OPTIMIZATION OF PLURONIC LECITHIN ORGANOGEL OF TERBINAFINE HYDROCHLORIDE USING DESIGN OF EXPERIMENTS FOR THE TREATMENT OF FUNGAL DISEASES

OPTIMIZATION OF PLURONIC LECITHIN ORGANOGEL OF TERBINAFINE HYDROCHLORIDE USING DESIGN OF EXPERIMENTS FOR THE TREATMENT OF FUNGAL DISEASES

Sourodip Saha, Bharat Kumar Reddy Sanapalli and Veera Venkata Satyanarayana Reddy Karri ^*

Department of Pharmaceutics, JSS College of Pharmacy, JSS Academy of Higher Education & Research, Ooty - 643001, Tamil Nadu, India.

ABSTRACT: The research work focused on the optimization of Terbinafine HCl (TBH) pluronic lecithin organogels (PLOs) for increasing the penetration and efficacy of the drug. The factors affecting the PLOs such as viscosity and percentage drug release, were screened and optimized using the Box-Behnken design. The optimum formulation based on desirability (0.995) exhibited 3139.92 Cp for viscosity and 93.4561% for drug release. The optimized PLO was evaluated for drug content, pH, in-vitro drug release and ex-vivo studies. The percentage cumulative release of TBH permeated from optimized PLOs and marketed cream (MC) at the end of 12 h after the application was found to be 60.29 ± 0.69% and 19.35 ± 2.19% respectively. The comparatively higher permeability TBH in optimized PLOs could be due to increased solubility and diffusion coefficient of the drug. In-vitro microbial assay, studies showed that the PLO reduced the fungal burden of Candida albicans as compared to M.C (0.34 ± 0.08 mm) in a shorter duration of time with a larger zone of inhibition (0.58 ± 0.12 mm). Hence, it was concluded that TBH PLOs had better skin targeting ability and may serve as a promising carrier in the treatment of fungal skin diseases.

Terbinafine HCl, Pluronic lecithin organogels, Box-Behnken design, Microbial assay, Fungal skin diseases

INTRODUCTION: Currently, the research is focusing its trends towards the noninvasive drug delivery system to overcome the painful strategies of drug delivery. Among all approaches, topical drug delivery gains the most attention due to its non-invasiveness, ease of use, larger surface area and, most important no risk of systemic adverse events and drug interactions ¹. In terms of the economic prospects, it increases patient compliance with reduction of the treatment cost.

Topical drug delivery is designed for the potent drugs to ensure therapeutic efficacy with the correct physicochemical properties into the skin, and as penetration enhancers ² to facilitate topical drug permeation. Invasive fungal infection is one of the common threats to human health in today’s world. The developing and underdeveloped countries are more prone to fungal diseases ³.

About 40 million people are affected by a fungal infection in those countries. The researcher found that, the invasive diseases caused by Candida spp. and Aspergillus spp. is 30-50% prime among all fungal conditions ⁴. Despite the advancement of diagnostics and therapeutic improved strategies, the overall mortality is still very poor. Terbinafine HCl (TBH) is most commonly prescribed topically as well as orally active allylamine antifungal drug which prevents fungal ergosterol biosynthesis via specific and selective inhibition of fungal squale oxidase ⁵. The research already proved that TBH is one of the best candidates for treating almost all forms of susceptible Candida infections in both immune-competent and immune compromised hosts. The safety profile and efficacy of the antifungal agent are important to decide oral or topical antifungal administration. Lower drug requirements and higher affectivity for many fungal or yeast skin infections are the main advantages of TBH. With the conventional tablet formulation, the drug is reported with gastrointestinal problems, hepatic problems, and sensory problems along with few adverse drug reactions possibly due to its extensive bio-distribution. So, delivery of TBH through the topical route is one of the best choices for the management of fungal infections ⁶. However, the poor solubility of TBH limits its permeability and efficacy through the topical route. Many of the researchers tried to increase the permeability of TBH by liposome ⁷, SLN⁸, NLC ⁹ nanoemulsion ¹⁰ and much more nanoformulation approach. However, these strategies are not economical and lack scale-up abilities.

Avramiotis et al., 2007 formulate PLO of bioactive compounds, and the result indicates that the PLOs used to incorporate model bioactive compounds with medical interests for the active molecules. The result also indicates that permeation was 35% after 24 h ¹¹. Wenqiang Ba et al., 2015 formulated sinomenine pluronic lecithin organogels system (PLO), and to evaluate the permeability of the optimized PLO in-vitro and in-vivo¹². The results recommend that PLO can be used as an advantageous transdermal delivery vehicle to enhance the permeation and skin deposition of sinomenine.

PLO is the biphasic system facilitated to accumulate both hydrophilic and lipophilic drugs and form a reversed polymer –micelles in specific critical micelles concentration (CMC) ¹³. The optimized CMC makes the PLO thermodynamically stable, and the ingredients use for the preparation of the reverse micelles are biocompatible and non-irritant. The visco-elastic gel has the advantage of rapid absorbance through the skin and produces localized action ¹⁴. The thermoreversible property modifies the skin and acts as a matrix system for the various types of potential drugs ¹⁵. The research article focused on the formulation of the TBH loaded PLO and optimized the formulation using the Box-Behnken method to provide better permeability and the skin deposition for the management of the fungal infection.

MATERIALS AND METHODS:

Materials: Terbinafine Hydrochloride, Pluronic F127, and Lecithin were purchased from Sigma Aldrich, Mumbai, Isopropyl Myristate (IPM) was purchased from S.D. Fine chemicals, Mumbai, Dialysis membrane (MWCO: 12 000–14 000) was purchased from Hi-media. All other reagents and solvents used were of reagent grade.

Preparation of TBH PLO System: PLOs were prepared, basically mixing the oil and aqueous phase. Generally, the first one is an aqueous phase (Pluronic gel) and the second one is the oil phase (lecithin phase). In cold temperatures, PLO gels are a liquid solution, and with increasing the temperature they changed their phase and became a gel ¹⁶.

For the preparation of lecithin phase first, a specific amount of soya lecithin was dissolved in IPM, which was used as dispersing, emulsifying, and stabilizing agent, and then the mixture needs to keep overnight for lecithin’s complete dissolution. Sorbic acid (0.2-0.3% w/w) was added in the mixture as a preservative ¹⁷. A weighed amount of Pluronic F-127 was dispersed in cold water to prepare the Pluronic gel. For the complete dissolution of Pluronic F-127, the mixture needs to keep overnight at 2-4 °C in a refrigerator. Then the drug was dissolved in ethanol and mixed with the aqueous phase. At last, in the final stage, 70% of aqueous phase needs to add slowly drop by drop in 30% of the oil phase with continuous stirring at 400 rpm by using a mechanical stirrer.

Optimization of PLOs:

Determination of Viscosity: A Brookfield digital viscometer (Brookfield Engineering Laboratories, Middleboro, MA) was used with Spindle no 7 to find out viscosities of PLO gels ¹⁸. All tests were performed at room temperature (25 ºC). The viscosity of the TBH loaded PLO gels were evaluated.

In-vitro Permeation Studies using Synthetic Membrane: Permeation studies of TBH loaded PLOs were performed using Franz diffusion cells with a diffusion area of 1.813 cm^{2 19}. The receptor chamber was filled with 27ml of Phosphate buffer (pH 7.4) solution and constantly stirred with a magnetic stirrer at 600 rpm. The water maintained a temperature of 32 ± 0.5 ºC. The known amounts (200 mg) of formulation were applied to the cellulose membrane surface. Four Franz diffusion cells were run simultaneously i.e. PLOs and blank. 2 ml of sample was withdrawn from each cell during the predetermined time intervals of 0, 0.50, 1, 3, 6, 9, 12 h respectively. The amount of penetrated drug in the collected samples was determined by the RP-UFLC method at 222 nm ²⁰.

2.4. Experimental Design of Pluronic Lecithin Organogels using Box-Behnken Design: The critical independent variables [pluronic F127 (X1), lecithin (X2) and IPM (X3)] influencing the transdermal properties of the produced PLO were selected, and a three-factor, three-level Box– Behnken design (BBD) was developed. The viscosity (Y1) and drug release from the skin (Y2) in vitro were selected as responses. The coded and actual values of the variables are given in Table 1.

The most influencing factors effects, as well as the interactive and quadratic factor significance of these formulation parameters on the dependent variables, studied using Design-Expert® software (Version 8.0.7.1, M/s Stat-Ease, Minneapolis, USA) ^{21, 22}. Seventeen experiments with five center points were designed by the software (in order to allow the estimation of pure error) and experiments were run in random order.  The nonlinear quadratic model generated by the design was:

Y = A0 + A1X1 + A2X2 + A3X3 + A4X1X2 + A5X2X3 + A6X1X3 + A7X1… + A8X22 + ...A9X3…..eq (1)

The Y and A0 – A9 represents the dependent variable and regression coefficients of the variables respectably. The interaction of the parameters computed from the observed experimental values of Y; while X1, X2, X3 represent the dependent variable with −1, 0, and +1 representation with analogous to low, middle, and high values shown in Table 1. The experimental matrix data produced by the software was shown in Table 2. BBD and contour plots were used to optimize the final PLO formulations ^{23, 24}. The responses (Y) for each experiment follow the eq (2), interactive eq (3) or quadratic model eq (4) carrying out multiple regression analysis. The F-test use to study the statistical significance.

Y = b0 + b1X1 + b2X2 + b3X3 linear eq (2)

Y = b0 + b1X1 + b2X2 + b3X3 +⋯ b12X1 X2+b13 X1 X3 +... b23X2X3 +... b123 X1X2 X3.....eq(3)

Y = b0 + b1X1 + b2X2 + b3X3 +⋯ b11X12 +⋯ b22X22 +⋯ b33X32 +⋯ b12X1 X2 +… b23X2X3 …+ b13X1X3 +... b123X1X2X3.....eq(4)

Seventeen runs of various combinations were generated and formulated using the data of selected variables X1, X2, and X3 at different levels. Mathematical modelling generated using Eq. (5) to get the second-order polynomial equation.

Y = b0 + b1X1 + b2 X2 + b3X3 + b11X12 + b22X22+ b33X32 + b12X1 X2+ b23X2 X3 + b13X1 X3+ b123X1 X2 X3.....eq (5)

TABLE 1: FACTOR LEVEL FOR BOX-BEHNKEN DESIGN FOR OPTIMIZATION OF PLOs

3D Response Surface Plots: The potential relationship between three variables studied by the 3D surface and 3D wireframe plots.

Desirability Criteria: The desirability lies between 0 and 1 and it represents the closeness of a response to its ideal value (Eq. 6). Desirability criteria use to optimize viscosity and drug release²⁵.

D = (dDrug release - dViscosity) ½…..eq (6)

Where D is the total desirability, drug release and viscosity are individual desirabilities.

Evaluation of PLOs:

Organoleptic Properties: Physical examinations were carried out by inspecting visually of PLO gels color, appearance, odor, texture and phase separation.

Homogeneity Test: The prepared PLO gel pressed between the fingers to study the consistency and the presence of any coarse particles.

Wash Ability: To study the washability of the formulated gel, the formulation was rubbed on the backside of hand skin and wash with tap water after drying. The washable property of the gel was observed.

Determination of pH: For the determination of pH, 1 g of drug-loaded PLO gel was directly determined using a digital pH meter (Eutech Instruments, Mumbai, India). This study performed in triplicate and average pH values were reported.

Drug Content Determination: 1 g of PLO was taken and dissolved in methanol and sonicated for 1 h. The resulted solution was filtered with a 0.22µ filter to obtain a clear solution ²⁶. Then the drug content was analyzed at 222nm by the RP-UFLC method.

Gel Transition Temperature: The gel-sol-gel mechanism is the key to the PLO gel therapeutic efficacy ²⁷. The gel transition temperature is the significance parameter for gel efficacy. The gel transition temperature was studied using the vial contacting the magnetic beads and PLO in water bath maintains the temperature of 40 ºC. The constant heat flow was maintained with the agitation of 60 rpm.

The gelation temperature was measured to study the phase transition temperature.

Spreadability: Spreadability is another important parameter to study the uniform distribution of the gel through the skin for uniform drug release ²⁸. The spreadability was measured using the modified wooden block method and compare with the marketed formulation. A measured amount of the gel was placed on the fixed glass plate and a moveable glass plate is attached to the pan ²⁹. The gel was a sandwich between the two glass slides. Spreadability was determined using the following formula:

S = mX l/t

Where S is spreadability, m is weighing of the upper slide, l represents the length of glass slides and t is time is taken to separate the slides.

Ex-vivo Permeation Studies Using Rat Skin: Permeation studies of optimized PLO and MC were performed using Franz diffusion cells with a diffusion area of 1.813 cm². The receptor chamber was filled with 27ml of phosphate buffer 7.4 solutions containing 0.5% of tween 80 and constantly stirred with a magnetic stirrer at 600 rpm. The water maintained a temperature of 32 ± 0.5 ºC. The known amount (200 mg) of the formulation was applied to the rat skin surface. Four Franz diffusion cells were run simultaneously i.e. optimized PLOs and, MC) and blank ³⁰. Two ml of the sample were withdrawn from each cell during the predetermined time intervals of 0.50, 1, 3, 6, 9, 12 h, respectively. The amount of terbinafine in the collected samples was determined by the RP-UFLC method at 222 nm.

Release Kinetics: The kinetic release model of zero order, first order, Higuchi, Pappas, and Hixson Crowell were generated using the in-vitro release of the formulation to study the best fit.

Microbiological Assay: The fungal stain (IMTECH Chandigarh) was grown in the nutrient agar medium for 18-24 h at 30 ºC. Every cell suspension containing the yield of 1-5.10⁶ organism/mL confirms with colony counter. A sterile hole bore was used to drug the 7 mm wells and the test and MC sample along with the pure drug in the equivalent weight of 2 mg was poured in a different well. After the incubation of 24 h at 35 ºC, the zones of inhibition were measured with the help of divider and scale and expressed in mm ³¹.

Acute Dermal Irritation Test: The test was performed on 20 weeks old albino rabbits, the fur was removed on the back of rabbit 24 h prior carefully to prevent abrading the skin. The test was done for 72 h. Test compound incorporated PLO was applied to an area of skin (5 cm x 5 cm) and untreated skin area served as the control. At the end of the contact time, the lasting preparation was removed using gauze and water without changing the prevailing response or integrity of the epidermis. The scoring system examined the presence of erythema and edema as per OECD 404 from 0 to 4 ³².

Statistical Analysis: Statistical analysis for all the descriptive statistics, values are given as mean ± SD. Statistical significance was checked by the independent samples t-test. p<0.05 was considered statistically significant. Other levels of significance are as noted.

RESULTS:

Effect of the Lecithin Concentration on Viscosity and Drug Release: The lecithin concentration found to be the most significant factor in the drug release and the viscosity of the POLs. The research was clearly confirmed that the increase in the concentration of the lecithin delay the drug release and enhance the viscosity due to the formation of the matrix structure. So, the optimization of the lecithin concentration is the key to the desired therapeutic efficacy of the PLOs.

Effect of Pluronic Concentration on Viscosity and Drug Release: Pluronic F127 (PF127), the triblock water-soluble copolymer has the thermal gelling characteristics and affect the three-dimensional packing of micelles due to amphiphilic molecule interactions. The structural orientation of the pluronic concentration affects the viscosity of the PLOs and alters the drug release. The gel-sol transition of the formulation showed a liner drop with the increase of the PF127 concentration. And its significantly affect the viscosity of the formulation.

Effect of IPM on the Viscosity and Drug Release: Isopropyl myristate (IPM) is a commonly used penetration enhancer in topical and transdermal formulations. The percentage concentrations of the IPM synergistically enhance the drug release by enhancing the flux of the formulation and enhance the permeation. Thus, it can well be concluded that the amount of drug released, and flux show potential synergism when formulated with isopropyl myristate.

Box- Behnken Design: A total no of 17 experiments were performed and tabulated in Table 2 to understand the effect of formulation variables on viscosity and drug release. The value of responses Y1 (viscosity) and Y2 (Drug release) ranges from 2200 to 3400 Cp and 85.4 to 95.4% respectively. The ratio of high and the low for responses Y1 and Y2 are 1.545 and 1.117 respectively. Therefore, the power transformation of values for viscosity and drug release was not required. Box-Cox plot provides a guideline for choosing the correct power transformation. Power transformation is required for responses if the ratio of maximum to minimum response is more than 10. If the ratio is less than 3 that means the transformation has less effect ³⁰. The study focused on the sequential model, sum of squares, and lack of fit tests and concludes the acceptability of the specific model to represent the maximum viscosity, drug release, and the results are shown in Table 3. The study recommends selecting the model for viscosity and drug release of Probability >F value of P<0.0001, low standard deviation, high R² and lower Predicted Residual Error Sum of Square (PRESS).  ANOVA results indicate that the model was significant (Model Prob>F less than 0.05). The Model F value using Fischer’s test for response y1 and y2 was 11.16 and 4.69 respectively, which implies model is significant and represented in Table 4. Lack of fit F-value for y1 and y2 were 3.78 and 0.033 respectively which implies Lack of fit was not significant relative to the pure error Table 4.

The multiple regression terms were also analyzed in Table 5. The coefficient of variation (C.V.) determines the precision of the experiment and its value for all responses was found to be low that indicates the deviation between actual and predicted values is low. It not only shows a high degree of accuracy but good reliability in conducting the experiments. The predicted R-squared values and Adjusted R-squared values for response y1 and y2 were 0.8329 and 0.8511 and 0.4364 and 0.4089 respectively which is in acceptable agreement and shows that the model has predicted the responses suitably. Adequate (Adeq.) precision for both responses y1, y2 was 10.610 and 7.135 respectively. The ratio of response to deviation was measured by the Adeq precision. The Suitable ratio should be greater than 4. Here in both cases, the ratio is greater than 4. To navigate the design space this model can be used. The final equation of coded factors for responses y1 and y2 is given in eq 7 and 8.

Viscosity = + 3000.00 + 262.50*A + 207.50*B + 70.00*C+125.00 * A * B - 50.00*A * C +10.00 *B*C - 142.50*A2-182.50*B2-157.50 *C2…..eq (7)

Drug release = + 90.54 + 2.78*A +1.25*B + 0.50*C…..eq (8)

The response of Y1, positive coefficient of A, B and C indicates that the higher the concentration of Pluronic F-127, lecithin, and IPM more the Viscosity. For response Y2, positive coefficients of A, B, and C indicate Drug release increase as the concentration of Pluronic F-127, lecithin and IPM increases. The theoretic data were compared with the experimental data of responses using predicted vs. actual response graph Fig. 1 was found to be in close arrangement.

FIG. 1: LINEAR CORRELATION PLOTS REPRESENTING THE RELATIONSHIP BETWEEN PREDICTED AND ACTUAL VALUES FOR DIFFERENT RESPONSES A) VISCOSITY AND B) DRUG RELEASE

To find out the factors which affect the response perturbation graphs were plotted Fig. 2. For response Y1, factors A, B, and C show steep slope which indicates that concentration of pluronic, lecithin and IPM have a significant effect on the Viscosity of PLOs. For response Y2, factors B and C were slight bends, which indicate that lecithin and IPM have a significant effect on the drug release.

FIG. 2: PERTURBATION GRAPH REPRESENTING THE EFFECT OF AN INDIVIDUAL FACTOR ON A) VISCOSITY AND B) DRUG RELEASE RESPONSE. A, B, AND C REPRESENT PLURONIC, LECITHIN AND IPM RESPECTIVELY

TABLE 3: FIT SUMMARY OF THE MEASURED RESPONSES

TABLE 4: ANOVA FOR RESPONSE SURFACE QUADRATIC MODEL OF MEASURED RESPONSES

TABLE 5: REGRESSION TERMS FOR VISCOSITY AND DRUG RELEASE

Response Surface Plot:

Desirability Criteria: The optimum formulation obtained after the run based on desirability was found at 33.55, 3.27 and 2.84 level of X1, X2, and X3. The calculated desirability factor was 0.995, indicating the appropriateness of the designed factorial model. The results of the dependent variables given by the run were 3139.92 Cp for viscosity and 93.4561% for drug release Fig. 3 and 4. The predicted value of responses Y1 and Y2 were 3139.92 Cp and 90.25%. After the final formulation, the actual values of Y1 and Y2 were found 3013 Cp and 90.25% TBH loaded PLOs which is in close agreement to the predicted values Table 6.

FIG. 3: 3D RESPONSE SURFACE PLOT SCREENING EFFECT OF INDEPENDENT VARIABLES A) PLURONIC CONCENTRATION (X1) AND LECITHIN CONCENTRATION (X2) B) PLURONIC CONCENTRATION (X1) AND IPM CONCENTRATION (X3) C) LECITHIN CONCENTRATION (X2) AND IPM CONCENTRATION (X3) ON VISCOSITY (Y3) (NOTE: REGION IN THE RED REPRESENTS MAXIMA AND REGION IN BLUE REPRESENTS MINIMA).

FIG. 4: 3D RESPONSE SURFACE PLOT SCREENING EFFECT OF INDEPENDENT VARIABLES A) PLURONIC CONCENTRATION (X1) AND LECITHIN CONCENTRATION (X2) B) LECITHIN CONCENTRATION (X2) AND IPM CONCENTRATION (X3) C) PLURONIC CONCENTRATION (X1) AND IPM CONCENTRATION (X3) ON DRUG RELEASE (Y3) (NOTE: REGION IN THE RED REPRESENTS MAXIMA AND REGION IN BLUE REPRESENTS MINIMA)

TABLE 6: PREDICTED AND ACTUAL RESPONSES FOR TERBINAFINE PLOs

Determination of Viscosity: The viscosities for the 17 PLO gel formulations at 60 rpm were found in a range of 2200 Cps to 3400 Cps Table 2.

In-vitro Permeation Studies using Synthetic Membrane: After the study for 12 h, the percentage drug release from the 17 PLO formulations was found in a range of 85.4-95.4% Table 2.

Evaluation of the PLOs:

Organoleptic Characteristics: The prepared formulations were found homogenous, odorless, off-white in color, greasy, and reluctant to wash and stable without any sign of phase separation.

pH: The pH of all the formulation was found within the range of 5.57-5.80, which similar to the normal skin pH. So, it can be concluded that the PLO gels are non-irritant to the skin.

Drug Content Determination: The drug content of optimized PLO formulation is 99.87%, which means the drug was dispersed homogeneously throughout the gels with good content uniformity.

Spreadability: The spreadability value indicates that the PLO gel formulation was easily spreadable, but the concentration of the lecithin and pluronic effect the spreadability of the formulation. The optimized formulation had the spreadability of 32. 67g.cm/S compare with the MC spreadability of 28.67g.cm/S.

The increased concentration of the polymer act as the resist of spreadability causes a decrease in the tendency of the spreadability.

Gel Transition Temperature: The gel transition temperature of PLO gels was found at 37.4-30.21 ºC. An increase in the concentration of the polymer increases the gel strength at a lower temperature.

TABLE 2: THE LAYOUT OF BOX-BEHNKEN DESIGN SHOWING THE VALUES OF DEPENDENT VARIABLES OF 17 FORMULATIONS

FIG. 5: COMPARATIVE EX-VIVO PERCENTAGE PERMEATION PROFILE OF TERBINAFINE HYDRO-CHLORIDE FROM OPTIMIZED PLO AND MARKETED FORMULATION THROUGH THE RAT SKIN AT pH 7.4. (PERMEATION TIME = 12 h, MEAN ± SD, n = 3).

Ex-vivo Permeation Studies using Rat Skin: The permeation profile was obtained by placing PLOs and MC (each containing equivalent weight of 2 mg TBH) on the rat skin in the Franz diffusion cell Fig. 5. It was observed that TBH concentration steadily increased in the receptor chamber with an increase in time. The results also indicate that the permeation profile followed Fick’s diffusion law. The % cumulative amount of TBH permeated from optimized PLOs and MC at the end of 12^th h after the application was found to be 60.29 ± 0.69 and 19.35 ± 2.19, respectively. The comparatively higher permeability of optimized PLOs could be due to the increased diffusion coefficient of the drug. The permeation was observed as a maximum for optimized PLO.

Release Kinetics: The in-vitro drug release data obtained from release studies were fitted into various kinetic models like zero order, first order, Higuchi, Hixson Crowell, Korsmeyer-Peppas, and represented in Table 7. The best linearity was obtained in Higuchi’s plot for optimized PLO formulation indicating the release as a square root of the time-dependent process.

TABLE 7: REGRESSION VALUE FOR VARIOUS KINETIC MODELS

Microbial Study: The microbial assay was performed using C. albicans strain. The maximum zone of inhibition for the PLO was found 0.58 ± 0.12 mm where the MC the zone of inhibition was 0.34 ± 0.08 mm shows in Fig. 6. The low viscosity of the PLO gel than MC, which helps in high penetration of TBH resulted in an increase in the inhibition zone. The result indicates that PLO could be more effective than the MC for the better management of the fungal infection.

FIG. 6: RESULT OF FUNGAL (CANDIDA ALBICANS) GROWTH INHIBITION ON NUTRIENT AGAR MEDIUM BY AGAR DIFFUSION METHOD AT THE INCUBATION TIME OF 24 h AT 35 ºC. INHIBITION EFFECTIVENESS OF THE ANTIFUNGAL A) MARKETED CREAM (MC) B) PURE DRUG SOLUTION C) OPTIMIZED PLO OF TERBINAFINE HYDROCHLORIDE (CONCENTRATION USED: 2mg EACH)

DISCUSSION: Gel-sol-gel phase transition of PLOs varies with the change of the temperature condition, which indicates that the optimization of the phase transition temperature is the critical factor for the PLO gels. The skin permeation study indicates the statically significant enhancement from the PLO compares to that of MC. The ingredients used for the preparation of the PLO are the key ingredient for the enhancement of the skin permeation, which alters the skin barrier function. Lecithin and pluronic interact and disorganize the lipid layers of the stratum corneum and enhance the drug permeation through the skin. The permeation also depends on the various factors like the micellar structure of the PLO, which contains both the hydrophilic and the hydrophobic ingredients and enhances the diffusion through both the lipid layer and the distribution and slight disorganization of skin. Lecithin is a major component of the cell membrane itself, which may contribute to the good adhesiveness to the skin and good delivery of the drug. Additionally, the IPM acts as an emollient, moisturizer and penetration enhancer. The PLO gels showed a better zone of inhibition compares to the marketed formulation due to the controlled release as well as low viscosity of the formulation which enhances the penetration to facilitate in increasing the zone of inhibition. The pH of the PLO gel shows a significant effect as the low pH favor the inhibition of the fungal strain. The bioadhesive property of PLO made it a better candidate as a control release carrier to enhance the surface action of the TBH by enhancing drug concentration above its minimum inhibition concentration (MIC). The adhesive property due to the ionic interaction of the PLO and the bacteria cell wall propose to enhance the antimicrobial activity of the PLO gel formulation. The acute dermal irritation studies also revealed that the prepared formulation is nonirritant as per OECD 404 and can be used topically.

CONCLUSION: The present study was the attempt to increase the skin permeability of poorly water-soluble drug-like TBH using PLO as the carrier for the fungal skin disorder using DOE to optimize the formulation. The basic results showed that the viscosity and the drug release from the PLOs was the most critical factor for the release of drug from the gel formulation and the therapeutic efficacy of the formulation. The study effectively proved the enhancement of the permeation of the TBH from PLOs through the skin. As well as the kinetic studies indicate the sustain action of the gel formulation which was essential for the fungal disease. In-vitro drug release study shows the control action of the formulation for up to 12 h resulting in prolonging the active time for the drug in the topical treatment for the antifungal action. The result clearly indicates that the incorporation of TBH in the PLO gels certainly enhance the antifungal activity of the drug. Even the used ingredients act synergistically to enhancing the antifungal activity by 1.5-fold compared with MC formulation. These results support the hypothesis that TBH PLOs are the promising candidate for the topical drug delivery system to overcome the limitation of the existing marketed formulations for enhancing skin permeability.

ACKNOWLEDGEMENT: The authors would like to thank Department of Science and Technology - Fund for Improvement of Science and Technology Infrastructure in Universities and Higher Educational Institutions (DST-FIST), New Delhi for their infrastructure support to our department.

CONFLICTS OF INTEREST: The authors declare that there are no conflicts of interest in this study. The authors alone are responsible for the content and writing of the paper.

AUTHOR’S CONTRIBUTION: Sourodip Saha was the lead author and synthesized the literature. Veera Venkata Satyanarayana Reddy Karri provided conceptual input. Bharat Kumar Reddy Sanapalli involved in drafting the paper and critical revision of the manuscript. All authors read and approved the final paper.

STATEMENT OF ANIMAL RIGHTS: In the present study no animals were used. Hence, no approval is required.

FUNDING: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

REFERENCES:

1. Mathias N and Sridharan S: Alternate Routes of Administration. Translating Molecules into Medicines: Springer 2017: 391-19.
2. Alkilani A, McCrudden MT and Donnelly R: Transdermal drug delivery: innovative pharmaceutical developments based on disruption of the barrier properties of the stratum corneum. Pharmaceutics 2015; 7(4): 438-70.
3. Singh SK: Infectious diseases, national security and globalisation. World Affairs: The Journal of International Issues 2019; 23(1): 10-23.
4. Siah A, Magnin-Robert M, Randoux B, Choma C, Rivière C, Halama P and Reignault P: Natural agents inducing plant resistance against pests and diseases. In Natural Antimicrobial Agents 2018; 121-59.
5. Phan CM, Subbaraman L and Jones L: Contact lenses for antifungal ocular drug delivery: a review. Expert Opinion on Drug Delivery 2014; 11(4): 537-46.
6. Tanrıverdi ST and Özer Ö: Novel topical formulations of Terbinafine-HCl for treatment of onychomycosis. European Journal of Pharmaceutical Sciences 2013; 48(4-5): 628-36.
7. Sudhakar B, Varma JR and Murthy KR: Formulation, characterization and ex-vivo studies of terbinafine HCl liposomes for cutaneous delivery. Current Drug Delivery 2014; 11(4): 521-30.
8. Kumar S, Narayan R, Ahammed V, Nayak Y, Naha A and Nayak UY: Development of ritonavir solid lipid nanoparticles by Box Behnken design for intestinal lymphatic targeting. Journal of Drug Delivery Science and Technology 2018; 44: 181-9.
9. Gaba B, Fazil M, Khan S, Ali A, Baboota S and Ali J: Nanostructured lipid carrier system for topical delivery of terbinafine hydrochloride. Bulletin of Faculty of Pharmacy, Cairo University 2015; 53(2): 147-59.
10. Karri VNR, Raman SK, Kuppusamy G, Mulukutla S, Ramaswamy S and Malayandi R: Terbinafine hydrochloride loaded nanoemulsion based gel for topical application. Journal of Pharmaceutical Investigation 2015; 45(1): 79-89.
11. Saha S, Shivarajakumar R and Karri VV: Pluronic lecithin organogels: an effective topical and transdermal drug delivery system. Int J Pharm Sci Res 2018; 11: 4540-50.
12. Ba W, Li Z, Wang L, Wang D, Liao W and Fan W: Optimization and evaluation of pluronic lecithin organogels as a transdermal delivery vehicle for sinomenine. Pharmaceutical Development and Technology 2016; 21(5): 535-45.
13. Gupta V and Trivedi P: Dermal Drug Delivery for Cutaneous Malignancies: Literature at a Glance. J Pharm Innov 2016; 11(1): 1-33.
14. Esposito CL, Kirilov P and Roullin VG: Organogels, promising drug delivery systems: an update of state-of-the-art and recent applications. Journal of controlled release. 2018; 271: 1-20.
15. Kathe K and Kathpalia H: Film forming systems for topical and transdermal drug delivery. Asian Journal of Pharmaceutical Sciences 2017; 12(6): 487-97.
16. Chen S, Li W and Zhu WH: Novel Ethene-Bridged Diarylethene Photochromic Systems: Self-Assembly, Photoswitcher, and Molecular Logic Gates. InPhoton-Working Switches 2017: 37-68. Springer, Tokyo.
17. Alsaab H, Bonam SP, Bahl D, Chowdhury P, Alexander K and Boddu SH: Organogels in drug delivery: a special emphasis on pluronic lecithin organogels. J of Pharmacy and Pharmaceutical Sciences 2016; 19(2): 252-73.
18. Jhawat V, Gupta S and Saini V: Formulation and evaluation of novel controlled release of topical pluronic lecithin organogel of mefenamic acid. Drug delivery 2016; 23(9): 3573-81.
19. Zhang Q, Song Y, Page SW and Garg S: Evaluation of transdermal drug permeation as modulated by lipoderm and pluronic lecithin organogel. Journal of Pharmaceutical Sciences 2018; 107(2): 587-94.
20. Moghddam SMM, Ahad A, Aqil M, Imam SS and Sultana Y: Optimization of nanostructured lipid carriers for topical delivery of nimesulide using Box–Behnken design approach. Artificial Cells, Nanomedicine, and Biotechnology 2017; 45(3): 617-24.
21. Singh B, Saini S, Lohan S and Beg S: Systematic development of nanocarriers employing quality by design paradigms. Nanotechnology-Based Approaches for Targeting and Delivery of Drugs and Genes: Elsevier; 2017; 110-48.
22. Ahad A, Aqil M, Kohli K, Sultana Y and Mujeeb M: Enhanced transdermal delivery of an anti-hypertensive agent via nanoethosomes: statistical optimization, characterization and pharmacokinetic assessment. International Journal of Pharmaceutics 2013; 443(1-2): 26-38.
23. Anderson M and Whitcomb, P: RSM Simplified. New York: Productivity Press 2017, https://doi.org/10.1201/ 9781315382326.
24. Krull DP, Thomovsky SA, Chen AV, Mealey KL and Papich MG: Evaluation of transdermal administration of phenobarbital in healthy cats. Journal of the American Animal Hospital Association 2019; 55(1): 1-7.
25. Khan AW, Kotta S, Ansari SH, Sharma RK, Kumar A and Ali J: Formulation development, optimization and evaluation of Aloe vera gel for wound healing. Pharmacognosy Magazine 2013; 9(Suppl 1): S6.
26. Abbas N, Parveen K, Hussain A, Latif S, Uz Zaman S, Shah PA and Ahsan M: Nanosponge-based hydrogel preparation of fluconazole for improved topical delivery. Tropical Journal of Pharmaceutical Research 2019; 18(2): 215-22.
27. Grela KP, Bagińska I, Burak J, Marciniak DM and Karolewicz B: Natural Gums as Viscosity-Enhancers in Pluronic® F-127 Thermogelling Solutions. Die Pharmazie-An International Journal of Pharmaceutical Sciences 2019; 74(6): 334-9.
28. Samarajeewa AD, Velicogna JR, Princz JI, Subasinghe RM, Scroggins RP and Beaudette LA: Effect of silver nanoparticles on soil microbial growth, activity and community diversity in a sandy loam soil. Environmental Pollution 2017; 220: 504-13.
29. Sanapalli BK, Kannan E, Balasubramanian S, Natarajan J, Baruah UK and Karri VV: Pluronic lecithin organogel of 5-aminosalicylic acid for wound healing. Drug Development and Industrial Pharmacy 2018; 44(10): 1650-8.
    

    ---

    How to cite this article:

    Saha S, Sanapalli BKR and Karri VVSR: Optimization of pluronic lecithin organogel of terbinafine hydrochloride using design of experiments for the treatment of fungal diseases. Int J Pharm Sci & Res 2019; 10(12): 5499-09. doi: 10.13040/IJPSR.0975-8232. 10(12).5499-09.

    ---

    2All © 2013 are reserved by the International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---