Paraffin oils

MaterialsDutasteride was obtained as a gift sample from Taj Pharmaceutical Ltd. (Mumbai, India) and was used as received. Propylene glycol, monocaprylic ester (sefsol 218) was obtained as a gift sample from Nikko Chemicals (Tokyo, Japan). Diethylenemonoglycol ether (carbitol) and castor oil were purchased from Sigma Aldrich (St. Louis, MO). Isopropyl myristate, glycerol triacetate (triacetin), castor oil, eucalyptus oil, oleic acid were purchased from E-Merck (Mumbai, India). Polyoxyethylenesorbitanmonolaurate (tween-20), polyoxyethylenesorbitanmonostearate (tween-60), polyoxyethylenesorbitanmonooleate (tween-80), ethanol, isopropyl alcohol, PEG 200, propylene glycol, pleurol oleic, brij 35, lecithin were procured from S.D Fine Chemicals (Mumbai, India). Milli Q water was used during the whole experiment. All chemicals and solvents were of analytical grade.
Screening of excipientsThe most important criterion for screening of components is the solubility of drug in oils, surfactants and co-surfactants.
Screening of oilThe solubility of dutasteride in various oils was determined by adding an excess amount of drug in 2 mL of the different oil separately in a 5 mL capacity stopper vials. The content of the vials were mixed using a vortex mixer. The mixture vials were then kept at 25±1.0 °C in an isothermal shaker for 72 h to achieve equilibrium. The equilibrated samples were removed from the shaker and centrifuged at 3,000 rpm for 15 min. The supernatant was taken and filtered through a 0.22-μm membrane filter. Supernatant 10 µL oil was taken and diluted with methanol and concentration of dutasteride was determined in oils using a UV spectrophotometer at 240 nm (10 ).
Screening of surfactant and co-surfactant for nanoemulsionTo find out the suitable surfactant and co-surfactant, the solubility of dutasteride was determined in various surfactants including tween-20, tween -60, tween-80, brij35, lecithin, plurol oleic acid and a combination of two surfactants was taken. The solubility of dutasteride was also checked in co-surfactants such as ethanol, isopropyl alcohol, PEG 200, and polyethylene glycol following the similar procedure as mentioned in oil selection.
Phase studiesOn the basis of solubility studies, eucalyptus oil: oleic acid (1.5:1) was selected as an oil phase. Tween-20 and ethanol were selected as surfactant and co-surfactant, respectively. Milli Q water was used as an aqueous phase. For the determination of existence zone of nanoemulsion, pseudo ternary phase diagrams were constructed using water titration method (spontaneous emulsification method) (11 ). Surfactant and co-surfactant (S _mix) were mixed in different weight ratios (1:1, 2:1, 3:1 and 1:2). These S _mix were chosen in increasing concentration of co-surfactant with respect to surfactant. For each phase diagram, oil and specific S _mix were mixed well in different ratios. Sixteen different combinations of oil and S _mix (1:9, 1:8, 1:7, 1:6, 1:5 1:4, 1:3.5, 1:3, 3:7, 1:2, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1) were made so that maximum ratio could be covered for the study to delineate the boundaries of the phases formed precisely in the phase diagrams (12 ). Slow titration with aqueous phase was done for each weight ratio of oil and S_mixunder moderate stirring, and visual observation was used for transparent and easily flowablenanoemulsion. Gels were claimed for those clear and highly viscous mixtures that did not show a change in the meniscus after being tilted to an angle of 90°. The physical state of nanoemulsion was marked on a pseudo three component phase diagram with one axis representing the aqueous phase, second representing oil, and the third representing a mixture of surfactant and co-surfactant at fixed weight ratio (S _mix ratio).
Selection of formulationsFrom the pseudoternary phase diagrams showing maximum nanoemulsion area, a number of nanoemulsions with different composition were selected covering the entire range of nanoemulsion occurrence in the phase diagrams with minimum surfactant and maximum water concentration. 0.5 mg dutasteride, which was kept constant in all the selected formulations, was added to the oil phase during the formulation of nanoemulsions. Selected formulations were subjected to various physical stability tests.
Thermodynamic stability testing of nanoemulsionsIn order to find out the stable nanoemulsion and to discard the unstable or metastable nanoemulsions the placebo nanoemulsions were subjected to following thermodynamic stability studies.
Freeze thaw cycleNanoemulsions were kept in deep freezer (at -20 °C) for 24 h. After 24 h the nanoemulsions were removed and kept at room temperature. The thermodynamically stable nanoemulsions returned to their original form within 2-3 min. 2-3 such cycles were repeated.
Centrifugation studiesNanoemulsions after freeze thaw cycle were subjected to centrifugation studies where they were made to undergo centrifugation for 30 min. at 5,000 rpm in a centrifuge. The stable formulations did not show any phase separation or turbidity.
Heating cooling cycleSix cycles between refrigerator temperature (4 °C) and 40 °C with storage of 48 h were performed. Those formulations which were stable at these temperature, subjected to further study.
Characterization of nanoemulsionsGlobule size analysisThe droplet size of the nanoemulsions was determined by photon correlation spectroscopy, which analyses the fluctuations in light scattering due to Brownian motion of the particles using a Zetasizer 1000 HS (Malvern Instruments, Worcestershire, UK). Light scattering was monitored at 25 °C at a 90° angle.
ViscosityViscosity of nanoemulsion was determined by using Brookfield LV rotational viscometer at 2.5, 5, 10 and 20 rpm. Each reading was taken after equilibrium of the sample at the end of two minutes. The samples were repeated three times. The viscosity values at 5 rpm were selected
Refractive indexThe refractive index of the system was measured by an Abbe refractometer (Bausch and Lomb Optical Company, Rochester, NY) by placing one drop of the formulation on the slide in triplicate at 25 °C.
pH MeasurementsThe apparent pH of the formulations was measured by a pH meter (Mettler Toledo MP 220, Greifensee, Switzerland) in triplicate at 25 °C.
Transmission Electron Microscopy (TEM)Morphology and structure of the nanoemulsion were studied using Morgagni 268D electron microscope (Fei Company, Netherlands) operating at 70 kV capable of point-to-point resolution. Combination of bright field imaging at increasing magnification and of diffraction modes was used to reveal the form and size of the nanoemulsion. In order to perform transmission electron microscopy (TEM) observations, a drop of the nanoemulsion was suitably diluted with water and applied on a carbon-coated grid, then treated with a drop of 2% phosphotungstic acid and left for 30 s. The coated grid was dried and then taken on a slide and covered with a cover slip and observed under the microscope.
Hydrogel thickened nanoemulsionThe very low viscosity often exhibited by nanoemulsion is not suitable for transdermal use. The viscosity can be increased by adding thickening agents, which also change the appearance of the system, usually influencing drug release. Recently, the gel matrices such as carbopol 934, sodium alginate, ethyl cellulose, and HPMC have been used to prepare the nanoemulsion based gel for improving the viscosity of nanoemulsion(13 (link), 14 (link)). The selection of polymer for preparing gel is normally based on the character of external phase (oil for w/o type and water for o/w type). Because dutasteridenanoemulsion is a type of o/w type, so carbopol 934 was selected for preparation of nanoemulsion gel. For preparation of nanoemulsion gel 1% carbopol 934 dispersed in sufficient quantity of distilled water. This dispersion was kept in dark for 24 h for complete swelling of carbopol 934. Prepared nanoemulsion was added slowly to carbopol 934 dispersion. 0.5% w/w of triethanolamine (TEA) was added in this mixture to neutralize carbopol 934. Then by mixing hydrogel thickened nanoemulsion was obtained.
In-vitro skin permeation studiesThe protocol to carry out in-vitro permeation studies was approved by the Institutional Animal Ethics Committee, S.B.S College of Pharmacy, Patti, Amritsar, Punjab, India. The committee's guidelines were followed for the studies. In-vitro skin permeation studies were performed on a fabricated Franz diffusion cell with an effective diffusional area of 5.24 cm² and 5 mL of receiver chamber capacity using rat abdominal skin. The full-thickness rat skin was excised from the abdominal region, and hair was removed with an electric clipper. The subcutaneous tissue was removed surgically, and the dermis side was wiped with isopropyl alcohol to remove adhering fat. The cleaned skin was washed with distilled water and stored in the deep freezer at -21 °C until further use. The skin was brought to room temperature and mounted between the donor and receiver compartment of the Franz diffusion cell, where the stratum corneum side faced the donor compartment and the dermal side faced the receiver compartment. Initially, the donor compartment was empty and the receiver chamber was filled with phosphate buffer (pH 7.4). The receiver fluid was stirred with a magnetic rotor at a speed of 100 rpm, and the assembled apparatus was placed in the oven and the temperature was maintained at 37 ± 1 °C. All the receiver fluid was replaced every 30 min to stabilize the skin. It was found that the receiver fluid showed negligible absorbance after 4.5 h and beyond indicating complete stabilization of the skin. After complete stabilization of the skin, 1 mL of nanoemulsion formulation (0.5 mg/mL dutasteride) was placed into each donor compartment and sealed with paraffin film to provide occlusive conditions. Samples were withdrawn at regular intervals (0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, and 24 h), filtered through a 0.45 membrane filter, and analyzed for drug content by UV spectrophotometer at λ_max of 240 nm (10 ).
Permeation and distribution data analysisThe cumulative amount of dutasteride permeated through the albino rat skin (Q, μg/cm²) was plotted as a function of time (t, h) for optimized nanoemulsion formulation A1, nanoemulsion gel of A1 and control. Control group represents 30% of S_mix (1:1) Tweeen-20 and Ethanol, solution containing 0.5 mg in mL of dutasteride without oil mixture.The permeation rate (flux) at the steady state (J_ss ,μg/cm² /h) and lag time were calculated from the slope and intercept of the straight line obtained by plotting the cumulative amount of dutasteride permeated per unit area of skin versus time at steady-state condition, respectively. Permeability coefficient (K _p) was calculated by dividing the flux by initial drug concentration (C₀) in the donor portion of cell as given below (15 (link)):
K _p = J_ss /C₀Enhancement ratio (E_r) was calculated by dividing the J_ss of the respective formulation by the J_ss of the control formulation as given below:
E _r = J_ss of formulation/J_ss of controlHistopathology studiesAbdominal skin of Wistar rats was treated with the optimized dutasteridenanoemulsion gel of A1. After 24 h, the rats were killed and skin samples were taken from untreated (control) and treated areas. Each specimen was stored in 10% formalin solution in phosphate buffer saline (pH 7.4). The specimens were cut into sections vertically. Each section was dehydrated using ethanol embedded in paraffin wax for fixing and stained with hematoxylin and eosin. These samples were then observed under light microscope (Motic, Japan) and compared with control samples.
Stability studies as per ICH guidelinesStability studies on optimized nanoemulsion were performed by keeping the sample at refrigerator temperature (4 °C) and room temperature (25 °C). These studies were performed for the period of 3 months. The droplet size, viscosity and refractive index were determined at 0, 1, 2 and 3 months. Accelerated stability studies were also performed on optimized nanoemulsion as per international conference on harmonization (ICH) guidelines. Three batches of optimized formulation were taken in glass vials and were kept at accelerated temperature of 30, 40, 50 and 60 °C at ambient humidity. The samples were withdrawn at regular intervals of 0, 1, 2 and 3 months. These samples were analyzed for drug content by stability-indicating HPLC method at a wavelength of 241 nm(16 ). The chromatographic column used was a reverse phase 25 cm X 4.6 mm, i.d., 5 im, C18 DB reversed phase column (Phenomenox). The mobile phase was methanol: water (90:10) with the flow rate of 1.25 mL/min. The retention time (Rt) of drug was 5.24 min. Zero time samples were used as controls (100% drug). Analysis was carried out at each time interval by taking 100 µL of each formulation and diluting it to 5 mL with methanol and injecting into the HPLC system at 241 nm. The solubility of sample in methanol was 63.8 mg/mL. In addition, samples of pure oil (combination of eucalyptus oil and oleic acid), pure surfactant and co-surfactant (S) were run separately to check interference of the excipients used in the formulations.
The amount of drug decomposed and the amount remaining (undecomposed drug) at each time interval was calculated. Order of degradation was determined by the graphical method (17 ). Degradation rate constant (K) was determined at each temperature. Arrhenius plot was constructed between log K and 1/T to determine the shelf-life of optimized nanoemulsion formulation. The degradation rate constant at 25 °C (K₂₅) was determined by extrapolating the value of 25 °C from Arrhenius plot. The shelf-life (T_0.9) for each formulation was determined by using the formula:
T_0.9 = 0.1054/ K₂₅

For each experiment, alive mites of all motile stages (n = 20) were placed in a plastic Petri dish (3 cm in diameter). In the first experiment, all the pure essential oils included in the present study were successively tested against mites. The following bioassays were conducted using the essential oils that killed all the mites within 1 h of direct contact. The selected essential oils were diluted with paraffin to get concentrations at 10, 5 and even 1% (for the most efficient ones). In each Petri dish, 1 ml of the diluted solution was added in direct contact with the mites. A control Petri dish was inoculated with 1 ml of paraffin oil. The mites were inspected under a stereomicroscope (Nikon©, SMZ645, Lisses, France) 10, 20, 30, 40, 50, 60, 90, 120, 150 and 180 min after inoculation. Mites were considered dead when no movement was seen even after touching it with a needle, and no gut movement was observed over 2 min [12 (link)].

Essential oils of clove (Syzygium aromaticum) (CAS No. 8000-34-8/84961-50-2), cinnamon (Cinnamomum sp.) (product code PO128351, Batch No. 990133) and citronella (Cymbopogon sp.) (Batch No. 0000065697) were purchased from the Union Science Co., Ltd. (Chiang Mai, Thailand). The dried fruit of cardamon (Amomum krervanh) was purchased from a herbal store in Chiang Mai and was extracted using a hydro-distillation method [33 (link)]. Their chemical compositions are shown in Table 1. Liquid paraffin (Labchem, Ajax Finechem Pty Ltd.), canola and sunflower oil, coconut oil (Naturel, Lam Soon (Thailand) Public Company Limited) and rice bran oil (King, Thai Edible Oil Co., Ltd.) were used to test for suitable solvents for the essential oils.

Three surfactants were used to perform the experiments, namely: $C_{12}$ AOS (Anionic surfactant, Alpha Olefin Sulfonate, Germany, >99.0 wt% pure), $C_{12}$ Betaine (Zwittterionic surfactant, Cocamidopropyl Betaine, Russia, >99.0 wt% pure), and FARUS (Nonionic surfactant, $C_2$ Oxyethylated phenols in monatomic alcohols, Russia,>99.0 wt% pure). In this paper, they are hereafter referred to as AOS, BETAINE and FARUS, respectively. Two binary surfactant systems were prepared from these surfactants: AOS and FARUS, hereafter referred to as AOSFAR, and BETAINE and FARUS, hereafter referred to as BETFAR. All surfactants were used as received without further treatment. The surfactant solutions were prepared in a high salinity brine (23.4 wt%) with the compositions listed in the Table 1 below. All the salt components were purchased from Chimmed, Moscow and are >99.0 wt% pure, as declared by the manufacturer.Table 1

Brine composition.

Salt	NaCl	KCl	CaCl2	MgCl2
Composition, g/L	172.056	4.261	42.172	15.7

The critical micelle concentrations of both single surfactants and their binary mixtures have been earlier determined in our previous work¹² . Thus, in this study, each surfactant was used at 1.5 times their original CMC to maximally harness their enhanced surface activity (Table 2).Table 2

Surfactant compositions and their concentrations.

Surfactant composition	Active content	Concentration, wt%
AOS - Alpha Olefin Sulfonate	35%	0.315%
FARUS - Oxyethylated phenols in monatomic alcohols	50%	0.480%
BETAINE - Cocamidopropyl betaine	75%	0.525%
AOSFAR - AOS + FARUS (1:2)	–	0.135%
BETFAR - BETAINE + FARUS (1:2)	–	0.120%

CO₂ gas with a purity of 99.98% was used to generate foam. Three mineral oils and one crude oil model were used to investigate the effect of the oil on the foam stability. The mineral oils used were n-Decane, n-Octadecane (liquid paraffin) and Toluene. The crude oil model was a sample from a producing oilfield in Russia. Their densities and molecular weights are shown in Table 3Table 3

Oil properties.

Oil	Density (g/cm3)	Molecular weight (g/mol)
Decane	0.83	142.29
Toluene	0.77	92.14
Octadecane	0.88	254.49
Crude oil	0.85	215.74

For mineral oils, 0.05 wt% of Sudan I dye was added to the oil phase to visualize the oil droplets in the foam column. Preliminary tests, including interfacial tension, density and foam columns, were performed to ensure that the dye had no influence on foaming behavior.