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Transcript
Chapter 1 Introduction CHAPTER-I INTRODUCTION 1.1. ORAL DRUG DELIVERY The desired site of drug action, systemic or localized, is a major factor for consideration in deciding the route of drug administration. Systemic routes of drug administration are traditionally classified into enteral and parenteral routes (Hughes et al., 2005). Enteral routes are those involving the gastrointestinal tract (GIT) which include oral ingestion, sublingual, buccal and rectal administration while parenteral drug delivery routes include intra-arterial, intravenous, intrathecal, intramuscular, intracardiac, cutaneous and subcutaneous injections, surgical site-specific implantations, and inhalational routes (Yadav and Prakashan, 2008). Drugs intended for local actions are administered through topical application on the skin and mucosal membranes of the eye, ear, rectum and the anus. The choice of route of drug administration is decided by a number of factors including the physicochemical properties of the drug, biopsychosocial condition of the patient, the desired site and onset of drug action, dosage frequency as well as ease and convenience of administration (Strang et al., 2006). For toxicological considerations, the possibility of reverse administration like gastric lavage and stimulation of emesis are extreme considerations in the choice of route of drug administration (Rathbone et al., 1994). The choice of route of drug administration has a significant influence on patient compliance, therapeutic efficacy and manifestation of side-effects (Liu et al., 1997; Sterling et al., 1997; Schwartzman and Morgan, 2004). Oral drug delivery remains the most favorable and preferred route of drug administration both by patient and physicians (Wong et al., 2006). Oral administration is generally accepted, easy and convenient; and offers the patient the possibility of self administration requiring no expertise (De Jong et al., 2007). Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 1 Chapter 1 Introduction Compared to injectables and implants, pharmaceutical products intended for oral administration are cheaper and currently >60% of drugs are marketed as oral products (Pretorius and Bouic, 2009). Researchers and pharmaceutical companies are increasingly aware of the need for earlier assessment of new drug entities for their potential as oral candidates (Davis and Wilding, 2001). The merits of new drugs are measured in their delivery convenience in addition to their therapeutic efficacy (Breimer, 1999). The possibility of self-administration, patient compliance and less risk of irreversible side-effects has placed the oral route as a standard in drug delivery (Sastry et al., 2000; Tong, 2007). Because it is the easiest and most convenient way of non-invasive administration, the oral route has always, been the preferred way of dosing. Oral drug delivery systems also being the most cost-effective to manufacture, they have always lead the worldwide drug delivery market. Figure 1.1. Percent sales of orally administered drugs for the 50 most sold pharmaceutical products in US and Europe (from IMS Health 2001) 1.1.1. Drug absorption from the gastrointestinal tract Drug transposition from its dosage form into the general circulation, when considering oral administration of immediate-release dosage forms, is a multi-step process involving: Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 2 Chapter 1 Introduction - Disintegration: the process by which the dosage form breaks up into primary particles, drug and excipients, when exposed to the dissolution media. - Dissolution: the process by which drug molecules leave the solid drug particle and enter into the nearby dissolution media to form a solution. - Absorption: the process by which dissolved drug molecules pass through the membranes of the gastrointestinal tract to reach the systemic circulation. Figure 1.2. Processes involved in getting a drug into solution in the gastrointestinal tract so that absorption may take place. Heavy arrows indicate primary pathways that the majority of drugs administered in a particular dosage form undergo. Dashed arrows indicate that the drug is administered in this state in the dosage form. Thin continuous arrows indicate secondary pathways, which are usually inconsequential in achieving therapeutic efficacy. (Reproduced from Hoener and Benet, 2002) As we can clearly see in Figure 1.2., drug absorption following its oral uptake can thus find a limitation either from the drug release from its pharmaceutical form (i.e. solubilization – dissolution) and/or in the permeation Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 3 Chapter 1 Introduction across the gastro intestinal tract (GIT) membranes; tablet or capsule disintegration being generally a well-controlled manufacturing parameter. As the overall rate of absorption of a drug is dictated by its solubility and permeability characteristics, a classification system based on those parameters has been introduced as the biopharmaceutical classification system (BCS). 1.1.2. Biopharmaceutical Classification System (BCS) The Biopharmaceutics Classification System is a guide for predicting the intestinal drug absorption provided by the United States Food and Drug Administration (USFDA CDER Guidance, 2000). The fundamental basis for the BCS was established by Dr. Gordon Amidon who was presented with a Distinguished Science Award at the August 2006, International Pharmaceutical Federation (IPF) congress in Salvador, Brazil. The “Waiver of In-vivo Bioavailability and Bioequivalence Studies for Immediate Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System” is an FDA guidance document, which allows pharmaceutical companies to forego clinical bioequivalence studies, if their drug product meets the specification detailed in the guidance (FDA CDER Guidance, 2000). The principles of the BCS classification system can be applied to NDA and ANDA approvals as well as to scale-up and post approval changes in drug manufacturing. BCS classification can therefore save pharmaceutical companies a significant amount of development time and reduce costs. The BCS is based on the scientific rationale that, if the highest dose of a drug candidate is readily soluble in the fluid volume on average present in the stomach (250 mL) and the drug is more than >90% absorbed, then the in-vitro drug product dissolution profiles should allow assessment of the equivalence of different drug formulations. Solubility and dissolution can be easily measured invitro. Extent of absorption has historically been determined by conducting mass Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 4 Chapter 1 Introduction balance studies both preclinically and clinically. However, the work of BCS and that of collaborators has demonstrated that the effective intestinal permeability (Peff) of therapeutic agents correlates well with total fraction absorbed in both humans, rats and to a lesser extent in-vitro tissue culture systems. Based on these studies a drug candidate can fall into one of four BCS categories, with category I, High Permeability and High Solubility, being the subject of the BCS guidance. The World Health Organization (WHO) has recently recommended bio waivers for Class III and some Class II drugs and American Association of Pharmaceutical Scientists (AAPS-FDA) scientific conferences have recommended bio waivers for Class III compounds as well. Figure 1.3. BCS Classification of Drugs There are products in the market with molecules from each class depending on the bioavailability requirement for the therapeutic response. However, currently 40% of the New Chemical Entities (NCE)'s fall in class II and class IV due to poor solubility. Formulating such a molecule into a suitable oral dosage form for a desired therapeutic response poses a challenge to the formulation scientist. There are a number of technologies to improve the solubility. Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 5 Chapter 1 Introduction Solubility: a drug substance is considered highly soluble when the highest dose strength is soluble in 250 mL or less of aqueous media over a pH range of 1 to 7.5 (equilibrium solubility at 37°C). Permeability: in the absence of evidence suggesting instability in the gastrointestinal tract, a drug substance is considered highly permeable when the extent of absorption in humans is determined to be 90% or more of an administered dose based on mass balance determination or in comparison to an intravenous reference dose (absolute bioavailability study). The permeability class of a drug may also be determined using in-vivo intestinal perfusion approaches (human or appropriate animal models) or in-vitro permeation studies (excised human or animal intestinal tissues or monolayers of cultured intestinal cells). Specifications regarding the dissolution of the immediate release (IR) drug product are also added to this classification. Dissolution: an immediate release drug product is considered rapidly dissolving when no less than 85% of the labelled amount of the drug substance dissolves within 30 min using USP type I apparatus at 100 rpm (or USP type II at 50 rpm) in a volume of 900 ml or less in each of the following media: (1) HCl 0.1N or USP SGF without enzymes, (2) a pH 4.5 buffer and (3) a pH 6.8 buffer or USP SIF without enzymes. An in vitro-in vivo correlation (IVIVC) has been defined by the FDA as a predictive mathematical model describing the relationship between an in-vitro property of a dosage form and an in-vivo response. The objective behind the development and the evaluation of an IVIVC is to establish the dissolution test as a surrogate for human bioequivalence studies (i.e. biowaver – permission to replace pharmacokinetic studies by dissolution testing). Biowavers can actually be requested for solid, orally administered immediate-release products meeting the dissolution requirements described above and containing highly soluble and highly permeable drugs (Class I compound). For information, IVIVC can be Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 6 Chapter 1 Introduction expected for Class I drugs if the dissolution rate is slower than the gastric emptying rate (otherwise limited or no correlation) and for Class II drugs if the invitro dissolution rate is similar to the in-vivo dissolution rate (unless the dose is very high); limited or no correlations being expected for Class III and IV compounds as permeability is the rate controlling step in drug absorption (Amidon et al., 1995; Löbenberg and Amidon, 2000). A well developed review by Lindenberg et al. made in association with the World Health Organization (WHO) (Lindenberg et al., 2004) establishes a classification of drugs belonging to the WHO model list of Essential Medicines. Examples of this classification are shown in Table 1.1. Table 1.1. Classification of orally administered drugs on the WHO model list of Essential Medicines according to the BCS (Lindenberg et al., 2004). (NB: Class III drugs in bold are drugs with permeabilities corresponding to at least 80% absorption). Although the Class IV is the most problematic, interest will be concentrated primarily on BCS Class II drugs, since it is the most common combination as poor solubility of many drugs is directly associated with good lipophilicity which in turn ensures good membrane permeability. For BCS Class II drugs, the dissolution of the drug product is the rate limiting step to absorption as it is the parameter that changes the actual drug concentration in solution over time and that there is no limitation permeability-wise. Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 7 Chapter 1 Introduction 1.1.3. Barriers to oral drug administration The most significant limitation to oral drug administration is the resulting poor systemic bioavailability of various drugs as a result of first-pass metabolism (DeMario and Ratain, 1998; Veber et al., 2002). In numerous instances, significant allowances are made for pre-systemic drug loss in the design of the dosage regimen and the drug delivery system. Previous studies on structurally diverse drugs have also revealed that subject variableness in bioavailability was indirectly proportional to the extent of bioavailability which implies higher subject variability for poorer bioavailable drugs (Hellriegel et al., 1996; Gidal et al., 2000; Ezzet et al., 2005). This variation and the resulting poor control of plasma drug concentrations would particularly be of concern for drugs that have a narrow therapeutic window or a precipitous dose-effect profile (Aungst, 2000). Prominent among impediments to oral absorption are intestinal efflux proteins (Chang et al., 2004; Takano et al., 2006; Yamagata et al., 2007), physicochemical stability of the drug in the various compartments of the gastrointestinal tract (Tong, 2007), insufficient contact time in transit (Severijnen et al., 2004), poor permeability across the gastrointestinal mucosa (Thanou et al., 2001; Wu and Benet, 2005) and digestive and metabolic enzyme activity (Jeong et al., 2005; Cao et al., 2006). The extremely poor solubility of certain drugs such as the bisphosphonates can make their oral delivery difficult. This challenge is even greater when the required dose is high (Veber et al., 2002; Hu et al., 2004). In addition to digesting and absorbing nutrients, the GIT wall forms a physiological barrier against the invasion of foreign substances including pathogens, antigens, toxins and poisons (Brenchley and Douek, 2008). This barrier comprises the cell membranes, tight junctions between adjacent epithelial cells, the mucus layer, catabolic enzymes and the efflux proteins that propel molecules back into the GIT lumen after oral administration (Wang et al., 2005). Drug molecules are often recognized as foreign substances and therefore, the Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 8 Chapter 1 Introduction absorption of drug is also inhibited (Hamman et al., 2007). Overcoming these challenges in oral drug delivery has been one of the most challenging endeavors facing the pharmaceutical industry for decades. When drugs are administered orally, apart from their exposure to possible physical degradation, chemical inactivation or microbial biotransformation, the anatomical proximity of the liver to the GIT necessitates the passage of absorbed drug through the liver where drugs are metabolized to varying degrees by a process known as the first-pass effect (Gibaldi et al., 1971; Kwan, 1997; Back and Rogers, 2007). The most important of all the factors responsible for poor oral drug bioavailability is the cytochrome P450 (CYP)-mediated first-pass metabolism. In a few cases, >90% of administered drug is lost to pre-systemic metabolism (Ghilzai, 2004; Hamman et al., 2005; Leonard et al., 2006; Majumdar and Mitra, 2006). The human CYP enzyme system present in the intestines and liver is responsible for the metabolism of a wider range of drugs (Fang and Xiao-yin, 2005). A sub-family of this enzyme system CYP 3A is responsible for the metabolism of >50% of marketed drugs to a large extent (Rendic, 2002). The ability of CYP 3A to metabolize numerous structurally unrelated compounds apart from being responsible for the poor oral bioavailability of numerous drugs is responsible for the large number of documented drug-drug and drug-food interactions (Quintieri et al., 2008). Successful inhibition of the metabolic activity of these enzymes on orally administered drug may enhance the drug oral bioavailability. Various strategies have been employed to improve the systemic availability of orally administered drugs (Gomez-Orellana, 2005). The principles are generally based on the modification of the physicochemical properties of the drug (Delie and Blanco-Prieto, 2005), addition of novel functionality to the molecular structure of the drug to enhance intestinal wall penetration (Hajduk and Greer, 2007) and modification of pharmaceutical formulation technology by the use of Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 9 Chapter 1 Introduction novel drug delivery carrier systems such as microparticles (Muhrer et al., 2006; Ozeki et al., 2005), nanoparticles (Mohanraj and Chen, 2006; Bawarski et al., 2008) and dry emulsions (Morishita and Peppas, 2006). Due to changes in the composition and thickness of the GIT mucus layer, the GIT regional pH as well as the surface area and enzyme activity, certain drugs undergo site-specific absorption (Hamman et al. 2005). The most significant site of GIT drug absorption is the small intestine (Lacombe et al. 2004, Masaoka et al., 2006). Although the small surface area and short residence time of most drugs in the stomach limits gastric absorption, gastro-retentive drug delivery systems have been used to enhance the local action of drugs in the stomach such as antidiarrheals (Connor et al., 2001), antacids (Fabregas et al., 1994), anti-ulcer agents like misoprostol (Oth et al., 2004), and to facilitate absorption of furosemide in the stomach and the upper small intestine (Streubel et al. 2006). Gastroretentive drug delivery systems are also crucial for drugs such as captopril and ranitidine that are unstable in the intestine and colon (Drummer and Jarrott, 2006) and diazepam that exhibits low solubility at high pH values (Castrol et al. 1999). 1.2. POORLY SOLUBLE DRUGS The advancement in high throughput screening technology in recent drug development era yields small molecules with characteristics like rocks. The clinical requirement for such molecules in order to elicit the therapeutic response in humans is that it needs to be dosed more than 20mg/kg/day. Because of the poor physiochemical and biopharmaceutical properties of such molecules, it is very difficult for the formulation scientist to come up with a suitable dosage form. In the case of therapeutic indications, such as HIV and cancer, it is more important to cure the disease or extend the life than the compliance. For such diseases, it can be possible to have a very high dose formulation. However, for diseases such as Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 10 Chapter 1 Introduction arthritis, diabetes and hypertension, it is very important to have a low dose formulation because it has to be taken every day for the rest of the patient's life. Over one third of drugs listed in the U.S. Pharmacopoeia and about 50% of new chemical entities (NCEs) are insoluble or poorly soluble in water. Over 40% of drug molecules and drug compounds are insoluble in the human body. In spite of this, lipophilic drug substances having low water solubility are a growing drug class having increasing applicability in a variety of therapeutic areas and for a variety of pathologies. There are over 2500 large molecules in various stages of development today, and over 5500 small molecules in development. Each of the existing companies focusing on these large and small molecules has its own restriction and limitations with regard to both large and small molecules on which they focus. Figure 1.4. Current market share of drugs based on BCS classification Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 11 Chapter 1 1.3. Introduction SOLUBILIZATION OF POORLY SOLUBLE DRUGS Therapeutic effectiveness of a drug depends upon the bioavailability and ultimately upon the solubility of drug molecules. Solubility is one of the important parameter to achieve desired concentration of drug in systemic circulation for pharmacological response to be shown. Currently only 8% of new drug candidates have both high solubility and permeability (Conference, June 2005, Thistle Marble Arch, London, UK). The solubility of a solute is the maximum quantity of solute that can dissolve in a certain quantity of solvent or quantity of solution at a specified temperature (Solubility. http://www.sciencebyjones.com/). In other words the solubility can also be defined as the ability of one substance to form a solution with another substance. The substance to be dissolved is called as solute and the dissolving fluid in which the solute dissolve is called as solvent, which together form a solution. The process of dissolving solute into solvent is called as solution or hydration if the solvent is water (http://en.wikipedia.org/wiki/Solubility). Table 1.2. Solubility definitions Definition Parts of solvent required for one part of solute Very soluble <1 Freely soluble 1 - 10 Soluble 10 - 30 Sparingly soluble 30 - 100 Slightly soluble 100 - 1000 Very slightly soluble 1000 - 10,000 Insoluble > 10,000 Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 12 Chapter 1 Introduction 1.3.1. Process of solubilisation The process of solubilisation involves the breaking of inter-ionic or intermolecular bonds in the solute, the separation of the molecules of the solvent to provide space in the solvent for the solute, interaction between the solvent and the solute molecule or ion. Step 1: Holes opens in the solvent Step 2: Molecules of the solid breaks away from the bulk Step 3: The freed solid molecule is intergrated into the hole in the solvent Figure 1.5. Steps involved in the process of solubilization 1.3.2. Factors affecting solubility The solubility depends on the physical form of the solid, the nature and composition of solvent medium as well as temperature and pressure of system (James, Solubility and related properties, Marcel Dekker). Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 13 Chapter 1 Introduction Particle Size The size of the solid particle influences the solubility because as a particle becomes smaller, the surface area to volume ratio increases. The larger surface area allows a greater interaction with the solvent. The effect of particle size on solubility can be described by Where, S is the solubility of infinitely large particles S0 is the solubility of fine particles V is molar volume γ is the surface tension of the solid r is the radius of the fine particle T is the absolute temp in degree kelvin R is the universal gas constant Temperature Temperature will affect solubility. If the solution process absorbs energy then the solubility will be increased as the temperature is increased. If the solution process releases energy then the solubility will decrease with increasing temperature. Generally, an increase in the temperature of the solution increases the solubility of a solid solute. A few solid solutes are less soluble in warm solutions. For all gases, solubility decreases as the temperature of the solution increases. Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 14 Chapter 1 Introduction Pressure For gaseous solutes, an increase in pressure increases solubility and a decrease in pressure decrease the solubility. For solids and liquid solutes, changes in pressure have practically no effect on solubility. Nature of the solute and solvent While only 1 gram of lead (II) chloride can be dissolved in 100 grams of water at room temperature, 200 grams of zinc chloride can be dissolved. The great difference in the solubilities of these two substances is the result of differences in their natures. Molecular size Molecular size will affect the solubility. The larger the molecule or the higher its molecular weight the less soluble the substance. Larger molecules are more difficult to surround with solvent molecules in order to solvate the substance. In the case of organic compounds the amount of carbon branching will increase the solubility since more branching will reduce the size (or volume) of the molecule and make it easier to solvate the molecules with solvent. Polarity Polarity of the solute and solvent molecules will affect the solubility. Generally non-polar solute molecules will dissolve in non-polar solvents and polar solute molecules will dissolve in polar solvents. The polar solute molecules have a positive and a negative end to the molecule. If the solvent molecule is also polar, then positive ends of solvent molecules will attract negative ends of solute molecules. This is a type of intermolecular force known as dipole-dipole interaction. All molecules also have a type of intermolecular force much weaker than the other forces called London Dispersion forces where the positive nuclei of Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 15 Chapter 1 Introduction the atoms of the solute molecule will attract the negative electrons of the atoms of a solvent molecule. This gives the non-polar solvent a chance to solvate the solute molecules. Polymorphs A solid has a rigid form and a definite shape. The shape or habit of a crystal of a given substance may vary but the angles between the faces are always constant. A crystal is made up of atoms, ions, or molecules in a regular geometric arrangement or lattice constantly repeated in three dimensions. This repeating pattern is known as the unit cell. The capacity for a substance to crystallize in more than one crystalline form is polymorphism. It is possible that all crystals can crystallize in different forms or polymorphs. If the change from one polymorph to another is reversible, the process is called enantiotropic. If the system is monotropic, there is a transition point above the melting points of both polymorphs. The two polymorphs cannot be converted from one another without undergoing a phase transition. Polymorphs can vary in melting point. Since the melting point of the solid is related to solubility, so polymorphs will have different solubilities. Generally the range of solubility differences between different polymorphs is only 2-3 folds due to relatively small differences in free energy (Singhal and Curatolo, 2004). 1.3.3. Techniques Of Solubility Enhancement There are various techniques available to improve the solubility of poorly soluble drugs. Some of the approaches to improve the solubility are (Pinnamaneni et al, 2002): I. Physical Modifications A. Particle size reduction a. Micronization Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 16 Chapter 1 Introduction b. Nanosuspension B. Modification of the crystal habit a. Polymorphs b. Pseudopolymorphs C. Drug dispersion in carriers a. Eutectic mixtures b. Solid dispersions c. Solid solutions D. Complexation a. Use of complexing agents E. Solubilization by surfactants: a. Microemulsions b. Self microemulsifying drug delivery systems c. Self nanoemulsifying drug delivery systems II. Chemical Modifications A. Salt Formation B. Co-crystallization C. Co-solvency D. Hydrotropic E. Solubilizing agent F. Nanotechnology Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 17 Chapter 1 Introduction I. Physical Modifications A. Particle size reduction Particle size reduction can be achieved by micronisation and nanosuspension. Each technique utilizes different equipments for reduction of the particle size. a. Micronization The solubility of drug is often intrinsically related to drug particle size. By reducing the particle size, the increased surface area improves the dissolution properties of the drug. Conventional methods of particle size reduction, such as comminution and spray drying, rely upon mechanical stress to disaggregate the active compound. The micronisation is used to increased surface area for dissolution (Chaumeil, 1998). Micronisation increases the dissolution rate of drugs through increased surface area, it does not increase equilibrium solubility (Blagden et al, 2007). Micronization of drugs is done by milling techniques using jet mill, rotor stator colloid mills etc. Micronization is not suitable for drugs having a high dose number because it does not change the saturation solubility of the drug. b. Nanosuspension Nanosuspensions are sub-micron colloidal dispersion of pure particles of drug, which are stabilised by surfactants. The advantages offered by nanosuspension is increased dissolution rate is due to larger surface area exposed, while absence of Ostwald ripening is due to the uniform and narrow particle size range obtained, which eliminates the concentration gradient factor. Techniques for the production of nanosuspensions: 1) Homogenization The suspension is forced under pressure through a valve that has nano aperture. This causes bubbles of water to form which collapses as they come out of valves. This mechanism cracks the particles. Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 18 Chapter 1 Introduction Three types of homogenizers are commonly used for particle size reduction in the pharmaceutical and biotechnology industries: conventional homogenizers, sonicators, and high shear fluid processors. 2) Wet milling Active drug in the presence of surfactant is defragmented by milling. Other technique involves the spraying of a drug solution in a volatile organic solvent into a heated aqueous solution. Rapid solvent evaporation produces drug precipitation in the presence of surfactants. The nanosuspension approach has been employed for drugs including tarazepide, atovaquone, amphotericin B, paclitaxel and bupravaquone. All the formulations are in the research stage. One major concern related to particle size reduction is the eventual conversion of the high-energy polymorph to a low energy crystalline form, which may not be therapeutically active one (Pinnamaneni et al, 2002, Aulton, Pharmaceutics, The science of dosage form design). Drying of nanosuspensions can be done by lyophilisation or spray drying. Other techniques for reduction of the particle size: 1) Sonocrystallisation Recrystallization of poorly soluble materials using liquid solvents and antisolvents has also been employed successfully to reduce particle size (Michael Hite, 2003). The novel approach for particle size reduction on the basis of crystallisation by using ultrasound is Sonocrystallisation. Sonocrystallisation utilizes ultrasound power characterised by a frequency range of 20–100 kHz for inducing crystallisation. It’s not only enhances the nucleation rate but also an effective means of size reduction and controlling size distribution of the active pharmaceutical ingredients (API) (Sheere Banga, 2004). Most applications use ultrasound in the range 20 kHz-5 MHz. Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 19 Chapter 1 Introduction 2) Supercritical fluid process Novel nanosizing and solubilization technology whose application has increased particle size reduction via supercritical fluid (SCF) processes (Vasu Kumar et al., 2004). A supercritical fluid (SF) can be defined as a dense noncondensable fluid (Irene Pasquali et al, 2006). Supercritical fluids are fluids whose temperature and pressure are greater than its critical temperature (Tc) and critical pressure (Tp). Through manipulation of the pressure of SCFs, the favorable characteristics of gases- high diffusivity, low viscosity and low surface tension may be imparted upon liquids to precisely control the solubilisation of a drug with a supercritical fluid. SCFs are high compressible, allowing moderate changes in pressure to greatly alter the density and mass transport characteristics of fluid that largely determine its solvents power. Once the drug particles are solubilised within SCFs, they may be recrystalised at greatly reduced particle sizes. A SCF process allows micronisation of drug particles within narrow range of particle size, often to sub-micron levels. Current SCF processes have demonstrated the ability to create nanoparticulate suspensions of particles 5 to 2,000 nm in diameter. The most widely employed methods of SCF processing for micronized particles are rapid expansion of supercritical solutions (RESS) and gas antisolvents recrystallisation (GAS), both of which are employed by the pharmaceutical industry using carbon dioxide (CO2) as the SCF due to its favourable processing characteristics like its low critical temperature (Tc = 31.1C) and pressure (Pc = 73.8 bar) (Hamsaraj Karanth et al, 2006). RESS involves solubilising a drug or a drug-polymer mixture in SCF and subsequently spraying the SCF solution into a lower pressure environment via a conventional nozzle or capillary tube. The rapid expansion undergone by the solution reduces the density of the CO2, correspondingly reducing its solvent power and supersaturating the lower pressure solution. This supersaturation results in the recrystallisation and precipitation of pure drug or drug-polymer particles of Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 20 Chapter 1 Introduction greatly reduced size, narrow size distribution and high purity. The solubility of nifedipine has been improved by RESS (Perrut et al, 2005). GAS processing requires the drug or drug-polymer mixture be solubilised via conventional means into a solvent that is then sprayed into an SCF; the drug should be insoluble in the SCF, while the SCF should be miscible with the organic solvent. The SCF diffuses into the spray droplets, causing expansion of the solvent present and precipitation of the drug particles. The low solubility of poorly water-soluble drugs and surfactants in supercritical CO2 and the high pressure required for these processes restrict the utility of this technology in the pharmaceutical industry (Bhupendra et al, http://www.pharmaquality.com/). 3) Spray drying Spray drying is a commonly used method of drying a liquid feed through a hot gas. Typically, this hot gas is air but sensitive materials such as pharmaceuticals and solvents like ethanol require oxygen-free drying and nitrogen gas is used instead. The liquid feed varies depending on the material being dried and is not limited to food or pharmaceutical products and may be a solution, colloid or a suspension. This process of drying is a one step rapid process and eliminates additional processing. Spray drying of the acid dispersed in acacia solutions resulted in as much as a 50% improvement in the solubility of poorly water soluble salicylic acid (Kawashima et al, 1975). B. Modification of the crystal habit Polymorphism is the ability of an element or compound to crystallize in more then one crystalline form. Different polymorphs of drugs are chemically identical, but they exhibit different physicochemical properties including solubility, melting point, density, texture, stability etc. Broadly polymorphs can be classified as enantiotropes and monotropes based on thermodynamic properties. In the case of an enantiotropic system, one polymorphs form can change reversibly Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 21 Chapter 1 Introduction into another at a definite transition temperature below the melting point, while no reversible transition is possible for monotropes. Once the drug has been characterized under one of this category, further study involves the detection of metastable form of crystal. Metastable forms are associated with higher energy and thus higher solubility. Similarly the amorphous form of drug is always more suited than crystalline form due to higher energy associated and increase surface area. Generally, the anhydrous form of a drug has greater solubility than the hydrates. This is because the hydrates are already in interaction with water and therefore have less energy for crystal breakup in comparison to the anhydrates (i.e. thermodynamically higher energy state) for further interaction with water. On the other hand, the organic (nonaqueous) solvates have greater solubility than the nonsolvates. Some drugs can exist in amorphous form (i.e. having no internal crystal structure). Such drugs represent the highest energy state and can be considered as super cooled liquids. They have greater aqueous solubility than the crystalline forms because they require less energy to transfer a molecule into solvent. Thus, the order for dissolution of different solid forms of drug is Amorphous >Metastable polymorph >Stable polymorph Melting followed by a rapid cooling or recrystallization from different solvents can be produce metastable forms of a drug. C. Drug dispersion in carriers The solid dispersion approach to reduce particle size and therefore increase the dissolution rate and absorption of drugs was first recognised in 1961 (Sekiguchi and Obi, 1961).The term “solid dispersions” refers 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 method, or fusion solventmethod (Chiou and Riegelman, 1971). Novel additional preparation techniques Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 22 Chapter 1 Introduction have included rapid precipitation by freeze drying (Emara et al, 2002) and using supercritical fluids (Juppo et al, 2003) and spray drying (Kai et al, 1996), often in the presence of amorphous hydrophilic polymers and also using methods such as melt extrusion (Forster et al, 2001). The most commonly used hydrophilic carriers for solid dispersions include polyvinylpyrrolidone (Ambike et al, 2004; Paradkar et al, 2004), polyethylene glycols (Doshi et al, 1967), Plasdone-S630 (Alazar et al, 2007). Many times surfactants may also used in the formation of solid dispersion. Surfactants like Tween-80, Docusate sodium, Myrj-52, Pluronic-F68 and Sodium Lauryl Sulphate used. The solubility of etoposide, glyburide, itraconazole, ampelopsin, valdecoxib, celecoxib, halofantrine can be improved by solid dispersion using suitable hydrophilic carriers. The eutectic combination of chloramphenicol/urea and sulphathiazole/ urea served as examples for the preparation of a poorly soluble drug in a highly water soluble carrier. 1) Hot Melt method Sekiguchi and Obi used a hot melt method to prepare solid dispersion. Sulphathiazole and urea were melted together and then cooled in an ice bath. The resultant solid mass was then milled to reduce the particle size. Cooling leads to supersaturation, but due to solidification the dispersed drug becomes trapped within the carrier matrix. A molecular dispersion can be achieved or not, depends on the degree of supersaturation and rate of cooling used in the process (Christian Leuner and Jennifer Dressman, 2000). An important requisite for the formation of solid dispersion by the hot melt method is the miscibility of the drug and the carrier in the molten form. When there are miscibility gaps in the phase diagram, this usually leads to a product that is not molecularly dispersed. Another important requisite is the thermostability of the drug and carrier. Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 23 Chapter 1 Introduction 2) Solvent Evaporation Method Tachibana and Nakumara, 1965 were the first to dissolve both the drug and the carrier in a common solvent and then evaporate the solvent under vacuum to produce a solid solution. This enabled them to produce a solid solution of the highly lipophilic β-carotene in the highly water soluble carrier polyvinylpyrrolidone. An important prerequisite for the manufacture of a solid dispersion using the solvent method is that both the drug and the carrier are sufficiently soluble in the solvent (Christian Leuner and Jennifer Dressman, 2000). The solvent can be removed by various methods like by spray-drying or by freeze-drying. Temperatures used for solvent evaporation generally lie in the range 23-65ºC. The solid dispersion of the 5- lipoxygenase/cyclooxygenase inhibitor ER34122 shown improved in-vitro dissolution rate compared to the crystalline drug substance which was prepared by solvent evaporation (Kushida et al, 2002). These techniques have problems such as negative effects of the solvents on the environment and high cost of production due to extra facility for removal of solvents (Serajuddin, 1999). Due to the toxicity potential of organic solvents employed in the solvent evaporation method, hot melt extrusion method is preferred in preparing solid solutions. 3) Hot-melt Extrusion Melt extrusion was used as a manufacturing tool in the pharmaceutical industry as early as 1971 (el-Egakey et al, 1971). It has been reported that melt extrusion of miscible components results in amorphous solid solution formation, whereas extrusion of an immiscible component leads to amorphous drug dispersed in crystalline excipient (Breitenbach, 2002). The process has been useful in the preparation of solid dispersions in a single step. Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 24 Chapter 1 Introduction 4) Melting –solvent method A drug is first dissolved in a suitable liquid solvent and then this solution is incorporated into the melt of polyethylene glycol, obtainable below 70ºC without removing the liquid solvent. The selected solvent or dissolved drug may not be miscible with the melt of the polyethylene glycol. Also polymorphic form of the drug precipitated in the solid dispersion may get affected by the liquid solvent used. Table 1.3. Carriers for Solid Dispersions Sr. No. Chemical Class Examples 1 Acids Citric acid, Tartaric acid, Succinic acid 2 Sugars Dextrose, Sorbitol, Sucrose, Maltose, Galactose, Xylitol 3 Polymeric Materials Polyvinylpyrrolidone, PEG-4000, PEG6000, Carboxymethyl cellulose, Hydroxypropyl cellulose, Guar gum, Xanthan gum, Sodium alginate, Methylcellulose, HPMC, Dextrin, Cyclodextrins, Galactomannan 4 Surfactants Polyoxyethylene stearate, Poloxamer, Deoxycholic acid, Tweens and Spans, Gelucire 44/14, Vitamine E TPGS NF 5 Miscellaneous Pentaerythritol, Urea, Urethane, Hydroxyalkyl xanthines Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 25 Chapter 1 Introduction D. Complexation Complexation is the association between two or more molecules to form a nonbonded entity with a well defined stichiometry. Complexation relies on relatively weak forces such as London forces, hydrogen bonding and hydrophobic interactions. There are many types of complexing agents and a partial list can be found in below table. Table 1.4. List of Complexing Agents Sr.No. Types Examples 1 Inorganic IB- 2 Coordination Hexamine cobalt(III) chloride 3 Chelates EDTA, EGTA 4 Metal-Olefin Ferrocene 5 Inclusion Cyclodextrins, Choleic acid 6 Molecular Complexes Polymers 1) Staching complexation Staching complexes are formed by the overlap of the planar regions of aromatic molecules. Nonpolar moieties tend to be squeezed out of water by the strong hydrogen bonding interactions of water. This causes some molecules to minimize the contact with water by aggregation of their hydrocarbon moieties. This aggregation is favored by large planar nonpolar regions in the molecule. Stached complexes can be homogeneous or mixed. The former is known as self Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 26 Chapter 1 Introduction association and latter as complexation. Some compounds that are known to form staching complexes are as follows: Nicotinamide, Anthracene, Pyrene, Methylene blue, Benzoic acid, Salicylic acid, Ferulic acid, Gentisic acid, Purine, Theobromine, Caffeine, and Naphthalene etc. Higuchi and Kristiansen, 1970 proposed a model according to which the compounds capable of undergoing stacking can be classified into two classes (classes A and B) based on their structure. The compounds in class A have higher affinity for compounds in class B than for those in class A and vice versa (Rajewski and Stella, 1996). 2) Inclusion complexation Inclusion complexes are formed by the insertion of the nonpolar molecule or the nonpolar region of one molecule (known as guest) into the cavity of another molecule or group of molecules (known as host). The major structural requirement for inclusion complexation is a snug fit of the guest into the cavity of host molecule. The cavity of host must be large enough to accommodate the guest and small enough to eliminate water, so that the total contact between the water and the nonpolar regions of the host and the guest is reduced. The most commonly used host molecules are cyclodextrins. The enzymatic degradation of starch by cyclodextrin-glycosyltransferase (CGT) produces cyclic oligomers, Cyclodextrins. Cyclodextrins are non-reducing, crystalline, water soluble, cyclic, oligosaccharides. Cyclodextrins consist of glucose monomers arranged in a donut shape ring. Three naturally occurring CDs are α-Cyclodextrin, β-Cyclodextrin, and γ- Cyclodextrin. The complexation with cyclodextrins is used for enhancement of solubility (Kaneto Uekama et al, 1998). Cyclodextrin inclusion is a molecular phenomenon in which usually only one guest molecule interacts with the cavity of a cyclodextrin molecule to become entrapped and form a stable association. The internal surface of cavity is hydrophobic and external is hydrophilic, this is due to the arrangement of hydroxyl group within the molecule. Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 27 Chapter 1 Introduction Molecules or functional groups of molecules those are less hydrophilic than water, can be included in the cyclodextrin cavity in the presence of water. In order to become complex, the "guest molecules" should fit into the cyclodextrin (CD) cavity. The cavity sizes as well as possible chemical modifications determine the affinity of cyclodextrins to the various molecules. The kinetics of cyclodextrin inclusion complexation has been usually analyzed in terms of a one-step reaction or a consecutive two-step reaction involving intracomplex structural transformation as a second step. Cyclodextrins is to enhance aqueous solubility of drugs through inclusion complexation. It was found that cyclodextrins increased the paclitaxel solubility by 950 fold (Anil Singla et al, 2002). Complex formation of rofecoxib, celecoxib, clofibrate, melarsoprol, taxol, cyclosporin A etc. with cyclodextrins improves the solubility of particular drugs. Factors affecting complexation: 1. Steric effects 2. Electronic effects a. Effect of proximity of charge to CD cavity b. Effect of charge density c. Effect of charge state of CD and drug 3. Temperature, additives and cosolvent effects E. Solubilization by surfactants Surfactants are molecules with distinct polar and nonpolar regions. Most surfactants consist of a hydrocarbon segment connected to a polar group. The polar group can be anionic, cationic, zwitterionic or non-ionic (Swarbrick and Boylan, 2002). When small apolar molecules are added they can accumulate in the hydrophobic core of the micelles. This process of solubilization is very important Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 28 Chapter 1 Introduction in industrial and biological processes. The presence of surfactants may lower the surface tension and increase the solubility of the drug within an organic solvent. Microemulsion The term microemulsion was first used by Jack H. Shulman in 1959. A microemulsion is a four-component system composed of external phase, internal phase, surfactant and cosurfactant. The addition of surfactant, which is predominately soluble in the internal phase unlike the cosurfactant, results in the formation of an optically clear, isotropic, thermodynamically stable emulsion. It is termed as microemulsion because of the internal or dispersed phase is < 0.1 μ droplet diameter. The formation of microemulsion is spontaneous and does not involve the input of external energy as in case of coarse emulsions. The surfactant and the cosurfactant alternate each other and form a mixed film at the interface, which contributes to the stability of the microemulsions (Lawrence and Rees, 2000). Non-ionic surfactants, such as Tweens (polysorbates) and Labrafil (polyoxyethylated oleic glycerides), with high hyrophile-lipophile balances are often used to ensure immediate formation of oil-in-water droplets during production. Advantages of microemulsion over coarse emulsion include its ease of preparation due to spontaneous formation, thermodynamic stability, transparent and elegant appearance, increased drug loading, enhanced penetration through the biological membranes, increased bioavailability (Tenjarla, 1999), and less interand intra-individual variability in drug pharmacokinetics (Kovarik et al, 1994). II. Chemical Modifications A. Salt Formation It is the most common and effective method of increasing solubility and dissolution rates of acidic and basic drugs. Acidic or basic drug converted into salt having more solubility than respective drug. Ex. Aspirin, Theophylline, Barbiturates (Sinko, 2006). Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 29 Chapter 1 Introduction B. Co-crystallisation The new approach available for the enhancement of drug solubility is through the application of the co-crystals, it is also referred as molecular complexes. If the solvent is an integral part of the network structure and forms at least two component crystal, then it may be termed as co-crystal. If the solvent does not participate directly in the network itself, as in open framework structures, then it is termed as clathrate (inclusion complex). A co-crystal may be defined as a crystalline material that consists of two or more molecular (and electrically neutral) species held together by non-covalent forces (Aakeröy, 1997). Co-crystals are more stable, particularly as the co-crystallizing agents are solids at room temperature. Only three of the co-crystallizing agents are classified as generally recognised as safe (GRAS) it includes saccharin, nicotinamide and acetic acid limiting the pharmaceutical applications. Co-crystallisation between two active pharmaceutical ingredients has also been reported. This may require the use of subtherapeutic amounts of drug substances such as aspirin or acetaminophen (Almarsson and Zaworotko, 2004). At least 20 have been reported to date, including caffeine and glutaric acid polymorphic co-crystals (Trask et al, 2004). Co-crystals can be prepared by evaporation of a heteromeric solution or by grinding the components together. Another technique for the preparation of cocrystals includes sublimation, growth from the melt, and slurry preparation (Zaworotko, 2005). The formation of molecular complexes and co-crystals is becoming increasingly important as an alternative to salt formation, particularly for neutral compounds or those having weakly ionizable groups. C. Co-solvency The solubilisation of drugs in co-solvents is another technique for improving the solubility of poorly soluble drug (Amin et al, 2004). It is wellknown that the addition of an organic cosolvent to water can dramatically change the solubility of drugs (Yalkowsky,S.H. and Roseman, 1981). Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 30 Chapter 1 Introduction Weak electrolytes and nonpolar molecules have poor water solubility and it can be improved by altering polarity of the solvent. This can be achieved by addition of another solvent. This process is known as cosolvency. Solvent used to increase solubility known as cosolvent. Cosolvent system works by reducing the interfacial tension between the aqueous solution and hydrophobic solute. It is also commonly referred to as solvent blending (Joseph, 2002). Most cosolvents have hydrogen bond donor and/or acceptor groups as well as small hydrocarbon regions. Their hydrophilic hydrogen bonding groups ensure water miscibility, while their hydrophobic hydrocarbon regions interfere with waters hydrogen bonding network, reducing the overall intermolecular attraction of water. By disrupting waters self-association, cosolvents reduce waters ability to squeeze out non-polar, hydrophobic compounds, thus increasing solubility. A different perspective is that by simply making the polar water environment more non-polar like the solute, cosolvents facilitate solubilization (Jeffrey et al., 2002). Solubility enhancement as high as 500-fold is achieved using 20% of 2pyrrolidone. D. Hydrotrophy Hydrotrophy designate the increase in solubility in water due to the presence of large amount of additives. The mechanism by which it improves solubility is more closely related to complexation involving a weak interaction between the hydrotrophic agents (sodium benzoate, sodium acetate, sodium alginate, and urea) and the solute. Example: Solubilisation of Theophylline with sodium acetate and sodium alginate E. Solubilizing agents The solubility of poorly soluble drug can also be improved by various solubilizing materials. PEG 400 is improving the solubility of hydrochlorthiazide85. Modified gum karaya (MGK), a recently developed excipient was evaluated as carrier for dissolution enhancement of poorly soluble Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 31 Chapter 1 Introduction drug, nimodipine (Murali Mohan Babu et al., 2002). The aqueous solubility of the antimalarial agent halofantrine is increased by the addition of caffeine and nicotinamide (Lee-Yong Lim and Mei-Lin Go, 2000). F. Nanotechnology approaches Nanotechnology will be used to improve drugs that currently have poor solubility. Nanotechnology refers broadly to the study and use of materials and structures at the nanoscale level of approximately 100 nanometers (nm) or less. For many new chemical entities of very low solubility, oral bioavailability enhancement by micronisation is not sufficient because micronized product has the tendency of agglomeration, which leads to decreased effective surface area for dissolution and the next step taken was Nanonisation. a. Nanocrystal A nanocrystal is a crystalline material with dimensions measured in nanometers; a nanoparticle with a structure that is mostly crystalline. The nanocrystallization is defined as a way of diminishing drug particles to the size range of 1-1000 nanometers. Nanocrystallization is thought to be a universal method that can be applied to any drug (Radtke, 2001). There are two distinct methods used for producing nanocrystals; ’bottomup’ and ’top-down’ development. The top-down methods (i.e. Milling and High pressure homogenization) start milling down from macroscopic level, e.g. from a powder that is micron sized. In bottom-up methods (i.e. Precipitation and Cryovacuum method), nanoscale materials are chemically composed from atomic and molecular components. 1) Milling Nanoscale particles can be produced by wet-milling process. In ball mills, particle size reduction is achieved by using both impact and attrition forces. The Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 32 Chapter 1 Introduction most common models are a tumbling ball mill and a stirred media mill. One problem of this method is the degradation of mill surfaces and subsequent suspension contamination. 2) High pressure homogenization In high pressure homogenization, an aqueous dispersion of the crystalline drug particles is passed with high pressure through a narrow homogenization gap with a very high velocity. Homogenisation can be performed in water (DissoCubes) or alternatively in non-aqueous media or water-reduced media (Nanopure). The particles are disintegrated by cavitation and shear forces. The static pressure exerted on the liquid causes the liquid to boil forming gas bubbles. When exiting from the gap, gas bubbles collapse under normal air pressure. This produces shock waves which make the crystals collide, leading to particle disintegration. A heat exchanger should be used when operating on temperature sensitive materials because high pressure homogenization causes increase in the sample temperature. The particle size obtained during the homogenization process depends primarily on the nature of the drug, the pressure applied and the number of homogenization cycles. 3) Precipitation In the precipitation method a dilute solution is first produced by dissolving the substance in a solvent where its dissolution is good. The solution with the drug is then injected into water, which acts as a bad solvent. At the time of injection, the water has to be stirred efficiently so that the substance will precipitate as nanocrystals. Nanocrystals can be removed from the solution by filtering and then dried in air. 4) Cryo-vacuum method In the cryo-vacuum method the active ingredient to be nanonized is first dissolved in water to attain a quasi-saturated solution (Salvadori et al., 2006). The method is based on sudden cooling of a solvent by immersing the solution in Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 33 Chapter 1 Introduction liquid nitrogen (-196ºC). Rapid cooling causes a very fast rise in the degree of saturation based on the decrease of solubility and development of ice crystals when the temperature drops below 0ºC. This leads to a fast nucleation of the dissolved substance at the edges of the ice crystals. The solvent must be completely frozen before the vessel is removed from the liquid nitrogen. Next the solvent is removed by sublimation in a lyophilization chamber where the temperature is kept at constant -22ºC and the pressure is lowered to 10-2 mbar. Cryo-assisted sublimation makes it possible to remove the solvent without changing the size and habit of the particles produced, so they will remain crystalline. The method yields very pure nanocrystals since there is no need to use surfactants or harmful reagents. b. NanoMorph The NanoMorph technology is to convert drug substances with low watersolubility from a coarse crystalline state into amorphous nanoparticles. A suspension of drug substance in solvent is fed into a chamber, where it is rapidly mixed with another solvent. Immediately the drug substance suspension is converted into a true molecular solution. The admixture of an aqueous solution of a polymer induces precipitation of the drug substance. The polymer keeps the drug substance particles in their nanoparticulate state and prevents them from aggregation or growth. Water redispersable dry powders can be obtained from the nanosized dispersion by conventional methods, e.g. spray-drying. Using this technology the coarse crystalline drug substances are transformed into a nanodispersed amorphous state, without any physical milling or grinding procedures. It leads to the preparation of amorphous nanoparticles. Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 34 Chapter 1 Introduction Table 1.5. Nanotechnology approaches to improve the solubility of hydrophobic drugs Company Nanoparticulate Description Technologies Elan NanoCrystal NanoCrystal drug particles (<1,000 nm) produced by wet-milling and stabilised against agglomeration through surface adsorption of stabilisers; applied to NMEs eg aprepitant/reformulation of existing drugs eg. sirolimus Eurand Biorise Nanocrystals/amorphous drug produced by physical breakdown of the crystal lattice and stabilised with biocompatible carriers (swellable microparticles or cyclodextrins) SkyePharma IDD Insoluble Drug Delivery: micro-nm particulate/droplet water-insoluble drug core stabilised by phospholipids; formulations are produced by high shear, cavitation or impaction BioSante CAP Calcium Phosphate-based nanoparticles: for improved oral bioavailability of hormones/proteins such as insulin; also as vaccine adjuvant Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 35 Chapter 1 American Introduction NAB Bioscience Nanoparticle Albumin-Bound technology: injectable suspension of biocompatible protein with drug improves solubility/removes need for toxic solvents; eg paclitaxel-albumin nanoparticles Nanoparticle Albumin-Bound technology: injectable suspension of biocompatible protein with drug improves solubility/removes need for toxic solvents; eg paclitaxel-albumin nanoparticles Baxter Nanoedge Nanoedge technology: drug particle size reduction to nanorange by platforms including direct homogenisation, microprecipitation, lipid emulsions and other dispersed-phase technology Company Nanostructuring Description Technologies pSivida BioSilicon Drug particles are structured within the nano-width pores of biocompatible BioSilicon microparticles, membranes or fibres; gives controlled release/improves solubility of hydrophobic drugs iMEDD NanoGate Silicon membrane with nano-width pores (10-100 nm) used as part of an implantable system for drug delivery and biofiltration Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 36 Chapter 1 Introduction PharmaSol NLC8 Nanostructured Lipid Carriers: nanostructured lipid particle dispersions with solid contents produced by highpressure homogenisation; lipid-drug conjugate nanoparticles provide highloading capacity for hydrophilic drugs for oral delivery 1.3.4. Need of Solubility Enhancement The better characterization of biochemical targets increasingly drives drug development; these targets are generally cell-based and access to them in these models is relatively straightforward. This has led to the widely discussed proliferation of highly active compounds that have physicochemical characteristics that are poorly suited to delivery to a whole organism: at the head of this list of undesirable characteristics is poor water solubility (Perrett and Venkatesh, 2006). According to recent estimates, nearly 40% of new chemical entities are rejected because of poor solubility i.e. biopharmaceutical properties. Solubility is one of the important parameter to achieve desired concentration of drug in systemic circulation for pharmacological response to be shown. Poor aqueous solubility is caused by two main factors 1) High lipophilicity and 2) Strong intermolecular interactions which make the solubilisation of the solid energetically costly. Solubility of active pharmaceutical ingredients (APIs) has always been a concern for formulators, since inadequate aqueous solubility may hamper development of products and limit bioavailability of oral products. Solubility Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 37 Chapter 1 Introduction plays an essential role in drug disposition, since the maximum rate of passive drug transport across a biological membrane, the main pathway for drug absorption, is the product of permeability and solubility. Among the five key physicochemical screens in early compound screening, pKa, solubility, permeability, stability and lipophilicity, poor solubility tops of the list of undesirable compound properties. Compounds with insufficient solubility carry a higher risk of failure during discovery and development since insufficient solubility may compromise other properties, influence both pharmacokinetic and pharmacodynamic properties of the compound, and finally may affect the ability of the compound to develop as API . Poorly water-soluble drug candidates often emerge from contemporary drug discovery programs, and present formulators with considerable technical challenges (Pouton, 2006). The poor solubility and low dissolution rate of poorly water soluble drugs in the aqueous gastro-intestinal fluids often cause insufficient bioavailability. Especially for class II substances according to the Biopharmaceutics Classification System (BCS), the bioavailability may be enhanced by increasing the solubility and dissolution rate of the drug in the gastro-intestinal fluids (Urbanetz, 2006). Consideration of the modified NoyesWhitney equation provides some hints as to how the dissolution rate of even very poorly soluble compounds might be improved to minimize the limitations to oral availability: dC/ dt = AD (Cs - C)/ h Where, dC/dt is the rate of dissolution, A is the surface area available for dissolution, D is the diffusion coefficient of the compound, Cs is the solubility of the compound in the dissolution medium, C is the concentration of drug in the medium at time t, h is the thickness of the diffusion boundary layer adjacent to the surface of the dissolving compound. Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 38 Chapter 1 Introduction The main possibilities for improving dissolution according to this analysis are to increase the surface area available for dissolution by decreasing the particle size of the solid compound and/or by optimizing the wetting characteristics of the compound surface, to decrease the boundary layer thickness, to ensure sink conditions for dissolution and, last but definitely not least, to improve the apparent solubility of the drug under physiologically relevant conditions. Larger the surface area, higher will be the dissolution rate. Since the surface area increases with decreasing particle size, which can be accomplished by conventional methods like trituration, grinding, ball milling, fluid energy micronization, salt formation and controlled precipitation. Although these conventional methods have been used commonly to increase dissolution rate of drug, there are practical limitation with these techniques as the desired bioavailability enhancement may not always be achieved. Therefore, formulation approaches are being explored to enhance bioavailability of poorly soluble drugs. One such formulation approach that has been shown to significantly enhance absorption of such drugs is to formulate self emulsifying drug delivery system (SEDDS). 1.4. SELF-EMULSIFYING/ MICROEMULSIFYING DRUG DELIVERY SYSTEMS Self-Emulsifying / Microemulsifying drug delivery systems (S(M)EDDS) are isotropic mixtures of oil, hydrophilic surfactant and/or a cosurfactant, and a solubilized drug. They can be encapsulated in hard or soft gelatin capsules or can be converted to solid state (Solid SEDDS/SMEDDS). These formulations spontaneously form a fine oil-in-water emulsion in case of SEDDS and a nanoemulsion in the case of SMEDDS upon dilution with water. In the GI tract, they are readily dispersed, where the motility of the stomach and small intestine provides the gentle agitation necessary for emulsification. SEDDS produces Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 39 Chapter 1 Introduction coarse emulsions while SMEDDS produces droplets of size less than 100 nm. This property of S(M)EDDS makes them a natural choice for delivery of hydrophobic drugs that have adequate solubility in oil-surfactant blends. S(M)EDDS improves the rate and extent of absorption of hydrophobic drugs, whose absorption is considered to be dissolution rate-limited. Upon aqueous dilution the drug remains in the oil droplets or as a micellar solution since the surfactant concentration is very high in such formulations (Pouton and Porter, 2008). The drug in the oil droplet may partition out in the intestinal fluid as shown in figure 1.6. Figure 1.6. Mechanism of drug partitioning in S(M)EDDS Potential advantages of these systems include; 1. Enhanced oral bioavailability enabling reduction in dose, 2. More consistent temporal profiles of drug absorption, 3. Selective targeting of drug(s) toward specific absorption window in GIT, 4. Protection of drug(s) from the hostile environment in gut. Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 40 Chapter 1 Introduction 5. Control of delivery profiles 6. Reduced variability including food effects 7. Protective of sensitive drug substances 8. High drug payloads 9. Liquid or solid dosage forms 1.4.1. Excipient selection for lipid based formulations Chemically, lipids are considered as one of the most versatile excipient classes available today. There are various subcategories of lipids available and there is a constant influx of new lipid based excipients in the market. This provides flexibility to the formulator in terms of selecting a suitable excipient, but at the same time the formulator should be cautious while selecting a particular excipient. Pouton et al. described few factors that should be considered while selecting a lipid excipient. They are: (a) regulatory issues-irritancy, toxicity (b) solvent capacity (c) miscibility (d) morphology at room temperature (e) selfdispersibility (f) digestibility and fate of digested products (g) capsule compatibility (h) purity, chemical stability and (i) cost. The following description on lipid based excipients is in relation to the S(M)EDDS. 1.4.1.1. Oils Oils play a critical role in S(M)EDDS because it is responsible for solubilization of the hydrophobic drug, aiding in self-emulsification and moreover contributes to the intestinal lymphatic transport of the drug. The emulsification property of the oil is said to be dependent on the molecular structure of the oil (Kimura et al., 1994). Oils used in self-dispersing systems can be classified into three categories. Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 41 Chapter 1 Introduction Triglyceride vegetable oils: They are easily ingested, digested and absorbed presenting no safety issues. Depending on the vegetable source, they can have different proportions of long chain triglycerides (LCT) and medium chain triglycerides (MCT). Generally vegetable oils are rich in unsaturated LCT with the exception of coconut oil and palm kernel oil which are rich in saturated MCT. They are highly lipophilic and their effective concentration of ester group determines its solvent capacity. MCT’s are preferred over LCT’s in lipid based drug delivery owing to its good solvent capacity and resistance to oxidation. Vegetable oils are not widely used in SEDDS because of their poor solubility for the hydrophobic drug and due to poor self dispersing property. Vegetable oils derivatives: Popular vegetable oil derivatives are hydrogenated vegetable oil, mixed glycerides, polyoxylglycerides, ethoxylated glycerides and esters of fatty acids with various alcohols. Hydrogenated vegetable oils are produced by hydrogenation of the unsaturated bonds present in the oil. Usually vegetable oils are hydrogenated before they are transformed into their derivatives since hydrogenation increases chemical stability. Examples of such oils are hydrogenated cottonseed oil (Lubritab), hydrogenated palm oil (Dynasan), hydrogenated castor oil (Cutina HR) and hydrogenated soybean oil (Lipo). Mixed Partial Glycerides: They are formed by partial hydrolysis of triglycerides present in the vegetable oil resulting in a mixture of mono-,di- and tri-glycerides. The physical state, melt characteristics, and the HLB of the partial glycerides depend on the nature of the fatty acid present and the degree of esterification. Glycerides with medium chain or unsaturated fatty acids are used for improving bioavailability, while ones with saturated long chain fatty acids are used for sustained-release purposes (Jannin et al., 2008). Examples of glycerides with medium chain fatty acids are glyceryl monocaprylocaprate (Capmul MCM) and Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 42 Chapter 1 Introduction ones with long chain fatty acids are glyceryl monoleate (Peceol) and glyceryl monolinoleate (Maisine 35-1). Polyoxylglycerides / Macrogolglycerides: They are formed by polyglycolysis of vegetable oil (hydrogenated or not hydrogenated) with polyethylene glycols (PEG) of a particular molecular weight. It has a fixed composition of a mixture of mono-, di- and triglycerides and mono and diesters of PEG. They are readily dispersible in water making them a good choice for SEDDS. Like glycerides, they may be composed of unsaturated long chain fatty acids such as oleyl polyoxylglycerides (Labrafil 1944CS) and linoleyl polyoxylglycerides (Labrafil M 2125CS) or medium chain fatty acids such as caprylocaproyl polyoxylglycerides (Labrasol) and lauroyl polyoxylglycerides (Gelucire 44/14). Ethoxylated glycerides: They are formed from ethoxylation (etherification) of ricinoleic acid (present in glyceride) of castor oil. This reaction makes the oil hydrophilic. Examples of such glycerides are ethoxylated castor oil (Cremphor EL) and ethoxylated hydrogenated castor oil (Cremophor RH40 and Cremophor RH 60). Because of its amphiphilic nature, Cremophor’s are widely used as surfactants in the formulation of SEDDS. Moreover, they can dissolve large quantities of drugs, have good self-emuslification property, and their degradation products are similar to those obtained from intestinal digestion (Gershanik and Benita, 2000; Constantinides, 1995). Polyalcohol esters of fatty acids: These are newer oil derivatives that possess surfactant properties because of its amphiphilic nature and are effective in replacing conventionally used oils. Their composition is based on nature of alcohol used. They can be polyglycerol (Plurol Oleique CC 497), and propylene glycol (Capryol), and polyoxyethylene glycol (Mirj). Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 43 Chapter 1 1.4.1.2. Introduction Surfactants Surfactants are surface active molecules which concentrate at the oil-water interface and stabilize the internal phase in an emulsion. Surfactants are critical components of S(M)EDDS systems since they are responsible for forming a stable emulsion upon aqueous dilution. Nonionic surfactants are commonly used in this type of formulation. Proper selection of the surfactant is based on its Hydrophilic Lipophilic Balance (HLB) value and safety considerations. Nonionic surfactants with high hydrophilicity are required for SEDDS. A surfactant with an HLB value of more than 12 is necessary in SMEDDS to spontaneously form a fine oil-inwater nanoemulsion when dispersed in the GI tract fluids. Surfactants used in lipid based drug delivery are usually polyethoxylated lipid derivatives (Pouton, 2007). These lipids can be fatty acids, alcohols or glycerides which are linked to a certain number of repeating polyexthylene oxide units through ester linkage (fatty acids and glycerides) and ether linkage (alcohols). The polyethylene groups provide hydrophilic characteristics to the surfactant. Examples of such surfactants are polyethoxylated fatty acid ester (Myrj and Solutol HS 15), polyethoxylated alkyl ethers(Brij), polyethoxylated sorbitan esters (Tweens), and polyethoxylated glycerides (Cremphors, Labrasol). The most commonly used surfactants in SMEDDS are Tweens, Cremophors, and Labrasols. Block copolymers such as Pluronics have also been used in SEDDS (Singh et al, 2009). Emulsifiers of natural origin are preferred due to safety considerations but are not widely used because of their poor self emulsification property (Constantinides, 1995). Nonionic surfactants are less toxic and possess good emulsion stability over wider range of ionic strength and pH than ionic surfactants (Tenjarla, 1999), but may cause changes in intestinal lumen permeability (Swenson et al, 1994). The surfactant concentration necessary to form a stable S(M)EDDS ranges from 30%w/w to 60%w/w (Fanun, 2010). The least possible surfactant concentration should be used so as to prevent gastric irritation. Extremely small droplet size Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 44 Chapter 1 Introduction produced in case of SMEDDS promotes rapid gastric emptying and low local concentration of surfactant, thereby reducing the gastric irritation (Charman et al., 1992). The surfactant concentration is shown to have varied effects on emulsion droplet size. Increase in surfactant concentration causes a decrease in droplet size associated with stabilization of surfactant molecules at the oil-water interface (Neslihan Gursoy and Benita, 2004), while the reverse is possible due to enhanced water penetration into oil droplets leading to breakdown of oil droplets (Pouton, 1997). The surfactants being amphiphilic can dissolve large quantities of the hydrophobic drug. They can contribute to the total solubility of the drug in S(M)EDDS, thus preventing drug precipitation upon aqueous dilution and keep the drug in solubilized state in GI tract for further absorption (Neslihan Gursoy and Benita, 2004). 1.4.1.3. Cosolvents Water soluble cosolvents are widely used in lipid based dosage forms. Ethanol, polyethylene glycol (PEG), propylene glycol, and glycerol are examples of cosolvents used. Their role is: (a) to increase the solvent capacity of the drugs which are freely soluble in them. But this is associated with the risk of drug precipitation when S(M)EDDS are dispersed in water, (b) to dissolve large quantities of the hydrophilic surfactant in the oil. S(M)EDDS requires use of high concentration of surfactants to ensure proper dispersion of the formulation, (c) to increase the stability of nanoemulsion by wedging themselves between surfactant molecules (Benita, 2006). There are several key issues that have to be considered before using a particular cosolvent. The cosolvents are miscible with the oil only up to a certain limit. There are some incompatibilities of using alcohol since it may penetrate into soft and hard gelatin shell causing precipitation of the drug. Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 45 Chapter 1 Introduction 1.4.2. Role of SEDDS/SMEDDS in improvement of oral absorption S(M)EDDS partially avoids the additional drug dissolution step prior to absorption in the GI tract. They increase the amount of solubilized drug in the intestinal fluids resulting in good drug absorption. Apart from this, absorption of the drug may also be enhanced by using lipid based excipients in the formulation. There are several mechanisms through which increased absorption can be achieved; the following schematic diagram describes these mechanisms. Figure 1.7. Pathways for drug absorption from lipid based formulations Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 46 Chapter 1 Introduction Retardation of gastric emptying time: Surfactants are believed to play a role in retardation of gastric transit time, thereby increasing the time available for the drug to dissolve and get absorbed. Surfactants may slow down gastric emptying for a period of time by formation of viscous mass in the gastric and intestinal lumen. Labrasol (a caprylocaproyl macrogolglyceride) was shown to improve bioavailability of an investigational compound by retarding gastric emptying time (Chang and Shojaei, 2004). Increase in effective drug solubility in lumen: When exogenous lipid excipients are encountered in the gastric environment, they are digested by gastric lipases. Triglycerides are digested to di-glycerides and fatty acids. The duodenum secretes bile salts (BS), phosphatidylcholine (PL) and cholesterol (Ch) from the gall bladder and pancreatic lipases from pancreas. These agents in combination with lipid digestion products get adsorbed to the surface of emulsion droplet and transform into small, stable droplets. They also produce a series of colloidal particles such as micelles, mixed micelles, and vesicles as shown in figure 1.6. The drug contained in the oil droplet partitions into these micellar structures making them a drug reservoir at the absorption site. This results in an increased solubilization capacity of the drug in the GI tract. This capacity is dependent on the type (medium chain or long chain triglycerides) and quantity of the lipids, presence of additional lipid excipients such as surfactants and cosurfactants, and the level of endogenous BS and PL present (Porter et al., 2008). The micelles and nanoemulsions can be absorbed through following mechanisms: pinocytosis, diffusion, or endocytosis. The partition of the drug from the oil droplets depends on their size and polarity. Nano sized droplets will result in faster partitioning since the drug can diffuse faster from smaller droplets (Shah et al., 1994). In case of SMEDDS, it has been shown that digestion of the resultant nanoemulsion acts independently of bile salts (Trull et al., 1995) and the polarity of the oil droplets is Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 47 Chapter 1 Introduction not significant because the drug reaches the capillaries within the oil droplets (Benita, 2006). Lymphatic transport of the drug: Most of the drugs delivered using S(M)EDDS are absorbed systematically via portal vein except for certain type of drugs. Lymphatic transport of the drug occurs when the drug is highly lipophilic (logP >5) and shows high solubility in triglycerides (>50mg/ml). Such drugs are absorbed via lymph vessels in the intestine which are responsible for absorption of lipids. Since the drug is cleared by the lymph vessels, they bypass the liver metabolism. This results in an increased bioavailability of these drugs. The bioavailability of Ontazolast, an extensively first-pass metabolized drug was improved when delivered in a lipid based formulation. The drug was absorbed via lymphatic pathway and thus bypassed first-pass metabolism (Hauss et al., 1998). Enterocyte based drug transport: Few endogenous lipid transporters have been identified which are responsible for intestinal passage of lipophilic drugs. At low lipid concentrations drugs are actively transported, while at high lipid concentrations drugs are passively permeated. P-glycoprotein (P-gp) is an efflux transporter present in enterocytes that acts as a substrate for many lipophilic drugs. Surfactants are reported to inhibit these P-gp efflux transporters resulting in an increase in permeability of poorly permeated drugs (Porter et al., 2007). Labrasol was identified as the most effective surfactant in inhibiting the P-gp. Increasing membrane permeability: Lipids are responsible for causing fluidization of intestinal cell membrane and opening of tight junctions resulting in increased membrane permeability. Labrasol has a dual property of increasing membrane permeability by both the mechanisms, while Cremphor EL and Tween 80 act by opening the tight junction barrier. Surfactants also penetrate into the Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 48 Chapter 1 Introduction intestinal cell membrane and disrupt the structural organization of the membrane leading to an increased permeability (Neslihan Gursoy and Benita, 2004). 1.4.3. Mechanism of Self-emulsification Conventional emulsions are formed by mixing two immiscible liquids namely water and oil stabilized by an emulsifying agent. When an emulsion is formed surface area expansion is created between the two phases. The emulsion is stabilized by the surfactant molecules that form a film around the internal phase droplet. In conventional emulsion formation, the excess surface free energy is dependent on the droplet size and the interfacial tension. If the emulsion is not stabilized using surfactants, the two phases will separate reducing the interfacial tension and the free energy (Craig et al., 1995). In case of S(M)EDDS, the free energy of formation is very low and positive or even negative which results in thermodynamic spontaneous emulsification. It has been suggested that self emulsification occurs due to penetration of water into the Liquid Crystalline (LC) phase that is formed at the oil/surfactant-water interface into which water can penetrate assisted by gentle agitation during self-emulsification. After water penetrates to a certain extent, there is disruption of the interface and a droplet formation. This LC phase is considered to be responsible for the high stability of the resulting nanoemulsion against coalescence (Groves and De Galindez, 1976; Wakerly et al. 1986). 1.4.4. Stability of SMEDDS SMEDDS is a thermodynamically stable system. The stability issue arises mainly due to the supersaturation of the system at RT and when stored at low temperature which may result in re-crystallisation of the active substance. This phenomenon is more common in liquid SMEDDS. Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 49 Chapter 1 Introduction The stability of the SMEDDS to is influenced by the chemical characteristics of the selected carriers at different storage conditions, the interaction between the carrier and the active substances. SMEDDS in the form of liquid crystal, semisolid and solid can remain stable at both room temperature and 4°C. The factors which affect the stability of SMEDDS are as following: Polarity of the lipophillic phase: The polarity of the lipid phase is one of the main factors that govern the drug release from the micro-emulsions. The polarity of the droplet is governed by the HLB, the chain length and degree of unsaturation of the fatty acid, the molecular weight of the hydrophilic portion and the concentration of the emulsifier. In fact, the polarity reflects the affinity of the drug for oil and/or water, and the type of forces formed. The high polarity will promote a rapid rate of release of the drug into the aqueous phase. This is confirmed by the observations of Sang-Cheol Chi, who observed that the rate of release of idebenone from SMEDDS is dependent upon the polarity of the oil phase used. The highest release was obtained with the formulation that had oil phase with highest polarity. Nature and Dose of the Drug: Drugs which are administered at very high dose are not suitable for SEDDS unless they have extremely good solubility in at least one of the components of SEDDS, preferably lipophillic phase. The drugs which have limited or less solubility in water and lipids are most difficult to deliver by SEDDS. The ability of SEDDS to maintain the drug in solubilised form is greatly influenced by the solubility of the drug in oil phase. As mentioned above if surfactant or co-surfactant is contributing to the greater extent in drug solubilisation then there could be a risk of precipitation, as dilution of SEDDS will lead to lowering of solvent capacity of the surfactant or co-surfactant. Equilibrium Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 50 Chapter 1 Introduction solubility measurements can be carried out to anticipate potential cases of precipitation in the gut. However, crystallisation could be slow in the solubilising and colloidal stabilizing environment of the gut. Pouton’s study reveal that such formulations can take up to five days to reach equilibrium and that the drug can remain in a super-saturated state for up to 24 hours after the initial emulsification event. It could thus be argued that such products are not likely to cause precipitation of the drug in the gut before the drug is absorbed, and indeed that super-saturation could actually enhance absorption by increasing the thermodynamic activity of the drug. There is a clear need for practical methods to predict the fate of drugs after the dispersion of lipid systems in the gastrointestinal tract. 1.4.5. Toxicity of SMEDDS Unfortunately, there is not enough research conducted to investigate toxicity of SMEDDS. Researchers measured proliferation of keratinocytes in one of the topical niosome formulations. The effect of surfactant type on toxicity was investigated. It was determined that the ester type surfactants are less toxic than ether type surfactants. This may be due to enzymatic degradation of ester bounds. In general, the physical form of niosomes did not influence their toxicity as evident in a study comparing the formulations prepared in the form of liquid crystals and gels. However, nasal applications of these formulations caused toxicity in the case of liquid crystal type niosomes. In some instances, encapsulation of the drug by niosomes reduces the toxicity as demonstrated in the study on preparation of niosomes containing vincristine. It decreased the neurological toxicity, diarrhoea and alopecia following the intravenous administration of vincristine and increased vincristine anti-tumor activity in S-180 sarcoma and Erlich ascites mouse models. Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 51 Chapter 1 1.5. Introduction ISOTRETINOIN- A POORLY SOLUBLE MODEL DRUG The evolution of high throughput combinatorial chemistry and efficient receptor based in-vitro activity screen has resulted in molecules with poor physicochemical properties for absorption across the GIT, like Isotretinoin. Isotretinoin was approved in the United States in 1982 as a treatment for severe recalcitrant nodular (cystic) acne that is unresponsive to conventional therapy including systemic antibiotics. Isotretinoin is 13-cis retinoic acid or 13 cis-Vitamin A, its isomers and some of its analogs are widely known to leave a therapeutic activity in the treatment of several skin disorders like acne, hypertropic lupus erythmatosus, keratinization disorders. Some evidences also have been brought about the activity of Isotretinoin in basal cell carcinoma and squamous cell carcinoma. Isotretinoin is a reddish orange powder. It decomposes in the presence of light and atmospheric oxygen. Isotretinoin is very poorly soluble in water what makes its bioavailability quite low after an oral intake of about 25% in fasted condition and 40% in fed conditions. The maximum concentration (Cmax) is reached after 2-4hrs, while the Cmax of the active metabolite, 4-oxo-isotretinoin is reached after 6hrs. The elimination half life of Isotretinoin is about 7 to 37 hrs while the half life t1/2 of active metabolite is of 11 to 50 hrs. The steady state concentration is reached after 1 week of treatment. It is increasingly being recognized by the pharmaceutical industry that for these molecules, drug delivery systems play an important role for improving bioavailability. Isotretinoin is characterized by a low absolute bioavailability and a high inter and intra individual variability. It also presents a wide range of side effects among which some are severe (ocular, skin anemia, hepatic). It is consequently of particular interest to dispose of a reliable, stable and highly bioavailable formulation of Isotretinoin. Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 52 Chapter 1 Introduction The drug is available in most markets in the form of a soft gelatin capsule containing a fatty liquid formulation of Isotretinoin. The reported literatures show a maximum in-vitro drug release of about 60-70% even after 5 h. The commercially available marketed product constitutes batch to batch variability which further contributes to the variability caused by the nature of the molecule itself. This inadvertently created further hurdles in developing the generic version of Roacutane 20mg soft gelatin capsule product. This variation occurs mainly due to the particle size and solid state characteristics of Isotretinoin. The particle size of the active is mainly controlled by the API manufacturer during the recrystallization or purification step. The size reduction of the bigger particles causes serious blow in the purity and its physical characteristics due to the energy intensive milling process. Hence such process is mostly discouraged in the formulation industry but the quality of the product is built through the input and In-process controls by controlling the API particle size in a narrow range at vendor level itself. This makes the condition more complex and increases the cost of the active material and ultimately adds on the cost of the final formulation and makes the treatment costlier. Isotretinoin is a highly unstable molecule. It decomposes in the presence of light and atmospheric oxygen. A novel composition which shall be manufactured through easily scalable process at the formulation manufacturer site without involving sophisticated and energy intensive milling process irrespective of the particle size of the active material procured from the API manufacturer, to enhance the oral bioavailability and to address the stability issues associated with the Isotretinoin molecule would be welcome to the class of poorly soluble drugs. Design and Development of a Self Nano Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced Oral Bioavailability and Improved Stability 53