Rakesh K. Patel is a professor at S.K. Patel College of Pharmaceutical Education and Research, Ganpat University in India.

Rakesh K. Patel

Conventional ophthalmic solutions frequently show poor bioavailability and a weak therapeutic response because they are often eliminated before they can reach the cornea, when patients blink or their eyes tear. Use of

 gel forming solutions may help improve performance and patient compliance. These solutions are delivered as eye drops, but undergo a sol-gel transition in the conjunctival sac (

). This article describes how an ion-activated 

gelling system was designed to deliver an ophthalmic formulation of the antibacterial agent, Levofloxacin. 

The delivery system uses gellan gum, a novel ophthalmic vehicle that gels in the presence of mono or divalent cations in the lacrimal fluid. This gum was used alone and combined with sodium alginate as a gelling agent and hydroxypropyl methylcellulose (HPMC) Methocel F4M as a viscosity enhancer. 

 full factorial design approach was used, with two polymers: Gelrite and HPMC, as independent variables. Gelling strength, bioadhesion force, rheological behavior, and in-vitro drug release after 10 h were selected as dependent variables

 release studies and rheological profile studies indicated that the combined Gelrite-HPMC solution retained the drug better than the gellan gum alone or a combination of gellan gum-alginate-HPMC. The developed formulations were therapeutically efficacious and provide sustained release of the drug over a 12-h period  

.  These results demonstrate that the Gelrite-HPMC Methocel F4M mixture can be used as an

gelling vehicle to enhance ophthalmic bioavailability and patient compliance.

	Opthalmic drug delivery systems, such as eye drops, ointments, and soft gel capsules, are typically used to treat diseases of the eye.  However, the eye’s protective mechanisms often reduce their therapeutic effect. When a drug solution is dropped into the eye, there is typically a 10-fold reduction in the drug concentration within 4-20 min, due to the effective tear drainage and blinking action (1). The cornea’s limited permeability contributes to the low absorption of ocular drugs. Due to tear drainage, most of the administered dose passes via the nasolacrimal duct into the gastrointestinal tract, leading to side effects (2). Rapid elimination of both the solutions and the suspended solid administered often results in blurred vision, poor patient acceptance, and short duration of the therapeutic effect, making more frequent dosing necessary (3)

	New preparations have been developed to prolong the contact time on the ocular surface and slow down drug elimination (4, 5). Ocular inserts (5) and collagen shields (6)  can also be used, but they pose challenges.

	These delivery challenges can be overcome by using 

 gel-forming ophthalmic drug delivery systems prepared from polymers that exhibit reversible phase transitions (sol-gel-sol) and pseudoplastic behavior.  Such formulations minimize interference with blinking (7).

	Changes to the gel phase (8) can increase pre-corneal residence time and enhance ocular bioavailability. Three types of systems have been used: pH-triggered systems including cellulose acetate hydrogen phthalate latex (9, 10) and carbopol (11-15); temperature-dependent systems including pluronics (7, 16-20), tetronics (21, 22), and polymethacrylates (23); and ion-activated systems including Gelrite (24-26), gellan (27-28), and sodium alginate (29).

 gelling system to deliver Levofloxacin, a fourth-generation fluoroquinolone anti-infective agent, which can be used to treat conditions including acute and subacute conjunctivitis, bacterial keratitis, and keratoconjunctivitis. The goal was to demonstrate prolonged action and show antibacterial activity against gram-positive and gram-negative bacteria directly at the site of infection without loss of dosage. The combination of Gelrite (gellan gum) and hydroxypropyl methylcellulose (HPMC) (Methocel F4M) was used to prepare the gelling system, which was used with and without sodium alginate to prepare Levofloxacin eye drops (0.5% w/v). These drops would undergo gelation when instilled into the cul-de-sac of the eye, and provide controlled release of the drug in treatment of ocular infections.

. Levofloxacin was obtained from Zydus Healthcare, Gelrite from CP Kelco, and HPMC (Methocel F4M) was provided by Colorcon Asia Pvt. Ltd. All other reagents, chemicals, and solvents were of analytical grade. 

 gelling systems were prepared by dissolving gellan, alone and combined with sodium alginate and/or HPMC in hot phosphate buffer (pH 7.4, 70 °C), by continuous stirring at 40-45 °C for 24 h, as shown in 

. Then the weighed quantities of levofloxacin (0.5% w/v), mannitol, and preservatives, such as methyl paraben and propyl paraben, were added to the solution and stirred until dissolved. The solutions were then transferred into previously sterilized amber-colored glass vials, capped, and sealed with aluminum caps. The formulations were sterilized by terminal autoclaving at 121 °C, 15 PSI for 20 min. The sterilized formulations were stored in a refrigerator at 4-8 °C until use.

 full factorial approach was taken to design the gelling system. Two factors were selected, and a total of nine  experimental trials were performed using all possible combinations. The concentrations of Gelrite (cation-sensitive in-situ gelling polymer) as X

(0.3, 0.4, and 0.5%, m/V) and HPMC (viscosity imparting agent) as X

 (0.3, 0.5, and 0.7%, m/V) were selected as independent variables. 

	Gel strength (GS in s), bioadhesion force (BF in N), viscosity (VI) in Pa.s, and cumulative percent drug release after 10 h (CR10) were selected as dependent variables. The design is shown in 

 summarizes the experimental design, using two independent variables and three levels (low, medium, and high) of each variable:

 is the mean response of the nine runs, and b

is the estimated coefficient for factor X

) represent the average result of changing a factor at a time. The interaction term (X

) shows how the response changes when the factors are simultaneously changed. Polynomial terms (X

) are included to investigate nonlinearity.

	Statistical analysis and two-way analysis of variance (ANOVA) were used to evaluate the significance of each factor to the response at different levels. Three-dimensional response surface plots and two-dimensional contour plots of the data were generated using Design Expert software (Version 8).


	The following were used to evaluate the formulation.

 were carried out in a vial containing the gelation solution and simulated tear fluid (STF) solution, composed of 0.670 g of sodium chloride, 0.200 g of sodium bicarbonate, 0.008 g of calcium chloride dehydrate, and purified water, quantum satis to 100 g.

	The preparation was carefully taken into the vial using a micropipette, and 2 mL of gelation solution (STF) was added slowly. Gelation was assessed by visual examination (26).

. Viscosities of sample solutions were measured in a Brookfield synchrolectric viscometer (LVDVI prime) at different angular velocities at a temperature of 37±1°C. The angular velocity was increased from 0.5 to 100 rpm with 6 s between two speeds. The sequence of the angular velocity was reversed. The average of two readings was used to calculate viscosity. Evaluations were conducted in triplicate (26).

. Vials containing the formulation were shaken for 2-3 min, and the preparation was transferred aseptically to sterile volumetric flasks. The final volume was made up with phosphate buffer pH 7.4. The concentration of Levofloxacin present was determined at 287 nm using UV spectrophotometry (26).

. The studies were carried out using a Franz diffusion cell, with STF (pH 7.4) as dissolution medium. The cell consists of glass donor and receptor compartments, separated by a dialysis membrane. The optimized formulation was placed in the donor compartment, and freshly prepared STF was placed in the receptor compartment. The whole assembly was placed in a temperature-controlled shaker water bath maintained at 37 °C ± 0.5 °C. A sample (1 mL) was withdrawn at predetermined time intervals up to 24 h and the same volume of fresh medium was replaced. The withdrawn samples were analyzed by UV spectrophotometer at 287 nm. 

. Freshly excised goat conjuctival membrane was used to measure bioadhesive strength. The membrane was placed in an aerated saline solution at 4 °C until used. It was tied to the lower side of the hanging polytetrafluoroethylene (PTFE) cylinder using thread, and the cylinder was fixed beneath of left pan of a pan balance. The formulation was placed into a sterile petri plate that was kept on the platform beneath the left pan. The two sides of the pan balance were balanced by keeping a 2-g weight on the right pan

	The 2-g weight was then removed, lowering the left pan and allowing the membrane to come in contact with the formulation. The membrane was kept in contact with the formulation for 5 min. Weight was slowly added to the right pan slowly, in increments of 0.5 g, until the formulation detached from the membrane surface. The excess weight on the right pan was taken as the measure of the bioadhesive strength. The force of adhesion was then calculated using the following formula (13).

	Force of adhesion = Bioadhesive strength x 9.81 / 1000.

. Infrared (IR) spectroscopy and differential scanning calorimetry (DSC) were then used to analyze the pure drug, gellan, HPMC, physical mixture of drug-gellan-HPMC, and optimized formulation. Resulting spectra were then compared with reference spectra.

. The solution’s antimicrobial efficacy was determined using agar diffusion and commercial Levofloxacin eyedrops as a control. The sterilized solutions were poured into cups bored into sterile agar nutrient seeded with test organisms (

	After allowing diffusion of the solutions for two hours, the plates were incubated at 37 ËC for 24 h, and the zone of inhibition (ZOI) was measured around each cup and compared with control’s ZOI. The entire process, except for the incubation, was carried out under laminar flow units in an aseptic area (Class 10,000). Each solution was tested three times. Both positive and negative controls were maintained throughout the study (26).

 ocular irritation studies were performed using the Draize technique (30) and guidelines set by the Organization for Economic Cooperation and Development (OECD) (31). Six albino rabbits, each weighing 2-3 kg, were used for this study. The sterile formulation was administered to the test rabbits twice a day for 21 days and the rabbits were observed periodically.

	International Council for Harmonization (ICH) guidelines were used to determine the optimized formulation’s stability. The gel was stored in a stability chamber at ambient humidity between 2 °C to 8 °C, ambient temperature at 40 °C ±0.5 °C  for six months. The samples were withdrawn at regular intervals and analyzed. The logarithms of percent drug remaining were calculated and plotted against time in days. The degradation rate constant was calculated using the equation: slope = k/2.303, where k is a degradation rate constant. The shelf life of the developed formulation was calculated using the Arrhenius plot. 


	Composition of various batches of the prepared 

. In the batch containing gellan alone, the concentration of gellan was kept at a maximum of 0.4% (w/v). Higher concentration beyond 0.4% caused gelation upon cooling to 40 °C.

	In the combination batches, the concentration of gellan was varied and the concentration of sodium alginate was kept at approximately 0.25% (by compensating the concentration difference due to reduction in viscosity after autoclaving for sodium alginate) to give a maximum of 0.65% polymer concentration, because an increase beyond this concentration resulted in gelation during formulation.

	To maintain the proper pseudoplastic behavior of formulation, HPMC was used with gellan alone and with combination of gellan and sodium alginate. The drug content and gelling capacity of the formulations were found to be satisfactory as mentioned in 

, and the formulations were liquid at both room temperature and when refrigerated. Viscosity and gelling capacity (speed and extent of gelation) are the most important criteria for any in-situ gelling system.

	All batches exhibited pseudoplastic behavior, as showed in 

. All batches showed low viscosity at high shear rate and high viscosity at low shear rate. Autoclave process had not affected the viscosity of the formulations, except for those containing sodium alginate where viscosity was reduced around 8-15%. Therefore, the concentration of sodium alginate was adjusted to compensate (25). All measurements were taken three times and showed good reproducibility.

shows the cumulative percentage of Levofloxacin released versus time profiles for batches LV1 to LV8. These results suggested that Levofloxacin was sustainably released from formulation LV8, when the content of gellan gum was 0.4% and 0.5% of HPMC Methocel F4M (

). A similar release pattern is reported for pilocarpine (32) from alginate systems, wherein an inverse relationship between drug release and polymer concentration was observed. 


	Based on studies of response variables, the polynomial relationships are expressed in 

	BF (bioadhesion force) = 3069.44 + 989.83*X

	CR10 (cumulative percentage drug  release after 10 hs) = 90.51 - 9.03*X

	All the polynomial equations were found to be statistically significant (P < 0.05) and in good agreement with results. From 

 it can be concluded that both gellan gum and HPMC significantly affect the gelling strength (25, 13).

	Formulation batches LF1 and LF2 showed poor gelation strength, which might be due to the minimum amount of gellan and/or HPMC.  The results are shown in 

The studies showed that, in the presence of HPMC, as the amount of gellan increased, gel strength increased as well; this effect must be due to the additional effect of concentration of polymer. 

shows the response surface plot illustrating the effect of gellan gum and HPMC on the gelling strength. Studies confirmed that both polymers significantly affect the gelling strength.

, it can be concluded that both polymers have a predominant effect on bioadhesive force. The formulation contained gellan gum, which is a mucoadhesive agent. Studies show that polymers with charge can serve as good mucoadhesive agents. It has also been reported that polyanion polymers are more effective bioadhesives than polycations or nonionic polymers (33, 34). This polymer adheres to the mucin of the eye, which leads to prolonged retention of the formulation inside an eye (13).

 depicts the response surface plot, showing the influence of both polymers on bioadhesion force. The studies confirmed that, as the concentration of gellan gum or HPMC is increased, bioadhesion also increases. 

shows that HPMC has a predominant effect on viscosity compared to gellan gum. Normally, water-soluble polymers such as HPMC produce two effects:

		Lowering surface tension and improving mixing with the precorneal tear film 

		Increasing viscosity and prolonging contact time, thereby resisting drainage of drug from eye (13).

	Gellan gum can significantly increase viscosity of the formulation upon exposure to lachrymal fluid. So, by optimizing the concentration of HPMC viscosity-enhancing agent, one can decrease the amount of gellan gum in the preparation to improve patient compliance.

	Figure 3(c) shows the response-surface plot of effect of gellan gum and HPMC on viscosity. From Equation 5, gellan gum and HPMC are inversely related to the amount of drug released.

 release studies show that the formulations retain drug for the duration of the study (12 h). The movement of the eyelid and eyeball provide shearing action for faster dissolution of gels in the cul-de-sac. Figure 3(d) depicts the response-surface plot, showing the influence of both polymers on drug release after 10 h respectively. Checkpoint batches LF10 and LF11 were prepared (

) to validate the evolved model.  The actual values of GS, BF, VI, and CR10 of batches LF10 and LF11 are given in 

	Checkpoint batches were found in good agreement with the actual values. Results of ANOVA are shown in 

	Release of an optimized batch fitted to a Higuchian matrix equation showed a high R-squared value (0.99), least SSR value, and F value {21} as compared to other batches. Thus, it can be concluded that release of drug was based on a Higuchian-matrix, diffusion-controlled mechanism. 

Bioadhesive strength and thermogram results


	The bioadhesive strength measurement of designed batches is shown in 

. Differential scanning calorimetry (DSC) thermograms showed characteristic peaks of Levofloxacin at 230.50 °C and 111.55 °C, gellan gum at 266.11°C, and HPMC at 288.55 °C and 79.79 °C (

	The peak of Levofloxacin was found to be reduced in intensity in physical mixture of drug, gelling agent, and polymer (

) and could not be seen in optimized formulations of DSC thermogram (

), indicating the entrapment of drug in the in-situ matrix gel system of gellan gum and HPMC.

	The optimized formulation (LF5) showed antimicrobial activity when tested microbiologically by the cup-plate technique. Clear ZOIs were obtained in the case of the optimized formulation and marketed eye drops.

	The diameters of the ZOIs produced by the optimized formulation against both test organisms were either on par or higher than those produced by marketed eye drops as shown in 

	The antimicrobial effect of Levofloxacin gel formulation is probably due to its rapid initial release into the viscous solution and followed by formation of a drug reservoir that attributed to the slow and prolonged diffusion from the polymeric solution due to its higher viscosity (26).

	Ocular irritation studies (35) indicated that the formulation is well tolerated by rabbit eyes (36). No ocular damage or abnormal clinical signs were observed (37).

	The optimized formulation of Levofloxacin was kept for stability studies at refrigeration temperature (4 °C), ambient temperature (25 °C), and elevated temperature (40 °C) for a period of six months. Samples were withdrawn at regular time intervals and were evaluated for appearance, gelation studies, drug content, and in-vitro drug release. 


	The formulation was found to be sterile at the end of six months. The drug degraded to a negligible extent and the degradation rate constant for optimized formulation was very low (1.12 x 10

). Because the overall degradation is <5%, a tentative shelf life of two years may be estimated the formulation (13). 

 gel formulation of Levofloxacin was successfully formulated using gellan gum in combination with HPMC. The formulation underwent gelation in the conjunctival sac (

), allowing for sustained drug release over a 12-h period without any adverse effect to the ocular tissues.

	Stability data confirmed that the formulation is stable for a six-month period in given storage conditions. This new formulation can enhance bioavailability through its sustained drug release, higher viscosity, longer pre-corneal residence time, and better miscibility with the lacrimal fluid. These benefits promise to improve patient acceptance and compliance.


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	2. D.L. Middleton, S.S. Leung, and J.R. Robinson, “Ocular bioadhesive delivery systems,” in 

, V. Lenaerts, and R. Gurny, Eds. (Boca Raton, FL: CRC Press, 1st ed., 1990), pp. 179-202.
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, A.K. Mitra, Ed. (Marcel Dekker, New York, 1993), pp. 179-193.
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 8 (1), 328-335 (1998).
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 A.K. Mitra, Ed. (Marcel Dekker, New York 1993), pp. 261-275.
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241 (1) 47-55 (2002).
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 207 (1-2) 109-116 (2000).
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73 (2-3), 205-211 (2001).
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127 (1) 183-191 (2007).
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69 (3) 379-388 (2000).
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26 (3) 195-201 (1993).
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Articles

Delivery systems that allow drugs to be administered as liquids, but to form gel within the eye, promise to improve efficacy and patient compliance.

Beyond the Blink: Using In-Situ Gelling to Optimize Opthalmic Drug Delivery

	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, 

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