2.1. Lipid based drug delivery systems (Pouton, 2000)

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1 In recent years, much attention has been focused on lipid-based formulations (Humberstone et al., 1997) to enhance the solubility of poorly water soluble drugs and improving bioavailability to administer them through oral route resulting in increasing their clinical efficacy, cost effectiveness and ease of preparation Lipid based drug delivery systems (Pouton, 2000) Lipid based drug delivery systems (LBDDS) are one of the most popular approaches to improve the oral bioavailability of poorly water soluble drugs (Charman, 2000; Gershanik et al., 2000). Lipid vehicles are known to enhance the absorption of lipophilic drugs and there are various mechanisms by which LBDDS enhance the absorption of lipophilic drugs: Enhanced dissolution/solubilization The presence of lipids in the GI tract stimulates gall bladder contractions, biliary and pancreatic secretions, including bile salts (BS), phospholipids (PL) and cholesterol (Ch) (Fleisher et al., 1999). These products, along with the gastric shear movement form a crude emulsion and promote the solubilization of the co-administered lipophilic drug (Tso, 1985). Surfactants present in the delivery system may also improve the solubilization of the lipophilic compound. Affecting intestinal permeability A variety of lipids have been shown to change the physical barrier function of the gut wall and hence, to enhance permeability. For BCS class II compounds, permeability through the GI wall is not a limiting step towards absorption and hence, this mechanism is not thought to be a major contributor for the absorption enhancement of lipophilic drugs. However, this may be helpful for BCS class IV drugs. Reduced metabolism and efflux activity Certain lipids and surfactants also reduce the activity of efflux transporters in the GI wall and hence, to increase the fraction of drug absorbed (Dintaman et al., 1999; Nerurkar et al., 1996). Because of the interplay between P-gp and CYP3A4 activity this mechanism may reduce intra-enterocyte metabolism as well. 5

2 Stimulation of lymphatic transport Bioavailability of lipophilic drugs may also be improved by the stimulation of the intestinal lymphatic transport pathway (Trevaskis et al., 2008; Charman et al., 1996). Prolongation of gastric residence time Gastric transit time is prolonged with the presence of lipids in GI tract (Van Citters et al., 1999). As a result, the residence time of the lipophilic drug in the small intestine increases. This improves dissolution of the drug and thereby improves absorption. Classification of lipid delivery systems (Pouton, 2000) The simplest lipid products are those in which the drug is dissolved in digestible oil, usually a vegetable oil or medium chain triglyceride (fractionated coconut oil). These are safe food substances and do not present a toxicological risk to formulators. Oil solution has been the standard way of administering oil-soluble vitamins (A and D) for many years. Bioavailability from oil solutions is likely to be very good because the triglycerides are rapidly digested to free fatty acids and 2-mono-glycerides, and these products are solubilised to form a colloidal dispersion within bile salt-lecithin mixed micelles. A hydrophobic drug is also likely to be solubilised in mixed micelles resulting in a reservoir of drug in colloidal solution from which it can partition, allowing efficient, passive (transcellular) absorption. The low solvent capacity of triglycerides often prevents formulation in oil, but oil solutions may be a realistic option for potent drugs. Solvent capacity for less hydrophobic drugs can be improved by blending triglycerides with other oily excipients which include mixed monoglycerides and diglycerides. Since these are similar to the natural degradation products of triglycerides (the difference being that their monoglyceride content is mostly 1 monoglyceride rather than 2- monoglyceride) they do not prevent efficient digestion. As excipients with GRAS status they have the same advantages as triglycerides, and are useful for blending with triglycerides or for use as an alternative. Formulations which comprise drug in solution in triglycerides and/or mixed glycerides are classified here as Type I (Table 2.1).When an appropriate dose of the drug can be dissolved, a Type I formulation may well be the system of choice, in view of its simplicity and biocompatibility. The inclusion of a lipophilic surfactant (HLB 12) may improve the solvent capacity of the formulation, but this approach has mainly been used to promote emulsification. Under 6

3 optimum conditions it is possible to formulate a self-emulsifying drug delivery system (SEDDS) which emulsifies in aqueous solutions under very gentle conditions of agitation, to result in a dispersion of colloidal dimensions. If the surfactant is insufficiently hydrophilic to dissolve and form micelles in aqueous solution, then it exists itself as a dispersed phase, either with or separated from the oily components. This type of formulation is likely to retain its solvent capacity for the drug after dispersion and is referred to as Type II (Table 2.3). The distinguishing features of Type II systems are (a) efficient self-emulsification and (b) absence of water-soluble components. Formulation of Type II SEDDS has been described in detail elsewhere (Pouton, 1982, 1984, 1985a, b; Wakerley et al., 1986a,b, 1987; Charman et al., 1992) and reviewed recently (Pouton, 1997). Hydrophilic surfactants (water soluble with HLB> 12) and/ or water-soluble cosolvents have also been blended with oils to produce selfemulsifying systems. When the surfactant content is high (for example 40%, w/w, or more) or co-solvents are included in addition to surfactants, it is possible to produce very fine dispersions <100 nm in diameter) under conditions of gentle agitation (Constantinides, 1995; Constantinides and Scalart, 1997). This approach was used for the reformulation of cyclosporin A as 'Neoral' (Vonderscher and Meinzer, 1994). Formulations which include water-soluble components are referred to in Table 2.1 as Type III formulations, and have been referred to as 'self microemulsifying' systems, due to the optical clarity which can be achieved with Type III systems. Table 2.1. Classification of lipid delivery system. (Pouton, 2000) Components Type I Type II Type III Type IV Triglycerides or <20 mixed glycerides Surfactants (HLB>11) (HLB>11) (HLB>11) Hydrophilic cosolvents Particle size of Coarse dispersion (nm) Significance of Limited Solvent capacity Some loss of Significant phase aqueous dilution importance unaffected solvent capacity changes Significance of Cruical Not crucial but Not crucial but Not likely to occur digestibility likely to occur may be inhibited Advantages GRAS status; Unlikely to lose Clear or almost Formulation has simple;excellent solvent capacity clear dispersion good solvent capsule compatibility on dispersion capacity for many drugs Disadvantages Poor solvent Turbid o/w Possible loss of Likely loss of capacity unless dispersion solvent capacity solvent capacity drug is highly on dispersion; on dispersion;may lipophilic less digested not be digestible 7

4 2.2. Self-emulsifying drug delivery systems (SEDDS) Self-emulsifying drug delivery systems (SEDDS) are defined as isotropic mixtures of natural or synthetic oils, solid or liquid surfactants, or alternatively, one or more hydrophilic solvents and co-solvents/surfactants (Wakerly et al., 1986; Craig et al., 1993; Constantinides, 1985; Craig, 1993). Upon mild agitation followed by dilution in aqueous media, such as GI fluids, these systems can form fine oil in water (o/w) emulsions or microemulsions (SMEDDS). Self-emulsifying formulations spread readily in the gastro intestinal tract and the digestive motility of the stomach and the intestine provide the agitation necessary for selfemulsification (Shah et al., 1994; Kommuru et al., 2001). SEDDS typically produce emulsions with a droplet size between 100 and 300 nm while SMEDDS form transparent microemulsions with a droplet size of less than 50 nm. An additional advantage of SEDDS over simple oily solutions is that they provide a large interfacial area for partitioning of the drug between oil and water. Thus, for lipophilic drug compounds that exhibit dissolution rate limited absorption, these systems may offer an improvement in the rate and extent of absorption and result in more reproducible blood time profiles (Charman et al., 1992) and have been shown to enhance the oral bioavailability of lipophilic drugs (Humberstone et al., 1997; Pouton et al., 1997) such as cyclosporine (Klauser et al., 1997), halofantrine (Khoo et al., 1998), ontazolast (Jayaraj et al., 1998), and progesterone (Solomon et al., 1998). The ease of dispersion and the very small particle size of the resultant colloidal microemulsion have been viewed as the principal reasons for their utility in the delivery of lipophilic drugs. Consequently, most of the commercially available lipid formulations are complex mixtures of lipids,surfactants, and cosolvents/cosurfactants constructed to improve drug solubility in the formulation (and therefore increase drug pay load) and also to maximize dispersion of the dose form on exposure of the capsule fill to the GI contents (Porter et al., 2004). When compared with emulsions, which are sensitive and metastable dispersed forms, SEDDS are physically stable formulations that are easy to manufacture. Microemulsions are readily distinguished from normal emulsions by their transperancy, their low viscosity, and more fundamentally their thermodynamic stability. The dividing line, however, between the size of a swollen micelle (approx nm) and a fine emulsion droplet (approx nm) is not well defined, although microemulsions are very labile systems and a microemulsion 8

5 droplet may disappear within a fraction of a second whilst another droplet forms spontaneously elsewhere in the system. In contrast, ordinary emulsion droplets, however small, exist as individual entities until coalescence or Ostwald ripening occurs Advantages of SEDDS Increased stability (Gursay, et al., 2003) SMEDDS present small particle sizes, thus avoiding the problems of large particle size in conventional composition. Efficient transport The particle sizes in the aqueous dispersions of the SMEDDS are much smaller (l00 nm-250 nm) than the larger particle characteristics of vesicular and emulsion phases. This reduced particle size enables more efficient drug transport through the intestinal aqueous boundary layer, and through the absorption brush border membrane. Less dependence on lipolysis (Chris de Smidt, et al., 2004) The smaller particle size triglyceride components make SMEDDS less dependent upon lipolysis and other factors, which affect the rate and extent of lipolysis. The reduced lipolysis dependence further provides transport, which is less prone to suffer from any lag time between administration and absorption caused by the lipolysis process, enabling a more rapid onset of therapeutic action and better bioperformance characteristics. Non-dependence on biles and meal food contents (Charman & Pouton, 1997) Food is a major factor affecting the therapeutic performance of the drug in the body. Due to the higher solubilization potential over bile salt micelles, the SMEDDS are less dependent on endogenous bile and bile related patient disease states and meal fat contents. These advantages overcome meal dependence with meal-dosage restriction. Superior solubilization SMEDDS enables efficient loading capacity over conventional formulations. In addition, the particular combination of surfactant used can be optimized for a specific therapeutic agent to more closely match the polarity distribution of the therapeutic agent, resulting in still further enhanced solubilization. 9

6 Faster dissolution and release Dissolution being the rate limiting step for absorption of drugs, it is a major factor that limits the bioavailability of numerous poorly water soluble drugs. Due to the robustness of SMEDDS to dilution, the therapeutic agent remains solubilized and thus does not suffer problem of precipitation of the therapeutic agent in the time frame relevant for absorption. In addition, the therapeutic agent is presented in small particle carriers and is not limited in dilution rate by entrapment in the emulsion carriers. These factors avoid liabilities associated with the poor partitioning of lipid solubilized drug to the aqueous phase, such as large emulsion droplet surface area, and high interfacial transfer resistance and enable rapid completion of the critical partitioning stage. Consistent performance Aqueous dispersions of the SMEDDS are thermodynamically stable for the time period relevant for absorption and can be predictably reproduced, thereby limiting variability in bioavailability a particularly important advantage for therapeutic agents with a narrow therapeutic index. Efficient release The composition of the SMEDDS are designed using components that help to keep the therapeutic agent or absorption promoter, such as permeation enhancer, an enzyme inhibitor, etc., solubilized for transport to the absorption sites, but readily available for absorption, thus providing a more efficient transport and release. Less prone to gastric emptying delays Unlike conventional triglycerides containing formulations, which form bigger droplets on dispersion, the SMEDDS are less prone to gastric emptying delays, resulting in faster absorption and avoid unwanted retention in the gastro-intestinal tract. Small size Because of small size in aqueous dispersion, the SMEDDS allow for the faster transport of the therapeutic agent through the aqueous boundary layer. 10

7 Ease of manufacture and scale up (Patraval, et al., 2003) Ease of manufacture and scale up are the most important advantages that make SMEDDS unique when compared to other drug delivery systems like solid dispersions, liposomes, nanoparticles, etc., for improving bioavailability. SMEDDS require very simple and economical manufacturing facilities like simple mixer with agitator and volumetric liquid filling equipment for large scale manufacturing. This explains the interest of industry in the SMEDDS. Self-emulsification is a phenomenon, which has been exploited commercially for many years in formulations of emulsifiable concentrates of herbicides and pesticides. These formulations are used to produce concentrates of crop sprays which are diluted by users, such as farmer or household gardner, allowing very hydrophobic compounds to be transported efficiently. In contrast SMEDDS, using excipients acceptable for oral administration to humans have not been widely exploited. There are number of reasons why self-microemulsifying drug delivery system (SMEDDS) is not in common use. Some relate to custom and habit of pharmaceutical development laboratories and some to genuine reasons (Pouton, C. W., 1997); 1. Most pharmaceutical companies have a commitment to tabletting machinery, which leads them to favor tablet formulation at early stage often before sufficient data are available on the bioavailability of the drug. Once the path to formulation of a solid dosage form is selected, it is difficult to revert to oily formulation at a later stage. 2. Encapsulation of an oily formulation may have to be outsourced. 3. The solvent capacity of oily formulation is not high unless the drug is very hydrophobic/lipophilic (log P>4), so that formulation in oils is usually limited to potent compounds. 4. There may be some suspicion that chemically the drug will be unstable in an oily formulation than in crystalline form, and this may be so, but at present there is insufficient data to predict chemical stability in self-micro emulsifying drug delivery system (SMEDDS). 5. The use of high concentration of surfactant is a legitimate concern from a toxicological standpoint. 11

8 Additionally, formulators favor utilizing tried and tested materials to prevent problems surfacing at a later stage of development of new chemical entity. The above are the issues which will tend to direct a formulator away from SMEDDS, though it is now accepted that there are a group of compounds for which SMEDDS, is the delivery system of choice, exemplified by cyclosporin A. The development of this compound by Sandoz has confirmed the potential of se1f-microemulsifying drug delivery system (SMEDDS) in bringing hydrophobic compounds to the market place. There are several drugs which are poorly bioavailable from marketed conventional formulation which could have been formulated in oily systems, the potential of SMEDDS will not be realized until more human bioavailability studies have been published and until more information is available on the chronic toxicology of self-micro emulsifying drug delivery system (SMEDDS) Applications of SEDDS (Tang et al., 2007) Supersaturable SEDDS (S-SEDDS) The high surfactant level typically present in SEDDS formulations can lead to GI side effects and a new class of supersaturable formulations, including supersaturable SEDDS (S-SEDDS) formulations, have been designed and developed to reduce the surfactant side-effects and achieve rapid absorption of poorly soluble drugs (Poelma et al., 1991; Gao et al., 2003; Gao et al., 2004). The S-SEDDS approach is to generate a protracted supersaturated solution of the drug when the formulation is released from an appropriate dosage form into an aqueous medium. Surpersaturation is intended to increase the thermodynamic activity to the drug beyond its solubility limit and, therefore, to result in an increased driving force for transit into and across the biological barrier (Gao et al., 2004). The S-SEDDS formulations contain a reduced level of surfactant and a polymeric precipitation inhibitor to yield and stabilize a drug in a temporarily supersaturated state. Hydroxypropyl methylcellulose (HPMC) and related cellulose polymers are well recognized for their propensity to inhibit crystallization and, thereby, generate and maintain the supersaturated state for prolonged time periods (Pellet et al., 1994; Pellet et al., 1997; Usui et al., 1997; Yamada et al., 1999; Raghavan et al., 2000; Lervolino et al., 2000). A supersaturable self-emulsifying drug delivery system (S- SEDDS) of paclitaxel was developed employing HPMC as a precipitation inhibitor with a conventional SEDDS formulation. In vitro dilution of the S-SEDDS formulation results in formation of a microemulsion, followed by slow crystallization of paclitaxel on standing. 12

9 This result indicates that the system is supersaturated with respect to crystalline paclitaxel, and the supersaturated state is prolonged by HPMC in the formulation. In the absence of HPMC, the SEDDS formulation undergoes rapid precipitation, yielding a low paclitaxel solution concentration. A pharmacokinetic study showed that the paclitaxel S-SEDDS formulation produces approximately a 10-fold higher maximum concentration (C max ) and a 5- fold higher oral bioavailability (F = 9.5%) compared with that of the orally administered Taxol formulation (F = 2.0%) and the SEDDS formulation without HPMC (F =1%) (Gao et al., 2003). It is worth emphasizing that the significantly reduced amount of surfactant used in the S-SEDDS formulation approach provides a better toxicity/safety profile than the conventional SEDDS formulations. However, the underlying mechanism of the inhibited crystal growth and stabilized supersaturation by means of these polymers is poorly understood even although several studies have been carried out to investigate this (Gao et al., 2003; Raghavan et al., 2001; Pellet et al., 1997; Hasegawa et al., 1988). Solid SEDDS SEDDS are normally prepared as liquid dosage forms that can be administrated in gelatin capsules. An alternative method is the incorporation of liquid self-emulsifying ingredients into a powder in order to create a solid dosage form (tablets, capsules). A pellet formulation of progesterone in SEDDS has been prepared by the process of extrusion/spheronization to provide a good in vitro drug release (100% within 30 min, T50% at 13 min). The same dose of progesterone (16 mg) in pellets and in the SEDDS liquid formulation resulted in similar AUC, Cmax and Tmax values (Tuleu et al., 2004). Attama et al. used goat fat and Tween 65 admixtures to formulate self-emulsifying tablets containing diclofenac by pour moulding using a plastic mould. The tablets showed good release profiles, as well as acceptable tablet properties. Under mild agitation, such as occurs under gastrointestinal conditions, the release rates are comparable with those of conventional tablets (Attama et al., 2003). Encapsulating the emulsion lipid droplets in HPMC by spray-drying has been demonstrated to produce an improved bioavailability following release of the lipid droplets from the powder in vivo. Tue et al. (Hansen et al., 2005) have investigated the oral bioavailability of a directly compressible dry emulsion as a tablet and compared it with an HPMC dry emulsion powder and a simple lipid solution. Four female beagle dogs received a single dose of each formulation containing the same amount of MCT and model drug, Lu Cyclodextrin solutions administered orally and intravenously were used as references. The absolute 13

10 bioavailability decreased in the order: cyclodextrin solution (0.14) > HPMC dry emulsion (0.11) > technically improved dry emulsion (0.10) > MCT solution (0.06). The directly compressible dry emulsion tablets were concluded to be comparable with the HPMC dry emulsion powder in terms of bioavailability (Woo et al., 1961). SEDDS for TCM Silybin, the principal component of a Carduus marianus extract, is known to be very effective in protecting liver cells from harmful effects caused by smoking, drinking, overworking, environmental contaminants, stress or liver damaging drugs. However, the bioavailability of orally administered silybin is very low due to its low solubility in water. Woo et al. discloses an oral microemulsion consisting of a Carduus marianus extract containing a major amount of silybin, or a silybin derivative as an active ingredient. The composition of the invention consists of miglyol 812 and ethyl linoleate as oils, HCO 50 and Tween 20 as surfactant, dimethyl isosorbide as co-surfactant and D-tocopherol as an antioxidant. The formulation provides a greatly increased level of in vivo bioavailability of silybin, the level being atleast 4-fold higher than that achievable by conventional formulations (You et al., 2005) Curcuma zedoaria (Berg.) Rose. (Zingiberaceae), also called er-zhu in Chinese, has long been used as a folk medicine. The essential oil, zedoary turmeric oil (ZTO), was extracted from the dry rhizome of C. zedoaria. A series of studies on ZTO indicated that it exhibits potent pharmacological actions including the suppression of tumors, antibacterial and antithrombotic activity, increased white blood cell count, and increased gastric motility (You et al., 2005). To increase the in vivo absorption of zedoary turmeric oil (ZTO) and develop new formulations of a water insoluble oily drug, Li formulated SEDDS using ZTO as the oil (Li et al., 2002). Pueraria lobata is a traditional Chinese medicinal herb. In China, its extract has been used for the treatment of hypertension, senile ischemic cerebrovascular disease and anginapectoris. Studies of its pharmacology and clinical application shave shown that the active constituents in the extractare isoflavones, mainly puerarin. It is known to dilate coronary arteries, reduce myocardial oxygen consumption and improve microcirculation in both animals and humans suffering from cardiovascular disease (Li et al., 2002). Yufengningxin tablets are a formulation of total isoflavones obtained from Pueraria lobata, and are available commercially in China. The dissolution rate of Yufengningxin tablets is very low and, therefore, a SMEDDS formulation of Pueraria lobata isoflavone was developed to improve the oral bioavailability. An optimized 14

11 formulation consisted ethyl oleate, Tween 80 and transcutol P as cosurfactant. The dissolution of SMEDDS after 10 min was more than 90%, and the dissolution of Yufengningxin tablets at 60 min was less than 30%. The absorption of puerarin from the SMEDDS of Puerarialobata isoflavone resulted in a 2.2-fold increase in bioavailability compared with Yufengningxin tablets Ginkgo biloba L., the last surviving member of a family of trees (Ginkgoaceae) that appeared more than 250 million years ago, has been mentioned in the Chinese Materia Medica for more than 2500 years. A standardized Ginkgo biloba extract (GBE) contains 5-7% terpene lactones (ginkgolides and bilobalide) and 22-27% ginkgo flavonol glycosides (eg., the flavones quercetin, kaempferol, and isorhamnetin) (Kleijnen et al., 1992). Many pharmacological and clinical studies have demonstrated that the extracts of Ginkgo biloba possess antioxidant, antiischemic, neuro protective, cardiovascular and cerebrovascular activities, and have beneficial effects oncognitive deficits, including Alzheimer s type and multi infarct dementia, as well as peripheral vascular disease (Kressmann et al., 2002). The dissolution and bioavailability of the active components from the oral solid preparations of different Ginkgo biloba brands were obviously different and irreproducible, due to the lower solubility of the active components (Kressmann et al., 2002). The SEDDS formulation of GBE was accordingly developed to increase the dissolution rate and thus improve oral absorption and acquire the reproducible blood-time profiles of the active components of GBE. The prepared SEDDS was compared with the conventional GBE tablets following administration to fasted dogs. The active components of GBE, terpene lactones, were determined using liquid chromatography with electrospray ionization mass spectrometric detection (Tang et al., 2006). The relative bioavailability of SEDDS for ginkgolide was 154%, compared with the reference tablets Formulation of SEDDS (Patel et al., 2010) SEDDS formulation containing following components 1) Oil phase 2) Surfactant 3) Co-surfactant/Co-Solvent Self-emulsification has been shown to be specific to the nature of the oil/surfactant pair, the surfactant concentration and oil/surfactant ratio and the temperature at which self- 15

12 emulsification occurs (Wakerly et al., 1986; Wakerly et al., 1987; Pouton et al., 1985). In support of these facts, it has also been demonstrated that only very specific pharmaceutical excipient combinations could lead to efficient self-emulsifying systems (Charman et al., 1992; Shah et al., 1994; Chanana et al., 1995; Kimura et al., 1994; Hauss et al., 1998; Karim et al., 1994). Oils The oil represents one of the most important excipients in the SEDDS formulation not only because it can solubilise marked amounts of the lipophilic drug or facilitate selfemulsification but also and mainly because it can increase the fraction of lipophilic drug transported via the intestinal lymphatic system, thereby increasing absorption from the GI tract depending on the molecular nature of the triglyceride (Gershanik et al., 2000; Lindmark et al., 1995; Charman et al., 1991; Holm et al., 2002). Both long and medium chain triglyceride oils with different degrees of saturation have been used for the design of selfemulsifying formulations. Furthermore, edible oils which could represent the logical and preferred lipid excipient choice for the development of SEDDS are not frequently selected due to their poor ability to dissolve large amounts of lipophilic drugs. Modified or hydrolyzed vegetable oils havebeen widely used since these excipients form good emulsification systems with a large number of surfactants approved for oral administration and exhibit better drug solubility properties (Constantinides et al., 1995; Kimura et al., 1994; Hauss et al., 1998). They offer formulative and physiological advantages and their degradation products resemble the natural end products of intestinal digestion. Novel semisynthetic medium chain derivatives, which can be defined as amphiphilic compounds with surfactant properties, are progressively and effectively replacing the regular medium chain triglyceride oils in the SEOFs (Constantinides et al., 1995; Karim et al., 1994). Surfactants Several compounds exhibiting surfactant properties may be employed for the design of selfemulsifying systems, the most widely recommended ones being the non-ionic surfactants with a relatively high hydrophilic lipophilic balance (HLB). The commonly used emulsifiers are various solid or liquid ethoxylated polyglycolyzed glycerides and polyoxyethylene 20 oleate (Tween 80). Safety is a major determining factor in choosing a surfactant. Emulsifiers of natural origin are preferred since they are considered to be safer than the synthetic 16

13 surfactants (Constantinides et al., 1995; Hauss et al., 199; Yuasa et al., 1994; Georgakopoulos et al., 1992). However, these excipients have a limited self-emulsification capacity. Non-ionic surfactants are less toxic than ionic surfactants but they may lead to reversible changes in the permeability of the intestinal lumen (Swenson et al., 1994; Wakerly et al., 1986). Usually the surfactant concentration ranges between 30 and 60% w/w in order to form stable SEDDS. It is very important to determine the surfactant concentration properly as large amounts of surfactants may cause GI irritation.the surfactant involved in the formulation of SEDDS should have a relatively high HLB and hydrophilicity so that immediate formation of o/w droplets and/or rapid spreading of the formulation in the aqueous media (good selfemulsifying performance) can be achieved. For an effective absorption, the precipitation of the drug compound within the GI lumen should be prevented and the drug should be kept solubilized for a prolonged period of time at the site of absorption (Serajuddin et al., 1988; Shah et al., 1994). Surfactants are amphiphilic in nature and they can dissolve or solubilize relatively high amounts of hydrophobic drug compounds. The lipid mixtures with higher surfactant and co-surfactant/oil ratios lead to the formation of SMEDDS (Constantinides et al., 1995; Karim et al., 1994; Meinzer et al., 1995; Vondercher et al., 1994). There is a relationship between the droplet size and the concentration of the surfactant being used. In some cases, increasing the surfactant concentration could lead to droplets with smaller mean droplet size such as in the case of a mixture of saturated C8-C10 polyglycolized glycerides (Labrafac CM-10). This could be explained by the stabilization of the oil droplets as a result of the localization of the surfactant molecules at the oil-water interface (Levy et al., 1990). On the other hand, in some cases the mean droplet size may increase with increasing surfactant concentrations (Wakerly et al., 1987; Kommuru et al., 2001; Craig et al., 1995). This phenomenon could be attributed to the interfacial disruption elicited by enhanced water penetration into the oil droplets mediated by the increased surfactant concentration and leading to ejection of oil droplets into the aqueous phase (Pouton et al., 1997). Co-surfactants Role of co-surfactant together with surfactant is to lower the interfacial tension to a very small, even transient, negative value. At this value the interface would expand to form fine dispersed droplets, and subsequently adsorb more surfactant and surfactant/co-surfactant until their bulk condition is depleted enough to make interfacial tension positive again. This process is known as spontaneous emulsification forms the microemulsion. However, the 17

14 use of co-surfactant is crucial not only to formation of microemulsion, but also to solubilisation in microemulsion. Other variables such as the chemical nature of oil, salinity, and temperature are also expected to influence the curvature of the interfacial film. Co-solvents The production of an optimum SEDDS requires relatively high concentrations (generally more than 30% w/w) of surfactants. Organic solvents such as, ethanol, propyleneglycol (PG), and polyethylene glycol (PEG) are suitable for oral delivery, and they enable the dissolution of large quantities of either the hydrophilic surfactant or the drug in the lipid base. These solvents can even act as co-surfactants in microemulsion systems. On the other hand, alcohols and other volatile co-solvents have the disadvantage of evaporating into the shells of the soft gelatin, or hard, sealed gelatin capsules in conventional SEDDS leading to drug precipitation. Thus, alcohol free formulations have been designed (Constantinides et al., 1995), but their lipophilic drug dissolution ability may be limited Mechanism of self-emulsification (Gursoy et al., 2004) Self-emulsification takes place when the entropy change favouring dispersion is greater than the energy required to increase the surface area of the dispersion (Reiss et al., 1975). The free energy of a conventional emulsion formulation is a direct function of the energy required to create a new surface between the oil and water phases. The two phases of the emulsion tend to separate with time to reduce the interfacial area and thus the free energy of the systems. The conventional emulsifying agents stabilize emulsions resulting from aqueous dilution by forming a monolayer around the emulsion droplets, reducing the interfacial energy and forming a barrier to coalescence. On the other hand, emulsification occurs spontaneously with SEDDS because the free energy required to form the emulsion is both low and positive or negative (Constantinides et al., 1995). It is necessary for the interfacial structure to show no resistance against surface shearing in order for emulsification to take place (Dabros et al., 1999). The ease of emulsification was suggested to be related to the ease of water penetration into the various LC or gel phases formed on the surface of the droplet (Wakerly et al., 1986; Groves et al., 1974; Rang et al., 1999). The interface between the oil and aqueous continuous phases is formed upon addition of a binary mixture (oil/non-ionic surfactant) to water (Wakerly et al., 1986). This is followed by the solubilisation of water within the oil phase as a result of aqueous penetration through the interface. This will occur until the solubilisation 18

15 limit is reached close to the interphase. Further aqueous penetration will lead to the formation of the dispersed LC phase. In the end, everything that is in close proximity with the interface will be LC, the actual amount of which depends on the surfactant concentration in the binary mixture. Thus, following gentle agitation of the self-emulsifying system, water will rapidly penetrate into the aqueous cores and lead to interface disruption and droplet formation. As a consequence of the LC interface formation surrounding the oil droplets, SEDDS become very stable to coalescence. Detailed studies have been carried out to determine the involvement of the LC phase in the emulsion formation process (Wakerly et al., 1986; Rang et al., 1999; Pouton et al., 1987; Wakerly et al., 1987). Also, particle size analysis and low frequency dielectric spectroscopy (LFDS) were utilized to examine the self-emulsifying properties of a series of Imwitor 742 (a mixture of mono and diglycerides of capric and caprylic acids)/tween 80 systems (Craig et al., 1993; Craig et al. 1995). The results suggested that there might be a complex relationship between LC formation and emulsion formation (Craig et al. 1995). Moreover, the presence of the drug compound may alter the emulsion characteristics, probably by interacting with the LC phase (Craig et al., 1993). Nevertheless, the correlation between the LC formation and spontaneous emulsification has still not been established (Craig et al., 1993; Gursoy et al., 2003) Biopharmaceutical aspects (Tang et al., 2007) The ability of lipids and/or food to enhance the bioavailability of poorly water soluble drugs has been comprehensively reviewed and the interested reader is directed to these references for further details (Humberstone et al., 1997; Charman et al., 1997). Although incompletely understood, the currently accepted view is that lipids may enhance bioavailability via a number of potential mechanisms, including (Porter et al., 2001). a) Alterations (reduction) in gastric transit, thereby slowing delivery to the absorption site and increasing the time available for dissolution (Porter et al., 2001). b) Increases in effective luminal drug solubility. The presence of lipids in the GI tract stimulates an increase in the secretion of bile salts (BS) and endogenous biliary lipids including phospholipid (PL) and cholesterol (CH), leading to the formation of BS/PL/CH intestinal mixed micelles and an increase in the solubilisation capacity of the GI tract. However, intercalation of administered (exogenous) lipids into these BS structures either directly (if sufficiently polar), or secondary to digestion, leads to 19

16 swelling of the micellar structures and a further increase in solubilisation capacity (Porter et al., 2001). c) Stimulation of intestinal lymphatic transport. For highly lipophilic drugs, lipids may enhance the extent of lymphatic transport and increase bioavailability directly, or indirectly via reduction in first pass metabolism (Porter et al., 1997; Porter et al., 2001; Muranishi et al., 1991). d) Changes in the biochemical barrier function of the GI tract. It is clear that certain lipids and surfactants may attenuate the activity of intestinal efflux transporters, as indicated by the p-glycoprotein efflux pump, and may also reduce the extent of enterocyte based metabolism (Benet at al., 2001; Dintaman et al., 1999; Nerurkar et al., 1996). e) Changes in the physical barrier function of the GI tract. Various combinations of lipids, lipid digestion products and surfactants have been shown to have permeability enhancing properties (Aungst et al., 2000; Muranishi et al., 1990). For the most part, however, passive intestinal permeability is not thought to be a major barrier to the bioavailability of the majority of poorly water soluble, and in particular, lipophilic drugs. Enhanced drug absorption by lymphatic delivery Charman et al. proposed that drug candidates for lymphatic transport should have a log P >5 and, in addition, a triglyceride solubility >50 mg/ml. The importance of lipid solubility was illustrated by comparing the lymphatic transport of DDT (log P 6.19) with hexachlorobenzene (HCB, log P 6.53). Khoo et al. showed significant lymphatic transport of the poorly lipid soluble (1 mg/ml) HCl salt of halofantrine (Hf-HCl), following oral post prandial administration to dogs. The authors suggest that the high level of lymphatic transport of Hf-HCl (43.7% of dose), which was similar to that of the lipid soluble Hf base, was due to conversion of Hf-HCl in the intestinal lumen, during lipolysis, to the more lipophilic free base, which then becomes associated with chylomicron production (Khoo et al., 1999) 20

17 Although enhanced lymphatic transport has been suggested as a potential mechanism of enhanced bioavailability, few studies have investigated the lymphotropic potential of SEDDS. Haus et al. investigated the effects of a range of lipid based formulations on the bioavailability and lymphatic transport of ontazolast, following oral administration to conscious rats. This drug undergoes extensive hepatic first pass metabolism and it has solubility in soybean oil of 55 mg/ml, and a log P of 4. The formulations of ontazolast investigated included a suspension (lipid free control), a 20% soyabean o/w emulsion, two SEDDS containing Gelucire 44/14 and Peceol in the ratios 50:50 and 80:20, respectively, and a solution of the drug in Peceol alone. All the lipid formulations increased the bioavailability of ontazolast relative to the control suspension, while the SEDDS promoted more rapid absorption. The Effect of excipients on efflux transport Drug efflux mediated by broad specificity xenobiotic transporters present in the intestinal epithelium may be an important factor in the poor or variable absorption of orally administered drugs (Makhey et al., 1998). Lo et al. have shown that bile salts, fatty acids, phospholipids, and surfactants were potent absorption enhancers and efflux reducing agents in Caco-2 cells and the rat intestine (Lo et al., 1998; Lo et al., 2000; Lo et al., 2000). Other researchers also investigated the non-ionic surfactants, such as Tween 80, Pluronic P85, and Cremophor EL in vitro and in vivo in animals and in humans for their potential ability to reverse MDR caused by p-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRP) (Batrakova et al., 1999; Yamazaki et al., 2000). Recently, Cremophor, Tween 80, and Solutol HS-15 have been proven to reverse the MDR phenotype in cultured cells at concentrations likely to be achieved clinically (Yamazaki et al., 2000; Dudeja et al., 1995) TPGS (d-alpha-tocopheryl polyethylene glycol 1000 succinate) has been shown to be an effective inhibitor of P-gp mediated drug resistance and has been used to enhance the bioavailability of CsA in liver transplant patients as well as significantly improving 21

18 absorption and reducing the daily drug cost (Dintaman et al., 1999). Inhibition of MDRrelated pumps by various excipients has been proposed to occur due to binding competition, ATP depletion, and membrane perturbation (Yamazaki et al., 2000; Friche et al., 1990). For example, Tween 80 has been shown to modulate anthracycline and vinca alkaloid resistance in MDR cells by inhibiting the binding of these drugs to P-gp (Yamazaki et al., 2000; Friche et al., 1990). The ability of Pluronic copolymer, one poly (ethylene oxide) block copolymer, to antagonize P-gp and sensitize MDR cells appears to be a result of ATP depletion, and inhibition of P-gp and MRP drug efflux proteins (Kabanov et al., 2002). Studies with MDR modifiers such as bile salts indicated that perturbations of the cell membrane structure may influence P-gp-mediated drug transport (Lo et al., 1998; Schuldes et al., 2001; Dolderer et al., 2000). These modifiers may influence cytotoxic drug action by producing structural changes to the lipid domains in the plasma membrane. The membrane perturbation caused by pharmaceutical excipients, such as Tween 20, Tween 80, Brij 30, and Myrj 52, may result in a change in the fluidity of Caco-2 cell membranes, and thus inhibit the activity of membrane spanning proteins, such as P-gp and MRPs which substantially reduce the basolateral to apical efflux of epirubicin across Caco-2 monolayers (Lo et al., 2003). Tween 20, Tween 80, Brij 30, and Myrj 52 may also inhibit protein kinase C (PKC) activity, reduce phosphorylation of P-gp, and modulate P-gp mediated drug efflux (Komarov et al., 1996). Inhibition of the efflux and/or enterocyte based metabolism will increase the concentration and residence time of the intact drug in the cell. This may result in increased drug available for partitioning into the lymphatics (O Driscoll et al., 2002). Role of lipolysis Digestion of dietary triglyceride in the small intestine is very rapid, and many other non-ionic esters, such as mixed glycerides and surfactants, will be substrates for pancreatic lipase (Embleton et al., 1997). Digestion of formulations will inevitably have a profound effect on the state of dispersion of the lipid formulation, and the fate of the drug (Macgregor et al., 1997). Fortunately, the liberation of free fatty acid during lipolysis can be titrated using NaOH in a ph stat, allowing quantitative data about the kinetics of digestion to be obtained. The location of the drug can be assayed in various fractions after ultracentrifugation of the products of digestion, which allows investigation of the likely fate of the drug after lipolysis (Pouton et al., 2000). The inclusion of highly lipophilic compounds in SEDDS is often reported to result in strongly enhanced oral absorption although it is still controversial 22

19 whether further lipolysis of the dispersed lipid material is required for final transfer to the enterocyte membranes. In order to assess the relative roles of lipid vehicle dispersion and vehicle digestibility in the oral absorption of penclomedine (Pcm), a series of formulations of Pcm in medium chain triglyceride (MCT)/TPGS was developed having three sizes (160 nm, 720 nm, and mm sized (crude oil); with or without the inclusion of tetrahydrolipstatin (THL), a known lipase inhibitor. Oral absorption of Pcm was studied after administration of small volumes of these formulations to conscious rats. Formulations with a particle size of 160 nm had the highest relative bioavailability (set at F = 1), whereas administration in particle 720 nm in size resulted in a slightly lower bioavailability (F = 0.79). Co-inclusion of THL yielded similar bioavailability for these two SEDDS. Crude oil formulations had an F= 0.62 (without THL) and 0.25 (with THL). Positively charged SEDDS Many physiological studies have proved that the apical potential of absorptive cells, as well as that of all other cells in the body, is negatively charged with respect to the mucosal solution in the lumen (Corbo et al., 1990; Rojanasakul et al., 1992). A novel SEDDS, which results in positively charged dispersed oil droplets upon dilution with an aqueous phase, showed an increase in the oral bioavailability of progesterone in young female rats (Gershanik et al., 1996). More recently, it has been shown that the enhanced electrostatic interactions of positively charged droplets with the mucosal surface of the everted rat intestine are mainly responsible for the preferential uptake of the model drug cyclosporine A (CsA) from positively charged droplets (Gershanik et al., 1998). The Caco-2 cell model was used for the investigation of the charge dependent interactions of the SEDDS with human intestinal epithelial cells. The positively charged emulsions affected the barrier properties of the cell monolayer at high concentrations and reduced the cell viability. However, at the dilution with aqueous phase used in the study (1:2000), the positively charged SEDDS did not produce any detectable cytotoxic effect. The binding of the fluorescent dye DiIC18(3) was much higher from the positively charged SEDDS, compared with the negatively charged formulation, suggesting increased adhesion of the droplets to the cell surface due to electrostatic attraction (Gershanik et al., 2000). 23

20 Table 2.2. List of SEDDS of various drugs. Compound Formulation Excipients Observations Simvastatin (Kang et SMEDDS 37% Capryol 90, 28% BA 1.5 fold higher al., 2004) Cremophor EL, 28% Carbitol from SMEDDS Indomethacin (Kim SEDDS 70% ethyl oleate, 30% BA significantly et al., 2000) Tween 85 increased from SEDDS Progesterone (Tuleu et al., 2004) SEDDS Mono-di-glycerides: polysorbate 80, 50/50 w/w BA 9 fold higher from SEDDS Danazol (Porter et SMEDDS Medium chain lipids, BA 6 fold higher al., 2004) Cremophor EL, ethanol from SMEDDS Carvedilol (Wei et al., 2005) SEDDS Labrafil M1944CS, Tween 80, Transcutol BA 4 fold higher from SEDDS Cyclosporin (Trull et SEDDS Corn oil, ethanol Increased BA and al., 1994) Cmax from SEDDS Vitamin E (Julianto et al., 2000) SEDDS Tween 80: Span 80: Palm oil in 4:2:4 BA 3 fold higher from SEDDS Coenzyme Q10 SMEDDS 40% Myvacet, 90% BA 2 fold higher (Kommuru et al., Labrasol, 10% Lauroglycol from SEDDS 2001) Silymarin (Wu et al., SMEDDS Tween 80,ethyl alcohol, BA 2 fold higher 2006) ethyl linoleate, PEG 400 from SMEDDS Win (Charman et al., 1992) SEDDS 40% Neobee M5, 25% Tagat To Increased Cmax, No difference in BA Halofantrine (Khoo SEDDS 47% Captex 355, 23% High BA et al., 1998) Capmul MCM, 15% Cremophor, Ethanol Itraconazole (Hong et SEDDS Transcutol, pluronic L64, Increased BA and al., 2006) tocopherol acetate reduced Food effect Atorvastatin (Shen et SMEDDS Cremophor RH 40, BA increased al., 2006) propylene glycol, Labrafil, Labrafac significantly Atovaquone (Sek et SMEDDS LC lipids, ethanol, BA 3 fold higher al., 2006) cremophor EL from SMEDDS PNU (Gao et SEDDS 6% LC lipids, 30% BA 5-6 fold increase al., 2004) Cremophor, 18% Pluronic L44, 9% PEG, 5% DMA, 20% HPMC from SEDDS 24

21 Characterization of SEDDS (Patel et al., 2010) Differential scanning calorimetry Differential scanning calorimetry for SMEDDS can be determined using DSC 60. Liquid sample and Solid sample should be placed in the aluminum pan and result can be recorded. Any type of chemical interaction should be determined using DSC (Taha et al., 2004) Fourier transform-infrared spectroscopy Fourier transform-infrared for SMEDDS can be determined using FT-IR. Liquid sample should be placed in the liquid sample holder and result can be recorded. Any type of chemical interaction should be determined using FT-IR (Nazzal et al., 2002). Macroscopic evaluation Macroscopic analysis is carried out in order to observe the homogeneity of microemulsion formulations. Any change in color and transparency or phase separation occurred during normal storage condition (37±2 ºC) was observed in optimized microemulsion formulation. Visual assessment To assess the self-emulsification properties, formulation is introduced into 100 ml of water in a glass Erlenmeyer flask at 25 C and the contents are gently stirred manually. The tendency to spontaneously form a transparent emulsion is judged as good and it is judged bad when there was poor or no emulsion formation. Phase diagrams are constructed identifying the good self-emulsifying region (Shen et al., 2006). Determination of self-emulsification time The emulsification time of SMEDDS is determined according to USP XXII dissolution apparatus. Each formulation is added drop wise to purified water at 37 ºC. Gentle agitation can be provided by a standard stainless steel dissolution paddle rotating at 50 rpm. Emulsification time is assessed visually (Wei et al., 2005). Solubility studies 25