Rakesh P. Patel is an associate professor in the Department of Pharmaceutics and Pharmaceutical Technology, S. K. Patel College of Pharmaceutical Education and Research, Ganpat University, Gujarat, India.

Rakesh P. Patel

	Berberine hydrochloride (BRB) is an isoquinoline-alkaloid derivative that can be isolated from medicinal herbs such as 

 huangbai and huanglian are described as heat-removing agents for fever reduction (2). BRB, the major ingredient of these herbs, possesses antimicrobial activity against Gram-positive and Gram-negative bacteria, as well as against other microorganisms (3, 4). BRB inhibits the growth of streptococci and appears to prevent them from adhering to host cells (5). BRB also exhibits antimalarial, antisecretory, anti-inflammatory, and anticancer activities with relatively low cytotoxicity to human cells (6).

	Topical and transdermal products are important classes of drug-delivery systems, and their use is becoming more widespread. The purpose of topical dosage forms is to deliver drugs conveniently to a localized area of the skin (7). Topical creams (e.g., cold cream), which are oil-in-water (O/W) emulsions, are less greasy and more acceptable to patients. Patients generally prefer creams for the treatment of mild or short-duration conditions (8).

	Many topical antifungals are now available, but not all are equally effective. Only a few topical antibiotics are available for treating skin diseases (9, 10). The authors investigated cream formulations of BRB because it possesses antifungal, antibacterial, and anti-inflammatory activity.

	Apifil (PEG-8 Beeswax, Gattefossé, St. Priest, France) and Plurol Stearique WL 1009 (polyglyceryl-6-distearate, Gattefossé) were selected as O/W emulsifiers. Both emulsifiers can be used to formulate creams with various concentrations of the oil phase without phase inversion. At higher concentrations (e.g., 5–15%), they form stable creams with a firm texture and a smooth, glossy appearance. The agents emulsify vegetable oils by as much as 15% are particularly well-suited to the emulsification of short-chain or fatty-acid esters. They perform well with other fatty-acid esters, silicone oils, mineral oils, and their substitutes.

	The authors attempted to develop safe topical formulations of BRB that could deliver the drug locally in an effective concentration for its antimicrobial and anti-inflammatory effects. The effectiveness of the cream formulation would likely depend on the nature and concentration of the emulsifier used, on the concentration of BRB, and on the storage time of the cream formulations. The authors designed an experiment to investigate the effects of these variables on the formulation of BRB as a topical drug-delivery system.

 BRB HCl powder (90% purity) was received as a gift from Yucca Laboratories (Mumbai) and used in the study without further purification procedures.

 The authors chose the emulsifiers Apifil and Plurol Stearique WL 1009 for formulating the BRB creams. Both emulsifiers were obtained from Gattefossé. The formulations were based on aqueous cream BP (

). The formulation for 100 g of aqueous cream BP was the following:

		Boiled and cooled purified water (100 g).

	Emulsifying ointment BP consists of emulsifying wax BP (30%), white soft paraffin BP (50%), and liquid paraffin BP (20%), and acts as a hydrophobic vehicle, structural-matrix former, and emulsifying agent. Menthol BP was used as a permeation enhancer. The detailed composition of different cream formulations is shown in Tables Ia and Ib.

 a Gram-positive bacterium, is associated with skin ailments such as boils, lesions, and infected burn sites. 

 a fungus, is responsible for about 70–80% of all candidal infections. Infections can occur anywhere and are most common in skin folds and web spaces, on the penis, and around fingernails (11).

 Cream formulations containing five different concentrations of BRB (0.5%, 1.0%, 2.0%, 5.0%, and 10% w/w BRB) were prepared based on aqueous cream BP. Two concentrations, 5% and 10% w/w, of the two emulsifiers were used to prepare the creams. Various concentrations of menthol (2.5%, 5.0%, 10%, and 12.5% w/w) also were incorporated in the cream formulation that contained 10% w/w BRB and the optimum concentration of emulsifier (Apifil 10%) as a permeation enhancer. Control formulations containing no BRB or permeation enhancers were also prepared for skin-irritation and anti-inflammatory studies. Samples of the test cream formulations were stored at room temperature and shielded from light for a period of three months to determine the stability of the creams.

 Drug content of the cream formulations was determined by dissolving an accurately weighed quantity of cream in about 50 mL of pH 7.2 phosphate buffer. These solutions were transferred quantitatively to volumetric flasks, and appropriate dilutions were made with the same buffer solution. The resulting solutions were passed through 0.45-mm membrane filters before undergoing spectrophotometric analysis for BRB at 345 nm (λ

 A rotational digital viscometer (DV II RVTDV-II, Brookfield Engineering, Brookfield, MA) was used to measure the viscosity (in cps) of the creams. The spindle was rotated at 10 rpm. Samples of the creams were allowed to settle over 30 min at the temperature of test (25±1 °C) before the measurements were taken.

 were grown in tryptone soy broth for 24 h at 37 °C and for 48 h at 28 °C, respectively. Bacteria (100 mL of a 1-in-100 dilution) of the overnight culture were used to inoculate 20 mL of molten sterile nutrient agar (Hi-Media Laboratories, Mumbai) at 45 °C and allowed to set. For the fungi, 20 mL of molten sterile sabouraud dextrose agar (Hi-Media Laboratories) containing 0.05 mg/mL cycloheximide and 0.5mg/mL chloramphenicol was poured into 9-cm sterile plates and allowed to set. The surface was inoculated with 0.2 mL of the culture. The surface of the set agar was allowed to dry at 37 °C and equidistant 6-mm wells were cut into the agar. Tetracycline and nystatin were used as positive controls for the bacteria and fungi, respectively.

Antimicrobial analysis of cream formulations.

 One gram of each formulation was mixed with 1 mL of sterile water using a whirl mixer until a slurry was formed. One hundred μL of each slurried cream was placed into prelabeled wells. The creams were allowed to diffuse for 1 h before incubating the bacteria plates at 37 °C for 24 h, and the fungal plates were incubated at 25 °C for a minimum of 48 h. The diameter of zone of inhibition was measured after the incubation period for each formulation.

 Experiments were designed separately on formulations containing different concentrations of BRB. To study how well the various cream formulations inhibited 

 growth, each of the three variables-type of emulsifier (

)-were used at a high level (denoted by a subscript 

) and a low level (denoted by a subscript 

 (= 8) factorial experimental design (12–14). Using the above nomenclature, the eight combinations between the variables were 

 is zero time in storage of cream preparations, and 

 is 12 months of storage of cream preparations. Samples of the test cream formulations were stored at room temperature and shielded from light for a period of 12 months to test their stability.

	The combinations were grouped into sets to assess the effect that each variable had on the effectiveness of the cream preparations against the relevant microorganisms and to determine whether the variables interacted or acted independently. For example, the effect of increasing a particular variable such as 

 activity of the cream formulations against the microorganism involved was assessed by adding all the zone of inhibition values of combinations containing a high level of 

 and subtracting the sum of all zones of inhibition for combinations containing a low level of 

 as illustrated by the following equation:

	This value, whether positive or negative, was a quantitative measure of the effect of 

 on the activity of the cream formulation against the microorganism involved. Similar expressions were used to find the effects of 

 on the activity of the cream formulation against the relevant microorganisms.

	To determine whether any two variables interacted with each other, the authors added the zone of inhibition results of the combinations in which the variables appeared and subtracted the corresponding sum of other combinations. For example, the authors used the following equation to find the interaction between 

	A result of zero indicated no interaction, but a difference implied that the two variables had interacted. Similar equations were used to estimate the interactions between 

 skin-permeation studies were performed using a Franz diffusion cell with a receptor compartment capacity of 21 mL and an effective diffusion area of 1.84 cm

. Excised rat abdominal skin was mounted between the donor and receptor compartments of the diffusion cell. One gram of each cream formulation was placed on the skin. The receptor compartment of the diffusion cell was filled with phosphate buffer (pH 7.2). The whole assembly was fixed on a magnetic stirrer (Remi, Mumbai), and the solution in the receptor compartment was continuously stirred using magnetic beads at 50 rpm. The temperature was maintained at 37 ± 0.5 °C. The samples were withdrawn at different time intervals and analyzed for drug content spectrophotometrically at a wavelength of 345 nm. The concentration of BRB in each sample was determined from a previously calculated standard curve. The receptor phase was replenished with an equal volume of phosphate buffer after each sample withdrawal. The cumulative percents of drug permeation per square centimeter of skin were plotted against time.

 The authors followed the "Guidelines of the Institutional Animal Ethics Committee" for this experiment. The hair on the dorsal side of Wistar albino rats was removed by clipping one day before the start of the experiment (15). The rats were divided into three groups (

=6). Group I was the control (i.e., cream without drug and permeation enhancers or Formulation P), Group II received cream with permeation enhancers (M5), and Group III received a 0.8% v/v aqueous solution of formalin as a standard irritant (16). A new cream or new formalin solution was applied daily for seven days. Finally, the application sites were graded according to a visual scoring scale, always by the same investigator.

 Anti-inflammatory studies of prepared formulations were compared by the carrageenan-induced rat-paw edema method in Wistar albino rats. The protocol was approved by the Institutional Animal Ethics Committee. Eighteen rats were divided into three groups of six rats each for the different treatments shown in Table IV. Group I served was the control (i.e., Formulation P), Group II received cream with permeation enhancers (M5), and Group III received diclofenac gel containing 1% w/w of diclofenac (Diclomol Gel, Win-medicare, Delhi) as a standard formulation. Animals were fasted for 24 h before the experiment and given free access to water. Approximately 0.1 mL of 1% carrageenan suspension in saline was prepared 1 h before each experiment and injected into the plantar side of the right hind paw of the rat. Next, 100 mg of cream containing 10% of BRB was applied to the plantar surface of the hind paw by gently rubbing 50 times with the index finger. Rats of the control groups received only the cream base. Diclofenac gel 1.0% w/w was applied in the same way as a reference. Active and placebo creams were applied 1 h before the carrageenan injection. The paw volume was measured initially and at 1, 2, 3, and 4 h after carrageenan injection using the plythesmographic method (17). The percentage of inflammation was calculated for the purpose of comparison.

 All prepared cream formulations of BRB contained 99.2–100.1% of the indicated amount of BRB. As the concentration of menthol increased in the formulations, the viscosity decreased in both gel formulations (see Table III).

 The results of the experiments on the antimicrobial effectiveness of the various cream formulations of BRB, as measured by the diameter of zone of inhibition (mm), are presented in Figure 1. The combination of variables significantly influenced the effectiveness of the cream formulations. All the creams generally showed considerable effectiveness against 

 creams with a BRB concentration of 0.5% and 1.0% w/w had little effectiveness. The effectiveness of the creams, however, increased considerably with an increase in BRB concentration.

	The relevant values of the zones of inhibition were used to calculate the independent and interaction coefficients of the variables' influence on the creams' effectiveness. The values of the coefficients are presented in Table II. The variables had positive and negative influences on the cream formulations' effectiveness. A positive influence was increased inhibition of bacterial growth, and a negative influence was a decreased inhibitory effect on bacterial growth.

	The independent coefficient values show the influence of individual variables on the effectiveness of the cream formulations (see Table II). The effects of the variables were highly dependent on the concentration of BRB in the formulation.

	The type of emulsifier had by far the greatest effects on the inhibitory value of the creams, especially at higher concentrations of BRB. The effects of 

 were generally positive, indicating that a change in emulsifier from Plurol, the low level of 

 increased the creams' antimicrobial effectiveness. 

 had a much greater effect on the creams' effectiveness against 

 generally had moderate effects on the inhibitory value of the creams, especially at higher concentrations of BRB, where these effects were generally positive. This result indicates that changing the concentration of emulsifier from 5 to 10% would increase the effectiveness of the creams that contained higher concentrations of BRB.

 generally had the least effects on the inhibitory value of the creams, and thus the time of storage should be the least influential variable. The effects were all negative, thereby showing that the creams' effectiveness decreases with time. This reduction in effectiveness is negligible, however, because the creams stored for 12 months showed a similar zone of inhibition to that of creams stored for no time.

	The interaction coefficient values indicated the effects of the variables in combination (see Table II). The authors had difficulty ranking the interaction effects, although the strongest interactions appeared to be between 

 is more susceptible to cream formulations that contain a high concentration of Apifil than is 

 More importantly, the various interactions appeared to be generally weak, suggesting that the variables were, to a large extent, acting independently of each other. This observation therefore implies that 

 is the most influential variable in this particular work because it had the largest effects.

 The authors investigated the penetration-enhancing effect of menthol on the permeability of cream formulations of BRB through the excised rat epidermis. Permeation parameters for BRB in the cream formulations are shown in Table III. The cumulative amount of drug permeation through the rat epidermis from cream formulations that contained various amounts of menthol is shown in Figure 2.

) of BRB that permeated during the 24 h of the study was 6.20 ±0.23 mg/cm

 from Formulation P prepared without menthol. The flux was obtained by dividing the cumulative amount of drug permeated per cm

 of the skin by time. The corresponding flux of BRB was 260.21 ±9.76 μg/cm

	The authors observed a marked effect of menthol on BRB skin permeation. The cumulative amounts (

) of BRB that permeated over 24 h increased from 9.43 ±0.34 to 35.07 ±0.95 mg cm

 for cream formulations containing 2.5 and 12.5 %w/w of menthol, respectively. The corresponding flux values ranged from 391.73 ±12.58 to 1485.75 ±42.93 μg cm

 . However, a lag period of 1 h was observed for both formulations. The drug-permeation data were consistent with zero-order kinetics from 2 to 24 h with a lag period of about 1 h for all cream formulations (see Table III).

As the menthol concentration increased from 0 to 12.5% w/w, the permeability of BRB also increased, as indicated by an increase in both the permeability coefficient and enhancement ration (ER) (see Figure 2 and Table III). The authors observed a fivefold increase in the permeability of the drug from the cream containing 12.5% w/w of menthol (see Table III). Terpenes increase the drug's percutaneous permeation mainly by disrupting the intercellular packing of the subcutaneous lipids (18–20).

	Formulation M5 (12.5% w/w menthol), prepared using Apifil, showed the highest 

 permeability of BRB, hence this formulation was selected for skin-irritation and anti-inflammatory studies.

 The skin-irritation test of Formulation M5 resulted in a score of less than 2 (see Table IV). Compounds that have scores of 2 or less are considered to be nonirritants (21). Hence, both types of topical cream formulations may be categorized as nonirritants.

 The results of anti-inflammatory activity studies after topical application are reported in Table V. For the control formulation, the paw volume increases arithmetically with time. Formulation M5 and the standard diclofenac gel formulation showed better reduction in paw-volume measurement than did the control formulation. Statistical analysis showed that the edema inhibition of Formulation M5 and the standard diclofenac gel formulation were much better than that of the control group (P < 0.05). However, there was no statistical difference between the anti-inflammatory effects of formulation M5 or the standard diclofenac gel (

	Topical applications have a great potential as an effective and safe way to administer BRB for local antimicrobial and anti-inflamatory effects. An antimicrobial study of a prepared cream formulation of BRB indicated good antibacterial activity against 

 permeation study using rat epidermal membranes and anti-inflammatroy studies by the carrageenan-induced rat-paw edema method showed that menthol enhanced the transdermal absorption of BRB formulations based on aqueous cream BP. The topical-cream formulations of BRB developed in this study have great utility and are a viable option for the effective and controlled management of inflammation and skin infections caused by 

 is an assistant professor in the department of pharmacognosy, 

 is a professor, all at S.K. Patel College of Pharmaceutical Education and Research, Ganpat University, Kherva, 382711 Mehsana, Gujarat, India, fax +91 2762 286082, shailyrakesh@yahoo.com.

	*To whom all correspondence should be addressed.

	Submitted: Feb. 20, 2009. Accepted: Mar. 23, 2009.

	2. K.C. Huang and W.M. Williams, "Antibacterial, Antiviral, and Antifungal Herbs," in 

 K.C. Huang, Ed. (CRC Press, New York, 2nd ed., 1999), pp. 381–383.

	3. A.H. Amin, T.V. Subbaiah, and K.M. Abbasi, 

	5. D. Sun, S.N. Abraham, and E.H. Beachey, 

 A.O. David and H.A. Anton, Eds. (Marcel Dekker, New York, 1st ed., 1990), p. 1.

	11. G. L. Darmstadt, J. G. Dinulos, and Z. Miller, 

	21. J.H. Draize, G. Woodward, and H.O. Calvery, 

Articles

The authors sought to prepare a topical formulation of berberine hydrochloride for the effective and controlled management of inflammation and skin infections.

The Formulation and Evaluation of Topical Berberine-Hydrochloride Products

Self-emulsifying drug delivery systems (SEDDSs) have gained exposure for their ability to increase solubility and bioavailability of poorly soluble drugs. SEDDSs are isotropic mixtures of oils and surfactants, sometimes containing cosolvents, and can be used for the design of formulations in order to improve the oral absorption of highly lipophilic compounds. SEDDSs emulsify spontaneously to produce fine oil-in-water emulsions when introduced into an aqueous phase under gentle agitation. SEDDS can be orally administered in soft or hard gelatine capsules and form fine, relatively stable oil-in-water emulsions upon aqueous dilution. This article presents an overview of SEDDSs and their applications.

In recent years, the formulation of poorly soluble compoundspresented interesting challenges for formulationscientists in the pharmaceutical industry. Up to 40% of newchemical entities discovered by the pharmaceutical industryare poorly soluble or lipophilic compounds, which leads topoor oral bioavailability, high intra- and inter-subject variability,and lack of dose proportionality (1).

In the oral formulation of such compounds, a number ofattempts-such as decreasing particle size, use of wettingagents, coprecipitation, and preparation of solid dispersions-have been made to modify the dissolution profile andthereby improve the absorption rate. Recently, much attentionhas focused on lipid-based formulations to improve thebioavailability of poorly water soluble drugs. Among manysuch delivery options, like incorporation of drugs in oils (2),surfactant dispersion (3), emulsions (4) and liposomes (5),one of the most popular approaches are the self-emulsifyingdrug delivery systems (SEDDSs).

SEDDSs are mixtures of oils and surfactants, ideally isotropicand sometimes containing cosolvents, which emulsify spontaneously to produce fine oil-in-water emulsions whenintroduced into an aqueous phase under gentle agitation. Self-emulsifying formulations spread readily in the gastrointestinal (GI) tract, and the digestive motility of the stomach and the intestine provide the agitation necessary for selfemulsification.These systems advantageously present the drug in dissolved form and the small droplet size provides a large interfacial area for the drug absorption (6). SEDDSs typically produce emulsions with a droplet size between 100–300 nm while self-microemulsifying drug delivery systems (SMEDDSs) form transparent microemulsions with adroplet size of less than 50 nm. When compared with emulsions,which are sensitive and metastable dispersed forms, SEDDSs are physically stable formulations that are easy tomanufacture. Thus, for lipophilic drug compounds that exhibit dissolution rate-limited absorption, these systems mayoffer an improvement in the rate and extent of absorption and result in more reproducible blood-time profiles (7).

The self-emulsifying process is depends on: (7)

The nature of the oilâsurfactant pair 

The temperature at which self-emulsification occurs. 

 Oils can solubilize the lipophilic drug in a specificamount. It is the most important excipient because it canfacilitate self-emulsification and increase the fraction of lipophilicdrug transported via the intestinal lymphatic system,thereby increasing absorption from the GI tract (9).Long-chain triglyceride and medium-chain triglyceride oilswith different degrees of saturation have been used in thedesign of SEDDSs. Modified or hydrolyzed vegetable oilshave contributed widely to the success of SEDDSs owing totheir formulation and physiological advantages (8). Novelsemisynthetic medium-chain triglyceride oils have surfactantproperties and are widely replacing the regular medium-chain triglyceride (9).

 Nonionic surfactants with high hydrophilic–lipophilic balance (HLB) values are used in formulation of SEDDSs (e.g., Tween, Labrasol, Labrafac CM 10, Cremophore,etc.). The usual surfactant strength ranges between30–60% w/w of the formulation in order to form a stable SEDDS. Surfactants have a high HLB and hydrophilicity, which assists the immediate formation of o/w dropletsand/or rapid spreading of the formulation in the aqueous media. Surfactants are amphiphilic in nature and they candissolve or solubilize relatively high amounts of hydrophobic drug compounds. This can prevent precipitation of the drug within the GI lumen and for prolonged existence of drug molecules (10).

 Cosolvents like diehylene glycol monoethyleether (transcutol), propylene glycol, polyethylene glycol,polyoxyethylene, propylene carbonate, tetrahydrofurfurylalcohol polyethylene glycol ether (Glycofurol), etc., mayhelp to dissolve large amounts of hydrophilic surfactantsor the hydrophobic drug in the lipid base. These solventssometimes play the role of the cosurfactant in the microemulsionsystems.

With a large variety of liquid or waxy excipients available,ranging from oils through biological lipids, hydrophobic andhydrophilic surfactants, to water-soluble cosolvents, thereare many different combinations that could be formulatedfor encapsulation in hard or soft gelatin or mixtures whichdisperse to give fine colloidal emulsions (11).The following should be considered in the formulation ofa SEDDS:

The solubility of the drug in different oil, surfactantsand cosolvents. 

The selection of oil, surfactant and cosolvent based onthe solubility of the drug and the preparation of thephase diagram (12). 

The preparation of SEDDS formulation by dissolvingthe drug in a mix of oil, surfactant and cosolvent. 

The addition of a drug to a SEDDS is critical because thedrug interferes with the self-emulsification process to a certainextent, which leads to a change in the optimal oil–surfactant ratio. So, the design of an optimal SEDDS requirespreformulation-solubility and phase-diagram studies. In thecase of prolonged SEDDS, formulation is made by adding the polymer or gelling agent (13).

According to Reiss, self-emulsification occurs when theentropy change that favors dispersion is greater than theenergy required to increase the surface area of the dispersion.The free energy of the conventional emulsion is a directfunction of the energy required to create a new surfacebetween the oil and water phases and can be described bythe equation:

Where, DG is the free energy associated with the process(ignoring the free energy of mixing), N is the number ofdroplets of radius r and s represents the interfacial energy.The two phases of emulsion tend to separate with time toreduce the interfacial area, and subsequently, the emulsion isstabilized by emulsifying agents, which form a monolayer ofemulsion droplets, and hence reduces the interfacial energy,as well as providing a barrier to prevent coalescence (14).

The primary means of self-emulsification assessment is visualevaluation. The efficiency of self-emulsification couldbe estimated by determining the rate of emulsification,droplet-size distribution and turbidity measurements.

 This may provide important informationabout the self-emulsifying and microemulsifying property of the mixture and about the resulting dispersion (15,16,17).

 This is to identify efficient self-emulsificationby establishing whether the dispersion reachesequilibrium rapidly and in a reproducible time.

 This is a crucial factor in self-emulsificationperformance because it determines the rate and extent of drug release as well as the stability of the emulsion (10,18). Photon correlation spectroscopy, microscopic techniques ora Coulter Nanosizer are mainly used for the determinationof the emulsion droplet size (10,19,20). The reduction of the droplet size to values below 50 µm leads to the formationof SMEDDSs, which are stable, isotropic and clear o/wdispersions (6).

 This is used to identify the chargeof the droplets. In conventional SEDDSs, the charge on anoil droplet is negative due to presence of free fatty acids(17).

 Self-emulsification time, dispersibility, appearance and flowability was observed andscored according to techniques described in H. Shen 

(21) used for the grading of formulations.

SEDDS formulation is composed of lipids, surfactants, andcosolvents. The system has the ability to form an oil-in-wateremulsion when dispersed by an aqueous phase under gentleagitation. SEDDSs present drugs in a small droplet size andwell-proportioned distribution, and increase the dissolutionand permeability. Furthermore, because drugs can beloaded in the inner phase and delivered by lymphatic bypassshare, SEDDSs protect drugs against hydrolysis by enzymesin the GI tract and reduce the presystemic clearance in theGI mucosa and hepatic first-pass metabolism. Table I showsthe SEDDSs prepared for oral delivery of lipophilic drugsin recent years.

Self-emulsifying drug delivery systems are a promising approachfor the formulation of drug compounds with pooraqueous solubility. The oral delivery of hydrophobic drugscan be made possible by SEDDSs, which have been shownto substantially improve oral bioavailability. With futuredevelopment of this technology, SEDDSs will continue toenable novel applications in drug delivery and solve problemsassociated with the delivery of poorly soluble drugs.

1. P.P. Bhatt, "Osmotic drug delivery systems for poorly solubledrugs," 

The Drug Delivery Companies Report Autumn/Winter 2004

2. D.L. Burcham et al., "Improved oral bioavailability of the hypocholesterolemicDMP 565 in dogs following oral dosing in oil and glycol solutions," 

3. T.M. Serajuddin et al., "Effect of vehicle amphiphilicity on the dissolution and bioavailability of a poorly water-soluble drug fromsolid dispersion," 

4. R.A. Myers and V.J. Stella, "Systemic bioavailability of penclomedine(NSC-338720) from oil-in-water emulsions administeredintraduodenally to rats," 

5. R.A. Schwendener and H. Schott, "Lipophilic 1-beta-d -arabinofuranosylcytosine derivatives in liposomal formulations for oral and parenteral antileukemic therapy in the murine L1210 leukemiamodel," 

6. N.H. Shah et al., "Self-emulsifying drug delivery systems (SEDDS)with polyglycolyzed glycerides for improving in vitro dissolution and oral absorption of lipophilic drugs," 

7. R.N. Gursoy and S. Benita, "Self-emulsifying drug delivery systemsfor improved oral delivery of lipophilic drugs," 

8. P.P. Constantinides, "Lipid microemulsion for improving drugsdissolution and oral absorption: physical and biopharmaceuticalaspects," 

9. S.M. Khoo et al., "Formulation design and bioavailability assessmentof lipidic silf-emulsifying formulation of halofanitrine,"

10. N.H. Shah et al., "Self-emulsifying drug delivery systems (SEDDS)with polyglycolized glycerides for improving in vitro dissolutionand oral absorption of lipophilic drugs." 

11. J.R. Crison and G.L. Amidon, "Method and formulation for increasingthe bioavailability of poorly water-soluble drugs," USPatent No. 5,993,858, issued November 30, 1999.

12. N. Farah, J.P. Laforet and J. Denis, "Self Micro Emulsifying DrugDelivery Systems for improving dissolution of drugs: In vitroevaluations," presented by Gattefosse Patented Technology atthe AAPS Annual Meeting in San Diego, November 1994.

13. S. Nazzal and M.A. Khan, "Controlled release of a self-emulsifyingformulation from a tablet dosage form: Stability assessmentand optimization of some processing parameters," 

14. P.P. Constantinides, "Lipid microemulsions for improving drugdissolution and oral absorption: physical and biopharmaceuticalaspects," 

15. D.Q.M. Craig et al., "An investigation into the mechanisms ofself-emulsification using particle size analysis and low frequencydielectric spectroscopy," 

16. N. Gursoy et al., "Excipient effects on in vitro cytotoxicity of anovel paclitaxel selfemulsifying drug delivery system," 

17. T. Gershanik and S. Benita, "Positively-charged self-emulsifyingoil formulation for improving oral bioavailability of progesterone,"

18. B.D. Tarr and S.H. Yalkowsky, "Enhanced intestinal absorptionof cyclosporine in rats through the reduction of emulsion dropletsize," 

19. S.A. Charman et al., "Self-emulsifying drug delivery system: Formulationand biopharmaceutic evaluation of an investigational lipophilic compound," 

20. R. Holm, I.H.M. Jensen, and J. Sonnergaard, "Optimization ofself-microemulsifying drug delivery system using D-optimaldesign and the desirability function," 

Drug Development and Industrial Pharmacy 

21. H. Shen and M. Zhong, "Preparation and evaluation of self-microemulsifyingdrug delivery system containing atorvastatin,"

22. P. Patil, P. Joshi, and A. Paradkar, "Effect of formulation variableson preparation and evaluation of gelled self emulsifyingdrug delivery systems of ketoprofen," 

23. H.R. Shen and M.K. Zhong, "Preparation and evaluation of selfmicroemulsifyingdrug delivery systems containing atorvastatin,"

24. Wei Ianlan et al., "Preparation and evaluation of SEDDS andSMEDDS containing Carvedilol," 

25. B.K. Kang et al., "Development of self-microemulsifying drug delivery systems for oral bioavailability enhancement of simvastatinin beagle dogs," 

26. J.Y. Hong et al., "A new self-emulsifying formulation of itraconazolewith improved dissolution and oral absorption," 

27. G.S. Chaea et al., "Enhancement of the stability of BCNU using self-emulsifying drug delivery systems (SEDDS) and in vitro antitumoractivity of self-emulsified BCNU-loaded PLGA wafer,"

28. T.R. Kommuru et al., "Self-emulsifying drug delivery systems(SEDDS) of coenzyme Q10: formulation development and bioavailabilityassessment," 

29. A.A. Attama et al., "The use of solid self-emulsifying systems inthe delivery of diclofenac," 

30. Mauro Serratoni et al., "Controlled drug release from pellets containingwater-insoluble drugs dissolved in a self-emulsifying system,"

European Journal of Pharmaceutics and Biopharmaceutics 

31. Mette Grovea et al., "Bioavailability of seocalcitol II: Developmentand characterisation of self-microemulsifying drug delivery systems(SMEDDS) for oral administration containing medium andlong chain triglycerides," 

European Journal of Pharmaceutical Sciences 

32. Wei Wu, Yang Wang and Li Que, "Enhanced bioavailability of silymarin by self-microemulsifying drug delivery system," 

33. A.A. Date and M.S. Nagarsenker, Design and evaluation of self nanoemulsifying drug delivery systems (SNEDDS) for cefpodoximeproxetil," 

34. E.I. Taha et al., "Preparation and in vitro characterization of self nanoemulsified drug delivery system (SNEDDS) of all-trans-retinolacetate," 

 is an assistant professor, both in the Department of Pharmaceutics and Pharmaceutical Technology, S. K. Patel College of Pharmaceutical Education and Research, Ganpat University, Gujarat, India, 

 is a professor in the Department of Pharmaceutics, Kalol Pharmacy College, Kalol, Gujarat, India.

*To whom all correspondence should be addressed.

This review article explains how self-emulsifying drug delivery systems can increase the solubility and bioavailability of poorly soluble drug.

Self-Emulsifying Drug Delivery Systems

Lovastatin (LVS) is a well-known compound for lowering plasma cholesterol levels. After oral administration, the inactive parent lactone is hydrolyzed to the corresponding hydroxyacid form. The hydroxyacid is the principal metabolite and a potent inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase. This enzyme catalyzes the conversion of hydroxymethylglutarate to mevalonate, which is an early and rate-limiting step in cholesterol biosynthesis (1, 2).  

LVS is white crystalline powder that is insoluble in water (0.4 g/mL). At room temperature, the partition coefficient of LVS in n-octanol/water system is approximately: K

  (3). Low aqueous solubility of LVS leads to inadequate dissolution in gastrointestinal fluids and, hence, poor absorption, distribution, and targeted organ delivery. The improvement of aqueous solubility in such a case is a valuable goal to improve therapeutic efficacy. 

Cyclodextrin (CD) is a cyclic (α-1, 4)-linked oligosaccharide made of α-D-gluco-pyranose units (see Figure 1). Hydroxypropyl-β-cyclodextrin (HPβ-CD) is more water soluble than the parent molecule and has hydroxypropylester groups attached to the hydroxyl groups in position 2. The molecule has a cone-like configuration with a hydrophilic surface and a lipophilic cavity. In this cavity, hydrophobic molecules interact with lipophilic molecules without forming any covalent bonds and can produce so-called "inclusion complexes," which increase the water solubility and stability of the drug substance (4–6). Complexation with CDs has been reported to enhance the solubility, dissolution rate,  and bioavailability of poorly water-soluble drugs. CDs first gained attention in marketed products as drug delivery technologies that enabled the development of various prostaglandins (7). The inclusion complex of rofecoxib/HPβ-CD (1:1 molar ratio) was prepared by Baboota 

 using a kneading method with a subsequent improvement in dissolution caused by amorphization (8). Several other drugs such as ganciclovir, nimesulide, itraconazole, and tolbutamide have been tested for CD inclusion to enhance solubility (9–12). 

 Figure 1: The structure formula for the Î²-cyclodextrin molecule.			

β-CD has ideal dimensions to complex a range of commonly used drugs. Unfortunately, it has the limitation of a high affinity for cholesterol, which may lead to crystallization of a poorly water-soluble β-CD–cholesterol complex in the kidney. This complex could cause nephrotoxicity. HPβ-CD, a chemical derivative of β-CD, similarly improves the aqueous solubility of many drugs, but it is more hydrophilic than β-CD, forms a less-stable complex with cholesterol, and, therefore, is less toxic (13). 

In this study, the authors compare the similarities of 

dissolution profiles of LVS from complexes, physical mixture, and pure LVS. Dissolution profiles can be compared by calculating a similarity factor (

) and the mean dissolution time (MDT). The method for calculating the similarity factor was first reported by Moore and Flanner (14). It also has been adopted by the US Food and Drug Administration's Center for Drug Evaluation and Research (15) and by the Human Medicines Evaluation Unit of the European Medicines Agency (16) as a criterion for assessing the similarity of two dissolution profiles (17, 18). A similarity factor of 100% suggests that the test and reference profiles are identical. Values between 50 and 100 indicate that the dissolution profiles are similar, whereas smaller values imply an increase in dissimilarity between release profiles (14). MDT reflects the time for the drug to dissolve and is the first statistical moment for the cumulative dissolution process that provides an accurate drug-release rate (15). A higher MDT value indicates greater drug-retarding ability (16).  

The present study was intended to improve the aqueous solubility and dissolution rate of LVS by preparing its complexes with HPβ-CD using various methods such as kneading, coevaporation, and physical mixing. The study further aimed to characterize the interaction between LVS and HPβ-CD. 

HPβ-CD was a gift sample from Roquette Frères, (Lestrem, France). LVS was received as a gift sample from Lincoln Pharmaceuticals Ltd. (Ahmedabad, India). The samples of sodium lauryl sulfate (SLS) were purchased from S.D. Fine Chemicals, (Vadodara, India). Directly compressible lactose, maize starch, sodium starch glycolate, colloidal silicon dioxide, and magnesium stearate were received as gift samples from Maan Pharmaceuticals Ltd. (Ahmedabad, India). All chemicals and solvents used in this study were of analytical reagent grade. Freshly distilled water was used throughout the work. 

Phase-solubility studies were performed according to the method reported by Higuchi and Connors (19). LVS, in amounts that exceeded its solubility, was transferred to screw-capped vials containing 25 mL of  an aqueous solution of HPβ-CD (molecular weight = 1500 g/mol) in various molar concentrations (0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, and 14.0 mM/L). The contents were stirred with an electromagnetic stirrer (Remi, Mumbai, India) for 36 h at 37 °C ± 0.1 °C and 350 rpm (this duration was previously tested to be sufficient to reach equilibrium). After reaching equilibrium, samples were filtered through a 0.22-μm membrane filter, suitably diluted, and analyzed spectrophotometrically for drug content at the wavelength of 238.2 nm using a spectrophotometer (Shimadzu-1601, ultraviolet-vis spectrophotometer, Shimadzu Corp., Kyoto, Japan). Solubility studies were performed in triplicate (

 = 3). The apparent stability constant (Kc), according to the hypothesis of 1:1 stoichiometric ratio of complexes, was calculated from the phase-solubility diagrams using the following equation: 

in which the slope is obtained from the initial straight-line portion of the plot of LVS concentration against HPβ-CD concentration, and S0 is the equilibrium solubility of LVS in water. 

Complexes of HPβ-CD and LVS were prepared in the molar ratio of 1:1 (on the basis of the phase solubility study) by various methods such as physical mixture, coevaporation, and kneading.  

A physical mixture of HPβ-CD and LVS was prepared by mixing the powders with a spatula for 15 minutes.  

Methanol and water were used as solvents to prepare the complex by a coevaporation method. The required quantities of LVS and HPβ-CD were dissolved in methanol and water, respectively. Both the solutions were mixed and solvents were evaporated by controlled heating at 45–50 °C. The resultant solid was pulverized and then put through a 120 # sieve.  

The required quantities of HPβ-CD and distilled water were mixed together in a motor to obtain a homogeneous paste. Then, LVS was added slowly; while grinding, a small quantity of methanol was added to assist the dissolution of LVS. The mixture was then ground for 1 h. During this process, an appropriate quantity of water was added to the mixture to maintain a suitable consistency. The paste was dried in an oven at 45–50 °C for 24 h. The dried complex was pulverized and then put through a 120 # sieve.   

The complexes prepared by kneading, coevaporation, and physical mixture were assayed for LVS content by dissolving a specific amount of the complex in methanol and analyzing for the LVS content spectrophotometrically at 238.2 nm on spectrophotometer (ultraviolet-vis spectrophotometer, Shimazdu-1601). The final moisture contents of all samples were measured with an electronic moisture balance (Sartorius, model MA-45, Goettingen, Germany). 

 The IR spectra of moisture-free powdered samples of LVS, HPβ-CD, the physical mixture

 and the complex prepared by the coevaporation and the kneading methods were obtained using a spectrometer (FTIR-8300, Shimadzu Co., Kyoto, Japan) with a potassium bromide (KBr) pellet method.  

Powder X-ray diffraction (PXRD) analysis. 

PXRD patterns of LVS, HPβ-CD, the physical mixture, and complexes prepared by the coevaporation and the kneading methods were determined using a scanner (Phillips PW 3710) and a generator (IW 1830) with a CuK α anode at 40 kV and 30 mA at a scan rate of 1° min

Differential scanning calorimetry (DSC) analysis. 

DSC scans of the powdered sample of LVS, β-CD, the physical mixture, and complexes prepared by the coevaporation and the kneading methods were recorded using a DSC instrument (Shimadzu 60) with TDA trend-line software. The samples (6–7 mg) were weighed accurately in crimped aluminum pans and heated from 50 °C to 300 °C at a scanning rate of 10 °C /min under dry nitrogen flow (100 mL/min). 

The wettability study was performed using open tubes containing LVS, the physical mixture, and complexes prepared by the coevaporation and the kneading methods. The tubes were placed with their lower capillary ends dipped into colored water (0.01% eosin in water). The upward migration of the colored front was registered as a function of time. The porosity of all samples also was measured using a mercury porosimeter (PoreMaster 60, Quantachrome Instruments,  Boynton Beach, FL). 

 is defined as the percentage of void space in a solid. Mercury density (ρ

) values often are used to evaluate % porosity (€) (20). Each test was repeated four times and the mean was calculated. 

Dissolution studies of LVS in powder form, the physical mixture, and complexes prepared by the coevaporation and the kneading methods were performed to evaluate the 

 drug-release profile. Dissolution studies were carried out using a 

 dissolution apparatus type II with a 500-mL dissolution medium at 37 °C ± 0.5 °C and 50 rpm for 3 h. In addition, 0.1 N HCl and a phosphate buffer (pH 6.8) containing 0.25% (weight/volune [w/v]) of sodium lauryl sulfate were used as other dissolution mediums. At fixed time intervals, 5-mL aliquots were withdrawn, filtered, suitably diluted, and assayed for LVS content by measuring the absorbance at 238.2 nm using a spectrophotometer. An equal volume of fresh medium at the same temperature was placed into the dissolution medium after each sampling to maintain its constant volume throughout the test. 

Table I: Tablet formulations of plain lovastatin (LVS), the physical mixture (PM), and complexes made with the coevaporation (CPC) and the kneading methods (CPK).			

Pure drug, the physical mixture, and complexes prepared by the coevaporation and the kneading methods were evaluated in dissolution-rate studies. Dissolution studies were performed in triplicate (

 = 3) and the calculated mean values of cumulative drug release were used while plotting the release curves. MDT values were calculated to compare the extent of improvement in the dissolution rate of the physical mixture, and the complexes prepared by the coevaporation and the kneading methods. Preliminary tests demonstrated that there was no change in the λmax of LVS because of the presence of HPβ-CD dissolved in the dissolution medium. 

Table II:  Gibbs free energy of transfer (ÎGtrÃÂ°) for the solubilization process of lovastatin in aqueous solutions of hydroxypropyl-Î²-cyclodextrin (HP-Î²CD) at 37 8C.			

Formulation excipients were selected on the basis of preliminary tests that demonstrated the excipients did not interfere with the λmax of LVS. The % compressibility and the angle of repose were measured to assess the tableting ability of the kneading method.  

The % compressibility was calculated using the following equation: 

 is the powder volume before tapping and V

 is powder volume after tapping infinitely. The powder volume was calculated from Kawakita's equation (21) using data from 200 tapping points obtained with a tapping density analyzer (bulk density test apparatus, DBK Instruments, Mumbai, India) equipped with a 20-mL cylinder. 

For the angle of repose measurement, a pile of powder was carefully made by dropping the powder material through a funnel tip from a height of 2 cm (22). The angle of repose was calculated by tangentially inverting the ratio of the formed pile's height and radius. 

Tablets containing 10 mg of LVS were made by direct compression using various formulation excipients such as directly compressible lactose, colloidal silicon dioxide, and magnesium stearate (see Table I). Tablets equivalent to 10 mg LVS were made similarly using the kneading method, but using less lactose. The blend was compressed on an eight-station, single-rotary machine (Cadmach, India) using round-shaped, flat punches to obtain tablets of 4–7 kg/cm

  hardness and 3.5–3.7 mm thickness (see Table I). For the assay, three tablets were crushed and a blend equivalent to 10 mg of LVS was weighed and dissolved in dissolution mediums. The tablets were studied in triplicate (

 = 3) for the drug-release profile using the same methodology described for the 

Figure 2: The phase solubility curve of lovastatin in an aqueous solution of hydroxypropyl-Î²-cyclodextrin (HP-Î²CD) at 37 8C.			

A model-independent, mathematical approach proposed by Moore and Flanner (14) for calculating  

The objectives of this study were to prepare and characterize inclusion complexes of lovastatin with hydroxypropyl-β-cyclodextrin (HPβ-CD) and to study the effect of the complexes on the dissolution rate of lovastatin (LVS). The findings suggest that LVS's poor dissolution profile can be overcome by preparing its inclusion complex with HPβ-CD.