Definitions And Properties Of Microemulsions Biology Essay

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Introduction

Many micellar structures such as multiple emulsions, leptosomes and especially microemulsions have received considerable attentions in the past few years due to their ability to solubilise poorly soluble drugs or materials thus lowering skin irritation and enchance drug permeation.

The aim of this thesis is to review the work that has been conducted over the previous years with specific types of micellar structures for the formulation of suitable pharmaceutical containers i.e. microemulsions. Microemulsions are optimal vehicles for drug deliverybecause they are easy to formulate, they are thermodynamically stable and have optimal solubilization properties. Up to this date microemulsions are used to deliver drugs via parenteral, percutaneous ocular and oral routes and the reports all agree to the result that there is improved bioavailability for the majority of compounds

The focus of this thesis is to review the application of microemulsions and show their importance as the most appropriate vehicle for transdermal drug delivery.

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The theories underlying microemulsion formulation and the methods used to describe and characterize these systems are outlined. Selected examples of microemulsion formulations are given with particular emphasis on anti-inflammatory and anaesthetic agents. The in vitro and in vivo studies used to evaluate these formulations are examined. Finally, the future prospects for microemulsions as dermal and transdermal drug delivery vehicles are discussed.

Definition and Structure

The term microemulsion has been indtroduced in 1943 by Hoar and schulman and was defined as a clear solution obtained by titrating an oil-in-water emulsion with an alcohol(2) Microemulsions consists of water and oil and are stabilized in the presence of a surfactand and if required a co-surfactant. This process gives them their main properties which defines them as thermodynamically stable colloidal dispersions. In the case of difficult formulation of microemulsions we use a cosurfactand thus lowering the terfacial tension by three to four orders of magnitude(1). Danielson and Lindman defined microemulsions as composed of water, oil and amphiphile molecules , which are single phase and thermodynamically stable isotropic solutions(3)

The differences between emulsions and microemulsions, which despite theis similar nomenclature are very different, are highlighted in table 1 .

There is a large variety in the range of composiotion for microemulsions and their structure vary from agglomerates to spherical droplets mainly depending on the surfactand choice of nature and the composition of the microemulsions. There have been many studies suggesting that that microemulsions are 'oil in water' or 'water in oil' droplets of spherical size but later it was suggested that the main properties may be retained in the form of cubic structures

Microemulsions depending on the ratio of oil and water may be:

Continuous water solution with dispersed oil droplets

Continuous oil solution formed by inverted micelles with water

Continuous (In the case of similar amounts of water and oil)

Middle phase(Microemulsion in the middle, upper layer of oil and lower level of water)

The term microemulsion therefore may be given to a wide variety of preparations ranging from droplet type micellar structures to more complex lamellar structures(8-10)

Properties of Microemulsions

One of the main differences between emulsions and microemulsions is that the later are much more stable. This means that the main characteristing which is droplet size has the ability to remain unchanged over a large period of time thus promoting self emulsification of the system hence there is no significant energy input required neither the use of complex equipment is involves and we haveuniform drug distribution in the formulation whith the minimal resourced possible.

For a dispersion to form spontaneously, the free energy of mixing ( ΔG ) must be negative, i.e. lower than that of the unmixed components. The free energy of mixing is defined in equation 1:

ΔG = γΔA - T Δ S

γ:Surface tension

Δ A:change in interfacial area on emulsification

ΔS: change in entropy of the system

T:temperature

In order for a dispersion to be ΔG , must show a minimum value. Studies have concluded that in order for microemulsion to form they depend on the free energy resulting from the low interfacial tension between oil and water ( -10 -2 -10 -3 mN/m) [7, 11, 12]

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Microemulsion formation may be depended mainly on the ability of the various surfactants to decrase surface tension. In practice though this is not ideal and many times we require the presence of a cosurfactant in roder tao get the acceptable low surface energy. If a cosurfactand is not present , CMC(critical micelle concentration) limits the interfacial tension. The use of a cosurfactant reduces both CMC and interfacial tension (14).

High fluidity is a main disadvantage in a transdermal application. Thickening agents such as carbopol, Aerosil, Gelatin and Guar based polymers may be gelled with a lipophilic substance

Microemulsion Formulation

Oil Phase

Oil phase of microemulsion covers a vast variety of components which may be

Esters(isopropyl myristate, isopropyl palmitrate, ethyl oleate)

Medium chain triglycerides( caprylic acid, capric acid)

Alcohol(octanol and decanol)

Termpenes(limonene, cineole, camphor and menthol)

Fatty acides(oleic acid with combination of other components)[38,39,40]

Aqueous Phase

The aqueous phase is typically composed of:

Viscosity enchancing agents

Buffer salts

Preservatives and penetration enchancers

Sodium chloride[19]

Surfactants and Cosurfactants

One major factor when using surfactants is their side effects on human tissue. Most non ionic surfacyants are considered safe where as anionic surfactants are causing irritation and cationic surfactants are equally irritating but even more cytotoxic than anionic surfactants[56]

Nonionic surfactant formulations are:

Plurol Isostearique _ (isostearic acid ester of polyglycerol, containing 30-35% of diglycerol, 20-25% of triglycerol, 15-20% of tetraglycerol, and 10% of pentaglycerol and higher oligomers)

Transcutol P _ (diethylene glycol monoethyl ether) are proposed as less irritant alternatives to the medium chain alcohols

Labrasol [a mixture consisting of 30% mono-, di- and triglycerides of C 8 and C 10 fatty acids, 50% of mono- and diesters of poly(ethylene glycol)] has also been used in microemulsions designed for topical delivery because it is nonirritant as well as capable of forming microemulsions with nonalcoholic cosurfactants [ 42] .

Toxicity aspects will be discussed in detail in a later chapter of this thesis

Mechanisms of Percutaneous Enhancement

While the exact mechanism in which the microemulsions permeate the skin is not totally understood we know from their complex composition that several factors are involved as shown on the figure 1

.

Fugure 1: Mechanisms by which microemulsion components enchance drug permeation

Microemulsions may enhance transdermal drug delivery primarily by the following effects:

Excibition of high solubilization capacity for the drug therefore more drug is 'in' the microemulsion and thus the concentration of the drug that reaches the skin is increased

The so called 'reservoir effect' which described the process by which the internal phase of the microemulsion continuously provides drug to the external phase which comes into contact with the skin and so the external phase remains saturated with the drug for a longer period of time

Formulation components such as surfactants, cosurfactants and oils act as permeation enhancers by increasing drug diffusion on the skin area

Chemical enhancers may be incorporated in the microemulsion, which will also improve dermal and transdermal delivery of drugs.

The very low interfacial tension required for the microemulsion formation ensures great surface contact between the vehicle and the membrane

Characterization of Microemulsions

Even though they are very easy to produce, characterization of microemulsions is not so. The wide variety of structure is the main problem when attempting to characterize them so they are devided in some categories depending on their properties, the factors affecting drug release, stability and structure. In order to evaluate all these properties and characterize a certain microemulsion formulation we may use several techniques including NMR spectroscopy, electrical conductivity, self diffusion measurements and fluorescence spectroscopy

Phase diagrams

Figure 2: Effect of surfactant:cosurfactant ratio of the microemulsion formation region

Phase diagrams show the limits of the different phases as a function of the component composition.These diagrams are constructed after carefull visual inspection of the microemulsion often by polarized light microscopy but this most often happens in the case of known composition. If required we can also map several different parameters such as conductivity, viscosity etc.

The construction of these diagrams involves a lengthy process in which the various physical parameters of the microemulsion are measures. Pre-determined ratios of surfactant mixture and oil are blended and titrated with the aqueous phase. After carefully altering the ratios by increasing or decreasing the aqueous phase the various proportions of each component are calculated and noted. Those proportions in which a microemulsion is stably formed are used to plot a pseudo-ternary phase diagram as shown on figure 2.

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Microscopy

Transmition electron microscopy has been the most succesfull in the study and characterization of microemulsions [72] .

The main issue with T.E.M is the sensitivity of various microemulsion to heat and also the potentially introduction of exogenous materials in the form of artifacts(dust, etc). Another issue is the chemical reactions that may be caused from the introduction of electron from the microscope which may alter the structure of the microemulsion and finally tha lack of contrast between microemulsion structure and it's environment

Laser Light and Non-Optical Scattering Techniques

In the last few year laser scattering techniques(static and dynamic) and non-optical methods(X-ray scattering and SANS) are used for the characterization of the size of the colloidal phase. These techniques do not come without their disadvantages though. It has been reported that that there may be a misinterpretation of the diffusion coefficient and therefore the droplet size

A slightly different technique called the neutron scattering technique is used to to investigate the structural properties of a microemulsion

NMR

Self dissusion can be defined as a random movement of a molecule made in the absence of any concentration gradient and the effect this movement has on the environment in which the molecule is localized.

In the case of microemulsion we have molecules confined in a close aggregate auch as micelles and therefore we expect and find out that they have a self diffusion value 2 to 3 times lower than a pure solvent. Therefore in w/o microemulsions the self diffusion of water is slow and the self diffusion of oil is fast. The exact opposite are the results of water and oil molecules in o/w microemulsions

In bicontinuous structures, both oil and water molecules exhibit high self-diffusion coefficients. Microemulsion structure has been characterized as using self-diffusion measurements of the components, obtained by proton Fourier transform pulse-gradient spin echo NMR (PGSE NMR)

Conductivity and Viscosity

The nature of the microemulsion and the detection of phase inversion phenomena can be determined using classical rheological methods and by conductivity measurements [74] . Viscosity measurement show how drug release may be influenced by colloidal structures. A colloidal structure may be a vesicle with multilamellar layer or rod-like and worm-like reverse micelles. Usually watery microemulsions have high conductivity values whereas oily systems have low or non existence conductivity

Fluorescence Spectroscopy

Fluorescence spectroscopy is used to measure how easy the molecules of the fluorescent probe move in the microemulsion. This process is controlled by diffusion which varies depending on the viscosity of the medium and the microemulsion type[78]. In water continuous microemulsions though due to to slow diffusion of the water insoluble fluorescent(e.g pyrene) the propagation of excitation is inhibited. On the contrary oilconinuous microemulsions are not affected

Topical application of microemulsions

Transdermal administration of drugs has many advantages over other rootes of administration especially over oral administration. It's main advantage is that it avoids systemic side effects. One disadvantage is that we get lower drug efficacy due to the transdermal penetration rate and this limits by much the amount of drugs that may be used by this route. The problem with the drug permeation occurs mainly in stratum corneum which is the outermost layer of the skin. The main use of the stratum corneum is to prevent the body from dehydrating but in this case prevent the absobsion of the water based microemulsions

The main structural characteristics of the stratum corneum which is lipids seem to be essential for this function but recent findings by Engblom and Engstr6m , showed that Azone which is a well known enhancer of penetration properties increased the passing rate of water in lipidsystems such as the stratum corneum

If we consider that microemulsions have a great solubilising capacity we can expect that this particular property can affect the stratum corneum assembly with consequences for drug penetration. Several studies have shown that we get penetration enchancment when using a microemulsion:

Azelaic acid(a bioactive substance used to treat skin disorders) was found that an O/W microemulsion formed by water-propylene glycol, decanol-dodecanol, Tween 20, 1-butanol, and Carbopol 934 gave significantly better penetration than the corresponding water- propylene glycol- Carbopol "gel" (Fig. 3)

Tetrachyline hydrochloride

Analogously, Ziegenmeyer and Ffihrer compared the transdermal penetration of tetracycline hydrochloride from a W/O microemulsion prepared from dodecane, decanol, water, and an ethoxylated alkyl ether surfactant with that from conventional formulations and found enhanced absorption from the microemulsion formulation. Similarly, Bhatnagar and Vyas investigated the bioavailability of transdermally administered propranolol, a/-receptor blocking drug, which normally undergoes extensive first hepatic pass effects. It was found that the bioavailability of this drug could be extensively improved by transdermal application from a lecithin-based W/O microemulsion. Moreover, Willimann et al. employed lecithin-containing (W/O) microemulsions for the transdermal administration of scopolamine and broxaterol and found that the transport rate obtained with the lecithin microemulsion gels was much higher than that obtained with an aqueous solution at the same concentration (Fig. 4).

Since transdermal penetration from microemulsion systems is likely to depend on the degree of perturbation of the layered structures of the stratum corneum and the generation of "channels," it could be expected to depend on the microemulsion composition and structure (see above). For example, Osborne et al. studied the transdermal penetration of glucose from W/O microemulsions prepared from octanol, dioctylsodium sulfosuccinate, and water and found that an increased microemulsion water content caused enhanced water penetration. Differences in percutaneous glucose transport were further shown to parallel differences in the diffusion of water within the microemulsion vehicles prior to application to the skin.

An important aspect of enhanced transdermal transport is that it is often accompanied by irritation. This irritation is likely to be due to the disruption of the stratum corneum structure. It is important to note, however, that the composition of an applied multicomponent formulation, specifically that of a microemulsion, changes over time due to evaporation of the most volatile component, usually water. If, for example, what remains on the skin after water evaporation is an oil solution of surfactants, a disordering of the stratum corneum lipid structure may occur, with skin irritation as a consequence. If, on the other hand, the remains form a liquid crystalline phase (notably a lamellar phase), the inherent risk for irritation is reduced. Note, however, that the detailed mechanisms of both transdermal penetration enhancement and the occasionally occurring skin irritation are unclear at present despite the rather extensive research in this field.

Figure 4 Transport of scopolamine through human skin from (C)) a lecithin-isopropyl

palmitate water microemulsion ([H20]/[lecithin]=3) and from (0) an aqueous buffer solution.

Dermal and Transdermal Microemulsion Formulations

Microemulsion systems have been extensively studied with regard to their application in the pharmaceutical field [10] . Improved dermal drug delivery in particular has been observed for these systems when compared to conventional topical formulations such as emulsions [41] and gels [17, 40, 41] . The cutaneous drug delivery potential of microemulsions is dependent not only on the constituents of the vehicle, but as already noted, to a significant extent on the composition/internal phase structure, which may retard drug diffusion in the vehicles [39, 83].

A variety of in vitro and in vivo studies has been conducted in an effort to evaluate the influence of microemulsions on dermal and transdermal flux and to elucidate the mechanisms by which they promote enhanced drug transport, which will be detailed further in this section. Since topical therapy with anti-inflammatory and anaesthetic agents is often limited by poor skin permeation and a slow onset of action, particular attention is paid to the microemulsion literature relevant to these classes of drugs.

Anti-Inflammatory Drugs

1)In vitro Studies

The topical application of non-steroidal anti-inflammatory drugs (NSAIDs) has been widely explored in the treatment of several disorders (e.g. osteoarthritis) as an alternative route to overcome the adverse side effects associated with the oral and rectal routes of administration, including gastrointestinal intolerance. In the literature, several reports have shown that microemulsions represent promising vehicles for improving the delivery, efficacy and bioavailability of several NSAIDs, such as ketoprofen [41, 44] , celecoxib [84] , rofecoxib [85] , aceclofenac [86] , piroxicam [87] , and diclofenac [88-90] . For example, Rhee et al. evaluated the efficacy of microemulsions prepared with oleic acid, Labrasol/Cremophor RH 40 and water for dermal delivery of ketoprofen through rat skin [44] . The permeation profile of ketoprofen was highly dependent on the composition of the microemulsion, namely the choice and content of the oil (internal phase) and the surfactant mixture. In addition, the permeation improved when the content of water increased from 5 to 64% (w/w) with a concomitant reduction in surfactant content from 80 to 30%. The same trend was observed with a number of other lipophilic drugs [23, 38, 90] . These observations suggest that the thermodynamic activity of the drug in the external phase increases as a result of decreased drug solubility in the external phase, especially when the external phase content represents a higher proportion of the microemulsion than the other constituents. In a later study, Paolino et al. observed that the permeation of ketoprofen through abdominal human skin could also be enhanced when loaded into a lecithin microemulsion, consisting of triglycerides as the oil phase, a mixture of lecithin and n -butanol as the surfactant/cosurfactant system and an aqueous solution as the external phase [41] . It was suggested that the improved flux reflected lipid destabilization by the lecithin component. Other reports have suggested a direct relationship between the colloidal structure and in vitro drug release from formulations prepared with lecithin [66] . Depending on the ratio of diclofenac, lecithin and water, various colloidal structures including liposomes, microemulsions and lamellar liquid crystals were obtained. The diffusion behavior of diclofenac through artificial model membranes was affected by the microstructure of the various systems. For example, the release of diclofenac from microemulsions was very fast, whereas the addition of phospholipids and the resulting phase transition decreased the drug release from the vehicle, as a consequence of the increased viscosity. With regard to human skin permeation, the same study showed that the microemulsions provided higher flux than a simple aqueous solution of diclofenac. Although a range of colloidal structures were formed with phospholipids, only microemulsions were shown to enhance the SC permeation of diclofenac. The authors suggested that phospholipids may interact with the structure of the SC when applied as microemulsions, but not when applied as gel or liposomal formulations. In a further study, the effects of a microemulsion gel, composed of soybean phosphatidylcholine (lecithin), isopropyl palmitate (IPP) and water, on the transdermal transport of indomethacin and diclofenac through isolated human SC were investigated using Fourier transform infrared spectroscopy, differential scanning calorimetry and low-temperature scanning electron microscopy. No definitive conclusions on the role of lecithin as a penetration enhancer could be found, since similar temperature shifts of the SC lipid transitions were observed independently, whether isopropyl palmitate alone or the lecithin microemulsion gel was administered [37] . Nevertheless, this study observed higher permeation fluxes of indomethacin and diclofenac from the microemulsion gels in comparison to neat IPP.

Using excised skin from different animal species, the flux values of diclofenac sodium from w/o microemulsions, containing nonionic surfactants (PEG-40 stearate and glyceryl oleate) and a nonirritant cosurfactant (tetraglycol), were significantly higher than from aqueous solutions of the drug [89] . The incorporation of chemical enhancers (dimethyl sulfoxide and propylene glycol) in w/o microemulsions resulted in enhanced topical penetration of diclofenac sodium when compared with microemulsions without enhancers [88] . Flux through rabbit skin was improved for the enhancer/microemulsion formulations compared with commercially available gel formulations, but their effect was dependent on the choice of cosurfactant (propanol or isopropanol). Although histopathological examination of the tissue was conducted, the authors could not determine any changes in the skin induced by the different formulations and enhancers.

2)In vivo Studies

In comparison to the literature detailing the evaluation of microemulsion vehicles using in vitro models, there are limited reports of in vivo studies of the pharmacokinetics and the pharmacological activity of NSAIDs from microemulsions administered by the transdermal route [84-86] .

A pharmacokinetic study of the transdermal delivery of diclofenac in rats showed an 8-fold higher permeation of diclofenac from microemulsions than the commercially available gel with plasma levels comparable to those obtained after subcutaneous administration of the same dose of diclofenac [89] . Transdermal administration of the drug in microemulsions maintained constant levels of 0.7-0.9 _ g/ml for at least 8 h, while subcutaneous administration resulted in a peak plasma level of 0.94 _ g/ml after 1 h of administration, with evidence of rapid clearance from the plasma.

The anti-inflammatory activity of the selective cyclooxygenase-2-inhibitors rofecoxib and celecoxib in o/w microemulsion-based formulations has been evaluated in vivo, using mice [84, 85] . These studies suggested that a more rapid anti-inflammatory activity is achieved for microemulsion type systems than with conventional formulations. The higher release rates of microemulsion vehicles for the rofecoxib formulations were correlated with increased droplet size of the microemulsion. However, this is contradicted by the celecoxib studies in which the greatest anti-inflammatory effect was observed for the microemulsion with smaller droplet size. Higher release rates for these latter systems were also observed in vitro relative to a comparable microemulsion formulation with larger droplet size. Dalmora et al. prepared cationic microemulsions of piroxicam complexed with _ -cyclodextrin and investigated their in vivo anti-inflammatory effect in rats [16] . Daily topical application of the piroxicam formulations significantly inhibited granulomatous tissue formation, relative to a control in line with the ability of the microemulsion to act as a reservoir with extended release of drug over time.

Local Anaesthetic Agents

In vitro Studies

The delivery of local anaesthetics from microemulsions has been widely explored with evidence of increased skin permeation [39, 91] . Recently, Sintov et al. assessed the permeability of lidocaine from liquid and patch microemulsion vehicles prepared with tetraglycol as the cosurfactant, and glyceryl oleate combined with polyoxyl fatty acid derivatives as the surfactant film, through ratskin [23] . The authors observed that the microemulsion vehicles increased the flux of lidocaine-base across rat skin by 1.7- and 1.9-fold relative to a commercially available gel. This study confirms the results obtained by Kreilgaard et al., who observed the same magnitude of\ enhancement in the transdermal flux of lidocaine when incorporated in microemulsions composed of Labrasol, isostearyl isostearate, and Plurol Isostearique [39] .

Using human skin, Escribano et al. have shown that 4% amethocaine microemulsions prepared with decane, water and lauromacrogol 300, produced a 1.5-fold higher flux and permeability coefficient value than commercially available gels [92] . The same authors suggested that these microemulsions promoted the skin deposition and flux of amethocaine via enhanced partitioning into the skin rather than by increasing their diffusivity in skin. Lee et al. observed that o/w microemulsions composed of IPM, Tween 80 and ethanol promoted the flux of lidocaine free base and lidocaine hydrochloride across human skin to a significantly greater extent than w/o microemulsions [93] . The differences were attributed to the presence of N-methyl pyrrolidone (a chemical enhancer), which improved the drug partitioning in the aqueous external phase by a factor of 2.6, thus promoting its availability for skin transport. Since these authors noted that the partition coefficient of NMP in IPM/water is 0.02, they suggested that NMP is expected to be a more effective enhancer from the aqueous phase of a microemulsion than from the organic phase [94] .

In vivo Studies

Based on results from in vitro studies which suggested that microemulsions may promote drug localization in the skin, Sintov and Shapiro investigated lidocaine accumulation in skin layers in vivo after application of microemulsions for 10, 30 and 60 min in rats [23] . The microemulsion liquids promoted higher lidocaine concentrations in the epidermis and dermis of rats compared with a commercial cream formulation following 30 and 60 min of treatment. Interestingly, dermal drug concentrations were doubled when the microemulsion was applied as a patch in comparison to the liquid application, whereas epidermal concentrations were comparable for liquid and patch. The authors postulated that one explanation for this might be a reduction in the concentration gradient of lidocaine across the skin because of the adhesive properties of the patch.

Using a microdialysis technique, Kreilgaard evaluated the cutaneous absorption coefficient (apparent drug absorption rate) and lag time of lidocaine and prilocaine from topically applied microemulsions in rats [91] . Microemulsions improved the dermal absorption coefficient of lidocaine more than 8-fold (753 _ g/l/min) compared with a commercial emulsion (89 _ g/l/min) with shorter lag times. The results correlated well with in vitro studies which indicated that drug mobility in the microemulsion vehicle critically influenced dermal drug delivery rates.

Despite promising results indicating that analgesia may be achieved in shorter periods of time with microemulsions, few reports have evaluated the pharmacological effects of local anaesthetics when administered in these formulations. The majority of these studies have used animal species as in vivo models [60, 92, 94] with only one in human volunteers [95] . In the latter study, the pharmacokinetics of lidocaine were assessed, using microdialysis in 8 volunteers after administration of a microemulsion, and were then compared with the profile obtained from a commercial emulsion. The microemulsion formulation increased the cutaneous absorption of lidocaine 2.9-fold and decreased the lag time [95] . The second part of this study evaluated the pharmacodynamics of both formulations in 12 volunteers (placebo controlled design) by mechanical stimuli. Despite the significant differences in the absorption coefficient and lag times, both formulations produced similar analgesic effects.

In vivo studies in rats showed that AOT/IPM/water microemulsions (w/o) of tetracaine hydrochloride produced an 8-fold enhancement in the analgesic response of a drug compared to an aqueous saturated solution [60] . Using the tail-flick method, the maximum analgesic effect was observed 10 min after application, after which the effect decreased slightly. The analgesic response was still remarkably high even after 180 min of application when compared to the control. The same study showed that the analgesic response increases as AOT concentration and the concentration of water increases, which, according to the authors, could be explained as a result of the interaction of anionic surfactant with the skin and the increased hydration of the SC, respectively. The analgesic activity of microemulsion formulations compared with a commercial amethocaine gel was investigated in rats using the carrageenan-induced inflammation test [92] . The onset of analgesic activity was faster after application of the microemulsion (4.2 vs. 13.8 min for the gel). Žabka and Benková have also studied the influence of w/o microemulsion vehicles on the in vivo effect of the local anaesthetic agent pentacaine in rabbits [94] . Microemulsions containing the highest content of surfactant and cosurfactant produced the highest anaesthetic activity in rabbits. The onset of the local anaesthetic activity was within 10 min of application with a duration of 50 min.

Miscellaneous Drugs

Azelaic Acid

Azelaic acid is a naturally occurring dicarboxylic acid approved for the treatment of acne and other skin conditions. High concentrations of azelaic acid applied topically have also been used for the treatment of lentigo maligna, a pigmentary disorder. Gasco et al. [17] investigated the in vitro transport of azelaic acid, from microemulsion and gel formulations through full thickness mouse abdominal skin. The percentage of azelaic acid transported from the microemulsion was several times higher than that from the gel. The effect of dimethyl sulfoxide, chosen as a model enhancer, on transport was also investigated on drug permeation. After 8 h, 43 and 64% of the initial amount of drug in microemulsion formulations had passed through hairless skin using 1 and 2% of dimethyl sulfoxide, respectively.

In a clinical study involving 72 patients, two formulations containing different concentrations of azelaic acid were applied twice daily; a cream containing 20% azelaic acid and an o/w microemulsion containing 6.4% of azelaic acid [96] . Both formulations led to the complete regression of the lesions; however, the period of remission was considerably shortened when microemulsions were applied, thus reducing the treatment period. The authors suggested that this reflected high and prolonged bioavailability of azelaic acid from microemulsion formulations because of its dissolution in the formulation and partition into the disperse phase which might also act as a drug reservoir.

Apomorphine

The transdermal administration of apomorphine, a potent dopamine agonist, has been investigated for the treatment of Parkinson's disease [21] . Microemulsions were formulated using isopropyl myristate-decanol as the oil phase and octanoic acid, 1,2 propanediol, sodium hexanoate (or octanoate), sodium glycocholate (or taurocholate)

and apomorphine hydrochloride as the aqueous phase components. Studies conducted in hairless mouse skin resulted in steady state fluxes of 100 and 88 _ g/h -1 / cm -2 , respectively. The apparent log P of apomorphine increased from 0.30 in the absence of octanoic acid to 2.77 in the presence of octanoic acid (at an apomorphine: octanoic acid molar ratio of 1: 2.5). The amount of apomorphine released from microemulsions also diminished when the amounts of octanoate in the microemulsions were lowered, and the authors proposed that the formation of an ion pair with octanoic acid facilitated apomorphine transport from the formulations. The microemulsion formulation was subsequently evaluated in Parkinson's disease patients after cutaneous administration under occlusion to evaluate the absorption, efficacy, and tolerability of the formulation. Pharmacokinetic analysis demonstrated that the formulation provided a rapid permeation and a sustained release of the drug, with prolonged therapeutic plasma levels when compared with oral dopamine agonists, and suggested that the formulation might be a promising add-on treatment for uncontrolled 'wearing off' phases in Parkinson's patients [64] .

β -Blockers

A novel concept of microemulsion formation in situ was explored for transdermal delivery of _ -blockers. This approach assumes that in situ microemulsions can be produced as a result of a water uptake by the water-free formulation under occlusive conditions [97] . The water uptake decreased the apolar drug solubility, leading to enhancement of the drug thermodynamic activity. The pharmacodynamic effects of bupranolol and timolol in vivo were investigated over 10 h in New Zealand albino rabbits after application of these microemulsions under an occlusive patch and compared with delivery of the same drugs from matrix patches. Different _ -blockers were used as model drugs and the suppression of tachycardia produced by a standard dose of isoprenaline was used as the pharmacodynamic parameter [98] . The formation of microemulsions in situ resulted in faster pharmacodynamic effects of bupranolol and timolol in comparison with the matrix patches. According to the authors, the increased pharmacodynamic effect in vivo is in good agreement with the decrease of the solubility of the drug caused by the water uptake [99] .

Toxixity aspects

An important aspect in all drug delivery is the toxicity of the drug as well as that of the drug carrier. Therefore, toxicity has to be assessed also for microemulsion formulations. In microemulsion systems, the main concern regarding toxicity has to do with the cosurfactants used. For example, the majority of the work on the pharmaceutical application of microemulsions has involved the use of short- or medium-chain alcohols, e.g., butanol. In a range of studies it has been shown that these cause toxic side effects. For example, inhalation studies of the toxicity of 1-butanol, 2 butanol, and tert-butanol in rats showed a dose-dependent reduction in fetal weight . Furthermore, aqueous solutions of ethanol, propanol, and butanol were shown to result in elongated mitochondria in hepatocytes after 1month of exposure . (In addition to the toxicity aspects of these alcohols, microemulsions formed in their presence are often destabilized on dilution of the continuous phase.) Furthermore, many studies so far have involved aliphatic or aromatic oils, such as hexane or benzene, which obviously are unsuitable for pharmaceutical use. Moreover, ionic surfactants could in themselves be toxic and irritant .

Nonionic surfactants, such as ethoxylated alkyl ethers and sorbitan esters, as well as nonionic block copolymers [e.g., poly(ethylene oxide)-blockpoly(propylene oxide)] are generally less irritant and toxic than ionic surfactants. Moreover, many nonionic surfactants have the advantage over charged surfactants in that they can form microemulsions even without cosurfactants. For example, this was used by Siebenbrodt and Keipert, who investigated the ophthalmic application ofmicroemulsions containing poloxamer L64 [a poly(ethylene oxide)-poly(propylene oxide) block copolymer], propyleneglycol, water, and triacetine and found an acceptable tolerance of this model formulation.The comparatively good biological acceptance of nonionic surfactants and block copolymers and the fact that cosurfactants may not be needed for the microemulsion formation constitute the two main motives for the rather extensive use of nonionic surfactants, particularly for topical applications of microemulsions.

Despite the reasonable tolerance of nonionic surfactants, particularly in topical

applications, microemulsions prepared from (phospho)lipids seem to be preferred over those prepared by synthetic surfactants from a toxicity point of view. As discussed by Shinoda et al., lecithin in water-oil systems does not spontaneously form the zero mean curvature amphiphile layers required for the formation of balanced microemulsions but rather forms reverse structures.

On decreasing the "polarity" of the aqueous phase by addition of a short-chain alcohol, e.g., propanol, lecithin was found to form microemulsions at low amphiphile concentrations over wide ranges of solvent composition. The structure of the microemulsions formed was investigated by NMR self-diffusion measurements, and it was found that with a decreasing propanol concentration there was a gradual transition from oil droplets in water, over a bicontinuous structure, to water droplets in oil .

Another way of forming balanced or O/W microemulsions in phospholipid-based systems is through the addition of surfactants that favor structures with a curvature toward the oil. Examples of such substances include nonionic surfactants and block copolymers with long oligo(ethylene glycol) chains, as well as single-chain (an)ionic surfactants . Although this packing concept has not been extensively used for microemulsions so far, it is well known for other surfactant systems

Despite some rather promising studies of the use of phospholipid-based

microemulsions in drug delivery [1], the extensive use of cosurfactants (particularly medium-chain alcohols) means that toxicity still remains a problem for many of these for mulations. Thus, there is a need to develop new phospholipids and phospholipid-base microemulsions that do not require cosurfactants in order for this type of system to gain wide applicability in drug delivery.

Another type of "biocompatible" microemulsion, formed by water, triglycerides, and monoglycerides has been studied, e.g., by Engstr6m (Fig. 8). It was found that this system formed a rather extensive Lz phase at 40°C. Based on X-ray diffraction, this phase was proposed to have a lipid bilayer structure even at high oil content. Considering the biocompatibility and the ease of biodegradation of these components, this type of microemulsion is particularly attractive for oral delivery of drugs, where toxicity otherwise limits the applicability of microemulsions.

Conclusions

Microemulsions are an attractive technology platform for the pharmaceutical formulator as they are thermodynamically stable, possess excellent solubilization properties, and their formulation is a relatively straightforward process. There is now a considerable body of literature which confirms their potential for topical and transdermal drug delivery. Although a number of microemulsions for cosmetic applications are in use, there are as yet no dermal or transdermal microemulsion formulations on the market for pharmaceutical actives. This may be attributed to a number of factors. There is a need to evaluate the interactions of these formulations with human skin, as many of the reports to date have employed animal skin or models which are not predictive of human skin permeation. Many authors have not addressed the irritation/ toxicity issues which may arise because of high surfactant/ cosurfactant content. No systematic investigation has been conducted on how the inclusion of other penetration enhancers besides surfactant/cosurfactant components might influence drug permeation. Finally, the synergistic effects of microemulsion vehicles with physical enhancement methods such as iontophoresis should be investigated more fully. With the advent of new and improved biophysical techniques to interrogate the effects of these formulations on the skin it is likely that many of these issues should be addressed and that the technology will emerge as a practical and inexpensive method for dermal and/or transdermal drug delivery.