Formulation and Characterization of Microemulsion Made by the Combination of Carvedilol and Fluconazole

Microemulsion

Microemulsions are isotropic, thermodynamically stable, transparent (or translucent) systems of oil, water and surfactant, frequently in combination with a co-surfactant with a droplet size usually in the range of 10-100 nm. These homogeneous systems, which can be prepared over a wide range of surfactant concentration and oil to water ratio, are all fluids of low viscosity. Microemulsion as drug delivery tool show favorable properties like thermodynamic stability (long shelf-life), easy formation (zero interfacial tension and almost spontaneous formation), optical isotropy, ability to be sterilized by filtration, high surface area (high solubilization capacity) and very small droplet size. The small droplets also provide better adherence to membranes and transport drug molecules in a thermo dynamically stable spontaneously formed mixtures of ‘oil’ and ‘water’, the term “oil” actually means any water insoluble (hydrophobic) organic liquid [53-56]. Though water has been predominantly used as polar phase for the formation of microemulsion, recent studies have also shown successful application of ethanol as polar phase for formulating hybrid fuel systems having aforementioned structural similarities with ‘microemulsions’. The higher GCV of ethanol than that of water may be another advantage of incorporating it into the system.  Micro emulsification approach is successfully applied in fields like separation, chemical reactions, nanomaterial preparations, and drug delivery. The preceding decades have witnessed dramatic increase in microemulsion based applications. Microemulsification today plays a pivotal role in thrust areas like fabricating nanoparticles, oil recovery, pollution control, food and pharmaceutical industries etc.

2 Experimental Work

2,1 List of materials.

Instruments List

Table 2. Details of Instruments used in the research work

2.3 Method of estimation of fluconazole and carvedilol.

Determination of UV absorption maxima for fluconazole and carvedilol.

10 mg of Ibuprofen was first dissolved in ethanol (10 ml) and was then carefully diluted upto 100 ml with phosphate buffer, pH 6.4 diluted to obtain a stock solution of 100 µg/ml concentration. Then from the stock solution, 1 µg/ml and 2 µg/ml test solutions were prepared using above diluting solvent, i.e., Ethanol to phosphate buffer (pH 6.4) (1:9). The solutions were scanned in spectrum mode for absorbance between 200-600 nm using UV-Visible Spectrophotometer. 10 mg of Zaltoprofen was similarly dissolved in 10 ml of ethanol and diluted upto 100 ml with distilled phosphate buffer, pH6.4 (100 ppm) and then scanned between 200 nm and 400 nm using UV visible spectrophotometer.

2.4 Preparation of Calibration curve. UV Method.

From the stock solution of fluconazole and carvedilol 100 µg/ml, test solutions of concentration 2, 4, 6, 8, 10, 12, 14 µg/ml were prepared by suitable dilution with hydroalcoholic solvent (Ethanol: PB pH 6.4, 1: 9). The absorbance of each test solution was then taken at the absorption maxima (222 nm) against hydroalcoholic solvent as a blank for Ibuprofen and 228 nm for Zaltoprofen. The above procedure was performed in triplicate and average of the absorbance was taken into consideration (Yousry M, 2021).

2.5 HPLC Method.

Ibuprofen in solubility study samples as well as in brain and blood samples was quantified using the developed and reported reversed phase high performance liquid chromatography (RP-HPLC) method (Albert K, 2021 and Choudhary S, 2022). HPLC analysis was performed by using Waters 515 Series pumps combined with a detector i.e., Waters PDA 2998 series photo diode array (DAD). The column so used was Agilent C18 column (150 mm×4.6 mm, particle size 5-micron Agilent, USA). Analysis was done isocratic at 0.8 ml/min flow rate with mobile phase i.e., ACN: Buffer (0.025M Potassium dihydrogen ortho phosphate) pH 4.5 at 11:9 v/v. First the mobile phase was prepared by mixing the above said components and filtered through a 0.2 µm nylon membrane filter followed by degassing by sonication before use. The elution of drug was done at 230 nm. For Zaltoprofen, the experimental conditions were Acetonitrile and Phosphate buffer pH3 (60:40 v/v), as mobile phase at a flow rate of 1 ml/min and at 254 nm. HPLC column used was 5µm intensil, C18 column (4.6 x 250mm x 5µm).

2.6 Selection of Mobile phase and Preparation of Solutions.

Based on the literature review and solubility study, the mobile phase with their ratio was selected.

2.7 Preparation of Standard Stock Solution.

Accurately weighed Carvedilol as well as Fluconazole (10 mg) was dissolved in 100 ml of respective mobile phase taken in 100 ml of calibrated volumetric flask to prepare a solution of 100 μg/ml the same was further diluted into 100 times. An aliquot of 5 ml was pipette out from the stock solution and transferred to the calibrated volumetric flask of 25 ml and diluted to the mark in border to prepare 200 ng/ml using their respective mobile phase as diluting solvent.

2.8 Preparation of Working Standard Solutions.

For both the drugs, different aliquots of 1.0, 2.0, 3.0, 4.0 and 5.0 ml from the prepared stock solution (100 ng/ml) were separately transferred to a 10 ml of calibrated volumetric flask and dilute up to mark to generate the solutions of 20, 40, 60, 80 and 100 ng/ml concentrations with mobile phase. The test solutions were sonicated and further filtered through 0.22 μm filter i.e., nylon paper.

2.9 System Suitability Testing.

It can be defined as the tests to ensure the accuracy and precision of the developed system. The defined parameters in USP are used i.e., plate number (N), tailing factor (tF), resolution Time (RT) and the relative standard deviation (% RSD) of the peak height or the peak area for repetitive injections (n=5). At least two of these criteria are necessary to demonstrate system suitability for any methd

Table 3. Parameters for System Suitability

2.10 Pre-formulation study.

Identification of fluconazole and carvedilol.

Melting Point.

Melting point of both drugs was determined by capillary method through a melting point apparatus. A small amount of drug was inserted into the capillary tube (closed at one end) and the same was then placed into the melting point apparatus. Finally, the temperature at which drug starts melting, was taken as melting point and was then compared with the reference data (Adamo F, 2009).

2.11 FTIR spectrum of Ibuprofen and Zaltoropfen.

FTIR spectroscopy is a useful tool for the structure elucidation of drug using KBr disc method. The pure drug sample was triturated and dispersed with micronized IR grade KBr powder followed by application of 8-12 kpa pressure in the hydraulic KBr press to prepare the pellets. Pure KBr powder was used as blank for baseline correction. The pellets were transferred to sample compartment and were then scanned in the region of 400-4000 nm by FTIR spectrometer. Comparision of obtained FTIR spectrum of test sample was made with reference Ibuprofen as well as Zaltoprofen spectrum (Matkovic S, 2005).

2.12 Percentage Purity.

Percentage purity was done as per procedure given in IP 2010, the given limits (85% to 115%).

2.13 Spectral analysis (Absorption maxima, λmax determination).

50 μg/ml of Ibuprofen as well as Zaltoprofen solution was prepared in hydroalcoholic solvent. The absorbance was determined by scanning the sample at entire range of UV range (200-400 nm). The same was compared with the reference (Hapse S, 2011 and Diana M, 2010).

5.14 Solubility profile of fluconazole and carvedilol.

5 ml of different solvents (distilled water, phosphate buffer pH 6.4, acetonitrile, ethanol and acetone) were taken separately in the test tubes and excess amount of drug was added to saturate the system. The dispersion was shaken in thermostatic orbital shaker for 48 hrs at 37 ± 2˚C (80 rpm). All samples were then centrifuged at 8000 rpm for 15 min. To obtain only solubilised Ibuprofen as well as Zaltoprofen, the supernatant was filtered through 0.45μm filters and the filtrate was finally analyzed using UV-VIS spectrophotometer at appropriate wavelength (Diana M, 2010 and Yan et al, 2009). This test was performed three times and results were taken as mean ± SD.

2.15 Screening of components.

The screening criterion to select mucoadhesive microemulsion compositions are as per the literature (Jing C, 2006 and Huabing C, 2006).

• All formulation compositions should be of GRAS (Generally recognized as safe) category for intranasal administration.
• Drug solubility for oil phase, surfactant and co-surfactant (Jing C, 2006).
• Non-irritating property of surfactant and co-surfactant.
• HLB value of surfactant i.e., 12-16 for the development of O/W microemulsion.
• Drug excipients compatibility property (Chen et al, 2006).

Basing on the biocompatibility profile of oils for nasal mucosa from the literatures, drug solubility was determined. Excess amount of drug (100mg) in 5 ml of selected oils (Isopropyl Myristate, Oleic acid, Maisine 35-1, Capryol 90, Labrafil WL 2609 BS, Capmul MCM EP, Capmul MCM, Labrafil M 1944CS, Labrafac PG and Labrafil M 2125CS) was taken in stopper vials (10 ml) and were then mixed by vortex mixer. The vials containing mixture were then kept at 37 ± 2.0 C and 80 rpm in orbital shaker at isothermal condition for 48 hr to reach equilibrium. The equilibrated samples were the centrifuged at 5000 rpm for 15 min. The solubility profile of drug in oil was finally determined from the supernatant by using UV-VIS spectrophotometer at its respective absorption maxima (Serajuddin A, 2021). Various surfactants with HLB from 12 to 16 (Tween-80, Tween-60, Labrafac CC, Acconon CC, Cremophor EL and Cremophor RH 40) were screened for drug solubility in the same procedure as described for oils (Jing et al, 2006 and Zhang P, 2008).

2.16 Drug-Excipient compatibility study.

Drug-Excipient compatibility study was performed to ensure that the formulation has relatively more biocompatibility and physical stability of the formulation. Adequate quantity of drug to individual formulation components mixed separately and was kept undisturbed for 7 days. At day 3 (D3) and day 7 (D7), the samples were checked for precipitation, phase separation and color change in order to determine their physical compatibility (Pani N, 2011). Chemical compatibility is basically regarded as the chemical stability of the drug in all formulation components. Surfactant, oil and cosurfactant were considered for further development only if physically and chemically compatibility with drug was observed. Adequate quantity of Ibuprofen and all formulation components mixed and was kept at room temperature and at 50 C for 7 days FT-IR spectra of the mixture at day 1 (D1) and day 7 (D7) were analysed for compatibility since FT-IR spectrum gives the identification of specific functional groups and so that from this any chemical incompatibility if occurred can be easily identified by change in the peak of functional group (Marini et al, 2003).

2.2.1 Preparation of fluconazole and carvedilol microemulsion formulations.

Water titration technique was used for developing mucoadhesive microemulsion with the selected oil phase, surfactant-cosurfactant mixture and aqueous mucoadhesive polymeric phase (Patel M, 2013 and Acharya S, 2013). Required quantity of fluconazole and carvedilol was added to adequate amount of oil separately in a cleaned and dry beaker having a small magnetic bead and was mixed completely using magnetic stirrer. Then, to the mixture, surfactant and co-surfactant at definite ratio (Smix from the pseudo-ternary phase diagram) was added and was mixed at same rpm. The above mixture was finally titrated using aqueous solution of mucoadhesive polymer under same experimental condition on a magnetic stirrer (Omar S, 2012).

Table 4. Levels of Process variables in 32 factorial design

2.3.1 Transparency (% Transmittance and Refractive index).

Both developed MMEIs and MMEZ were diluted 50 and 100 times with distilled water. Optical transparency of these formulations was quantified at 560 nm by UV-VIS spectrophotometer against water. Results were taken in triplicate and the average was taken in to consideration (Chandra A, 2009). Abbe’s refractometer was used to determine the refractive index of the formulation. Results were taken in triplicate and the average was taken into consideration (Mandal S, 2011).

5.3.2 Globule size and Zeta potential analysis.

Both developed MMEIs and MMEZs were diluted 50 times and 100 times with distilled water by gentle agitation for 5 min spin vibrtor. In addition, globule size distribution (PdI) were determined using dynamic light scattering technique while zeta potential or surface charges of MMEIs was determined by Malvern zetasizer (NANO ZS). Results were taken in triplicate and the average was taken in to consideration (Joakim B, 2010 and Patel R, 2012).

5.3.3 Viscosity analysis.

2.4.3 In-vitro drug release study.

In-vitro drug release     study    of microemulsion formulations [MMEI(C) and MMEI(L) as well as MMEZ] were carried out in Franz diffusion cell having volume of 30 ml and an effective permeation area of 7.06 cm2 containing 30 ml of dissolution media i.e., ethanolic phosphate buffer saline (PBS), pH 6.4, as used in the UV determination (Pathak R, 2014 and Sharma G, 2009). Temperature was maintained at 37 ± 0.5 0C and rpm was set 50. Dialysis membrane of cut off weight 10,000 D was soaked PBS (pH 6.4) overnight prior to experiment.

2.4.4 Release kinetics.

In order to study the release kinetics of fluconazole and carvedilol from developed MMEs, the release data so obtained were fitted to the following equations as per the literature (Starychova et al, 2014 and Carvalho et al, 2010) and reference data is given in Table-4.6.

Zero kinetic equation: Qt = k0t, Where, Qt- % drug released at time t and k0 - Release rate constant.

First kinetic equation: ln (100-Qt) = ln 100 – k1t, Where, k1- Release rate constant.

Higuchi’s kinetic: Qt = kHt1/2, Where, kH - Higuchi release rate constant.

Hixson-Crowell kinetic: (100-Qt) 1/3 = 1001/3 – kHCt, kHC – Hixson-Crowell rate constant.

Korsmeyer Peppa’s model: Qt/Q∞= kKPtn, Where, Qt/Q∞ - Fraction of drug released at time t,  kKP a constant and n, the release exponent indicating the drug release mechanism.

Table-5. Release kinetics (Diffusional exponent v/s type of release)

2.5.9 Stability studies.

Stability of fluconazole and carvedilol mucoadhesive microemulsion.

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