0% READ
FULL TEXT
INTRODUCTION
A system with two immiscible phases is referred to as an emulsion, and it consists of an external phase that is continuous and an internal phase that is scattered as droplets. In order to properly transport the medicine, nanoemulsion as a topical drug delivery method faces various problems (Che Marzuki et al., 2019). An essential nanoemulsion feature is rheology. The nanoemulsion formulation's low viscosity and spreadability make it difficult to employ. Therefore, the restriction has limited the use of nanoemulsion in therapeutic settings. Therefore, the strategy of combining a nanoemulsion with a gelling system can aid in solving this issue. Oil in water (O/W) or water in oil (W/O) nanoemulsions with gelling agent are gelled (Costa and Santos, 2017). Fungal infection of nail is referred to as onychomycosis or Tinea ungium. Nail discolouration (White or yellow), nail detachment and nail thickness are symptoms. Nails or fingernails may be impacted; however, toe nails are more frequently impacted. The lower leg could develop cellulitis as a complication. Onychomycosis can be brought on by a variety of fungi, including Fusarium and dermatophytes (Boonme et al., 2009; Barry, 1991). Athlete's foot, other nail conditions, contact with others who have the ailment, peripheral vascular disease, and weakened immune system are risk factors (Kong et al., 2011). Nail discoloration, which can be white, black, yellow, or green and thickening are most typical sign of a fungal nail infection. Worsening of infection results in nail brittleness, breaking off in parts or fully falling away from the toe or finger (Mason et al., 2006). The fungal kingdom is home to dermatophytes, Candida (yeast), and non-dermatophyte moulds, which are the pathogens responsible for onychomycosis. While yeast and moulds are more typically implicated in the subtropics (hot-humid climate) and tropics, fungi dermatophytes are causative for onychomycosis in warmer western countries (Kale and Deore, 2017). The first topical triazole created especially for the treatment of onychomycosis is efinaconazole. In vitro, it exhibits strong antifungal activity against the most prevalent onychomycosis pathogens and a broad spectrum of activity (Talegaonkar et al., 2008).
Mixture design would be selected when the goal is the optimization of proportion or ratio of components and when factors are components of a mixture, the total of all components is a constant and response is a function of components (Souto et al., 2022). If 2 or 3 mixture components are involved and the ranges of all components are the same then use a simplex lattice mixture design. If 3 or 5 components are involved and the ranges of all components are the same then simplex centroid design could be used. If 2-4 components are involved and the ranges of all components are different then use constrained mixture design also known as D-Optimal design (Costa et al., 2014). In the present study statistical optimization of efinaconazole nanoemulsion was done by D-Optimal mixture design. Optimized nanoemulsion based gel for ease of application and anti-fungal studies against Candida albicans and Candida glabrata were studied for its efficacy.
MATERIALS AND METHODS
Efinaconazole is gifted from Suven life sciences, from moly chem manufacturer oleic acid is procured. From local market Olive oil, castor oil, and sunflower oil is obtained. Capmul MCM and captex is gifted from 200 ABITEC corporation. Tween 40, Tween 20, Tween 60, Tween 80, Span 80, and Span 20 is gifted from Sisco research laboratories Pvt. Ltd., PEG 400, PEG 600 is obtained from S.D fine chemicals Ltd., Methanol is procured formed Alfa Aesar-A Johnson Matthey Company. Transcutol is gifted from research Lab Fine Chem Industries. Neem oil is gifted from the chemical company, BASF. From S.D. Fine Chemicals Ltd., Hydrochloric acid and from In-house, Distilled water.
Solubility studies
Drug in excess with excipients was added to 2 mL Eppendorf tubes. The tubes were shaken for 48-72 hr to achieve a homogenous slurry in an orbital shaker. Then subjected to centrifugation for 10 min at 4500 rpm. Supernatant sample using a syringe filter were collected, diluted with water and analyzed by UV spectrophotometer (Tata et al., 1997).
Drug-excipient compatibility study Interactions by FTIR
Compatibility studies by FTIR spectrophotometer were studied by Potassium bromide (KBr) disks method and the IR spectrum was recorded from 4000cm-1 to 500 cm-1 in a scan time of 12 min. The resultant spectrum was compared for any spectra changes.
Optimization using D-Optimal mixture design
Phase Titration Method
Pseudo-ternary phase diagrams of different weight ratios of oil: emulgent (1:9 to 9:1) from which the better formulations ratios were selected based on that the design was applied (Liu et al., 2008; Gursoy and Benita, 2004). Oil was heated at 60°C in a beaker on continuous stirring, add a mixture of emulgent and drug with gradual stirring slowly dropwise to above mixture. On attaining room temperature, water drop wise was added with continuous stirring for diluting the sample to the total content, it results in formation of nano emulsion (Porras et al., 2005; Uskokovic and Drofenik et al., 2005). D-Optimal Mixture Design with levels minimum 20,20,10 and maximum 80,60,40 was used to analyse and construct quadratic response surfaces. These three parameters’ levels were determined from preliminary trials. Drug content, R1, and drug release in 8 hr, R2, were set as dependent factors (Aucouturier et al., 2002). The formulations were prepared by phase titration method according to Table 1, which was generated by Design Expert Version 11 software. In Table 1, 16 runs representing oil phase (component 1), emulgent (component 2), water (component 3), responses (Drug content, drug release) were given by the D-optimal mixture design.
| Run | FC | Component 1 A: oil | Component 2 B: emulgent | Component 3 C: water | Response 1 drug content % | Response 2 drug release % |
|---|---|---|---|---|---|---|
| 1 | NEP1 | 55.6821 | 34.001 | 10.3169 | 100.4±0.90 | 81.72±0.15 |
| 2 | NEP2 | 40 | 20 | 40 | 98.6±0.83 | 54.36±0.22 |
| 3 | NEP3 | 41.9277 | 33.4198 | 24.6525 | 100.2±0.76 | 80.41±0.16 |
| 4 | NEP4 | 20 | 41.5 | 38.5 | 98.61±0.45 | 45.92±0.12 |
| 5 | NEP5 | 41.9277 | 33.4198 | 24.6525 | 100.2±1.26 | 80.41±0.18 |
| 6 | NEP6 | 54.0816 | 20 | 25.9184 | 98.6±1.03 | 54.78±0.24 |
| 7 | NEP7 | 20 | 60 | 20 | 99.42±0.11 | 75.69±0.13 |
| 8 | NEP8 | 20 | 60 | 20 | 99.42±0.14 | 75.69±0.15 |
| 9 | NEP9 | 41.9277 | 33.4198 | 24.6525 | 100.2±0.18 | 80.41±0.84 |
| 10 | NEP10 | 42.9711 | 47.0289 | 10 | 100.7±0.24 | 82.85±0.25 |
| 11 | NEP11 | 67.4062 | 20 | 12.5938 | 96.54±0.34 | 59.52±0.36 |
| 12 | NEP12 | 31.7723 | 32.2222 | 36.0055 | 96.11±0.40 | 45.23±0.15 |
| 13 | NEP13 | 29.6215 | 46.2254 | 24.153 | 98.56±0.17 | 54.63±0.54 |
| 14 | NEP14 | 31.6123 | 58.3877 | 10 | 100.6±0.20 | 76.24±0.56 |
| 15 | NEP15 | 20 | 41.5 | 38.5 | 98.61±0.12 | 45.92±0.66 |
| 16 | NEP16 | 67.4062 | 20 | 12.5938 | 96.54±0.23 | 59.52±0.78 |
EVALUATION PARAMETERS OF NANO EMULSION
Thermodynamic stability studies
The diluted nanoemulsion formulations were centrifuged for 15 min at 3500 rpm, and observed for any phase separation visually. They were subjected to six heating and cooling (4ºC and 45ºC) cycles, for 48 hr (Kumar and Singh, 2012).
Visual observation and phase separations
The contents were taken in a beaker and gently swirled magnetically at 100 rpm at room temperature. Later after 24 hr, the generated emulsion was inspected for precipitation (Montilla et al., 2002).
Robustness to dilution
The nano emulsion was diluted upto 1000 times with water and solution of 0.1N HCl. Any separation of phases and precipitation was checked over.
Dispersibility test
0.5 mL formulation dispersibility in 500 mL distilled water using paddle type dissolution apparatus at 50 rpm, 37ºC (±0.5ºC) temperature was assessed visually and graded.
Identification of type of emulsion
Emulsion type was identified by standard tests, conductivity test, dye test and dilution test.
Globule size dimensions
Determined using a microscopic approach using stage micrometre and eye piece micrometre. The size and frequency of 100 globules was measured.
Transmittance %
Measured by UV spectrophotometer at 261 nm. Percentage transmittance against distilled water as a blank.
pH
pH was measured by digital pH meter three times, and standard values were determined.
Viscosity
An LVD II+ pro Brookfield viscometer using spindle 61, RPM 60, torque 91.2% at room temperature was used to determine viscosity of the nanoemulsion formulation.
Determination of drug content
Formulation containing efinaconazole 1 mL was added to 100 mL PBS 7.4 with tween 20 (1.5%), 5 mL methanol and sonicated. The solution was filtered, diluted and estimated by UV spectrophotometry at 261 nm (Koroleva and Yurtov, 2012).
In vitro drug diffusion
Using dialysis membrane, diffusion studies were performed. 1 mL of sample was taken in dialysis membrane, 200 mL of phosphate buffer saline (pH 7.4) with tween 20 (1.5%) as dissolution medium at 37±1ºC. Samples of 5 mL were withdrawn periodically and replaced with fresh media for every 1 hr till eight hours, to maintain sink condition. Drug content was analysed by UV- spectrophotometer at 261 nm (Kumar, 2014).
Drug kinetics
The data was analysed for zero order, first order, Higuchi release, Korsemeyer Peppas equation.
Statistical Analysis
Optimisation of D-Optimal mixture design, is done by establishing a mathematical relationship between factors and responses in the form of model by Design Expert Version 11 software. Model is selected by ANOVA with Lack of Fit and Goodness of Fit Statistics, where F value greater than 1 and p value less than 0.05 is preferred. Lack of Fit should be insignificant where p value should be greater than 0.1, the difference between precision value and adequate precision should be greater than 4 and difference between adjusted R2 and Predicted R2 should be greater than 0.2. Once model relating the factors and responses is selected, it is analysed by graphical indicators like Actual plot, Predicted plot, Contour plot and Response surface plot. Then design space is developed by Overlay plot and verified by minimum three confirmatory experimental runs.
Droplet size measurement
Poly Dispersibility Index (PDI), Mean globule size of the optimized nanoemulsion formulations were determined using zetasizer (Delsa Max analytics) at temperature of 25ºC (Kumar and Singh, 2012; Jain et al., 2013).
Zeta Potential (ZP)
Zeta potential for the optimized nanoemulsion formulation was determined using Zetasizer at 25ºC.
Formulation of Efinaconazole nanoemulsion gel
Efinaconazole nanoemulsion gel was prepared by using Carbopol 971, triethanolamine and distilled water. The required quantity of Carbopol was dispersed in water, using three-blade stirrer, required quantity of ethanol, propyl paraben and triethanolamine added drop wise. Add efinaconazole nanoemulsion to another portion of ethanol, and this solution is added dropwise to the above mixture. The prepared gel was evaluated for clarity, homogeneity, extrudability, pH, spreadability and drug content (physico-chemical properties) as per standard procedures.
Ex vivo permeation studies
This study was conducted according to the IAEC approval no: GPRCP/IAEC-1/27/03/2023/PCE/AE-1. Male Wistar rats were sacrificed by cervical dislocation, abdominal hair was removed and observed for presence of cuts or wounds. Skin section was cleaned with water and studies were performed using diffusion cell (Sugishita et al., 1981).
In vitro antifungal activity studies
Antifungal activity against test organism Candida albicans, Candida glabrata using Sterile Dextrose Agar plates was studied. 0.1 mL of the test organism was spread on agar plates, 5 mm diameter holes contained Efinaconazole (pure drug) 100 mg/mL, marketed product (luliconazole 1%) 1 mL, pure drug in gel form 1 mL, nanoemulsion gel 1 mL separately in each plate. The plates were kept at 4ºC for 4 hr and incubated at 28ºC for 48 hr. After the incubation, the inhibition zones were measured.
Stability
As per ICH guidelines, stability of optimized formulation (G2) stored in glass bottles in a humidity chamber at 40ºC±2ºC and 75%±5% RH for one-month period was studied. The samples were evaluated for drug content, physical appearance, and dissolution every week (Jaiswal et al., 2015).
RESULTS
Solubility studies
Oil and emulgent in which drug is more soluble was selected to prepare nanoemulsion. Efinaconazole concentration in different oils, surfactants as emulgents were determined by UV spectroscopy (Karthikeyan et al., 2012). Efinaconazole drug was more soluble in Oleic acid (3.261±0.02 mg/mL), castor oil (2.65±0.0 mg/mL), neem oil (1.195±0.07 mg/mL), Tween 80 (1.148±0.1 mg/mL) and Transcutol (5.1±0.01 mg/mL) as depicted in Figure 1, Aqueous solubility was found to be 0.016±0.02 mg/mL.
Compatibility studies by FTIR
IR peaks at region 3703.07 and 3749.36 shows OH stretching (hydroxyl group), 2939.31 and 2889 C-H stretching (Alkyl group), 1612.38 and 1616.24 C=O stretching (carbonyl), 1380 and 1350 C-F stretching (Fluro compound) characteristic of drug were present in excipient-drug (1:1) combination spectrum.
Evaluation parameters of nanoemulsion
Nanoemulsions according to Table 1 were prepared and evaluated for physico-chemical evaluations. Formulations NEP-(3,5,9) NEP-(4,15) NEP-(7,8) NEP-(11,16) are the repetitions. From Table 2, thermodynamic studies by heating and cooling cycle indicated NEP 6 and NEP 13 were unstable, NEP-(3,5,9), NEP-(7,8), NEP10, NEP 14 showed phase separation and on centrifugation NEP-(11,16) showed phase separation and NEP 12 precipitation. Dispersibility test, Conductivity test, dye test and dilution test indicated NEP 2, NEP (4,15), NEP 12, NEP 13 formulations are O/W type rest formulations W/O type. % Transmittance was in the range of 68.6% to 98.42%. Globule size by optical microscopy was in range of 54.3 µm to 162.9 µm. Viscosity of nanoemulsion was in range of 93.41±0.24 cps to 254.8±0.27 cps and pH 5.08 to 5.72.
| FC | Thermodynamic studies | Dispersibility test# | % Transmittance | Globule size (µm) | ||
|---|---|---|---|---|---|---|
| Heating and cooling cycle | Visual observation and phase separation | Centrifugation | ||||
| NEP 1 | Stable | Stable | Stable | B(W/O) | 98.42 | 90.5 |
| NEP 2 | Stable | Stable | Stable | A(O/W) | 94.12 | 72.4 |
| NEP 3,5,9 | Stable | Phase separation | Stable | C(W/O) | 68.56 | 162.9 |
| NEP 4,15 | Stable | Stable | Stable | A(O/W) | 97.24 | 72.4 |
| NEP 6 | Precipitation | Stable | Stable | C(W/O) | 91.41 | 181 |
| NEP 7,8 | Stable | Phase separation | Stable | D(W/O) | 95.6 | 54.3 |
| NEP 10 | Stable | Phase separation | Stable | A(O/W) | 97.84 | 126.7 |
| NEP 11,16 | Stable | Stable | Phase separation | D(W/O) | 93.25 | 108.6 |
| NEP 12 | Stable | Stable | Precipitation | B(O/W) | 92.76 | 54.3 |
| NEP 13 | Precipitation | Stable | Stable | A(O/W) | 90.15 | 90.5 |
| NEP 14 | Stable | Phase separation | Stable | D(W/O) | 93.21 | 72.4 |
In vitro diffusion studies
After performing diffusion studies the data depicts that percent drug release for NEP 1, NEP 3, NEP 5, NEP 9, NEP 10 shows highest drug release compare to other formulation as shown in Figure 2a up to 80% release within 8 hr. Formulation NEP 12, NEP 15 display less drug release during 8 hr, as shown in Figure 2b.
Model-dependent kinetics
All 16 formulations exhibit first order release and Korsemeyer Peppas with a "n-value" of greater than 0.6. As a result, all the formulations exhibit an anomalous transport mechanism.
Optimization: D-Optimal Mixture Design
For drug content (R1) response lack of fit was insignificant (p>0.10), F value was 5.26 which is greater than 1 for reduced quadratic model as shown in Table 3. Similarly, for % drug release (R2) response, F value was 6.33 and lack of fit was insignificant, which implies linear model is significant as there is only 1.2% chance of this large F value due to noise.
| Response | Drug content (R1) | Percentage drug release (R2) |
|---|---|---|
| Model | Quadratic | Linear |
| Sum of square | 18.93 | 1558.73 |
| Degree of freedom | 3 | 2 |
| F value | 5.26 | 6.33 |
| P value | 0.0151 | 0.012 |
| Adjusted R2 | 0.4601 | 0.4156 |
| Predicted R2 | 0.3084 | 0.2994 |
| Adeq. Precision | 6.9021 | 7.1927 |
| Lack of fit | 2.06 | 199.99 |
| Pure error | 0 | 0 |
The difference between predicted R2, adjusted R2 should be less than 0.2 and Adequate precision which measures signal-to-noise ratio, greater than 4.0 is desirable. For R1 and R2 responses with reduced quadratic model and linear model the difference is less than 0.2 and ratio of 6.9021 and 7.192 respectively as depicted in Table 3.
From counterplots and response surface plots it was observed with increase in X1 (Oil phase), X2 (emulgent), the drug release percentage was increased, but increase in X3(water) there is decrease in drug release (R2). Increase in X1 (oil phase), X2 (emulgent) resulted more drug content and increase in X3 water, less drug content (R1) as shown in Figures 3A, 3B. Three dependent variables have mixed effects on drug content. The Contour and response surface plot shows that when emulgent concentration (B) is increased drug content increases. With increment in the quantity of emulgent the percentage drug release was also enhanced, with increased oil concentration, there was increased drug release.
The optimum variables according to the criterion of desirability were acquired by the numerical analysis. Linearity of Predicted vs. Actual responses as shown in Figure 3C for drug content R2 is 0.9688 and for Drug Release percentage, R2 is 0.985. From overlay plot (Figure 3D) for graphical optimization, an optimized formulation based on maximum drug release and drug content is produced with desirability co-efficient 0.951. Optimized nanoemulsion formulation (NEP-17) prepared by 31.29 mL oil, 58.70 mL emulgent, and 10.0 mL water, has shown rapid emulsion formation with transparent appearance, good thermodynamic stability, drug content R1, 101.7±0.12% and % drug release R2, 82.09±0.01% as shown in Table 4.
| Optimized Formulation Nep -17 | |
|---|---|
| Parameters | Inference |
| Type of emulsion | W/O |
| PH | 5.69 |
| Drug content | 100.7% |
| % drug release | 82.19% |
| Zeta potential | - 42.1mv |
| Globule size | 229.7nm |
| Viscosity (spindle 61 Rpm 60) | 94.3 ±2.41 cps |
| Thermodynamic studies | Pass |
| Visual observation | Pass |
| Robustness to dilution | Pass |
Zeta potential and globule size
The formulation has shown a Zeta-potential of -42.1 mv (Figure 4B). Nanoemulsion is confirmed by its size 229.7 nm (Figure 4A) and with poly dispersibility index of 0.284. As shown in Figure 4.
Evaluation of Gel
The optimized NEP-17 formulation was formulated into gel according to procedure in methodology and the gel was evaluated. Gel formulations (G1 (pure drug), G2 (nanoemulsion)) were evaluated for clarity (++), Homogeneity (++), Extrudability (++), pH (5.12±0.3, 5.38±0.4), Spreadability (33±0.32 gm.cm/sec, 41±0.21 gm.cm/sec) and drug content (98.34± 0.09%, 98.45±0.01%) respectively which were found to be ideal. There were no particles present in the gel and was uniform, with good spreadability, and the pH was found to be suitable for the application.
Ex vivo permeation studies
Nanoemulsion-based gel (G2) after evaluating physicochemical parameters showed ex vivo permeation of 86.42±0.14% and for Pure drug gel (G1) it was found to be 56.76±0.34% as depicted in Figure 2C. And different permeability parameters are reported as, steady state flux 0.896±1.67 to 0.8831±1.69μg/cm2/hr, Lag time 0.2±0.4 to 0.16±0.3 hr, Permeability coefficient 4.48±0.93 to 4.41±0.87cm/hr.x10-3, Enhancement ratio 1.286±0.36, Skin deposition 0.53±0.24 0.69±0.36 μg/cm2. Model dependent kinetics shows that it follows first order kinetics (0.981) as it shows the R2value higher than the zero order kinetics (0.9783) and shows higuchi release mechanism with the value of 0.9868 which is greater than Korsemeyer peppas.
Antifungal studies
Antifungal studies against Candida glabrata and Candida albicans were performed and results are shown in Figure 5. In Figure 5a, Candida glabrata antifungal studies, it is observed in control (A) presence of white spots representing fungal growth, randomly spread in whole area. In pure drug solution form (B), white spots were seen at 10 mm from center, in gel form of pure drug (C), at 11 mm from center, in marketed (D) at 7 mm, whereas in efinaconazole nanoemulsion gel (E) at 13 mm from center.
Similarly in Figure 5b, Candida albicans antifungal studies, it is observed in control (A) presence of white spots representing fungal growth, randomly spread in whole area. In pure drug solution form (B), white spots were seen at 8 mm from center, in gel form of pure drug (C), at 10 mm from center, in marketed (D) at 15 mm, whereas in efinaconazole nanoemulsion gel (E) at 13 mm from center.
Stability study
Nanoemulsion-based gel G2, formulation in ambered colored bottle was tested. It passed through accelerated conditions as per ICH guidelines (40ºC±2ºC/75%±5% RH) for one month. Physical appearance, drug content, and in vitro drug release were evaluated every week. It was stable, with insignificant change in the physical appearance, % drug content and % drug release (Table 5).
| Sl. No. | Parameter | Initial | 1st week | 2nd week | 1 month |
|---|---|---|---|---|---|
| 1 | Appearance | Clear gel | Clear gel | Clear gel | Clear gel |
| 2 | Drug content | 100.07±0.1 | 99.04±0.2 | 99.03±0.3 | 99.03±0.41 |
| 3 | % drug release | 82.19±0.1 | 82.12±0.21 | 81.23±0.37 | 81.23±0.36 |
DISCUSSION
Solubility of efinaconazole in different oils and emulgents were determined to select oil phase and emulgent in nano emulsion preparation by phase titration method. Among oils selected highest solubility was observed in oleic acid and among emulgents, transcutol followed by Tween 80. FTIR studies indicated drug excipient compatibility. Preliminary trials were performed with oleic acid, transcutol, water and oleic acid, Tween 80, water as nanoemulsion and evaluated for thermodynamic stability studies. Among the trials oleic acid, transcutol, water nanoemulsion was more stable. So, these were considered as independent factors in D-optimal design with varying ratios in a mixture. Nanoemulsions were prepared and evaluated for type of emulsion, thermodynamic stability, % transmittance, globule size, viscosity and pH. Except NEP 2, NEP (4,15), NEP 12, NEP 13 formulations all are W/O type, thermodynamically stable, clear transparent nanoemulsions with optimum globule size, viscosity and pH. In vitro diffusion studies were performed for 8 hr and formulation NEP 10 showed maximum drug release. All formulations followed first order and anomalous transport.
On optimization by D-optimal mixture design, it is observed the dependent variables, drug content followed reduced quadratic model and was dependent on each component oil, emulgent, water and interaction of oil and emulgent. Percent drug release followed linear model and had positive effect with oil and emulgent component, negative effect with water component. Predicted vs Actual responses showed linearity with R2 nearly 1. With the criteria of maximum drug content and maximum drug release, design has given two solutions of desirability 0.951. The optimized formulation NEP 17 was prepared and evaluated for physico-chemical properties. The formulation has Zeta-potential of –42.1 mv, globule size 229.7 nm and poly dispersibility index of 0.284 indicating stability of formulation and uniformity of globules in nanosize.
Optimized nanoemulsion NEP 17 was formulated into gel for ease of application for onchomycosis. Gel was evaluated for physico-chemical properties, ex vivo permeation studies and compared with pure drug in gel form. Nanoemulsion gel had better permeability than pure drug gel.
Antifungal studies of G2 (nanoemulsion gel) against Candida albicans and Candida glabrata indicated antifungal activity similar to marketed Luliconazole preparation (LUZU). It was found to be efficient at very low dose, which was reduced to 10 times than the official dose where indication can be made that the solubility has been enhanced.
Stability studies of G2 showed no changes in drug content and % release. We conclude that the nanoemulsion-based gel was stable.
CONCLUSION
This study was aimed to enhance the solubility and bioavailability of efinaconazole. As the solubility of the drug is enhanced, efinaconazole 10% is efficacious, thus 10 times less than official dose could be administered. Further, determination of pharmacokinetics parameters and commercialization of optimized formulation would be beneficial.