) thereby ensuring the aqueous solubility of the PWSD. Aqueous solubility is an important molecular property that strongly determines drug accessibility to biological membranes, the major pathway for drug absorption, (Amol et al., 2009; Obitte et al., 2011).

Vergote et al (2001) reported complete drug release from sustained release formulations containing ketoprofen in nanocrystalline form. Different formulation approaches that have been sought to achieve sustained release, increase the bioavailability, and decrease the gastric irritation of ketoprofen which include preparation of matrix pellets of nanocrystalline ketoprofen, sustained release ketoprofen 46 and microparticles formulations (Yamada et al., 2001) floating oral ketoprofen systems, and transdermal systems of ketoprofen. This formulation enhanced bioavilability due to increase solubility of drug and minimizes the gastric irritation. Also, incorporation of gelling agent in SEDDS sustained the release of Ketoprofen. In SEDDS, the lipid matrix interacts readily with water, forming fine particulate oil-in-water (o/w) emulsion. The emulsion droplets will deliver the drug to the gastrointestinal mucosa in the dissolved state readily accessible for absorption. Therefore, increase in AUC i.e. bioavailability and Cmax is observed with many drugs when presented in SEDDS.

Protection against Biodegradation

Many drugs are degraded in physiological system, which may be due to acidic pH in stomach, enzymatic degradation or hydrolytic degradation, etc. Such drugs when presented in the form of SEDDS can be well protected against these degradation processes as liquid crystalline phase in SEDDS might be acting as barrier between degradating environment and the drug. Acetylsalicylic acid (Log P = 1.2, Mw=180) is a drug that degrades in the GI tract because it is readily hydrolyzed to salicylic acid in an acid environment. When the drug was formulated in a Galacticles™ Oral Lipid Matrix

System (SEDDS formulation) and compared with a commercial formulation, it showed the good plasma profile as compared to the reference formulation. The oral bioavailability of undegraded acetylsalicylic acid is improved by 73 % by the

Galacticles™ Oral Lipid Matrix System formulation compared to the reference formulation. This means that the SEDDS formulation has a capacity to protect drugs from degradation in the GI tract (Shah et al., 1994). Self emulsifying liquid crystalline nano-particles (LCNPs) are excellent solubilizers. Compared with conventional lipid or nonlipid carriers, LCNPs show high drug carrier capacity for a range of sparingly water-soluble drugs. For drugs susceptible to in vivo degradation, such as peptides and 47 proteins, LCNP vehicles protect the sensitive drug from enzymatic degradation. The

LCNP systems also address permeability limitations by exploiting the lipid-mediated absorption mechanism. For water-soluble peptides typical bioavailability enhancements range from twenty to more than one hundred times. In an alternative application large proteins have been encapsulated for local activity in the gastrointestinal tract. LCNP carriers can be combined with controlled release and targeting functionalities. The particles are designed to form in situ at a controlled rate, which enables an effective in vivo distribution of the drug. LCNP carriers can also be released at different absorption sites, for example in the upper or lower intestine, which is important for drugs that have narrow regional absorption windows.

1.3.10.1 LIQUID SELF EMULSIFYING DRUG DELIVERY SYSTEM

SEDDS has been used for the administration of PWSD due to its thermodynamic stability, high drug solubilization capacity, improvement in oral bioavailability and protection against enzymatic hydrolysis. However, its poor palatability due to the lipid content leads to poor patient compliance. Liquid selfemulsifying oil formulation is not without the common disadvantage possible with liquid encapsulated systems, which is leakage from capsule, stability and handling problems. The problems are addressed by the formulation of solid forms of SEDDS (Bo et al., 2008).

1.3.10.2 SOLID SELF EMULSIFYING DRUG DELIVERY SYSTEM

Lipid formulations may react with the capsule resulting to brittleness or softness of the gelatin shell. To prevent this limitation, liquid lipid formulations could be transformed into free flowing powder by loading the formulation on a suitable solid carrier. Liquid lipid loading onto solid carriers combines the features of a lipid based drug delivery 48 system and solid dosage form (Vikas and Akhtar, 2008; Rajesh et al., 2010). Solid

SEDDS is an improvement or alternative to conventional liquid SEDDS. It is superior in reducing production cost, simplifying industrial manufacture, and improving stability and also patient compliance. Solid SEDDS are very flexible to develop various solid dosage forms for oral and parenteral administration. Also, GI irritation is avoidable and controlled/sustained release of drug is obtained. Formulating a solid dosage form requires the use of semi‐solid excipients. SEOF have been transformed into solid dosage forms using techniques such as melt granulation, where the lipid excipient acts as a binder and solid granules are produced on cooling. Solvents or supercritical fluids can be used with semi‐solid excipients, which are solubilized and then the solvent evaporated to produce a waxy powder. Spraying techniques can be used to produce powder form formulations. These techniques enable the production of granules or powders that can then be compressed into a tablet form or filled into capsules. In all cases, the lipidic excipients used must be semi‐solid at room temperature. Another concept is by the adsorption/absorption of a liquid SEOF onto a neutral carrier (i.e., neutral silicate). Developing this solid dosage form technique has required extensive investigation of critical success parameters including:

• Extensive screening of different neutral carriers to evaluate their ability to

adsorb maximum levels of the liquid SEOF.

• Maximum loading value of the carrier and effect on tablet compression.

• Absorption onto the carrier and effect on flowability (an essential feature for

tablet compression).

• Evaluation of the integrity of the system with a poorly soluble API to examine

the effect of transforming a liquid into a powder on drug solubility and

dissolution rate. 49

Techniques for developments of solid self emulsifying delivery system

The techniques facilitate the transformation of liquid or semi-solid formulations into solid particles (powders, granules or pellets) which could subsequently be filled into capsules, sachets or compressed into tablets. Techniques are chosen on the basis of properties of lipid excipient. Solidification techniques for transforming liquid/ semisolid SEDDS to Solid‐SEDDS include:

Spray drying: This technique is defined as a process by which a liquid solution is sprayed into a hot air chamber to evaporate the volatile fraction. Polyoxylglycerides

(lauroyl or stearoyl) have been used alone or in combination with a solid carrier (silicon dioxide) to form microparticles of etoricoxib and glibenclamide (Chauhan et al., 2005).

This technique involves the preparation of a formulation by mixing lipids, surfactants, drug, solid carriers, and solubilization of the mixture before spray drying. The solubilized liquid formulation is then atomized into a spray of droplets. The droplets are introduced into a drying chamber, where the volatile phase (e.g. the water contained in an emulsion) evaporates, forming dry particles under controlled temperature and airflow conditions. Such particles can be further prepared into tablets or capsules. The atomizer, the temperature, the most suitable airflow pattern and the drying chamber design are selected according to the drying characteristics of the product and powder specification.

Spray Cooling

The molten droplets are sprayed into cooling chamber, which will congeal and re- crystallize into spherical solid particles that fall to the bottom of the chamber and subsequently collected as fine powder. The fine powder may then be used for development of solid dosage forms tablets or direct filling into hard shell capsules.

Many types of equipment are available to atomize the liquid mixture and to generate 50 droplets: rotary, pressure, two-fluid or ultrasonic atomizers (Rodriguez et al, 1999).

Adsorption to solid carriers: Free flowing powders may be obtained from liquid

SEDDS formulations by adsorption to solid carriers. The adsorption process is simple and just involves addition of the liquid formulation onto carriers by mixing in a blender

(Pouton, 2006). The resulting powder may then be filled directly into capsules or, alternatively, mixed with suitable excipients before compression into tablets. A significant benefit of the adsorption technique is good content uniformity (Fabio and

Elisabetta, 2003). SEDDS can be adsorbed at high levels (up to 70 % (w/w)) onto suitable carriers. Solid carriers can be microporous inorganic substances, high surface‐ area colloidal inorganic adsorbent substances, cross‐linked polymers or nanoparticle adsorbents, for example, silica, silicates, magnesium trisilicate, magnesium hydroxide, talcum, crospovidone, cross‐linked sodium carboxymethyl cellulose and cross linked polymethy methacrylate.

Melt granulation/Pelletization: Melt granulation is a process in which powder agglomeration is obtained through the addition of a binder that melts or softens at relatively low temperatures. As a ‘one‐step’ operation, melt granulation offers several advantages compared with conventional wet granulation, since the liquid addition and the subsequent drying phase are omitted. Moreover, it is also a good alternative to the use of solvent (Seo, 2003). The main parameters that control the granulation process are impeller speed, mixing time, binder particle size, and the viscosity of the binder.

Nucleation (onset of granule formation) is largely affected by binder viscosity at high impeller speed and binder particle size at low speed (Seo and Schaefer, 2001).

Depending on the combination of process parameters, two distinct mechanisms namely

“distribution” and “immersion” may be at play in the development of granules. Fine or atomized excipients with low viscosity at high impeller speed favour a homogenous 51 distribution of the binder onto the surface of the powder. Immersion of the powder on the other hand is the preferred mechanism which is assisted by combination of large binder particles possessing high viscosity and mixing under low impeller speed. The granule size distribution is controlled by the combined effect of the impeller and chopper speeds. The melt granulation process was usually used for adsorbing SEDDS

(lipids, surfactants, and drugs) onto solid neutral carriers (mainly silica and magnesium aluminometa silicate) (Gupta et al., 2001; Gupta et al., 2003). Generally, lipids with low HLB and high melting point are suitable for sustained release applications. Semi- solid excipients with high HLB on the other hand may serve in immediate release and bioavailability enhancement. The progressive melting of the binder allows the control of the process and the selection of the granule's size. Hydrogen bonding with adsorbent during storage governs drug dissolution from solid dispersion granules. The main advantages of melt granulation/pelletization with lipids are process simplicity (one- step), absence of solvents, and more importantly the potential for the highest drug loading capacity 85% theoretically, and up to 66% actually reported in the literature

(Chauhan et al., 2005 ).

Melt extrusion/extrusion spheronization: Melt extrusion is a solvent‐free process that allows high drug loading (60%) (Jannin, 2008) as well as content uniformity. Extrusion is a procedure of converting a raw material with plastic properties into a product of uniform shape and density, by forcing it through a die under controlled temperature, product flow, and pressure conditions (Verreck and Brewster, 2004; Breitenbach,

2002). The size of the extruder aperture will determine the approximate size of the resulting spheroids. The extrusion–spheronization process is commonly used in the pharmaceutical industry to make uniformly sized spheroids (pellets). This approach has been successfully tried for 17, β-estradiol and two model drugs with surfactants such as 52 sucrose monopalmitate, lauroylpolyoxylglycerides and polysorbate 80 (Tween® 80)

(Serratoni et al., 2007). An innovative “system-incylinder” molding technique was recently employed to develop a dual purpose (enhanced bioavailability and controlled release) formulation with propranolol hydrochloride (Mehuys et al., 2005).

The extrusion–spheronization process requires the following steps:

• Dry mixing of the active ingredients and excipients to achieve a homogeneous

powder; wet massing with binder.

• Extrusion into a spaghetti‐like extrudate.

• Spheronization from the extrudate to spheroids of uniform size.

• Drying sifting to achieve the desired size distribution and coating (optional)

(Newton, 2001).

Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid Carriers (SLC)

SLN and NLC are two types of submicron size particles (50–1000 nm) composed of physiologically tolerated lipid components. SLN are produced by high-pressure homogenization of the solid matrix and drug with an aqueous solution of the glyceryldibehenate as solid lipid matrix and poloxamers 188 or polysorbates 80 as surfactants. They typically contain a liquid lipid excipient such as medium chain triglycerides in addition to classic components of SLN. They have been mainly used for controlled-release applications in oral, intravenous or topical route (Souto and Muller,

2005).

Supercritical Fluid Based Method

Lipids may be used in supercritical fluid based methods either for coating of drug particles, or for producing solid dispersions. For environmental reasons, the preferred supercritical fluid of choice is supercritical carbon dioxide. Examples include 53 controlled release applications using glyceryltrimyristate (Dynasan™ 114) and stearoylpolyoxylglycerides (Gelucire® 50/02) (Dos Santos et al., 2003).

SOLID SEDDS COULD BE DEVELOPED IN DIFFERENT DOSAGE FORMS

Self emulsifying (SE) capsules: Solid SEDDS solves the leakage limitation problems of liquid SEDDS formulations. Capsule filling is the simplest and the most common technology to encapsulate liquid or semi-solid lipid-based formulations. Oral administration of SEDDS capsules has been found to enhance patient compliance compared with the previously used parenteral route. For instance, low molecular weight heparin (LMWH) used for the treatment of venous thrombo‐embolism was clinically available only via the parenteral route. So, oral LMWH therapy was investigated by formulating it in hard capsules. LMWH was dispersed in SMEDDS and thereafter the mixture was solidified to powders using three kinds of adsorbents: micro-porous calcium silicate (FloriteTM RE); magnesium aluminum silicate (NeusilinTM US2) and silicon dioxide (SylysiaTM 320). Eventually these solids were filled into hard capsules

(Ito et al., 2006). In another study, such adsorbents were also applied to prepare

SEDDS tablets of gentamicin that, in clinical use, was limited to administration as injectable or topical dosage forms (Ito et al., 2005).

Capsule filing of semisolid formulations, involves four‐step process:

• Heating of the semisolid excipient to at least 20 °C above its melting point,

• Incorporation of the active substances (with stirring) and

• Capsule filling with the molten mixture and Cooling to room temperature (Cole,

2008).

While capsule filing for liquid formulations involves two‐step process: o Filling of the formulation into the capsules and 54 o Sealing of the body and cap of capsule, either by banding or by microspray

sealing (Jannin, 2008).

Self emulsifying sustained/controlled release tablets: To reduce significantly the amount of solidifying excipients required for transformation of SEDDS into solid dosage forms, a gelled-SEDDS has been developed (Patil et al., 2004 and Obitte et al.,

2009). In their study, colloidal silicon dioxide (Aerosil 200) and carbosil were selected respectively as a gelling agent for the oil‐based systems, which served the dual purpose of reducing the amount of required solidifying excipients and aiding in slowing down of the drug release. This system has outstanding features such as stable plasma concentrations and controllable drug release rate, allowing a bioavailability of 156.78% relative to commercial carvedilol tablets (Wei et al., 2007).

Self emulsifying dry emulsions: Dry emulsions can be useful for further preparation of tablets and capsules. The formulations are typically prepared from oil/ water (O/W) emulsions containing a solid carrier (lactose, maltodextrin, and so on) in the aqueous phase by rotary evaporation ( Myers and Shively, 1992), freeze‐drying (Bamba et al.,

1995) or spray drying. Myers and Shively obtained solid state glass emulsions in the form of dry ‘foam’ by rotary evaporation, with heavy mineral oil and sucrose. Such emulsifiable glasses have the advantage of not requiring surfactant. In freeze‐drying, a slow cooling rate and the addition of amorphous cryoprotectants have the best stabilizing effects, while heat treatment before thawing decreases the stabilizing effects.

The technique of spray drying is more frequently used in preparation of dry emulsions.

This formulation consisted of a surfactant, a vegetable oil, and a pH‐responsive polymer, with lyophilization (Toorisaka, 2005). Recently, Cui (2007) prepared dry emulsions by spreading liquid O/W emulsions on a flat glass, then dried and triturated to powders. Dry emulsion may be redispersed into water before use. Medium chain 55 triglycerides are commonly used as oil phase for these emulsions (Christensen et al.,

2001).

Self emulsifying sustained/controlled release pellets: Pellets, as a multiple unit dosage form, possess many advantages over conventional solid dosage forms, such as flexibility of manufacture, reducing intra- and inter-subject variability of plasma profiles and minimizing gastrointestinal irritation without lowering drug bioavailability

(Gandhi et al., 1999). Serratoni et al (2007) prepared SE controlled release pellets by incorporating drugs into SEDDS that enhanced their rate of release, and then by coating pellets with a water‐insoluble polymer that reduced the rate of drug release. Pellets were prepared by extrusion/ spheronization and contained two water‐insoluble model drugs (methyl and propyl parabens); SEDDS contained monodiglycerides and

Polysorbate 80.

Self emulsifying (SE) solid dispersions: Although solid dispersions could increase the dissolution rate and bioavailability of poorly water soluble drugs, some manufacturing difficulties and stability problems existed. Serajuddin pointed out that these difficulties could be surmounted by the use of self emulsifying excipients (Serajuddin, 1999;

Vasanthavada and Serajuddin, 2007). These excipients have the potential to increase further the absorption of poorly water‐soluble drugs relative to previously used PEG solid dispersions and may also be filled directly into hard gelatin capsules in the molten state, thus obviating the former requirement for milling and blending before filling

(Serajuddin et al., 1988). Self emulsifying excipients like Gelucire® 44/14, Gelucire®

50/02, Labrasol®, Transcutol P® and TPGS (tocopheryl polyethylene glycol 1000 succinate) have been widely used in this field (Khoo et al., 2000). Gupta et al (2002) prepared SE solid dispersion granules using the hot-melt granulation method. 56

Self emulsifying beads: To transform SEDDS into a solid form with minimum amounts of solidifying excipients, Patil and Paradkar (2006) investigated loading

SEDDS into the micro‐channels of porous polystyrene beads (PPB) using the solvent evaporation method. PPB with complex internal void structures is typically produced by copolymerizing styrene and divinyl benzene. They are inert, stable over a wide pH range and to extreme conditions of temperature and humidity.

Self emulsifying sustained release microspheres: Zedoary turmeric oil (ZTO; a traditional Chinese medicine) exhibits potent pharmacological actions including tumor suppressive, antibacterial, and antithrombotic activity. ZTO release behavior could be controlled by the ratio of hydroxylpropyl methylcellulose acetate succinate to Aerosil

200 in the formulation.

Self emulsifying nanoparticles: Solvent injection is a nanoparticle technique. In this method, the lipid, surfactant, and drugs are melted together, and injected drop wise into a stirred non‐solvent. The resulting SEDDS nanoparticles are thereafter filtered out and dried. This approach yielded nanoparticles (about 100 nm) with a high drug loading efficiency of 74 % (Attama and Nkemnele, 2005).

Self emulsifying suppositories: Some investigators proved that solid SEDDS could increase not only gastrointestinal adsorption but also rectal/vaginal adsorption (Kim and Ku, 2000). Glycyrrhizin, which, by the oral route, barely achieves therapeutic plasma concentrations, can obtain satisfactory therapeutic levels for chronic hepatic diseases by either vaginal or rectal SE suppositories. The formulation included glycyrrhizin and a mixture of a C6–C18 fatty acid glycerol ester and a C6–C18 fatty acid macrogol ester.

Self emulsifying implants: Research into SE implants has greatly enhanced the utility and application of solid SEDDS. As an example, 1, 3‐bis (2‐chloroethyl) ‐ 1‐ 57 nitrosourea (carmustine, BCNU) is a chemotherapeutic agent used to treat malignant brain tumors. However, its effectiveness was hindered by its short half‐life. Loomis invented copolymers having a bio-resorbable region, a hydrophilic region and at least two cross‐linkable functional groups per polymer chain. Such copolymers show

SEDDS property without the requirement of an emulsifying agent. These copolymers can be used as good sealants for implantable prostheses (Loomis, 2002).

1.3.10.3 SUPERSATURABLE SELF EMULSIFYING DRUG DELIVERY

SYSTEM

Supersaturable SEDDS is another approach for enhancing the oral bioavailability of poorly soluble drugs. The supersaturatable self emulsifying drug delivery system (S-

SEDDS) represents a new thermodynamically stable formulation approach wherein it is designed to contain a reduced amount of a surfactant and a water-soluble cellulosic polymer (or other polymers) to prevent precipitation of the drug by generating and maintaining a supersaturated state in vivo. S-SEDDS formulations differ from the conventional SEDDS formulations as they contain a reduced amount of surfactant and a polymeric precipitation inhibitor (e.g., water-soluble cellulosic polymers, such as hydroxypropylmethylcellulose (HPMC), in order to generate and maintain a supersaturated state of the drug following mixing with water. The S-SEDDS formulations can result in enhanced oral absorption and the reduced surfactant levels may minimise gastrointestinal surfactant side effects. Higuchi (1960) recognized the need for increasing the thermodynamic activity of drug formulations and, thereby increasing the bioavailability of poorly soluble drugs, through supersaturation, more than four decades ago. Supersaturable formulations differ from supersaturated formulations. Supersaturated formulations are not thermodynamically stable and drugs 58 in supersaturated formulations can crystallize on storage. Therefore, the physical stability of such formulations is fundamentally challenging and this limits their practical utility. Supersaturation is intended to increase the thermodynamic activity of the drug beyond its solubility limit and, therefore, to result in an increased driving force for transit into and across the biological barrier (Gao et al., 2004). The S-SEDDS formulations contain a reduced level of surfactant and a polymeric precipitation inhibitor to yield and stabilize a drug in a temporarily supersaturated state. Crystal growth is believed to take place in three steps (Macie and Grant, 1986; Rodriguez-

Hornedo and Murphy, 1999; Raghavan et al., 2001a):

• diffusion of the molecule from the bulk media to the solid crystal interface;

• the adsorbed molecule, through a surface reaction, becomes part of the crystal lattice

and the heat of crystallization is released and

• the heat of crystallization is conducted to the bulk media. Materials that may inhibit

nucleation or crystal growth have been reported.

The crystal growth inhibitors have several potential actions including:

• altering bulk properties such as surface tension or saturation solubility;

• changing the adsorption layer at the crystal-medium interface;

• Selectively adsorbing to the crystal interface thereby blocking crystal growth;

• being adsorbed into growth layers and thereby disrupting growth layers across the

surface;

• adsorbing into surface imperfections causing rough surfaces to become flat;

• altering the surface energy of the crystal face which may change the level of

solvation.

Rheological polymers such as HPMC and PVP are thought to interact through a number of mechanisms including adsorbing to the crystal (via hydrogen bonding) and 59 collecting at the growing crystal-bulk media interface and thereby providing diffusion resistance (Raghavan et al., 2001a). Some reports also suggest that these polymers can form complexes with the drug of interest, increase their saturation solubility and therefore reduce the extent of supersaturation (Rodriguez-Hornedo and Murphy, 1999).

Studies of the mechanism responsible for inhibiting crystallization of drugs in aqueous solutions containing HPMC suggest that the HPMC polymer chain may inhibit nucleation as well as crystal growth by adsorption onto the surface of the nuclei or on to the surface of crystals. Ziller and Rupprecht, (1988) suggested that the polymer inhibits the introduction of drug molecules from solution into the crystal lattice by occupying adsorption sites such that the adsorbed polymer forms a mechanical barrier that inhibits crystallization. Raghavan et al (2001b) proposed that the mechanism of nucleation and growth is based on the interaction between the drug and the polymer molecules through hydrogen bonding. The hydrodynamic boundary layer surrounding the crystal, resulting from adsorption of the polymer molecules onto the crystal surface, leads to crystal growth inhibition as well as habit modification of the crystals.

Cyclodextrins can solubilize material through the formation of dynamic inclusion complexes (Loftsson et al., 2004; Loftsson and Brewster, 1996; Thompson, 1997;

Rajewski and Stella, 1996). Additional data suggest that cyclodextrins can also inhibit nucleation and crystal growth through non-complex based mechanisms which may be similar to those associated with the pharmaceutical polymers described above

(Brewster et al., 1989; Torres-Labandeira et al., 1990; Uekama et al., 1992). PEG4000 or Polyox® may affect supersaturated solutions through various mechanisms (Li et al.,

2006; Urbanetz and Lippold, 2005).

60

Figure 2: The role of HPMC as PPI in inhibiting the formation of crystalline materials. The hydrophobic polymer chains may preferably adsorb on the surface of the primary nuclei in the nucleation phase and, thus, result in amorphous nuclei due to polymer chains occupying the lattice sites. Amorphous nuclei undergo aggregation and precipitate as drug/ polymer composites.

Most of the work on supersaturation reported in the literature deals with topical delivery (Higuchi, 1960; Megrab et al., 1995; Ma et al., 1996; Schwarb et al., 1997;

Raghavan et al., 2001a; Pellet et al., 1997b; Iervolino et al., 2001) with less attention on the use of supersaturation for improving the oral delivery of poorly soluble drugs

(Simonelli et al., 1970; Sekikawa, 1978; Suzuki and Sunada, 1998; Yamada et al.,

1999; Kohri et al., 1999; Hasegawa et al., 1985). Polyvinylpyrrolidone (PVP) was found to be useful in generating a supersaturated state with a number of poorly soluble drugs (Megrab et al., 1995; Ma et al., 1996; Schwarb et al., 1997; Raghavan et al.,

2001b; Simonelli et al., 1970; Sekikawa, 1978; O’Driscoll and Corrigan, 1982). Other studies reported the use of the water-soluble cellulosic polymers, such as HPMC

(Iervolino et al., 2001; Suzuki and Sunada, 1998; Usui et al., 1997), hydroxypropyl methylcellulose phthalate (Kohri et al., 1999; Hasegawa et al., 1985) and sodium carboxymethylcellulose (Hasegawa, 1988). The cellulosic polymers are excellent crystal growth inhibitors and are effective in prolonging the supersaturated state of the drugs as shown by a number of in vitro studies (Raghavan et al., 2001; Pellet et al., 61

1997b; Usui et al., 1997; Hasegawa, 1988). Based on the above-cited literature, a promising approach for enhancing the oral bioavailability of poorly soluble drugs is the use of the principle of supersaturation in the development of supersaturatable formulations (Gao et al., 2004). The creation of a supersaturated state with HPMC with the S-SEDDS formulations may be due to the formation of a widely spaced cellulosic- polymer network that is generated by the HPMC chains in water. According to the literature, solutions of HPMC consist of ‘cellulosic bundles resulting in a tenuous network of swollen clusters with hydrophobic substituents surrounded by sheaths of structured water (Haque and Morris, 1993; Haque et al., 1993). Studies on the mechanism responsible for inhibiting crystallization of drugs in aqueous solutions containing HPMC suggest that the HPMC polymer chain may inhibit nucleation, as well as crystal growth by adsorption of the HPMC molecules onto the surface of the nuclei, or onto the surface of crystals (Raghavan et al., 2001; Ziller and Rupprecht,

1988). The general applicability of cellulosic polymers in inhibiting crystallisation of many pharmaceutical substances is widely reported (Raghavan et al., 2001a; Pellet et al., 1997; Iervolino et al., 2001; Suzuki and Sunada, 1998; Kohri et al., 1999;

Hasegawa, 1985; Usui et al., 1997). Simonelli et al (1970) suggested that the polymer at the crystal surface forms a net-like structure, which allows the drug to grow out in finger-like protrusions leading to a growth with a rough surface. Ziller et al (1988) suggests that the polymer inhibits the introduction of drug molecules from solution into the crystal lattice by occupying adsorption sites and, thus, the adsorbed polymer forms a mechanical barrier that inhibits crystallisation.

Mechanisms of drug precipitation inhibition

Precipitation inhibitors may act by a number of possible mechanisms that can be inferred from the theory of drug precipitation, (Joachim et al., 2008) including: 62 o reducing the degree of supersaturation by increasing the solubility (decrease in

both nucleation and crystal growth); o increasing the viscosity, resulting in a reduced molecular mobility (decreasing

nucleation), and diffusion coefficient (decreasing crystal growth); o increasing the cluster–liquid interfacial energy (decreasing nucleation); o changing the adsorption layer at the crystal-medium interface by, for example,

adsorbing onto the crystal surface thereby hindering crystal growth; this may be

accompanied with crystal habit modifications; o changing the level of solvation at the crystal– liquid interface, thereby affecting

the integration of drug molecules into the crystal.

Obviously, these mechanisms depend on properties of the inhibitor, the drug and the medium. It should be taken into account that inhibitory effects will depend not only on the concentration of the excipient, but also on the initial degree of supersaturation

(Raghavan et al., 2003; Overhoff et al., 2007).

Hydrogen bonds between drug molecules and the polymer, which were confirmed by means of infrared spectroscopy (Raghavan et al., 2001a; Joachim et al., 2008) increase the activation energy for nucleation.

63

1.4 LIPID FORMULATION CLASSIFICATION SYSTEM (LFCS)

Table 1: Lipid formulation classification system

Formulation Type of materials Characteristics Advantages Disadvantages Type 1 Oils without Non-dispersing, GRAS status, has poor solvent Surfactants requires digestion simple, excellent capacity unless drug (eg. Mono-, di-.and capsule is highly tri-glyceride) compartibility hydrophobic

Type 2 Oils and water SEDDS formed Unlikely to lose Turbid o/w insoluble surfactants without water solvent capacity dispersion (particle soluble component on dispersion size 0.25-2 μm)

Type 3 Oils, surfactants, SEDDS and Clear or almost Possible loss of cosolvents (both water SMEDDS formed clear dispersion, solvent capacity on insoluble and soluble with water soluble drug absorption dispersion; less excipients) components without digestion easily digested

Type 4 Water soluble Formulation Formulation has Likely loss of surfactants and disperses typically good solvent solvent capacity on coslvents (no oils) to form a micellar capacity for many dispersion, may not solution drugs be digested

Lipid based systems are defined as emulsions, microemulsions, SEDDS, micellar suspensions, and oil solutions. The LFCS briefly classifies lipid-based formulations into four types according to their composition and the possible effect of dilution and digestion on their ability to prevent drug precipitation.

Lipid formulation classification system (LFCS) as described by Pouton showing typical compositions and properties of lipid-based formulations (Pouton, 1985 and

2000). CAVITRON® and CAVASOL®

Type I lipid formulation: Non-dispersing systems

Type I systems consist of formulations where the drug is in a solution of triglycerides and/or mixed glycerides or in an oil-in-water emulsion stabilised by low concentrations of emulsifiers such as 1% w/v polysorbate 60 (Carrigan and Bates, 1973) and 1.2 % w/v lecithin (Myers and Stella, 1992). These systems exhibit poor initial aqueous 64 dispersion and require digestion by pancreatic lipase/co-lipase in the GIT to generate more amphiphilic lipid digestion products and promote drug transfer into the colloidal aqueous phase. Type I lipid formulations represent a relatively simple formulation option for potent drugs or highly lipophilic compounds where drug solubility in oil is sufficient to allow incorporation of the required “payload” dose.

Type II-SEDDS: Non-water soluble component systems

Type II lipid formulations, referred as a self emulsifying drug delivery systems,

(SEDDS) are isotropic mixtures of lipids and lipophilic surfactants with HLB < 12 that self-emulsify to form fine oil-in-water emulsions in aqueous media (Pouton, 1997).

Self-emulsification is generally obtained at surfactant content above 25%w/w.

However, at a surfactant content of 50-60%w/w, the emulsification process may be compromised by the formation of viscous liquid crystalline gels at the oil/ water interface (Pouton, 1985; Cuine et al., 2008). Type II lipid-based formulations offer the advantage of overcoming the slow dissolution step typically observed with solid dosage forms and they are able to generate large interfacial areas which in turn allow efficient partitioning of drug between the oil droplets and the aqueous phase from which absorption occurs.

Type III-SEDDS: Water soluble component systems

Type III lipid-based formulations, commonly referred to as self-microemulsifying drug delivery systems (SMEDDS), are defined by the inclusion of hydrophilic surfactants with HLB > 12 and co-solvents such as ethanol, propylene glycol and polyethylene glycol. Type III formulations can be further divided into Type IIIA and Type IIIB formulations in order to identify more hydrophilic systems. In Type IIIB, the content of hydrophilic Surfactants and co solvents are increased and the lipid content is reduced.

Type IIIB formulations typically achieve greater dispersion rates when compared with 65

Type IIIA although the risk of drug precipitation on dispersion of the formulation is higher owing to the lower lipid content. However, recent suggestions of a possible inhibitory effect of some lipid excipients, like polyoxyethylene sorbitan fatty acid esters, polyoxyethoxylated castor oil and D-α-tocopheryl polyethylene glycol 1000 succinate, on CYP3A and P-glycoprotein functionality involves the increase in bioavailability observed after oral administration of Neoral and many other SMEDDS formulations.

Type IV-dispersion systems: non-oil micellar Systems

The type IV category was recently added to the Lipid Formulation Classification

System (Pouton, 2006). Type IV formulations do not contain natural lipids and represent the most hydrophilic formulations. These formulations commonly offer increased drug payloads due to higher drug solubility in the surfactants and co-solvents.

When compared with formulations containing simple glyceride lipids they also produce very fine dispersions when introduced in aqueous media. It has been suggested that they produce rapid drug release and increased drug absorption. However, little is known about the solubilisation capacity of these systems in vivo and, in particular, whether they are equally capable of maintaining poor water soluble drugs (PWSD) in solution during passage along the GIT when compared with formulations consisting of natural oils (Type II and Type III). An example of a Type IV formulation is the current capsule formulation of the HIV protease inhibitor amprenavir (Agenerase) which contains TPGS as a surfactant and PEG 400 and propylene glycol as co-solvents.

1.5 COMPOSITION OF SELF EMULSIFYING OIL FORMULATION

SEDDS formulation is composed of lipids, surfactants, cosurfactants/ cosolvents and the drug. The system has the ability to form a fine oil-in-water emulsion when 66 dispersed by an aqueous phase under gentle agitation. SEDDS present drugs in a small droplet size and well-proportioned distribution, which increase the dissolution and permeability. Furthermore, since drugs can be loaded in the inner phase and delivered by lymphatic bypass share, SEDDS protect drugs against hydrolysis by enzymes in the

GI tract and reduce the presystemic clearance in the GI mucosa and hepatic first pass metabolism. The self‐emulsifying process depends on:

• The nature of the oil–surfactant pair

• The surfactant concentration

• The temperature at which self emulsification occurs.

1.5.1 LIPOPHILIC BASE

The realization that the oral bioavailability of Poor Water Soluble Drugs (PWSD) may be enhanced when co-administered with meal rich in fat has led to the increasing recent interest in the formulation of PWSD in lipids. Lipophilic base is one of the most important components in the formulation of SEDDS. It is a carrier to dissolve the lipophilic drug. Oils solubilize the lipophilic drug in a specific amount. Oil base assists self emulsification. Lipid based formulations can enhance the bioavailability of poorly soluble drug substances by keeping the compound in solution. Medium chain triglycerides (MCT) and long chain triglycerides (LCT) are commonly used for the lipid based formulations. Several major differences exist between MCT and LCT with respect to their in vivo fate, such as lipolytic products, differences in the modulation on gastric emptying (Hunt and Knox, 1968; Fatouros et al., 2007) and the contraction of the gallbladder in humans (Ladas et al., 1994). Long chain lipolytic products delay gastric emptying and facilitate the contraction of the gallbladder to a larger extent than the medium chain lipolytic products (Hunt and Knox, 1968). The type of lipid 67 component of the delivery system has a great influence on its capability to enhance absorption. Non-digestible lipids, including mineral oils, sucrose polyesters and others, are not absorbed from the gut lumen. They remain in the gastrointestinal lumen, tend to retain the lipophilic drug within the oil, and thus, may limit the absorption of the drug.

Naturally occurring oils and fats are mixtures of triglycerides, which contain fatty acids of varying chain lengths and degrees of unsaturation. The melting point is directly proportional to the degree of unsaturation which also increases the relative susceptibility to oxidation. Also naturally-derived oils are not frequently used as the oil fraction in SEDDS due to their poor ability to dissolve a large amount of lipophilic drug. Hydrogenation is done synthetically to decrease the degree of unsaturation and conferring resistance to oxidative degradation. Thus, synthetic or chemically modified oils, such as hydrolyzed vegetable oils are widely used in SEDDS. Besides having better drug solubility properties, chemically modified oils also have better ability to facilitate self-emulsification because they possess surfactant properties of their own

(Gursoy and Benita, 2004). Digestive lipids, including triglycerides, diglycerides, phospholipids, fattyacids, cholesterol and other synthetic derivatives, are suitable oils for drug delivery systems of lipophilic compounds (Arik Dahan and Amnon Hoffman,

2008). Moreover, lipids affect the oral bioavailability of drugs by exerting their effect through some mechanisms, including protection of the drug from enzymatic or chemical degradation in the oil droplets and activation of lipoproteins promoting the lymphatic transport of lipophilic drugs (Pouton, 2000). Generally, lipids with a low

HLB and high melting point are suitable for sustained release applications. Semi-solid excipients with high HLB, on the other hand, may be used for immediate release and bioavailability enhancement. Some examples of oil include; Cotton seed oil, Soybean oil, Corn oil, Sunflower oil, Castor oil, Sesame oil, Peanut oil, Labrafac, Labrafil. 68

Although incompletely understood, the currently accepted view is that lipids may enhance bioavailability via a number of potential mechanisms, including (Porter and

Charman, 2001): o Alterations (reduction) in gastric transit, thereby slowing delivery to the

absorption site and increasing the time available for dissolution. o Increases in effective lumenal drug solubility. The presence of lipids in the GI

tract stimulates an increase in the secretion of bile salts (BS) and endogenous

biliary lipids including phospholipid (PL) and cholesterol (CH), leading to the

formation of BS/PL/CH intestinal mixed micelles and an increase in the

solubilisation capacity of the GI tract. However, intercalation of administered

(exogenous) lipids into these BS structures either directly (if sufficiently polar),

or secondary to digestion, leads to swelling of the micellar structures and a

further increase in solubilisation capacity.

Effects of Lipids on the GIT

Various physiological mechanisms have been proposed to explain the effect of oils on the absorption of water‐insoluble compounds, including altered gastrointestinal motility, increased bile flow and drug solubilization, increased mucosal permeability, enhanced mesenteric lymph flow, and increased lymphatic absorption of PWSD and also increase in the bioavailability of hydrophobic drug. Therefore, the presence of lipids in the GI tract stimulates increased secretion of bile salts (BS) and endogenous biliary lipids (phospholipid (PL)) and cholesterol (CH)), leading to the formation of

BS/PL/CH intestinal mixed micelles and increased solubilisation capacity of the GI tract. However, inclusion of administered (exogenous) lipids into these micelles either directly (if sufficiently polar), or secondary to digestion, leads to swelling of the micellar structures and a further increase in solubilisation capacity (Porter and Chaman, 69

2001). The lipids play a significant role since they can increase the drug solubility in the lumen, can change the physical (Aungst, 2000) and the biochemical barrier function

(Benet, 2001) of the GI tract and they can stimulate the lymphatic transport (Porter and

Charman, 1997).

1.5.2 SURFACTANT

Surfactant molecules can be classified based on the nature of hydrophilic group within the moleecule. The four main groups of surfactants are defined as follows, Anionic surfactants, Cationic surfactants, Ampholytic surfactants and Nonionic surfactants:

• Anionic surfactants, where the hydrophilic group carries a negative charge such

as carboxyl (RCOO-), sulphonate (RSO3-) or sulphate (ROSO3-). E.g:

Potassium laurate, sodium lauryl sulphate.

• Cationic surfactants where the hydrophilic group carries a positive charge. E.g:

Quarternary ammonium halide.

• Ampholytic (zwitterionic) surfactants contain both positive and negative charge.

E.g: Sulfobetaines.

• Nonionic surfactants, where the hydrophilic group carries no charge but derives

its water solubility from highly polar groups such as hydroxyl OCH2CH2O).

E.g. Sorbitan polyoxyethylene esters (spans), polysorbates (Tweens).

They facilitate the selfemulsification process of the SEDDS, which helps to improve the bioavailability of PWSD. The mechanisms involved are complex and were described as “diffusion and stranding”. Surfactants are classified according to their hydrophile-lipophile balance (HLB) values. This HLB value indicates the hydrophilicity of surfactants where surfactants which are more hydrophilic possess higher HLB values. Oil-in-Water emulsions can be formed using surfactants which 70 have HLB values in the range of approximately 8-18. It was reported that surfactants with high HLB values will provide rapid emulsification, with excellent spreading properties and rapid cloud formation, leading to the formation of very fine o/w droplets.

Droplets formed could either be positively or negatively charged. As the mucosal linings are negatively charged, it was observed that the positively charged particles penetrate deeply into the ileum. Therefore, it is observed that cationic emulsion has greater bioavailability than anionic emulsion.

There is a relationship between the droplet size and the concentration of the surfactant being used. In some cases, increasing the surfactant concentration could lead to droplets with smaller mean droplet size. This could be explained by the stabilization of the oil droplets as a result of the localization of the surfactant molecules at the oil-water interface (Karim et al., 1994). On the other hand, in some cases the mean droplet size may increase with increasing surfactant concentrations (Georgakopoulos et al., 1992).

This phenomenon could be attributed to the interfacial disruption elicited by enhanced water penetration into the oil droplets mediated by the increased surfactant concentration and leading to ejection of oil droplets into the aqueous phase. The surfactants used in these formulations are known to improve the bioavailability by various mechanisms including: improved drug dissolution, increased intestinal epithelial permeability, increased tight junction permeability and decreased/inhibited p- glycoprotein drug efflux. However, the large quantity of surfactant may cause moderate reversible changes in intestinal wall permeability or may irritate the GI tract.

Selection of the type of surfactant to be used in the formulation of SEDDS will depend on factors such as its emulsification performance, safety, as well as the stability of the emulsion formed upon contact with aqueous medium. Naturally derived surfactants are safe to consume but they display limited selfemulsification capacity. As for synthetic 71 surfactants, non-ionic surfactants are widely recommended as compared to ionic surfactants because the former are less toxic, more stable, less irritating and biodegradable (Melmstein, 1999). To form stable SEDDS, the concentration of surfactants to be used was suggested to be within the range of 30-60% (w/w)

(Gershanik and Benita, 2000). At improper surfactant content self emuslification may be poor but fine emulsions can be prepared with sufficient shear by homogenization

(Pouton, 1985). It is important to note that compositional variables (oil, presence of other amphiphiles, hydrophilic and hydrophobic molecules (ie glycerol, sorbitol) or electrolyte) as well as temperature may have influence on hydrophilic and hydrophobic properties and geometry of the surfactant molecule and the efficiency of a surfactant to generate microemulsion (Kahlweit, 1999; Sjoblom et al., 1996; Djekic et al., 2008).

Research has determined efficient experimental temperature of Labrasol® to be 25 ± 1 oC which corresponds to common conditions of preparation, storage and application of pharmaceutical microemulsions, and expressed as the minimal concentration of the surfactant required to obtain a single phase microemulsion (Djekic et al., 2008).

1.5.3 CO-SURFACTANT/CO-SOLVENT

The production of an optimum SMEDDS requires relatively high concentrations

(generally more than 30%w/w) of surfactants, thus the concentration of surfactant can be reduced by incorporation of co-surfactant. Role of the co-surfactant together with the surfactant is to lower the interfacial tension to a very small even transient negative value. At this value the interface would expand to form fine dispersed droplets, and subsequently adsorb more surfactant and surfactant/co-surfactant until their bulk condition is depleted enough to make interfacial tension positive again. This process called ‘spontaneous emulsification’ forms the microemulsion. However, the use of co- 72 surfactant in SEDDS is not mandatory for many non-ionic surfactants. The selection of surfactant and co-surfactant is important not only to the formation of SMEDDS, but also to solubilization of the drug in the SMEDDS. Organic solvents, suitable for oral administration (ethanol, propylene glycol (PG), polyethylene glycol (PEG), etc) may help to dissolve large amounts of either the hydrophilic surfactant or the drug in the lipid base and can act as co-surfactant in the self emulsifying drug delivery systems, although alcohol- free self-emulsifying microemulsions have also been described in the literature. Therefore, such systems may exhibit some advantages over the previous formulations when incorporated in capsule dosage forms, since alcohol and other volatile co-solvents when incorporated in self emulsifying formulations are known to migrate into the shells of soft gelatin or hard sealed gelatin capsules resulting in the precipitation of the lipophilic drug. On the other hand, the lipophilic drug dissolution ability of the alcohol free formulation may be limited. Hence, proper choice has to be made during selection of components.

Co‐surfactant/Co‐solvents like Spans® 20 and 80, Capyrol® 90, Capmul®,

Lauroglycol®, diethylene glycol monoethyl ether (Transcutol® P ), propylene glycol, polyethylene glycol, polyoxyethylene, propylene carbonate, tetrahydrofurfuryl alcohol polyethylene glycol ether (Glycofurol®), etc., may help to dissolve large amounts of hydrophilic surfactants or the hydrophobic drug in the lipid base. Diethylene glycol monoethyl ether, Transcutol® HP, as a co-solvent, is considered as a component that decreases the fluidity of SEDDS, enhances drug incorporation into the SEDDS, improves self-emulsification properties, and possesses penetration enhancement effect

(Shen and Zhong, 2006; Cui et al., 2005; Hong et al., 2006).

73

1.6 LITERATURE REVIEW OF EXCIPIENTS

1.6.1 LABRASOL®

Labrasol® is a water dispersible surfactant composed of well-characterised polyethylene glycol (PEG) esters, a small glyceride fraction and free PEG. It is produced by the alcoholysis of vegetable oils by polyethyleneglycols. It has small fraction of glycerides and great surfactant power. It is able to selfemulsify on contact with aqueous media forming a fine dispersion, microemulsion.

Functions of labrasol®

Solubilizer and wetting agent: Surfactive power improves the solubility and wettiability of active pharmaceutical ingredients in vitro and in vivo.

Bioavailability enhancer: Increased bioavailability is reported to be associated with strong inhibition of the enterocytic efflux transporter (known as P-gp inhibition).

Promote drug penetration and permeation: Topically, it is used to solubilize active pharmaceutical ingredients.

Generally, it is used in human pharmaceutical products, veterinary products excluding food producing animals. It has a hydrophilic-lipophilic balance (HLB) of 14.

Formulation techniques and dosage forms

•Suitable for hard gelatin and soft gelatin capsules.

•Suitable for adsorption onto neutral carrier powders for use in tablets, capsule filling and sachets.

•Use in topical ointments, microemulsions, emulsions and gels.

•Use in transdermal patches.

1.6.2 TRIACETIN®

The triglyceride 1, 2, 3-triacetoxypropane is more generally known as triacetin or glycerin triacetate. Triacetin® is triester of glycerol and acetic acid. It is a clear, 74 combustible and oily liquid with a bitter taste and a fatty odor. It is slightly soluble in water but soluble in alcohol and ether. It has properties of both glycerol and acetate just as Diacetin® and Nonoacetin® are glycerin diacetate and glycerin monoacetate respectively. It is the second simplest fat after triformin. Triacetin is found in some food like butter as it is used as a food additive for the solvency of flavourings for the function of humectant. It is used in perfumery and cosmetics for these applications. It is used as an antifungal agent in external medicine for topical treatment of superficial fungal infections of the skin. Triacetin is applied to cigarette filter as a plasticizer. It is used as a gelatinizing agent in explosives.

Properties and Benefits

• excellent suitability for the solidification of acetyl cellulose fibers

• very good dissolving power for a number of organic substances

• good plasticizing effect for various plastics and cellulose-based paints

• good compatibility with natural and synthetic rubber

• good light resistance

1.6.3 TRANSCUTOL®P

Transcutol® P, highly purified diethylene glycol monoethyl ether EP/NF, has good solvent properties for PWSD. It enhances drug penetration, permeation, and produces a drug depot effect. It is used as a co-solvent in the formulation of SMEDDS (Stuhlmeier et al., 1999). Transcutol® P is manufactured from raw materials of strictly petrochemical origin. It is a colorless limpid liquid with a faint odor. It can be associated to Labrafils® and vegetable oils.

75

Applications

• Solubilizer of many active ingredients such as trinitrine, indomethacin,

nifedipine, hormones, sterols, etc.

• Absorption enhancer.

• It can be used in topical, transdermal and oral pharmaceutical preparations.

1.6.4 HYDROXYPROPYLMETHYLCELLULOSE

Hydroxypropylmethylcellulose (HPMC) is a cellulose derivative polymer andit is also known as Hypromellose. Basically, HPMC is a methylcellulose modified with propylene glycol ether groups in small amount attached to the anhydroglucose of the cellulose (Fatimi et al., 2008). HPMC is a white or creamy white coloured granular or fibrous, tasteless and odorless powder (Kibbe, 2000). It is soluble in cold water and forms a viscous colloidal solution. Moreover, HPMC solution changes into gel form when it is heated at temperatures between 50–90°C (Chen et al., 2007).

HPMC is widely used in various oral and topical pharmaceutical preparations, food products and has good mechanical and non-toxic properties. HPMC possess special characteristic for controlled release drug preparations and its applications based on the four features, i.e., surface activity, film forming ability, the capacity to form thermal gels that convert to liquid on cooling and efficient thinking. These properties are mainly due to the strong hydrophobic zones of the methyl substitutes with backbone of cellulose and hydroxypropyl group which are hydrophilic in nature (Perez et al.,

2006)). HPMC is widely used as a thickener, flow and texture enhancers, emulsifiers, stabilizers and thixotropic agents (Clasen and Kulicke, 2001). Concentrations between

2-5%w/v may be used as binder in dry or wet granulation of tablets and 2-20 % concentrations used to form film coating for tablets (Kibbe, 2000; Harwood, 2006). It is 76 also used in topical formulations such as ophthalmic preparations, ointments and tropical gels (Kibbe, 2000; Harwood, 2006). It is also for used as moisture retention and oil reduction in food products and as stabilizer and emulsifiers in cosmetics (Kibbe,

2000; Harwood, 2006).

Properties:

• Almost insoluble in ethanol, ether and acetone; quickly dispersed in 80-90

centigrade water; Aqueous solution is very stable at room temperature.

• Has good wetting/dispersing/adhesive/thickening/emlusifying/water

preserving/film-forming properties.

• Can prevent the infilration of grease.

• Film formed has excellent flexibility and transparency.

• Has good compatibility with other emulsifier.

• Easy salting-out.

• Its solution is stable at pH 2-12.

• Apparent density: 0.30-0.70 g/cm3, density is 1.3 g/cm3

1.7 MALARIA

Malaria is an important cause of morbidity and mortality in children, pregnant mothers and adults in tropical countries. Malaria is a parasitic infection and the most life threatening disease and accounts for 1 to 2 million deaths round the globe every year

(Greenwood and Mutabingwa, 2002). It is still one of the major endemic infectious diseases in many developing countries, especially in Asia, Africa, and South America.

Some regions have a fairly constant number of cases throughout the year and these regions are termed "malaria endemic". In other areas there are "malaria seasons" usually coinciding with the rainy season. Parasitic diseases are of immense global 77 significance as around 30 % of world’s population experiences parasitic infections. The tropical countries such as India are more prone to malaria and around 2 million cases are reported annually (Joshi et al., 2008).

In humans, malaria is caused by these distinct species of parasites: Plasmodium vivax,

Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi also exist (WHO guidelines for treatment of malaria, 2010). Amongst these, the most severe malaria is caused by blood-borne apicomplexan parasite P. falciparum, which is responsible for almost all malaria related deaths. Malaria is also more common in pregnant than non-pregnant women. The pattern of infection in pregnancy is comparable to that observed in infants and children (Brabin, 1983). Pregnant women are especially attractive to the mosquitoes, and malaria in pregnant women is an important cause of stillbirths, infant mortality and low birth weight, particularly

P.falciparium. In Africa, malaria in pregnancy is usually caused by strains of

Plasmodium falciparum that express unique variant surface antigen which allows the parasite to sequester in the placenta by binding to chondroitin sulphate A (Bozdech and

Llinas, 2003). Plasmodium falciparum causes a large majority of the clinical cases and mortalities (Bozdech and Llinas, 2003). P. knowlesi malaria (monkey malaria that can infect humans occasionally) can also cause life threatening illness (Daily et al., 2007).

Malaria is both preventable and curable. Malaria control requires an integrated approach made up of prevention including vector control and treatment with effective antimalarials (WHO Guidelines for Treatment of Malaria, 2010).

Malaria occurs by transmission of Plasmodium sporozoites via a bite from an infected anopheline mosquito. When a mosquito pierces the skin of an infected person, it potentially picks up gametocytes within the blood. Fertilization and sexual 78 recombination of the parasite occurs in the mosquito's gut, thereby defining the mosquito as the definitive host of the disease.

Malaria in humans develops via two phases: an exoerythrocytic and an erythrocytic phase. The exoerythrocytic phase involves infection of the hepatic system, or liver, while the erythrocytic phase involves infection of the erythrocytes, or red blood cells.

When an infected mosquito pierces a person's skin to take a blood meal, sporozoites in the mosquito's saliva enter the bloodstream and migrate to the liver. Within 30 min. of being introduced into the human host, they infect hepatocytes, multiplying asexually and asymptomatically for a period of 6–15 days. Once in the liver these organisms differentiate to yield thousands of merozoites which, following rupture of their host cells, escape into the blood and infect red blood cells, thus beginning the erythrocytic stage of the life cycle. The parasite escapes from the liver undetected by wrapping itself in the cell membrane of the infected host liver cell.

Within the red blood cells the parasites multiply further, again asexually, periodically breaking out of their hosts to invade fresh red blood cells. Several such amplification cycles occur. Thus, classical descriptions of waves of fever arise from simultaneous waves of merozoites escaping and infecting red blood cells. The merozoites mature successively from ring forms to trophozoites to mature red cell schizonts (asexual forms) over 24 hours (P. knowlesi), 48 hours (P. vivax, P. ovale, and P. falciparum) or

72 hours (P. malariae). Most released merozoites continue in the asexual cycle and infect new red cells, although a few differentiate into male or female gametocytes

(sexual forms) which cause no symptoms but can circulate in the bloodstream until they are ingested by a blood-feeding anopheline mosquito. These sexual forms complete their life cycle within the mid-gut of the Anopheles mosquito, and the sporozoites that form then migrate to the salivary glands of the mosquito, from where they can reinfect 79 humans. The infection gets further transmitted via gametocytes back to the mosquitoes when the next mosquito bites and the whole cycle follows.

Some P. vivax and P. ovale sporozoites do not immediately develop into exoerythrocytic-phase merozoites, but instead produce hypnozoites that remain dormant for periods ranging from several months (6–12 months is typical) to as long as three years (Imwong et al., 2007). After a period of dormancy, they reactivate and produce merozoites. Hypnozoites are responsible for long incubation and late relapses in these two species of malaria. In the setting of P. falciparum and P. malariae infection, hypnozoite parasites do not develop in the liver. However, P. malariae can cause very late relapse due to subpatent infection that can become symptomatic years to decades later. The parasite is relatively protected from attack by the body's immune system because for most of its human life cycle it resides within the liver and blood cells and is relatively invisible to immune surveillance. However, circulating infected blood cells are destroyed in the spleen. To avoid this fate, the P. falciparum parasite displays adhesive proteins on the surface of the infected blood cells, causing the blood cells to stick to the walls of small blood vessels, thereby sequestering the parasite from passage through the general circulation and the spleen. This "stickiness" is the main factor giving rise to hemorrhagic complications of malaria. High endothelial venules

(the smallest branches of the circulatory system) can be blocked by the attachment of masses of these infected red blood cells. The blockage of these vessels causes symptoms such as in placental and cerebral malaria. In cerebral malaria the sequestrated red blood cells can breach the blood brain barrier possibly leading to coma. The intracellular parasites modify the erythrocyte in several ways. They derive energy from anaerobic glycolysis of glucose to lactic acid, which may contribute to clinical manifestations of hypoglycemia and lactic acidosis (Brattig et al., 2008). They 80 also make the red cell membrane less deformable, resulting in hemolysis and accelerated spleenic clearance, which ultimately contribute to anemia. Alterations to uninfected red blood cells, such as the addition of P. falciparum glycosylphosphatidylinositol (GPI) to the membrane, may play a role in increased clearance of uninfected cells and contribute to anemia (Prato et al., 2005). Ultimately, new daughter merozoites are released from the infected erythrocytes. The remnants of cell membrane and the hemozoin crystal are phagocytized by circulating macrophages, an important first stimulus in the activation of the immune cascade (Prato et al., 2008;

Ferreira et al., 2008). In addition, free heme is released into the peripheral blood, an important stimulus for endothelial activation; endothelial cell damage also occurs in some patients (Prato et al., 2008; Ferreira et al., 2008; Bedu-Addo and Bates, 2002).

Red cell lysis stimulates release of pro-inflammatory cytokines, including tumor necrosis factor (TNF). TNF suppresses hematopoiesis, which also contributes to the anemia. The liver and spleen enlarge over time; the spleen may become massively enlarged (Wassmer et al., 2008). Thrombocytopenia is caused by a combination of hypersplenism (increased splenic sequestration and decreased platelet survival time) and, in the case of P. falciparum, consumption of platelets in microvascular sequestration (Wassmer et al., 2008; Wassmer et al., 2006c).

1.8 ARTEMETHER (ARM)

Artemether is available as white crystals or a white, crystalline powder which melts at

86-90˚C. Its chemical name is (3R,5aS,6R,8aS,9R,10S,12R,12aR)-Decahydro-10- methoxy-3, 6, 9-trimethyl-3, 12-epoxy-12H-pyrano(4, 3-j)-1, 2-benzodioxepin and it has a molecular mass of 298.4. The compound is practically insoluble in water; very 81 soluble in dichloromethane, chloroform and acetone; freely soluble in ethyl acetate, dehydrated ethanol and methanol.

Empirical Formula: C16H26O5

Chemical name:

[3R-(3R,5aS,6S,8aS,9R,10R,12S,12aR**)]-Decahydro-10-methoxy-3,6,9-trimethyl- 3,

12-epoxy-12H-pyrano [4, 3-j]-1,2-benzodioxepin.

Molecular weight: 298.4

Melting range: 86-900c

Structural Formula:

Figure 3: The Structural formular of artemether

In the synthesis of artemisnin derivatives, Artemisinin is reduced with sodium borohydride to produce dihydroartemisinin as a mixture of epimers (Olaniyi, 2005). To produce Artemether, the mixture is treated with methanol and an acid catalyst (Haynes and Vonwiller, 1994). Artemether can also be prepared from dihydroartemisinin using boron trifluoride. Artesunate is produced by esterification of dihydroartemisinin using succinic anhydride under basic conditions (Chekem and Wierucki, 2006). 82

Figure 4: Synthesis of artemisinin derivatives

Artemether (ARM) is a lipid soluble methylether of dihydroartemisinin or O-methyl ether prodrug of dihydroartemisinin. It is a derivative of artemisinin (qinghaosu), the principal antimalarial constituent of the Chinese herb Artemisia annua (Asteraceae)

(Qinghaosu Antimalaria Coordinating Research Group, 1979; Klayman, 1985).

Artemisinin is a novel sesquiterpene lactone, extracted from the leaves of the shrub

Artemesia annua and possesses an endoperoxide bridge which is a rare feature in natural products. Endoperoxide bridge is essential for its antimalarial activity. It is generally believed that the mechanism of action of artemisinin involves the formation of free radical intermediates resulting from interaction of the endoperoxide moiety with heme (Meshnick et al., 1996; Cumming et al., 1997; Robert and Meunier, 1998; Avery et al., 1999). However, it is not entirely clear on how the free radicals cause the parasite death (Bhisutthibhan et al., 1998; Olliaro et al., 2001).

Artemether is active against the erythrocytic stage of multidrug-resistant strains of

Plasmodium falciparum. Artemisinin and its semisynthetic derivative artemether are currently used to treat severe or multidrug-resistant P. falciparum malaria, including cerebral malaria. The schizonticidal activity of Artemther is due to the destruction of 83 the asexual erythrocytic forms of P. falciparum and P. vivax. A synthetic compound with an endoperoxide function like arteflene, Ro 42-1611 (Jaquet et al., 1994), also exhibits strong antimalarial activity. Artemisinin derivatives are active during the intraerythrocytic stage of infection. The inhibition of hemozoin formation by ARM has been proposed (Orjih, 1996). It has been proposed that the intraparasitic heme liberated during hemoglobin digestion might play an important role in the selective toxicity of artemisinin toward the parasite (Meshnick et al., 1991), and the reductive activation of artemisinin or other endoperoxide-based antimalarial drugs by Fen heme is probably a key point in the mechanism of action of these drugs (Meshnick and Taylor, 1996;

Cumming et al., 1997; Robert and Meunier, 1998).

The prevalence of a multidrug resistant P. falciparum strain against antifolates and standard quinoline antimalarial drugs, together with the decline of support for antimalarial development from pharmaceutical companies, makes it imperative to search for new antimalarial drugs (Morel, 2000). The characteristic peroxide lactone structure is indispensable for antimalarial activity.

1.8.1 ASSAY OF ARTEMETHER

The artemisinin derivatives do not have any significant light absorption in the workable wavelength region of the UV-VIS spectroscopy and they do not have particular chemical groups that easily react with certain reagents to yield coloured products.They lack strongly absorbing chromophores. Based on the lack of such chromophore groups, artemisinin and its derivates absorb weakly in the low wavelength region and this makes their quantification difficult. The available UV Spectrophotometric methods for the analysis of Artemether make use of base or acid treatment. They can be transformed by acid or base treatment to more reactive compounds such as enolate/carboxylates or 84

α-β unsaturated decalone and absorbs at a wavelength of 254 nm (Thomas et al., 1992;

Zhao and Zeng, 1986; Olajire et al., 2010). This transformation has been used as the basis for the determination of these drugs in dosage forms and biological fluids (Zhao,

1987; Idowu et al., 1989) using HPLC. Though this product absorbs strongly at the said wavelength, it requires very vigorous conditions for its formation. The IP method for the assay of Artemether (both as the pure sample and in formulations) requires the addition of 1M ethanolic HCl solution to an aliquot of Artemether in ethanol solution followed by heating at 55 ˚C for five hours (IP, 2008). The time demands as well as the heating required by these methods make them uneconomical. Green et al., (2001) have also described a method for the assay of Artemether and other artemisinins by the reaction of the acid decomposition product with a dye to yield a coloured derivative which absorbs at 420 nm. This method requires a period of one hour for the formation of the product prior to reaction with the dye (Green et al., 2001). The reactive methylene centres generated by acid or base treatment has also been used for the colorimetric detection of counterfeit artesunate, dihydroartemisinin (Green et al., 2000) and artemether (Green et al., 2001). Some other previously described methods for the assay of artemisinins are thin layer chromatography using spray reagents such as acidified 4-methoxybenzaldehyde reagent in methanol-water (Gabriels and Plaizier- vercammen, 2004) or p-dimethylamino benzaldehyde and heated at 80oC; HPLC without prior derivatization using electrochemical detectors (Acton et al., 1985;

Thomas et al., 1992) or with mass spectrometric detection (Ortelli et al., 2000; Christen and Veuthey; 2001, Naik et al., 2005).

However, majority of the HPLC techniques involved precolumn derivatization (Zhao and Zeng, 1986; Zhao, 1987; Zhou et al., 1987) or post-column derivatization (ElSohly et al., 1987; Liu et al., 2008). Some other methods include capillary electrophoresis 85

(D’Hulst et al., 1996) and bioassay techniques (Jaziri, 1993; Ferreira and Janick, 1996) and recently HPTLC (Agarwal et al., 2009). Majority of these techniques previously reported suffers from the disadvantage of using high acid concentration and carrying out the reaction at high temperatures for a prolonged period of time. Zollinger (1991) observed that the reactive methylene centres generated by the acid or base decomposition can readily react with diazonium salts and this procedure has been utilized by Green (2000) for the alkali decomposition product of artesunate with the diazonium salt fast red TR. The avidity with which diazonium ions react with active methylene centres depend on the reactivity of the diazonium ion. It has been demonstrated that the high reactivity of the diazonium; 4-carboxyl-2,6- dinitrobenzene diazonium (CDNBD) ion as a highly reactive diazo coupling reagent for secondary amino derivatives (Idowu et al., 2002; Idowu et al., 2006) and phenol ethers (Idowu et al., 2004, Adegoke et al., 2007b). Majority of the previously described methods especially HPLC techniques are difficult to adopt in poor-resource economies.

Figure 5: Structure of α, β-unsaturated decalone

86

1.8.2 PHARMACOLOGICAL USES OF ARTEMETHER

1.8.2.1 ANTIMALARIAL EFFECT OF ARTEMETHER

In many parts of the malaria endemic areas, particularly the African region, the only effective method of preventing the mortality and reducing the morbidity caused by the disease is through the use of antimalarial drugs (Olaniyi, 2005). An ideal antimalarial drug should have the following characteristics:

• Rapidly relieve symptoms of the disease.

• It should be harmless to the patient and have no unpleasant side-effects

• It should preferably destroy all the stages of development of plasmodium

species including the gametocytes

• It should be economically cheap and easy to administer

Mechanism of Action of Antimalarial Effect

The antimalarial activity has been attributed to chemical activation of the drug within the food vacuole of the intraerythrocytic stage of the parasite; it is proposed that reductive cleavage of the peroxide bridge (C-O-O-C) by heme liberated during digestion of hemoglobin generates free radicals (such as tert-butylperoxide) that can themselves kill malaria parasites, albeit in comparatively high concentrations (Hunt et al., 1984), which induce oxidative stress and alkylate heme and vital parasite proteins

(Cumming et al., 1997). In the presence of intra-parasitic iron, these drugs are converted into free radicals and other electrophilic intermediates which then alkylate specific malaria target proteins. An alternative mechanism of action for artemisinins based on inhibition of the malarial parasite’s calcium ATPase (sarcoplasmic endoplasmic reticulum calcium ATPase, SERCA) has also been suggested. Their potency has been proven to be similar to thapsigargin which is another sesquiterpene lactone, a highly specific SERCA inhibitor (Eckstein-Ludwig and Webb et al., 2003). 87

An interaction with membrane phospholipids has also been suggested (Basco and

Bras, 1993). The peroxide group (clears the peripheral blood of parasites more rapidly than other available drugs do) in these compounds appears essential for activity, and the peroxide group is retained in the active metabolite, dihydroartemisinin (Thomas et al.,

1992).

1.8.2.2 ANTISCHITOSOMIASIS EFFECT OF ARTEMETHER

Chinese scientists discovered that artemether was not only an antimalarial agent, but also effective against the blood flukes around 1980. Eventually, laboratory experiments have confirmed the broad spectrum of activity against different trematodes, including all human schistosomes, Clonorchis sinensis, Fasciola hepatica and Opisthorchis viverrini (Keiser and Utzinger, 2007). These studies revealed that artemether exhibits the highest activity against juvenile stages of the trematodes, while adult worms are significantly less susceptible. In addition, there is no indication of neurotoxicity following repeated high doses. Randomized controlled clinical trials confirmed that artemether, orally administered at a dose of 6 mg/kg once every 2–3 weeks, results in no drug-related adverse effects, and significantly reduces the incidence and intensity of schistosome infections, including those of Scistosoma mansoni, S. japonicum and S. haematobium (Xiao et al., 2002; Hou et al., 2008).

Human Schitosomiasis is a chronic and debilitating disease that remains one of the most prevalent parasite infections in the humid tropics. Schitosoma haematobium causes urinary schitosomaisis in most African countries. Adult S. haematobium worm lives in the veins of the vesicle plexus around the bladder especially the upper trigone, and along the ureters. Human pathology is caused by those eggs trapped within the urogenital system and kidneys are involved when reached by adult frequently causes 88 mild degrees of periportal fibrosis (grade 1) as well as hepatomegally and splenomegally as evidenced by both histopathologic examination and ultrasound

(Nafeh et al., 1992). The pathogenic mechanisms of some disorders of S. haematobium infection revealed accumulation into biharzioma of the liver and granulomatous hepatitis surrounded by biharzial pigment deposition (Voung et al., 1996; Mahmoud et al, 2006). Jacob et al (1999) inferred that the in vivo, S. haematobium can positively modulate S. mansoni egg antigen-induced granuloma formation and hepatic fibrosis resulting in more severe liver pathology. ARM which is a derivative of artemisinin has already widely been used against malaria (Danso-Appiah and De Vlas, 2002). ARM has also shown activity against S. japonicum (Colley, 2001), S. mansoni (Danso-

Appiah and De Vlas, 2002), and S. haematobium (Xiao et al., 2000; Mahmoud et al.,

2006).

Mechanism of Antischitosomial Effect of Artemether

Glutathione S-transferase (GST) and superoxide dismutase (SOD) are major antioxidant enzymes of schistosomes that are involved in detoxification processes. The inhibition of GST and, to a lesser extent also SOD enzymes, could lead to increased schistosome susceptibility to oxidant attacks and might be linked with the antischistosomal action of artemether.

1.8.2.3 ANTITUMOR EFFECT OF ARM

Artemether has been shown to have significant anticancer and antitumor activities. It is demonstrated that artemether caused strong inhibitory effects on brain glioma growth and angiogenesis in rats (Wu et al., 2009). It exhibits a dose- and time-dependent cytotoxicity, and induced apoptosis and G2 cell cycle arrest in ovarian cancer cell lines,

(Jiao et al., 2007), human leukemia HL60 cells, (Zhou et al., 2008) and human 89 pancreatic cancer BxPC-3 and AsPC-1 cells (Chen et al., 2009). Woerdenbag et al

(1993) were the first to document the cytotoxicity of artemisinins to tumour cells. It was found that artemisinin had activity in the micromolar range, whereas semi- synthetic analogues such as sodium artesunate had more potent activities in the low micromolar range. Further studies suggested that these compounds exert their effect on tumour cells by growth inhibition (Beekman et al., 1997). This cytotoxicity was also shown to be endoperoxide-dependent (Beekman et al., 1996). Selective activation of artemisinin by tumor cells has led to proposal of an iron dependent hypothesis due to the understanding that tumor cells maintain a high intracellular iron concentration to sustain continued proliferation in addition to an increased capacity to synthesize heme

(Kwok and Richardson, 2002).

Cancer cells exposed to artemisinin demonstrate decreased proliferation, increased levels of oxidative stress, induction of apoptosis and inhibition of angiogenesis

(Krishna et al., 2008). Interestingly, artemisinins have also shown cytotoxicity against drug and radiation resistant cell lines suggesting a different mechanism to traditional anti-cancer therapies (Efferth et al., 2003). The activation of antitumor immune responses is believed to suppress tumor growth, therefore, suppression of these responses by artemisinins may counteract anticancer activity. However recent results using a transgenic mouse melanoma model indicate that the cytostatic and apoptotic effects of artesunate are not diminished by simultaneous immunosuppression

(Ramacher et al., 2009). Whereas the monomeric forms of artemisinin have superior activity in the treatment of malaria, it is the dimeric forms of artemisinin that have shown enhanced anticancer activity (Stockwin et al., 2009).

With respect to bioactivation of artemisinin in tumor cells the actual mechanism is still unclear. However, the current consensus involves the iron (II)-mediated release (Efferth 90 et al., 2004) of reactive oxygen species (ROS) (Efferth, 2006) and/or carbon centered radicals (Mercer et al., 2007). Both may play an important role in inducing DNA damage, mitochondrial depolarisation and apoptosis. However, other factors may also come into play such as the ability of the tumor cell to transport ferrous iron and maintain supplies. In a study to explore the response of iron transporter proteins of tumor cells to artesunate, nearly a third of tumor cell lines showed no enhancement or even decreased activity upon addition of ferrous iron (Kelter et al., 2007).

Enhancement of artesunate response by ferrous iron was found to depend on the expression of two genes: the iron-binding transferrin receptor (TfR) and ATP-binding transporter (ABCB6). Hence, pretherapeutic detection of TfR and ABCB6 expression may predict the response of tumor cells towards artesunate. Consequently, this result could be exploited for individualized tumor therapy with artemisinins.

1.9 SELF EMULSIFYING DRUG DELIVERY SYSTEM (SEDDS)

1.9.1 PREPARATION OF SEDDS

The method of preparing self-emulsifying drug delivery system which improves the bioavailability of a drug by emulsifying the drug with the self-emulsifying excipient includes various steps as described below (US Patent 5993858, 1999; Himani Bajaj,

2011).

• Preparation of phase diagram (Farah, 1993)

• Solubilizing a PWSD in a mixture of surfactant, cosurfactant with or without

solvent.

• Mixing of the oil phase suitably prepared, if necessary, by heating or other

preparatory means, to the solubilized drug formulation and thoroughly mixed. 91

• The emulsion can then be added to a suitable dosage form such as soft or hard-

filled gelatin capsules and allowed to cool.

1.9.2 MECHANISMS OF SEDDS AND ITS APPLICATIONS

The mechanism of self-emulsification is yet to be fully elucidated. Self emulsification is related to free energy. Emulsification process occurs when the entropy change that favours dispersion is greater than the free energy required to increase the surface area between the oil and aqueous phases of the dispersion. The change in free energy (∆G) associated with the process of emulsification, ignoring the free energy of mixing, can be expressed by (Reiss, 1975; Jayvadan and Shah, 2008; Rajesh et al., 2010).

2 ∆G = ΣNi4πri σ ……………………………………………………………………3

‘N’i, is the number of droplets with radius (ri),

Whereas, σ, is the interfacial energy.

Selfemulsification will occur spontaneously only when the interfacial energy is low.

However, emulsions are not thermodynamically stable as the oil phase and the aqueous phase will tend to separate with time to reduce the interfacial area and also free energy of the system. Therefore, the presence of emulsifiers will help to reduce interfacial tensions by forming a barrier around the oil droplets and hence decreasing the free energy of the systems. On the other hand, Groves et al (1974) related emulsification with the formation of liquid crystalline phase. Liquid crystalline phase is the phase between liquid and crystal phase. A liquid crystal has both the properties of a crystal as well as liquid. When additional energy is exerted onto the liquid crystalline phase, it will turn into liquid phase. For selfemulsifying systems, when the oil phase is introduced into the aqueous phase with gentle agitation, the aqueous phase will penetrate through the interface into oil phase until the interface of the two phases is 92 disrupted. Consequently, oil droplets are formed resulting in emulsification. Thus, the ease of emulsification is governed by the ease of water penetration into the various liquid crystal or gel phases formed on the surface of droplets. The liquid crystal formation surrounding the oil droplets will increase the stability of the emulsion.

Nevertheless, the relationship between liquid crystal formation and emulsion formation could be more complicated as it appeared to be (Craig et al., 1995). Various factors can affect the process of self-emulsification, such as the nature of oil/surfactant pair, the surfactant concentration used as well as the temperature at which self-emulsification occurs. Moreover, the presence of drug compound will alter the emulsion characteristics, probably by interacting with the liquid crystalline phase.

Groves and Mustafa (1974) developed a method of quantitatively assessing the emulsification by monitoring the turbidity of the oil surfactant system in a water stream using phosphated nonylphenoxylate and phosphate fatty alcohol ethoxylate in n hexane and suggested that the emulsification process may be associated with the ease with which water penetrates the oil/water interface, with formation of liquid crystalline phase resulting in swelling at the interface, thereby resulting in greater ease of emulsification. Consequently, the authors were able to relate the phase behavior to the spontaneity of emulsification, with liquid crystals formation, tending to form emulsion more readily, as indicated by the lower equilibration times (Groves et al., 1974).

Pouton has argued that the emulsification properties of the surfactant may be related to phase inversion behavior of the system (Pouton, 1997). For example, if one increases the temperature of the oil in the water system stabilized by using non ionic surfactants, the cloud point of the surfactant will be reached followed by phase inversion

(Friedman, 2007, US Pat 20070190080). The surfactant is highly mobile at the phase 93 inversion temperature; hence the o/w interfacial energy is minimized, leading to a reduction in energy required to bring about emulsification (Pouton, 1997).

Other mechanisms by which SEDDS enhance the absorption profile

Prolongation of gastric residence time:

Presence of lipids in the GIT cause delay in the gastric emptying thereby increasing the gastric transit time. As a result of the delay the gastric transit time of the embedded drug will also be increased. This will enhance dissolution, absorption and improve bioavailability.

Enhanced dissolution/ solubilization:

Lipids in the GIT stimulate gall bladder contractions, biliary and pancreatic secretions including bile salts (BS), phospholipids (Pl) and cholesterol. These products with gastric shear movement form a crude emulsion that enhances the solubilization of the co-administered drug.

Stimulation of lymphatic transport:

Lipids cause stimulation of lymphatic transport pathway which improves bioavailability of hydrophobic drugs.

Increased intestinal permeability:

Research has shown that presence of bile and lipids possess the ability of changing the physical barrier function of the GIT which enhances intestinal membrane permeability

(Charman and Porter, 1997).

Reduced metabolism and efflux activity:

Some lipids and surfactants have been shown to reduce the inhibitory effects of p- glycoproteins and CYP3A4 thereby increasing absorption of the hydrophobic drugs as well as reducing intra-enterocyte metabolism.

94

1.9.3 CHARACTERIZATION OF SEDDS

The primary means of self-emulsification assessment is visual evaluation. The efficiency of self-emulsification could be estimated by determining the rate of emulsification, droplet size distribution and turbidity measurements, etc. Development and characterization of SEDDS and SMEDDS have been extensively reviewed by

(Pouton, 1997; Gershanik and Benita, 2000; Gursoy and Benita, 2004; Pouton, 2006).

1.9.3.1 Droplet Size

This is a crucial factor in self-emulsification performance because it determines the rate and extent of drug release as well as the stability of the emulsion. Photon correlation spectroscopy, microscopic techniques or a Coulter Nanosizer are mainly used for the determination of the emulsion droplet size. The reduction of the droplet size to values below 50 nm leads to the formation of SMEDDS, which are stable, isotropic and clear o/w dispersions (Crison and Amidon, 1999).

1.9.3.2 Equilibrium Phase Diagram

Although self-emulsification is a dynamic non-equilibrium process involving interfacial phenomena, information can be obtained about self-emulsification using equilibrium phase behavior. There seems to be a correlation between emulsification efficiency and region of enhanced water solubilization and phase inversion region, formation of lamellar liquid crystalline dispersion phase on further incorporation of water. An equilibrium phase diagram enables comparison of different surfactants and their synergy with cosolvent or cosurfactant (Pouton, 1987). The boundaries of one phase region can easily be assessed visually. The phase behavior of a three component system can be represented by a ternary phase diagram. 95

1.9.3.3 ZETA POTENTIAL MEASUREMENT

This is used to identify the charge of the droplets. In conventional SEDDS, the charge on an oil droplet is negative because of the presence of free fatty acids (Gershanik and

Benita, 1996).

1.9.3.4 DETERMINATION OF EMULSIFICATION TIME

Pouton et al (1987) quantified the efficiency of emulsification of various compositions of the Tween 85 and medium-chain triglyceride systems using a rotating paddle to promote emulsification in a crude nephelometer. This enabled an estimation of the time taken for emulsification. Once emulsification was complete, samples were taken for particle sizing by photon correlation spectroscopy, and selfemulsified systems were compared with homogenized systems. The process of selfemulsification was observed using light microscopy. It was clear that the mechanism of emulsification involved erosion of a fine cloud of small particles from the surface of large droplets, rather than a progressive reduction in droplet size.

1.9.3.5 LIQUEFACTION TIME

This test is designed to estimate the time required by solid SEDDS to melt in vivo in the absence of agitation to simulated GI conditions. One dosage form is covered in a transparent polyethylene film and tied to the bulb of a thermometer by means of a thread. The thermometer with attached tablets is placed in a roundbottom flask containing 250 ml of simulated gastric fluid without pepsin maintained at 37 ± 1oC

(Attama et al., 2003). The time taken for liquefaction is subsequently noted.

96

1.9.3.6 RELEASE STUDIES

Drug release from SEDDS has been reported to take place by interfacial transfer and vehicle degradation (De Smidt et al., 2004; Porter and Charman, 2001; Obitte et al.,

2011). Drug release through interfacial transfer mechanism takes place when the drug diffuses from the formulation into the bulk medium or directly over the intestinal membrane while vehicle degradation involves mainly the lipase-catalysed lipolytic degradation of the SEDDS resulting into a drug release from the dosage form (Fatouros et al., 2007). SEDDS-loaded drug release should be a controlled uniform profile.

Ideally, complete release of the active ingredient will give absolute drug absorption which is important for effective plasma concentration to be obtained.

1.9.3.7 THERMODYNAMIC STABILITY STUDIES

The physical stability of a lipid –based formulation is also important to its performance, which can produce adverse effect in the form of precipitation of the drug in the excipient matrix. The poor physical stability of the formulation can lead to phase separation of the excipient, which affects not only formulation performance but also the visual appearance of formulation. Also incompatibilities between the formulation and the gelatin capsules shell can lead to brittleness or deformation, delayed disintegration, or incomplete release of drug.

For thermodynamic stability studies we have performed three main steps, they are;

Heating cooling cycle: Six cycles between refrigerator temperature (4oC) and 45oC with storage at each temperature of not less than 48 h is studied. Those formulations, which are stable at these temperatures, are subjected to centrifugation test.

Centrifugation: Passed formulations are centrifuged thaw cycles between 21oC and

25oC with storage at each temperature for not less than 48 h is done at 3500 rpm for 30 97 min. Those formulations that do not show any phase separation are taken for the freeze thaw stress test.

Refigeration cycle test: Three freeze for the formulations. Those formulations that passed this test showed good stability with no phase separation, creaming, or cracking

(Shafiq et al., 2007).

1.10 OBJECTIVES OF THE STUDY

Artemether (ARM), a hydrophobic drug belonging to Biopharmaceutics Classification

II, is one of the most commonly used antimalarial agents.

It is thought that if artemether is formulated into an optimum dosage form for oral administration, its poor solubility may be improved and its bioavailability made consistent. Since SEDDS approach has been reported to be one of the possible formulation options capable of addressing poor aqueous solubility, we intend to adopt it as a carrier for ARM formulation. Artemether loaded SEDDS formulation is expected to mask its extreme bitter taste, improve its aqueous solubility, avert GIT disturbances, afford opportunity for avoidance of first pass effect via lymphatic absorption, and improve bioavailability. Therefore, the objectives of the research work were to:

• formulate artemether loaded-SEDDS,

• evaluate some of its in vitro properties,

• improve its poor aqueous solubility of ARM,

• improve the antimalarial activity and

• prevent drug precipitation through supersaturation approach.

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CHAPTER TWO

2.0 MATERIAL AND METHODS

2.1 MATERIALS AND APPARATUS

Artemether (CAS 71963-77-4 Hangzhou Dayangchem. Co., Limited, free gift);

Triacetin®, 99% (free gift from Acros Organies);

Labrasol® (EP: Caprylocaproyl Macrogolglycerides and USP NFCaprylocaproyl/ polyoxylglycerides), Capryol PGMC® (USP NF: Propyleneglycol Monocaprylate (type

1)), Gelucire® 33/01 (EP/USP NF: Hard fat); Maisine 35-1(EP/ USP NF: Glycerol

Monolinoneate), Lauroglycol FCC® (EP/USP NF: Propylene glycol monolaureate (type

1)), Peceole® (EP: Glycerol mono-oleate (40)), Capryol 90® (USP NF: Propylene glycol monocarprylate (type II)), Transcutol P® (EP/USP NF: Diethylene Glycol monoethyl ether), Labrafac Lipophile® WL 1349 (EP: Triglycerides medium-chain/

USP NF: medium-chain Triglycerides), Labrafil M® 1944 CS (EP: Oleoyl polyoxyl-6

Glyceride) all is free gift from Gateffosse Company;

Conc HCl, and Methanol (Sigma-Aldrich®, Germany);

Sodium hydroxide, and Monobasic potassium phosphate (BDH, Poole, England)

All chemicals were of analytical grades.

Laboratory centrifuge (SM800B), Filter Paper (Whatman size, Cat No 100-125mm) and Magnetic Stirrer/hot plate (Gallenkamp) from England;

Zetasizer (NANO ZS90 zetasizer, Malvern Instruments Corp) and UV/VIS

Spectrophotometer (Spectrumlab 752s) from U.K;

Viscometer (micro Ostwald viscometer) and Dissolution apparatus (Veego dissolution apparatus) from India; 99

Sonicator (Fisher Scientific Co Llc) and Microscope (Hund® binocular microscope

Weltzlar), Oven (SchutzartbDin 40050-IP20). and Separating funnel (Fortuna W. G.

Co, W.) from Germany;

Electronic Thermoregulated Themostat (HH.W21.Cr42II);

Electronic weighing balance (Ohaus Adventure, SNR-1121 R5386 and Digital Camera

(Kodak, Easyshare C1450 KCGRD13462391) China); pH meter (Mettler Toledo MP 220, Greifensee, Switzerland);

Mice (Department of Pharmacology and Toxicology, UNN).

2.2 METHODS

2.2.1 MELTING POINT DETERMINATION

A 40 mg of pure ARM drug was dried in the oven (SchutzartbDin 40050-IP20,

Germany) set at 40oC for 10 min. Using three capillary tubes sealed one ends, the drug was filled into them to about three-quarter volume. The tubes were fixed and assembled with thermometer in the Melting point apparatus. The temperature range at which melting occurred was recorded.

2.2.2 STANDARD BEER-LAMBERT PLOT

Calibration curves of ARM were determined using different media such as distilled water, simulated gastric fluid (SGF) and simulated intestinal fluid (SIF).

Accurately weighed 10 mg of ARM was transferred to a 100 ml volumetric flask. To it,

25 ml of 1 N HCl was added and this solution was heated on the thermoregulated water bath (HH.W21.Cr42II) for 25 min at temperature 80 ± 2 ºC. The solution was allowed to cool at room temperature, filtered and volume was then made up to the mark (100 ml) with distilled water to get concentration of 100 μg/ml and used as a stock solution 100

(Prepard et al., 2011). The stock solution was further diluted with distilled water to get different concentrations (0.1-0.9 mg/10 ml). One of the solutions was then scanned in the range of 200 – 400 nm. A 2.5 ml aliquot of 1 N HCl diluted with the medim (water,

SGF and SIF) to 10 ml mark was treated the same way was used as a blank. The wavelength of maximum absorbance of ARM was obtained.

2.2.3 SOLUBILITY OF DRUG IN DIFFERENT VEHICLES

Different oils, surfactants and cosurfactants/cosolvents were screened for solubilization of Artemether by saturation solubility method. The solubility of artemether in

Triacetin®, Peceol®, Labrafac lipophile® 1349, Labrafil M® 1944, Maisine, Labrasol®,

Capryol® 90, Capryol PGMC, Gelucire,® Transcutol P®, Lauroglycol® 90 and

Lauroglycol FCC® was determined by dissolving excess amount of ARM in 5 ml of each of the selected excipients in a test tube. The test tubes were shaken at time intervals for 24 h under ambient temperature. The supernatants were taken and assayed for artemether content using UV/VIS spectrophotometer (Spectrumlab 752s, UK). The successful ones (Triacetin®, Labrasol® and Transcutol P®) were used for further studies.

2.2.4 CONSTRUCTION OF PHASE DIAGRAM

The Pseudoternary phase diagrams of oil, surfactant/cosurfactant and water were constructed using the titration method by admixing the surfactant and cosurfactant

(Smix) at the following ratios: 1:0, 1:0.5, 1:1, 1:2, 1:3, 3:1 and 3:2. Thereafter, the oil is also admixed with each of the Smix ratios at the following ratios: 1:9, 1:8, 1:7, 1:6, 1:5,

1:4, 1:3.5, 1:3.0, 1:2.5, 1:2.0, 1:1.5, 1:1.0, 1:0.5, 1:0.3, 1:0.2 and 1:0.1. SEDDS were kept at ambient conditions for 5 h and then evaluated for visual isotropicity, turbidity, 101 and quality of emulsion formed after dilution with water. Then the optimized SEDDS were selected.

2.2.6 SOLUBILITY STUDIES OF THE OPTIMIZED SEDDS BATCHES

Excess amount of artemether was introduced into each of the SEDDS batches contained in different test tubes. The tubes were shaken at time intervals for 24 h under ambient temperature. The supernantants were collected and assayed for ARM content using

UV/VIS spectrophotometeric (Spectrumlab 752s, UK).

2.2.7 PREFORMULATION ISOTROPICITY TESTS

The appropriate quantities of the optimized SEDDS resulting from the phase diagram were weighed out, mixed in different test tubes, stored further for 24 h at ambient temperature and observed for phase separation and stability.

2.2.8 FORMULATION OF ARM-LOADED SEDDS

Further studies were limited to only the SEDDS that were optimized by Pseudoternary

Phase Diagram. Appropriate constituents of the SEDDS were measured and introduced into 10 ml test tubes and different drug concentrations (40 mg, 50 mg and 55 mg) were incorporated and set up on a thermoregulated water bath maintained at a temperature of

50oC and stirred for 10 min (Obitte et al., 2011). These formulations were called non-

HPMC batches.

102

2.2.9 FORMULATION OF ARM-LOADED SUPERSATURABLE SEDDS (S-

SEDDS)

The above method of SEDDS formulation was adopted in addition, 5% HPMC was incorporated into the different concentrations at a temperature between 50-60oC. All formulations were stored at room temperature before use. This formulation was called

HPMC batch.

Table 2: The percent composition ratios of the SEDDS and S-SEDDS formula

OIL:SURF:COSURF Triacetin Labrasol Transcutol

(%)

33.33: 44.44: 22.22 1 1.3 0.7

28.6: 47.6: 23.8 1 1.7 0.8

22.22: 58.33: 19.44 1 2.6 0.9

20: 60: 20 1 3 1

SURF= Surfactant, COSURF= Cosurfactant

2.3 EVALUATION OF THE SEDDS AND S-SEDDS

2.3.1 DROPLET SIZE, POLYDISPERSITY INDEX AND ZETA POTENTIAL

The particle size, polydispersity and zeta potential were determined by photon correlation spectroscopy (NANO ZS90 zetasizer, Malvern Instruments Corp, U.K) at

25oC and fixed angle of 90o in disposable polystyrene cuvettes. Each capsule was dispersed in 0.1 N HCl. Droplet size was evaluated using curvette containing 2.5 ml of the nano/microemulsion, and zeta potential was measured using 1 ml in capillary cuvettes (Obitte et al., 2013). 103

2.3.2 POSTFORMULATION ISOTROPICITY/ STABILITY TEST

The HPMC and non-HPMC batches loaded with different concentrations of ARM were stored for 72 h at ambient temperature and observed for isotropicity (homogeneity).

The SEDDS were stored for a further 3 months, and observed for phase separation and/or drug precipitation.

2.3.3 EMULSIFICATION TIME TESTS

The emulsification time tests of the two different batches (HPMC and non-HPMC) were determined by introducing one capsule in a 250 ml beaker containing 0.1 N HCl.

The beaker was set up on a hot plate-magnetic Stirrer (Gallenkamp, England) assembly set at 50 rpm and 37 ± 1oC. The time for complete emulsification, as indicated by constant turbidity, was recorded. It was repeated in triplicate.

2.3.4 REFRIGERATION CYCLE TEST

Three capsules from each batch were wrapped in a polyethylene material and kept in the refrigerator for 12 h at 4oC. The cycle was repeated three times and thereafter observed for physical changes and drug precipitation.

2.3.5 CENTRIFUGATION

The formulated SEDDS and S-SEDDS batches were centrifuged at 3000 rpm for 30 min using centrifuge machine (SM800B, England) and observed for any physical changes.

2.3.6 AQUEOUS DILUTION TEST

SEDDS containing the equivalent of one dose was emulsified in 100 ml of 0.1N HCl prior to dilution to up to 1 L. It was stored for 24 h and observed for drug precipitation. 104

2.3.7 VISCOSITY OF ARM-LOADED SEDDS

Viscosities of the SEDDS ratios in different batches were determined and recorded using Ostwald viscometer (mini Ostwald viscometer, India) at 25 oC.

2.3.8 DETERMINATION OF pH

The pH of the different batches was determined for 3 consecutive months using pH meter (Mettler Toledo MP 220, Greifensee, Switzerland) in triplicate at 25°C.

2.3.9 LOADING EFFICIENCY

Unit dose containing different concentrations of ARM from different batches were introduced in different 100 ml volumetric flasks. Into each was added 1ml of methanol and 25 ml of 1N HCl. The mixtures were shaken and heated in a thermoregulated oven set at 80oC for 25 min. It was allowed to cool and volume made up to 100 ml with distilled water and called stock solution. From the stock solution 5 ml was collected and made up to 10 ml and then assayed using UV Spectrophotometer (Spectrumlab,

752s, UK).

2.3.10 CRYSTALLIZATION/PRECIPITATION STUDIES

2.3.10.1 PHOTOMICROGRAPH OF STANDARD DISPERSION OF ARM,

SEDDS AND S-SEDDS

The standard dispersion of ARM was prepared with 55 mg Artemether. A 55 mg quantity of artemether was dissolved in 0.7 ml ethanol. To the solution 0.4 ml SGF was added and the crystals examined using an optical microscope (Hund® binocular microscope Weltzlar, Germany). 105

Similarly, SEDDS and S-SEDDS dispersions were examined using the same microscope to monitor time of in vitro drug precipitation. In brief, a beaker containing

100 ml SGF was set up on a magnetic stirrer hot plate assembly maintained at 37 ± 1oC and 50 rpm. Then one capsule containing a unit dose of artemether was placed in the beaker. The capsule was allowed to emulsify. The aqueous emulsion was introduced into a separating funnel and drops collected every hour on a microscopic slide for microscopy. It was examined for the presence/absence of drug crystals/precipitates.

2.3.11 DRUG RELEASE STUDIES

The USP Paddle Method was adopted. The dissolution medium consisted of 900 ml of freshly prepared SIF (pH 6.8) or SGF (pH 1.2). The temperature was maintained at 37

± 1oC. 40 mg ARM-loaded SEDDS of non-HPMC and HPMC batches equivalent to one dose were introduced into the 900 ml dissolution medium. The speed of rotation was maintained at approximately 100 rpm. At predetermined time intervals 5 ml aliquots of the dissolution medium was collected and treated as previously described with HCl. It was heated in 1 N HCl, allowed to cool, filtered and assayed using UV spectrophotometer (Spectrumlab, 752s, UK). The volume of dissolution medium was kept constant by replacing with 5 ml of a fresh dissolution medium. The amount of drug released at each time interval was determined.

2.3.12 IN VIVO STUDIES

In vivo antimalarial study was carried out on 25 mice that weighed between 20 and 27 g. They were grouped into five (5) as Test (treated with ARM-loaded SEDDS), chloroquine, placebo, negative (untreated), and positive (treated with aqueous dispersion of pure ARM) with five mice in each group. The mice were fed and allowed 106 to acclimmatize for one week preinfection. Peter’s four day suppressive test was adopted using Plasmodium berghei (Peter et al., 1993; Paula Melariri et al., 2011).

Malaria infection was established in mice by the intraperitoneal (i.p) inoculation of 200

μl of 1 x 106 parasitized cells/ ml on the first day (D0) of the experiment. Each mouse in the groups received appropriate dose of the formulation 24 hours post infection. The dosing was maintained for four days. To ascertain the parasitemia level 24 h post treatment, 2 slides containing thin blood smear were made and stained with 10%

Giemsa in Phosphate buffer (pH, 7.2) for 20 min. The slide was examined under microscope at 100X. The percentage parasitemia was determined by counting the parasitized red blood cells on at least 100 red blood cells in random fields of the two

Giemsa stained slides. The percentage antimalarial activity was determined as follows

(Patravale et al., 2010) in Equation 4.

= 100 –

× 100……… (4)

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CHAPTER THREE

RESULTS AND DISCUSSION

3.1 MELTING POINT

The melting point range of ARM was recorded to be 86-87oC after triplicate determinations.

3.2 SOLUBILITY (MG/ML) OF ARM IN DIFFERENT VEHICLES

180

160

140

120

100

80

Solubility (mg/ml) 60

40

20

0

Vehicles

Figure 6: Solubility profile of ARM in different vehicles 108

The development of microemulsion systems for poorly water soluble drugs is critical and drug loading per formulation is a very important design factor that depends on drug solubility in various formulation components (Natesan et al., 2004; Obitte et al., 2008).

Solubility studies were carried out to identify oil, surfactant and cosurfactant that possess good solubilising capacity for ARM. Identifying the suitable oil, surfactants and cosurfactants having maximal solubilising potential for drug under test is very important to achieve optimum drug loading. The ability of oil to accommodate large amount of hydrophobic drug can be improved in the presence of corsurfactants. The high solubilization capacity of the microemulsion during the dilution and hydrolysis process is helpful in the dispersion and high absorption of the drug. The combination of surfactant with oils also offers an advantage for microemulsions in terms of drug solubilization capacities for lipophilic compounds because of extra locus for solubilization provided by the oil phase (Hu et al., 2011). From the graph in figure 6,

ARM showed high solubility in Triacetin® (136 ± 0.09 mg/ml), Labrasol® (156 ± 0.10 mg/ml) and Transcutol P® (166 ± 0.02 mg/ml) and were selected as oil, surfactant and co-surfactant respectively. The solubility of ARM in the other components was less in comparison with the aforementioned components. Triacetin®, labrasol® and

Transcutol P® were therefore, selected as components of the formulation due to their high solubilising capacities as shown in Fig 6.

109

3.3 PSEUDOTERNARY PHASE DIAGRAM

Smix 0 100 10 90 20 80 30 70 40 60 50 50 60 40 70 30 80 20 90 10 100 0 Water 0 10 20 30 40 50 60 70 80 90 100 Oil

Figure 7: The phase diagram of the Smix ratio 1:0. 110

Smix

0 100 10 90 20 80 30 70 40 60 50 50 60 40 70 30 80 20 90 10 100 0 Water 0 10 20 30 40 50 60 70 80 90 100 Oil

Figure 8: The phase diagram of the Smix ratio 1:0.5. 111

Smix 0 100 10 90 20 80 30 70 40 60 50 50 60 40 70 30 80 20 90 10 100 0 Water 0 10 20 30 40 50 60 70 80 90 100 Oil

Figure 9: The phase diagram of the Smix ratio 1:1. 112

Smix 0 100 10 90 20 80 30 70 40 60 50 50 60 40 70 30 80 20 90 10 100 0 Water 0 10 20 30 40 50 60 70 80 90 100 Oil

Figure 10: The phase diagram of the Smix ratio 1: 2. 113

Smix 0 100 10 90 20 80 30 70 40 60 50 50 60 40 70 30 80 20 90 10 100 0 Water 0 10 20 30 40 50 60 70 80 90 100 Oil

Figure 11: The phase diagram of the Smix ratio 1:3. 114

Smix 0 100 10 90 20 80 30 70 40 60 50 50 60 40 70 30 80 20 90 10 100 0 Water 0 10 20 30 40 50 60 70 80 90 100 Oil

Figure 12: The phase diagram of the Smix ratio 3: 1. 115

Smix 0 100 10 90 20 80 30 70 40 60 50 50 60 40 70 30 80 20 90 10 100 0 Water 0 10 20 30 40 50 60 70 80 90 100 Oil

Figure 13: The phase diagram of the Smix ratio 3:2. 116

The relationship between the phase behaviour of a mixture and its composition could be captured with the aid of phase diagram (Lawrence and Rees, 2000; Baboota et al.,

2007). Phase digrams were constructed to obtain the proportion of components that can result in maximum nano- or micro-emulsion area so that the emulsion formulations could be optimized. The areas of large microemulsion existence were selected in figure s8 and 12 of Smix ratios 1:0.5 and 3:1 respectively from which the following optimized batches of Oil:Smix ratios were selected as 1:2.0 and 1:2.5 of Smix 1:0.5 and 1:3.5 and

1:4 of Smix 3:1.

117

PHOTOGRAPH OF AQUEOUS DISPERSIONS OF SEDDS

Figure 14: A typical aqueous SEDDS dispersion

118

3.4 SOLUBILITY STUDIES OF OPTIMIZED BATCHES

120

100

80

60 Solubility (mg/ml)

40

20

0 BATCH 1:2:0 BATCH 1:2:5 BATCH 1:3.5 BATCH 1:4:0 SEDDS

Fig ure 15: Solubility of ARM in the optimized SEDDS batches

119

The solubility studies of the optimized SEDDS batches were carried out to note the amount of ARM to load per unit dose. This prevents the issue of precipitation while on the shelf.

3.5 PREFORMULATION ISOTROPICITY TEST

In the Preformulation isotropicity test result, after 24 h, it was observed that the Oil:

Smix ratios 1:2.0, 1: 2.5, 1:3.5 and 1:4 were isotropically stable without phase separation. This test is a prerequisite to further formulation steps. Preformulation isotropicity test gives a clue to the optimum ratios of the ingredients that would be required for the formulation of stable SEDDS. It is critical to avoid postformulation drug partitioning consequent upon phase separation. It has been reported that the concentration of surfactant that will form stable SEDDS formulation ranges between

30-60%w/w (Gursory and Benita, 2004).

120

Figure 16: Capsules of ARM loaded SEDDS and S-SEDDS containing different concentrations (40, 50 and 55 mg) at ratios of Oil: Smix (1:2.0, 1:2.5, 1:3.5 and

1:4.0).

121

3.6 DROPLET SIZE (DS), POLYDISPERSITY INDEX (PDI) AND ZETA POTENTIAL(Z) Table 3: Droplet size, polydispersity and zeta potential of the SEDDS formulation

S. Ratio DSAverage(μm) PDI Z-Potential(-)(mV)

Placebo

1:2.0 1.278 0.63 13.5

1:2.5 3.520 1.00 15.5

1:3.5 0.395 0.36 28.8

1:4.0 3.522 0.82 10.4

40 mg ARM-loaded SEDDS

1:2.0 2.598 0.63 11.6

1:2.5 1.852 0.71 9.0

1:3.5 1.112 0.61 11.9

1:4.0 3.689 0.57 8.6

50 mg ARM-loaded SEDDS

1:2.0 1.254 0.50 11.8

1:2.5 2.986 0.82 8.9

1:3.5 3.698 1.00 9.1

1:4.0 4.856 1.00 6.2

55 mg ARM-loaded SEDDS

1:2.0 5.211 0.91 6.9

1:2.5 2.934 0.91 16.5

1:3.5 3.578 0.92 4.9

1:4.0 4.111 1.00 12.1

122

Droplet size is an important factor in self-emulsification as it determines the rate and extent of drug release as well as the stability of the emulsion. The droplet sizes were found out to be in micrometer ranges as shown in Table 3. It was statistically shown that the presence of drug and its concentration were not significant on the droplet sizes.

SEDDS with larger particle sizes possess lower rate of emulsification in vivo than

SEDDS with smaller particle sizes and consequently, larger emulsion droplets may be formed as a result (Attama and Nkemnele, 2005). Droplet sizes as well as the rate and extent of lipolysis of the emulsion products formed have also been shown to affect the bioavailability of trocotrienol administered from self emulsifying formulations (Yap and Yuen, 2004).

The polydispersity index is the ratio of standard deviation to the mean droplet size. It signifies the uniformity of droplet size within the formulation. The higher the value of polydispersity, the lower is the uniformity of droplet size within the formulation. Low polydispersity indices are preferable as emulsions with higher values may be prone to instability. The polydispersity index values of the formulations were low which indicates uniformity of droplet size within the formulations.

The zeta potential measurement was determined to identify the charges of the droplets.

In conventional SEDDS (o/w), the charges on the oil droplet is negative due to presence of free fatty acids except when cationized (Gershank and Benita, 1996; Patel and Patel, 2008; Obitte et al., 2009). Therefore, the negative zeta potential associated with oil-in-water emulsions had minor variations amongst the batches.

123

3.7 POSTFORMULATION ISOTROPICITY TEST

Postformulation isotropicity test also showed that the Oil: Smix ratios 1:2.0, 1: 2.5,

1:3.5 and 1:4.0 were stable without phase separation. This test is to ascertain if the presence of ARM would cause any instability to the formulation. The confirmation of

SEDDS stability in the presence of drug was needed to ensure formulation stability during storage. The efficiency of addition of drugs into a SEDDS is specific in each case depending on the physicochemical compatibility of the drug/system (Gursory and

Benita, 2004; Obitte et al., 2008). More often the drug interferes with the self- emulsification process to a certain degree leading to a change in the optimal oil/surfactant ratio (Gursory and Benita, 2004; Obitte et al., 2008). Every oil: Smix ratio often has a certain solvent capacity for drug, beyond which drug precipitation or phase separation could occur. Better solvent capacities for drugs are common with most of the synthetic oils.

3.8 EMULSIFICATION TIME

The emulsification times (ETs) were obtained in non-HPMC and HPMC batches between 18-19 sec and 29-31 sec respectively. Emulsification is a rate determining step to absorption, which is similar to disintegration test in tablet (Obitte et al., 2010). This process takes place when the SEDDS is gently agitated in vitro by any mechanical stirring means or in vivo by gastric motility, thus dispersing the o/w emulsion with the drug partitioned between the surfactant and the oil. The process begins by forming liquid crystals and gel phases, the properties of which significantly affect droplet formation and availability of interface for drug partitioning (Craig et al., 1995; Porter and Charman, 2001; Obitte et al., 2011). The presence of HPMC increased the emusificstion time of the HPMC batches. SEDDS with higher viscosities are likely to 124 experience lower emulsification rate (Obitte et al., 2011). Emulsification rate is known to be an important index for emulsification efficiency assessment. Some researchers

(Pouton, 1985; Obitte, 2008; Khoo et al., 1998) reported 2 min as an upper emulsification time limit. Therefore, values less than 2 min recorded by the batches are within the acceptable time limit. Emulsification time could be affected by factors such as viscosity, admixture of oils and free energy of the system (Lanlan et al., 2005).

Increase in viscosity of SEDDS formulation will prolong emulsification time (Obitte et al., 2008). The presence of the polymeric material, HPMC prolonged the emulsification time of the SEDDS when compared to non-HPMC batches though the difference was not significant. This may be because of the concentration of HPMC used.

3.9 REFRIGERATION CYCLE TEST

Refrigeration cycle test carried out was to test the robustness and stability of the formulations. The products were observed to retain their physical properties and showed no sign of colour change, odour change, or phase separation. This shows that storage under refrigeration conditions could be done without the formulation losing its stability as particle growth which is a sign of physical instability in transparent solution could be induced by refrigeration.

3.10 CENTRIFUGATION

The SEDDS and S-SEDDS batches remained stable after centrifugation at 3000 rpm for 30 min. There was no phase separation.

125

3.11 AQUEOUS DILUTION TEST

Dilution study was done to evaluate effect of aqueous dilution on SEDDS, because dilution may mimic the condition of stomach after oral administration. When microemulsions are administered orally, they become diluted with water in the gastrointestinal tract, which could lead to drug precipitation after some time. Thus, the stability of the solubilization capacity in the diluting process is very important. Yin et al (2009) reported that the microemulsions of Docetaxel® exhibited satisfactory solubilization capacity for at least 24 h. In the results of aqueous dilution test, phase separation was not noticed after dilution of the SEDDS to a litre. This shows that the formulations could be dispensed for reconstitution to even 1 L without thermodynamically losing its stability after 24 h.

3.12 VISCOSITY STUDIES

The SEDDS system is generally administered in soft gelatin or hard gelatin capsules and therefore, can be easily pourable into capsules and such system should not be too thick to create a pourability problem. The rheological properties of the micro-emulsion are evaluated using a viscometer. The viscosity determination confirms whether a system is w/o or o/w. If system has low viscosity then it is o/w type of the system and if a high viscosity then it is w/o type of the system (Shah, 1994; Shafiq, 2007). There was significant difference (P ˂ 0.05) between the viscosities of the HPMC and non-HPMC batches (Fig 17-22). HPMC which forms hydrogel in aqueous phase was responsible for the viscosity difference.

126

60

50

40

30

Viscosity (cst) 20

10

0 Batch 1:2.0 Batch 1:2.5 Batch 1:3.5 Batch 1:4.0 SEDDS batches

Figure 17: Viscosity of non-HPMC batches of 40 mg ARM-loaded SEDDS

127

90

80

70

60

50

40 Viscosity(cst)

30

20

10

0 Batch 1:2.0 Batch 1:2.5 Batch 1:3.5 Batch 1:4.0 SEDDS batches

Figure 18: Viscosity of non-HPMC batches of 50mg ARM SEDDS

128

80

70

60

50

40 Viscosity(cst)

30

20

10

0 Batch 1:2.0 Batch 1:2.5 Batch 1:3.5 Batch 1:4.0 SEEDS batches

Figure 19: Viscosity of non-HPMC batches of 55mg ARM-loaded SEDDS

129

120

100

80

60 Viscosity(cst)

40

20

0 Batch 1:2.0 Batch 1:2.5 Batch 1:3.5 Batch 1:4.0 SEDDS batches

Figure 20: Viscosity of HPMC batches of 40mg ARM-loaded SEDDS

130

120

100

80

60 Viscosity(cst)

40

20

0 Batch 1:2.0 Batch 1:2.5 Batch 1:3.5 Batch 1:4.0 SEDDS batches

Figure 21: Viscosity of HPMC batches of 50 mg ARM-loaded SEDDS

131

120

100

80

60 Viscosity(cst)

40

20

0 Batch 1:2.0 Batch 1:2.5 Batch 1:3.5 Batch 1:4.0 SEDDS batches

Figure 22: Viscosity of HPMC batches of 55mg ARM-loaded SEDDS

132

3.13 pH STUDIES

The results of the pH determination of non-HPMC and HPMC batches showed that there was no significant difference in the pH of the formulatins batches after three months shown in Fig 23 -28. It might be as a result of high stability of the SEDDS formulation. The batches showed pH at acidic region.

133

7

6

5

4 pH 3

2

1

0 M 0 M 1 M 2 M 3 Time (month)

Batch 1:2.0 Batch 1:2.5 Batch 1:3.5 Batch 1:4.0

Figure 23: Effect of storage time (month) on pH of 40 mg ARM-loaded SEDDS of non-HPMC batches 134

7

6

5

4 pH

3

2

1

0 M0 M1 M2 M3 Time (month) Batch 1:2.0 Batch 1:2.5 Batch 1:3.5 Batch 1:4.0

Figure 24: Effect of storage time (month) on pH of 50 mg ARM-loaded SEDDS of non-HPMC batches 135

7

6

5

4 pH

3

2

1

0 M0 M1 M2 M3 Time (month) Batch 1:2.0 Batch 1:2.5 Batch 1:3.5 Batch 1:4.0

Figure 25: Effect of storage time (month) on pH of 55 mg ARM-loaded SEDDS of non-HPMC batches

136

7

6

5

4 pH

3

2

1

0 M0 M1 M2 M3

Time (month)

Batch 1:2.0 Batch 1:2.5 Batch 1:3.5 Batch 1:4.0

Figure 26: Effect of storage time (month) on pH of 40 mg ARM-loaded SEDDS of HPMC batches

137

7

6

5

4 pH

3

2

1

0 M0 M1 M2 M3 Time (month)

Batch 1:2.0 Batch 1:2.5 Batch 1:3.5 Batch 1:4.0

Figure 27: Effect of storage time (month) on pH of 50 mg ARM-loaded SEDDS of HPMC batches

138

7

6

5

4 pH

3

2

1

0 M0 M1 M2 M3

Time (month) Batch 1:2.0 Batch 1:2.5 Batch 1:3.5 Batch 1:4.0

Figure 28: Effect of storage time (month) on pH of 55 mg ARM-loaded SEDDS of HPMC batches

139

3.14 LOADING EFFICIENCY OF ARM-LOADED SEDDS AND S-SEDDS

The ability of the SEDDS to accommodate active molecules is an important property

and this is expressed by the Loading efficiency (LE). In Tables 4-9 show the result of

LE (%) which indicate that the HPMC batches improved the solubilisation of the drug

more in the SEDDS when compared to non-HPMC batches although there was no

significant variation.

Table 4: The loading efficiency of 40 mg ARM-loaded SEDDS of non-HPMC batches

SEDDS L. E (%) STD

BATCH 1:2.0 100.315 0.08

BATCH 1:2.5 100.367 0.109

BATCH 1:3.5 100.473 0.199

BATCH 1:4.0 98.745 0.137

L.E=Loading efficiency and STD= standard deviation

140

Table 5: The loading efficency of 50 mg ARM-loaded SEDDS of non-HPMC batches

SEDDS L.E (%) STD

BATCH 1:2.0 100.731 0.073

BATCH 1:2.5 94.951 0.007

BATCH 1:3.5 92.72 0.007

BATCH 1:4.0 100.89 0.036 L.E=Loading efficiency and STD= standard deviation

Table 6: The loading efficiency of 55 mg ARM-loaded SEDDS of non-HPMC batches

SEDDS L.E (%) STD

BATCH 1:2.0 88.324 0.001

BATCH 1:2.5 99.470 0.004

BATCH 1:3.5 93.333 0.036

BATCH 1:4.0 91.213 0.005 L.E=Loading efficiency and STD= standard deviation

141

Table 7: The loading efficiency of 40 mg ARM-loaded S-SEDDS of HPMC batches

SEDDS L.E (%) STD

BATCH 1:2.0 96.234 0.197

BATCH 1:2.5 100.908 0.131

BATCH 1:3.5 100.707 0.036

BATCH 1:4.0 95.607 0.116 L.E=Loading efficiency and STD= standard deviation

Table 8: The loading efficiency of 50 mg ARM-loaded SEDDS of HPMC batches

SEDDS L.E (%) STD

BATCH 1:2.0 100.669 0.045

BATCH 1:2.5 99.331 0.008

BATCH 1:3.5 92.259 0.023

BATCH 1:4.0 100.172 0.061 L.E=Loading efficiency and STD= standard deviation

142

Table 9: The loading efficiency of 55 mg ARM-loaded SEDDS of HPMC batches

SEDDS L.E (%) STD

BATCH 1:2.0 100.883 0.027

BATCH 1:2.5 100.134 0.089

BATCH 1:3.5 100.188 0.084

BATCH 1:4.0 100.607 0.024 L.E=Loading efficiency and STD= standard deviation

143

3.15 CRYSTALLIZATION/PRECIPITATION STUDIES

The effect of HPMC on drug crystallization was studied using optical microscopy. The results showed that after 3 h, the presence of drug crystals was observedin the non-

HPMC batches while no crystal was observed in the HPMC batches. This effect was achieved due to high droplet sizes of the formulated SEDDS that were in micrometer range. Surpersaturation is intended to increase the thermodynamic activity of the drug beyond its solubility limit and, therefore, to result in an increased driving force for transit into and across the biological barrier (Gao et al., 2004). The general applicability of cellulosic polymers in inhibiting crystallization of many pharmaceutical substances has been widely reported and are effective in prolonging the supersaturated state of the drugs for prolonged time periods as shown by a number of in vitro studies

(Gao, 2004; Gao and Morozowich, 2005; Suzuki and Sunada., 1998; Yamada et al.,

1999; Usui et al., 1997; Raghavan et al., 2001a; Ziller and Rupprecht, 1988; Raghavan et al., 2001b). Studies of the mechanism responsible for inhibiting crystallization of drugs in aqueous solutions containing HPMC suggest that the HPMC polymer chain may inhibit nucleation as well as crystal growth by adsorption onto the surface of the nuclei or onto the surface of crystals (Suzuki and Sunada., 1998; Ziller and Rupprecht,

1988; Raghavan et al., 2001b). Ziller and Rupprecht (1988) suggested that the polymer inhibits the introduction of drug molecules from solution into the crystal lattice by occupying adsorption sites such that the adsorbed polymer forms a mechanical barrier that inhibits crystallization. Raghavan et al (2001b) proposed that the mechanism of nucleation and growth is based on the interaction between the drug and the polymer molecules through hydrogen bonding. The hydrodynamic boundary layer surrounding the crystal, resulting from adsorption of the polymer molecules onto the crystal surface, leads to crystal growth inhibition as well as habit modification of the crystals. In order 144 to take advantage of the creation of intraluminal supersaturation, this state should be stabilized for a time period allowing sufficient transepithelial transport by temporaryly inhibiting precipitation. This may require the intraluminal presence of the so called precipitation inhibitors.

145

3.15.1 PHOTOMICROGRAPHIC RESULTS

Figure 29: Photomicrograph of ARM crystals

146

Figure 30: Photomicrograph of ARM-loaded SEDDS of non-HPMC batches after

3 h

147

Figure 31: Photomicrograph of ARM-loaded S-SEDDS after 3 h

148

3.16 RELEASE STUDIES OF ARM LOADED SEDDS AND S-SEDDS

The in vitro release studies were carried out in order to determine the rate of drug release from SEDDS and S-SEDDS and also to compare the SEDDS with pure encapsulated ARM in SIF and SGF. The results are presented below in Figs 32–35.

The release profile of ARM from different batches; 1:2.0, 1:2.5, 1:3.5, and 1:4.0 in SGF

(pH, 1.2) and SIF (pH, 6.8) for HPMC and non-HPMC batches was obtained. The drug release profiles of non-HPMC batches showed T50 and T85 values that ranged between

3-8 min and 14-22 min respectively in SIF, and 3-4 min and 5-18 min respectively in

SGF. On the other hand, the drug release profiles of HPMC batches showed T50 and

T85 values that ranged between 8-35 min, and 53- 153 min in SGF and T50 and T85 values that ranged between 4-13 min and 92-213 min respectively in SIF.

According to US-FDA guidance for immediate release dosage form, T85 of the labelled amount of the drug should be 30 min. In both media (SGF and SIF), the non-HPMC released 85 % of its drug content before 30 mins while the HPMC released 85 % drug content within 3-4 hours. Therefore, all the non-HPMC batches which released immediately conformed to it while the HPMC batches due to the presences of HPMC had a sustaininig effect. The formulations could be summarized as having had optimum drug release showing that SEDDS is a formulation of choice for improving the release and anticipated bioavailability of ARM. The release of compound from SEDDS based formulation is thought to take place by two major pathways: Interfacial transfer and degradation of vehicle (Porter et al., 2004). Interfacial transfer is noted to be a concentration gradient driven process in which the compound diffuses from the formulation into the bulk or directly over the intestinal membrane. The rate and extent of interfacial transfer is thought to be governed by partition coefficient and solubility in the donor (formulation) and recipient phase particle size and hence surface area of 149 formulation (Armstrong and James, 1980). The second pathway is degradation of the vehicle inducing the release of the compound out of the vehicle. As mentioned above, for lipid based formulations, the most important degradation is the lipolysis catalyzed by pancreatic lipase. The release rate is thought to be dependent on the solubility of the compound in the formulation and rate and extent of the degradation of the vehicle.

Lipolysis of triacylglycerols (TG) by the pancreatic lipase–colipase complex releases monoacylglycerols, diacylglycerols and free fatty acids. These lipolysis products are amphiphiles that will further assist the solubilization of poorly soluble compounds in the GI fluids. The presence of lipids in the GI tract stimulates an increase in the secretion of bile salts (BS) and endogenous biliary lipids including phospholipid (PL) and cholesterol (CH), leading to the formation of BS/PL/CH intestinal mixed micelles and an increase in the solubilisation capacity of the GI tract

The lipids play a significant role since they can increase the drug solubility in lumen, can change the physical (Aungst, 2000) and the biochemical barrier function (Benet,

2001) of the GI tract and they can stimulate lymphatic drug transport (Charman and

Porter, 1997).

150

120

100

80

60

AMOUNT RELEASED(%) 40

20

0 0 5 10 15 20 25 30 35 40 45 50 55 60 90 120 150 180 210 240 270 300 Time (min)

BATCH 1:2.0 BATCH 1:2.5 BATCH 1:3.5 BATCH 1:4.0

Figure 32: Release profile of HPMC batches in SIF

151

120

100

80

60

AMOUNT RELEASED(%) 40

20

0 0 5 10 15 20 25 30 35 40 45 50 55 60 90 120 150 180 210 240 270 300

TIME(min)

BATCH 1:2.0 BATCH 1:2.5 BATCH 1:3.5 BATCH 1:4.0

Figure 33: Release profile of HPMC batches in SGF.

152

120

100

80

60

40 AMOUNT RELEASED(%)

20

0 0 5 10 15 20 25 30 35 40 45 50 55 60

Time (min)

Batch 1:2.0 Batch 1:2.5 Batch 1:3.5 Batch 1:4.0 Pure ARM

Figure 34: Release profile of non-HPMC batches in SIF 153

120

100

80

60

AMOUNT RELEASED(%) 40

20

0 0 5 10 15 20 25 30 35 40 45 50 55 60 TIME(min)

Batch 1:2.0 Batch 1:2.5 Batch 1:3.5 Batch 1:4.0 Pure ARM

Figure 35: Release profile of non-HPMC batches in SGF

154

3.16 RELEASE MECHANISMS OF HPMC BATCHES IN SGF AND SIF

The drug release kinetics and mechanisms were studied using four kinetic models including zero order, first order, Higuchi and Ritger–Peppas models. The results are shown in Figs 36-43.

The zero order shows a system where the drug release rate is independent of its concentration. Here, a plot of amount of drug released versus time showed linearity.

The dosage forms following this profile, release the same amount of drug by unit time and it is the ideal method of drug release in order to achieve a prolonged pharmacological action (Chime et al., 2013). This relationship can be used to determine the drug dissolution from various types of modified release dosage forms such as matrix tablets with low soluble drugs, coated tablets and capsules and osmotic systems

(Kalam et al., 2007; Varles et al., 1995; Chime et al., 2013. The first order describes the release of drug from a system where release rate is concentration dependent

(Gilbaldi and Feldman, 1967; Wagner, 1967). The first order plot also resulted in a linear graph with a negative slope (Kabir et al., 2009). The Ritger–Peppas plot (Ritger–

Peppas, 1987) showed a Fickian diffusion release mechanism for all the HPMC batches as their n value ≤ 0.5. Higuchi (1961 and 1963) plot of amount of drug release against square root of time for all the batches of HPMC were linear showing that the processes of drug release from the batches, also followed diffusion controlled mechanisms. The plot of Log Q versus Log t gave n value below 0.5, indicating that diffusion was not the only predominant mechanism of release for all the batches (Ofoefule and Chukwu,

2002).

The correlation coefficients obtained for Higuchi plots of HPMC batches were found to be superior to those obtained for the other models. Therefore, it is concluded that the release kinetic of the HPMC batches follows the Higuchi model most. 155

120

y4 = 1.105x + 53.43 R² = 0.873 100

y 3= 0.867x + 41.20 R² = 0.927

80 y2 = 0.660x + 43.31 R² = 0.971

60 Q (%)

y1 = 0.382x + 34.31 R² = 0.933

40

20

0 0 10 20 30 40 50 60 t (min)

Figure 36: Zero order plot of HPMC batches in SGF. Where Q=amount released, t=time, y1=batch 1:2.0, y2=batch 1:2.5, y3=batch 1:3.5 and y4=1:4.0 156

2

y1 = -0.003x + 1.829 R² = 0.932

y2 = -0.007x + 1.779 1.5 R² = 0.942

y3 = -0.011x + 1.829 R² = 0.974

1 y4 = -0.017x + 1.697 R² = 0.899 Q - Log 100 0.5

0 0 10 20 30 40 50

-0.5 t (min)

Figure 37: First order plot of HPMC batches in SGF. Where Q=amount released, t=time, y1=batch 1:2.0, y2=batch 1:2.5, y3=batch 1:3.5 and y4=1:4.0

157

140

120 y4 = 19.32x - 14.11 R² = 0.925

100

y3 = 9.401x + 18.71 R² = 0.981 80

y2 = 6.365x + 29.53 R² = 0.945 Q (%) 60

y1 = 3.790x + 25.83 R² = 0.942 40

20

0 0 2 4 6 8 √t (min0.5)

Figure 38: Higuchi plot of HPMC batches in SGF. Where Q= amount released, t=time, y1=batch 1:2.0, y2=batch 1:2.5, y3=batch 1:3.5 and y4=1:4.0

158

2.5

y4 = 0.263x + 1.547 2 R² = 0.941 y3 = 0.334x + 1.353 R² = 0.978

y2 = 0.221x + 1.481 R² = 0.920

y1 = 0.189x + 1.390 R² = 0.948 1.5 Log Q

1

0.5

0 0 0.5 1 1.5 2 Log t

Figure 39: Ritger–Peppas plot of HPMC batches in SGF. Where Log Q=amount released in logarithm, Log t=time in logarithm, y1=batch 1:2.0, y2=batch 1:2.5, y3=batch 1:3.5 and y4=1:4.0

159

250

y 4= 0.504x + 49.67 R² = 0.902 200 y3 = 0.421x + 59.98 R² = 0.960

y 2= 0.456x + 44.78 R² = 0.903

150 Q (%)

100

y1 = 0.120x + 50.68 R² = 0.904

50

0 0 50 100 150 200 250 300 350 t (min)

Figure 40: Zero order plot of HPMC batches in SIF. Where Q=amount released, t=time, y1=batch 1:2.0, y2=batch 1:2.5, y3=batch 1:3.5 and y4=1:4.0

160

2.5

2

1.5

y1 = -0.001x + 1.704 R² = 0.910 Q - 1 log 100

0.5

0 0 50 100 150 200 250 300 350

y 3= -0.006x + 1.617 R² = 0.923

y4 = -0.008x + 1.827 -0.5 R² = 0.906 t (min) y2 = -0.009x + 1.930 R² = 0.928

Figure 41: First order plot of HPMC batches in SIF. Where Q=amount released, t=time, y1=batch 1:2.0, y2=batch 1:2.5, y3=batch 1:3.5 and y4=1:4.0

161

120

y3 = 3.041x + 56.47 R² = 0.903

100 y4 = 2.481x + 49.7 R² = 0.961

y1 = 2.019x + 44.66 80 R² = 0.907

y 2= 1.719x + 48.43 R² = 0.971

60 Q (%)

40

20

0 0 5 10 15 20 √t (min 0.5)

Figure 42: Higuchi plot of HPMC batches in SIF. Where Q=amount released, t=time, y1=batch 1:2.0, y2=batch 1:2.5, y3=batch 1:3.5 and y4=1:4.0

162

2.5

y4 = 0.216x + 1.484 R² = 0.901 2 y 3= 0.131x + 1.677 R² = 0.932 y1 = 0.108x + 1.612 R² = 0.921 y2 = 0.111x + 1.597 R² = 0.908

1.5 Log Q

1

0.5

0 0 0.5 1 1.5 2 2.5 3 Log t

Figure 43: Ritger–Peppas plot of HPMC batches in SIF. Where Q=amount released, T=time, y1=batch 1:2.0, y2=batch 1:2.5, y3=batch 1:3.5 and y4=1:4.0

163

3.17 THE RESULTS OF IN VIVO STUDIES OF ARM-LOADED SEDDS

120

100

80

60

40 Antimalarial Activity(%) 20

0 SEDDS CQ Placebo Pure ARM Batches

Figure 44: Chart showing antimalarial activity with 40 mg ARM loaded SEDDS

164

In the antimalarial studies carried out, the following percent activities were obtained:

SEDDS-treated group (94%), chloroquine-treated (59%), placebo-treated group (16%), and aqueous dispersed ARM-treated group (47%). The SEDDS-treated group had a significantly (P < 0.05) higher activity than other groups. This might be due to the following important facts; (i) SEDDS and SMEDDS present hydrophobic drugs to GIT in solubilised and micro emulsified form and are associated with the generation of large surface area dispersions that provide optimum conditions for the increased absorption of poorly soluble drugs (Patil and Patil, 2007; Tao and Jiangling, 2008; Sagar et al.,

2008; Wei et al., 2006). (ii) High drug solubilisation capacity and self- microemulsification efficiency of the SEDDS, (iii) Some very lipophilic drugs associate with lipoproteins within the enterocytes and reach the systemic circulation via the intestinal lymph and it was agreed that the gut associated lymphoid tissue (GALT), including the isolated lymphatic follicles or Peyer’s patches, is important in the particulate absorption process, (Hauss, 1998; Trevaskis et al., 2008; Wu et al., 2011) with the advantage of a reduced first-pass metabolism, (iv) It is clear that certain lipids and surfactants (labrasol) are potent absorption enhancers and efflux-reducing agents in

Caco-2 cells and the rat intestine and therefore, attenuate the activity of intestinal efflux transporters, as indicated by the P-gp efflux pump, and also reduce the extent of enterocyte-based metabolism (Benet, 2001; Dintaman and Silverman, 1999; Nerurkar et al, 1996). (v) SEDDS protection of drugs against biodegradation: Many drugs are degraded in physiological system, which may be due to acidic pH in stomach (e.g.

ARM), enzymatic degradation or hydrolytic degradation, etc. Such drugs when presented in the form of SEDDS can be well protected against these degradation 165 processes as liquid crystalline phase in SEDDS might be acting as barrier between degradating environment and the drug.

166

CHAPTER FOUR

CONCLUSION AND RECOMMENDATION

CONCLUSION

Artemether-loaded SEDDS and S-SEDDS were formulated to improve the poor aqueous solubility of artemether. The formulations were evaluated for solubility studies, droplet size, pre/postformulation visual isotropicity, emulsification time, refrigeration cycle, centrifugation, aqueous dilution, viscosity, pH, loading efficiency, microscopy, in vitro drug release and antimalarial studies. SEDDS batches with droplet sizes in the micrometer range were selected for crystallization inhibition studies.

Artemether showed high solubility in Triacetin® (136 ± 0.09 mg/ml), Labrasol® (156 ±

0.10 mg/ml) and Transcutol P® (166 ± 0.02 mg/ml). The emulsification time of less than one minute recorded by the optimized formulations was within acceptable limit of

2 min. Refrigeration cycle test showed high stability of the formulations. There was no significant change in pH throughout the 3-month evaluation period. The incorporation of HPMC (a polymeric material) resulted in significantly (p < 0.05) higher viscosity than non-HPMC batches. Inhibition of crystallization was only achieved in the presence of HPMC. Over 80% of drug was released within 30 min from non-HPMC batches while the artemether-loaded supersaturable SEDDS showed about 60% of drug release and extended drug release profile of about 3-4 h. Artemether SEDDS recorded the highest antimalarial activity. It is therefore concluded that the poor water solubility and antimalarial activity of artemether were improved using SEDDS technique; while HPMC inhibited drug crystallization.

167

RECOMMENDATION

The fact that over 40 % of new chemical moieties are hydrophobic in nature means that studies with SEDDS and S-SEDDS will continue, and more drugs will be formulated as

SEDDS and widely get circulated in the market.

We, therefore, recommend formulation of artemether-sustained release dosage form using HPMC as a polymeric material.

168

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190

APPENDIX 1

Solubility studies in different vehicles

Vehicles Solubility ± SDV

Labrasol 156 ± 0.10

Gelucire 44/14 60 ± 0.08

Transcutol P 166 ± 0.02

Triacetin 136 ± 0.09

Capryol 90 130 ± 0.14

Capryol PGMC 108 ± 0.24

Lauroglycol 90 153 ± 0.20

Lauroglycol FCC 129 ± 0.10

Peceol 119 ± 0.07

Labrafac™ Lipophile WL 1349 106 ± 0.04

Labrafil 1944 94 ± 0.18

Maisine 137 ± 0.16 SDV= standard deviation

APPENDIX 2

The percentage compositions of the pseudoternary phase diagram of different Smix ratios.

OIL: Smix OIL (%) Smix (%) WATER (%)

1:9 0.328 2.951 96.721 191

1:8 0.271 2.171 97.559

1:7 0.352 2.465 97.183

1:6 0.397 2.375 97.228

1:5 0.617 3.092 96.291

1:4 0.645 2.581 96.774

1:3.5 0.698 2.476 96.825

1:3.0 13.793 41.379 44.828

1:2.5 15.481 38.769 45.750

1:2.0 29.684 59.190 11.126

1:1.5 37.647 56.470 5.880

1:1.0 47.059 47.059 5.880

1:0.5 62.641 31.485 5.870

1:0.3 74.670 22.303 3.030

1:0.2 80.880 16.078 3.045

1:0.1 88.230 8.736 3.033

Percent composition of pseudoternary phase diagram of Smix (1:0) Smix = surfactant/cosurfactant mix

OIL: Smix OIL (%) Smix (%) WATER (%) 192

1:9 0.392 2.529 96.078

1:8 0.435 3.490 96.075

1:7 0.500 3.500 96.00

1:6 0.572 3.420 96.008

1:5 0.680 3.408 95.913

1:4 0.870 3.478 95.650

1:3.5 9.643 34.182 56.169

1:3.0 12.121 36.364 51.515

1:2.5 12.343 30.914 56.745

1:2.0 22.256 44.378 33.367

1:1.5 30.476 45.714 23.810

1:1.0 43.243 43.243 13.514

1:0.5 66.550 27.758 9.361

1:0.3 72.471 21.647 5.882

1:0.2 80.877 16.077 3.045

1:0.1 88.231 8.736 3.033

Percent composition of pseudoternary phase diagram of Smix (1:0.5) Smix = surfactant/cosurfactant mix

OIL: Smix OIL (%) Smix (%) WATER (%) 193

1:9 0.388 3.492 96.120

1:8 0.529 4.238 95.234

1:7 0.646 4.519 94.835

1:6 0.753 4.505 94.747

1:5 0.876 4.394 97.361

1:4 1.053 4.211 94.737

1:3.5 10.353 36.706 52.941

1:3.0 13.333 40.000 46.667

1:2.5 16.558 41.757 41.685

1:2.0 20.542 40.961 38.497

1:1.5 27.234 40.910 31.915

1:1.0 39.024 39.024 21.951

1:0.5 54.620 27.453 17.927

1:0.3 70.400 21.029 8.571

1:0.2 78.480 15.603 5.910

1:0.1 88.230 8.735 3.033

Percent composition of pseudoternary phase diagram of Smix (1:1) Smix = surfactant/cosurfactant mix

194

OIL: Smix OIL (%) Smix (%) WATER (%)

1:9 1.000 9.000 90.00

1:8 1.233 9.888 88.879

1:7 1.785 12.500 85.714

1:6 7.271 43.473 49.256

1:5 8.587 43.064 48.350

1:4 10.492 41.967 47.541

1:3.5 11.932 42.305 45.763

1:3.0 14.286 42.857 42.857

1:2.5 16.914 42.355 40.729

1:2.0 20.154 40.202 39.659

1:1.5 25.600 38.400 36.00

1:1.0 36.364 36.364 27.273

1:0.5 56.056 28.175 15.770

1:0.3 72.471 21.647 5.882

1:0.2 80.877 16.078 3.045

1:0.1 88.231 8.7357 3.033

Percent composition of pseudoternary phase diagram of Smix (1:2) Smix = surfactant/cosurfactant mix

195

OIL: Smix OIL (%) Smix (%) WATER (%)

1:9 5.978 53.799 40.164

1:8 7.306 58.562 34.134

1:7 8.228 57.662 34.145

1:6 9.444 56.501 34.054

1:5 10.041 50.384 39.575

1:4 11.852 47.408 40.741

1:3.5 13.037 46.222 40.741

1:3.0 14.546 32.124 41.818

1:2.5 16.909 42.375 40.717

1:2.0 20.148 40.205 39.647

1:1.5 25.185 37.778 37.038

1:1.0 34.783 34.783 30.435

1:0.5 55.178 27.776 17.046

1:0.3 68.895 22.369 10.527

1:0.2 78.725 15.698 5.577

1:0.1 87.144 8.628 4.228

Percent composition of pseudoternary phase diagram of Smix (1:3) Smix = surfactant/cosurfactant mix

196

OIL: Smix OIL (%) Smix (%) WATER (%)

1:9 0.400 3.600 96.000

1:8 0.493 3.955 95.551

1:7 0.556 3.950 95.553

1:6 0.650 3.934 95.461

1:5 0.757 3.797 95.446

1:4 1.026 4.103 94.872

1:3.5 11.000 39.000 50.000

1:3.0 19.048 57.143 23.810

1:2.5 21.227 53.197 25.578

1:2.0 26.701 52.851 20.016

34.595 51.892 13.514 1:1.5

1:1.0 45.714 45.714 8.571

1:0.5 62.700 31.562 5.880

1:0.3 74.667 22.303 3.030

1:0.2 80.838 16.119 3.044

1:0.1 88.231 8.742 3.033

Percent composition of pseudoternary phase diagram of Smix (3:1) Smix = surfactant/cosurfactant mix

197

OIL:SC OIL (%) Smix (%) WATER (%)

1:9 0.385 3.462 96.154

1:8 0.463 3.708 95.829

1:7 0.532 3.723 95.745

1:6 0.622 3.718 95.660

1:5 0.757 3.795 95.448

1:4 1.081 4.324 94.595

1:3.5 11.541 40.918 47.541

1:3.0 12.121 36.364 51.515

1:2.5 17.564 43.987 48.062

1:2.0 21.365 42.601 36.035

1:1.5 28.444 42.667 28.889

1:1.0 38.095 38.092 23.810

1:0.5 60.854 30.587 8.560

1:0.3 72.471 21.647 5.882

1:0.2 78.487 15.603 5.910

1:0.1 88.231 8.736 3.033

Percent composition of pseudoternary phase diagram of Smix (3:2) Smix = surfactant/cosurfactant mix

198

APPENDIX 3

Solubility studies in different optimized SEDDS batches

SEDDS A1 A2 A3 Mean A Conc.(mg/ml)

1:2.0 0.669 0.747 0.736 0.717 68.205

1:2.5 0.605 0.771 0.800 0.725 103.448

1:3.5 0.632 0.761 0.929 0.774 110.440

1:4.0 0.730 0.987 0.884 0.867 123.710

A= absorbance; Conc. = concentration

199

Appendix 4

The formular for 1 capsule containing 300 mg SEDDS

SEDDS Oil Surfactant Co-surfactant

1:2.0 0.083 0.117 0.062

1:2.5 0.071 0.126 0.066

1:3.5 0.055 0.0157 0.054

1:4.0 0.050 0.158 0.056

200

APPENDIX 5

Viscosity in centistokes (cst) and density g/ml of non-HPMC and HPMC batches

40 mg ARM 50 mg ARM 55 mg ARM

Density Viscosity Density Viscosity Density Viscosity

(g/ml) (cst) (g/ml) (cst) (g/ml) (cst) SEDDS batches 1:2.0 1.018 30.500 1.076 77.420 1.036 52.170

1:2.5 1.030 32.100 1.072 77.080 1.052 69.360

1:3.5 1.038 52.260 1.077 78.110 1.052 69.380

1:4.0 1.039 52.950 1.046 62.690 1.052 69.360

Viscosity and density of non-HPMC batches

201

40 mg ARM 50 mg ARM 55 mg ARM

Density Viscosity Density Viscosity Density Viscosity

(g/ml) (cst) (g/ml) (cst) (g/ml) (cst) SEDDS batches 1:2.0 1.075 94.080 1.087 97.760 1.0891 104.450

1:2.5 1.090 105.170 1.067 90.150 1.054 88.420

1:3.5 1.058 88.760 1.067 90.150 1.052 88.310

1:4.0 1.084 101.360 1.060 88.970 1.040 79.760

Viscosity and density of HPMC batches

202

APPENDIX 6

The result of the percent parasitemia and day of death (DOD) of the mice in in vitro studies

SEDDS Placebo Aqueous Untreated dispersion of ARM Chloroquine treated

Parasitemia % Parasitemia DOD % Parasitemia DOD % Parasitemia DOD % DOD % Parasitemia DOD 2.0 31 12 26 29.5 10 13 20 37.5 8

2.0 31 11.5 27 20.0 13 13.0 20 25.0 12

2.5 39 11.5 29 29.5 13 18.0 20 38.5 13

1.0 39 8.0 29 23.5 21 18.5 27 27.0 20

1.0 - 13.0 30 19.0 23 12.0 31 20.0 20

DOD= Date of death

203

APPENDIX 7

The pH studies of both non-HPMC and HPMC batches

SEDDS M 0 M 1 M 2 M 3 Mean STD

Batch 1:2.0 5.7 5.7 5.6 5.4 5.6 0.1

Batch 1:2.5 5.6 5.6 5.5 5.3 5.5 0.1

Batch 1:3.5 5.4 5.4 5.4 5.3 5.375 0.1

Batch 1:4.0 5.4 5.4 5.4 5.2 5.35 0.1

pH of 40 mg ARM loaded-SEDDS of non-HPMC batches M=month (0-3), STD= standard deviation

SEDDS M0 M1 M2 M3 Mean STD

Batch 1:2.0 5.7 5.7 5.6 4.5 5.4 0.6

Batch 1:2.5 5.6 5.6 5.5 5.3 5.5 0.1

Batch 1:3.5 5.5 5.4 5.4 5.1 5.4 0.2

Batch 1:4.0 5.6 5.4 5.4 4.9 5.3 0.3

pH of 50 mg ARM-loaded SEDDS of non-HPMC batches M=month (0-3), STD= standard deviation

204

SEDDS M 0 M 1 M 2 M 3 Ave.M STD

Batch 1:2.0 5.7 5.7 5.6 4.5 5.4 0.6

Batch 1:2.5 5.6 5.6 5.5 5.3 5.5 0.1

Batch 1:3.5 5.5 5.4 5.4 5.1 5.4 0.2

Batch 1:4.0 5.6 5.4 5.4 4.9 5.3 0.3

pH of 55 mg ARM-loaded SEDDS of non- HPMC batches M=month (0-3), STD= standard deviation

SEDDS M 0 M 1 M 2 M 3 mean STD

Batch 1:2.0 5.8 5.6 5.5 5.1 5.5 5.5

Batch 1:2.5 5.8 5.1 5.1 4.8 5.2 5.2

Batch 1:3.5 5.8 5.3 5.2 4.9 5.3 5.3

Batch 1:4.0 5.9 5.8 5.5 4.8 5.5 5.5

pH of 40 mg ARM-loaded S-SEDDS of HPMC batches M=month (0-3), STD= standard deviation

205

SEDDS M 0 M 1 M 2 M 3 Mean STD

Batch 1:2.0 5.8 5.6 5.3 4.8 5.4 0.5

Batch 1:2.5 5.8 5.6 5.5 4.5 5.4 0.6

Batch 1:3.5 5.8 5.5 5.3 5.1 5.4 0.3

Batch 1:4.0 5.9 5.7 5.3 4.8 5.4 0.5

pH of 50 mg ARM-loaded S-SEDDS of HPMC batches M=month (0-3), STD = standard deviation

SEDDS M1 M2 M3 M4 Mean STD

Batch 1:2.0 5.8 5.4 5.4 5 5.4 0.3

Batch 1:2.5 5.8 5.4 5.3 5.1 5.4 0.3

Batch 1:3.5 5.8 5.4 5.2 4.9 5.3 0.4

Batch 1:4.0 5.8 5.7 5.5 5.4 5.6 0.1

pH of 55mg ARM-loaded S-SEDDS of HPMC batches M=month (0-3), STD= standard deviation

206

APPENDIX 8

Regression coefficient data of release kinetic modelss of HPMC batches in SGF and SIF

Batches Q VS √t Log Q Vs Log 100-Q Q Vs

Log t Vs t

R2 N R2 n R2 n R2

1: 2.0 0.942 3.790 0.948 0.189 0.932 0.003 0.933

1: 2.5 0.945 6.365 0.920 0.221 0.942 0.007 0.971

1: 3.5 0.981 9.401 0.978 0.334 0.974 0.011 0.927

1: 4 0.925 19.32 0.941 0.263 0.899 0.017 0.873

Regression coefficient data of release kinetic models of HPMC batches in SGF

Batches Q VS √t Log Q Vs Log 100-Q Vs Q Vs

Log t t

R2 n R2 n R2 n R2

1: 2.0 0.907 2.019 0.921 0.108 0.910 0.001 0.904

1: 2.5 0.971 1.719 0.908 0.111 0.928 0.009 0.903

1: 3.5 0.903 3.041 0.932 0.131 0.923 .0.006 0.960

1: 4 0.961 2.481 0.901 0.216 0.906 0.008 0.902

Regression coefficient data of release kinetic models of HPMC batches in SIF 207

APPENDIX 9

PREPARATION OF 1 N HYDROCHLORIC ACID (HCl)

Given that conc. HCl (37 %) is 12 N. Therefore, using the equation C1V1=C2V2 where

C1=12 N, V1=vol. of the stock to use in the preparation (unknown), C2=1 N, and V2= vol.of the preparation (i.e 1 litre). After the calculation, 83.33 ml of conc HCl was measured out and the volume made up to 1 L with distilled water.

PREPARATION OF SGF WITHOUT PEPSIN

A 2.4 ml volume of conc. hydrochloric acid 37 % (HCl) was measured and transferred into 2 L of distilled water and 10 g sodium chloride (NaCl) was weighed and mixed with the HCl until it completely dissolved. The solution was made up to 5 L with distilled water. The pH was adjusted with HCl to pH 1.2 (USP, 1995).

PREPARATION OF SIF WITHOUT PANCREATIN

SIF was prepared using sodium hyhroxide (NaOH), monobasic potassium phosphate, distilled water and HCl. A 40 g quantity of NaOH and 34 g of mono potassium phosphate were accurately weighed and transferred into 2 L of distilled water. This was mixed properly until both completely dissolved. The solution was made up to 5 L with distilled water and the pH adjusted with HCl to 6.8 (USP. 1995).

208

The results of calibration curve of ARM determined using different media such as

1 N HCl, SGF and SIF are as follow:

1.2

1 y = 2.393x 0.8 R² = 0.997 0.6

Absorbance 0.4

0.2

0 0 0.1 0.2 0.3 0.4 0.5 Concentration (mg/10 ml)

The calibration curve of ARM in 1 N HCL at wavelength of 250 nm.

0.16

0.14

0.12 y = 0.240x 0.1 R² = 0.997

0.08

Absorbance 0.06

0.04

0.02

0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Concentration (mg/10 ml)

The calibration curve of ARM in SGF at wavelength of 254 nm. 209

1.2

1 y = 2.393x R² = 0.997 0.8

0.6

Absorbance 0.4

0.2

0 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 Concentration (mg/10 ml)

The calibration curve of ARM in SIF at wavelength of 260 nm.