Evaluation of copaiba oil as enhancer of ibuprofen skin permeation

medications on the skin through transcutaneous routes is a practice that has been used by mankind for millennia. Some studies have been reporting the use of terpenes and natural oils rich in terpenes as an enhancer of cutaneous penetration. Copaiba oil, due to its rich content of terpenes, presents itself as a great choice of penetration enhancer for drugs administered on the skin. In this study, we developed two cream formulations containing 5% of ibuprofen (IBU) and copaiba oil: IBCO5 and IBCO10 with 5% and 10% of copaiba oil respectively. Ex vivo cutaneous penetration/permeation studies of IBU were performed using pig ear skin as biological membrane in the Franz-type diffusion cells. The steady-state flux of IBU samples, IBCO5 (35.72 ± 6.35) and IBCO10 (29.78 ± 2.41) were significantly higher when compared with control without copaiba oil (10.32 ±1.52) and with a commercial product (14.44 ± 2.39). In the penetration analysis, the amount of IBU found in the samples IBCO5 and IBCO10 was markedly higher in the dermis than epidermis. Our results showed that copaiba oil possesses attracting properties in promoting skin penetration and permeation of IBU when added into cream formulations.


INTRODUCTION
Transcutaneous formulations are an interesting approach to transport several drugs, as an alternative to overcome issues related to the pharmacokinetics and pharmacodynamics when drugs are administered by other routes (Simon et al., 2016;Hussain et al., 2012). The advantages of the percutaneous delivery reside not only in reducing fluctuations in plasma drug concentrations and frequency of administration but also reducing variability of oral absorption and mitigating the presystemic metabolism. Furthermore, this route allows abrupt discontinuation of treatment and constitutes an attracting alternative to intravenous administration (Obata et al., 2010).
Among factors that have the potential to perturb percutaneous absorption are skin hydration state and its biological characteristics, as well as the physical and chemical properties of the drug, including its molecular mass, aqueous solubility, melting point, partition coefficient (Log P) and dissociation constant (pKa). In addition, pharmaceutical and adjuvant forms used to convey drugs could also affect drug absorption through transdermal delivery (Hussain et al., 2012;Pontrelli, Monte, 2014).
The use of permeation enhancers, incorporated in different types of formulations, improve drug flux through diverse membranes. These chemicals are usually pharmacologically inactive and are able to permeate or interact with the constituents of the stratum corneum when incorporated into a transcutaneous formulation, reducing skin resistance to the diffusion of a drug (Jiang et al., 2017). Several studies has been shown that the use of natural oils rich in terpenes as permeation enhancers are safe and suitable to promote the percutaneous absorption of drugs from topical formulation into lower skin layers (Herman, Herman, 2015).
Among natural compounds with potential applications as permeation enhancer, stands out copaiba oil. This oil is obtained by tapping the trunk of the trees from species of Copaifera L. (Leguminosae), popularly known as "copaiba" or "pau-de-óleo" (Oliveira et al., 2010). Copaiba oil is from natural source, rich in terpenes, mainly sesquiterpenes and constitutes one of the most important renewable sources of natural remedy for population of the Amazon region and can be found in drugstores and markets all over Brazil (Leandro et al., 2012). Furthermore, copaiba oil has been reported to increase skin permeation of drugs, suggesting its use as a penetration enhancer in topical dosage forms (Veiga-Junior et al., 2007).
Topical drug delivery of Ibuprofen using natural oils as permeation enhancers have been reported in the literature (Chen et al., 2016;Khan et al., 2011). Ibuprofen is a classical non-steroidal anti-inflammatory drug (NSAID) which is used for the symptomatic treatment of rheumatoid arthritis, ankylosing spondylitis, dysmenorrhea, etc. However, an effective permeation of Ibuprofen, is difficult to achieve by transdermal delivery due to its poor skin permeability (Bello, Kuwornu, 2014;Padula, Nicoli, Santi, 2011). Thus, the purpose of the present study was to develop and evaluate a novel, simple and stable topical cream formulation containing 5% of IBU, using copaiba oil (5% and 10%) as skin permeation enhancer, in comparison with commercial cream formulation containing 5% of ibuprofen.

GC-MS of copaiba oil
Copaiba oil was analyzed by GC-MS on a Shimadzu QP2010 Plus (Shimadzu Corporation, Kyoto, Japan) system equipped with an AOC-20i auto sampler operating in the electron ionization (EI) mode at 70 eV under the following conditions: Restek Rtx-5MS fused silica capillary column (30 m x 0.25 mm i.d. x 0.25 µm film thickness) composed of 5%-phenyl-95%methylpolysiloxane; carrier gas helium (99.999%) at a constant flow rate of 1.46 mL/min; injected volume of 0.1 µL of a solution containing copaiba oil (2 µL) and standard β-caryophyllene (2 µL) in 1000 µL of hexane without flow divider; injector temperature 245 o C; ionsource temperature 280°C. The oven temperature was programmed to increase from 60 to 240 °C at 3 °C/min. Mass spectra were recorded with a scan interval of 0.5 s within the mass range 40-600 Da. Quantification of each constituent was estimated by internal normalization (%). The identification of the copaiba oil components was based on their retention indices, relative to a homologous series of n-alkanes (C 8 -C 20 ) measured on an Rtx-5MS capillary column under the same operating conditions. Computer matching was accomplished with the aid of the Wiley 7 and NIST 08 spectra libraries (McLafferty, 2008). The mass spectra of the constituents were also compared to those reported in the literature (Adams, 2007).

Sample preparation
VersaPro TM cream, a pre-made oil-in-water (O/W) emulsified base, was purchased in order to make a cream formulation containing: (1) ibuprofen 5% as control; (2) ibuprofen 5% and copaiba oil 5% (IBCO5) and; (3) ibuprofen 5% and copaiba oil 10% (IBCO10), where copaiba oil was used as cutaneous penetration enhancer. This base was chosen because of its excellent stability for incorporation of water and oil-soluble drugs (Sarkar et al., 2011). Finally, a commercial cream containing Ibuprofen 5% was used as reference for permeation and penetration studies as well as for the stability tests.

Preliminary stability studies
Samples were prepared in triplicate and evaluated after 24 hours (t 0 ) and 12 days (t 12 ) of heatingcooling cycles for the following parameters: color, odor, consistency, brightness, absence of clumps and precipitates, pH analysis, lack of homogeneity after mechanical stress (centrifuge) and IBU content (Zamarioli et al., 2015). This 24 hours heating-cooling cycle test on the formulation samples was performed by storing the samples in the fridge/oven (-5 ± 2°C and 45 ± 2°C) as required for 12 days. The organoleptic properties such as consistency, color, odor, glare and lack of clumps and precipitates were evaluated by direct visual perception.
The pH of the formulations was measured using a digital pH meter Model PG2000 (Gehaka, São Paulo, SP, Brazil) previously calibrated with solutions of pH 4.0 acetate and pH 7.0 phosphate buffers. 10% aqueous solution (w/v) samples were prepared and their pH was measured, in triplicate, at room temperature.
For the physical stability test, samples were placed in tubes and subjected to conditions of mechanical stress by centrifugation at a speed of 1370 g for 15 min in triplicate. Samples were evaluated visually for precipitation, coalescence and for separation of the emulsified preparations.
Additionally, ibuprofen assay was performed by liquid chromatography (HPLC), according to the method described in British Pharmacopoeia, 2014. The chromatographic system consisted of an Alliance 2695 solvent delivery module (Waters, USA) equipped with auto sampler, degasser, column oven and a dual wavelength UV-Vis detector 2489. The chromatographic conditions were: X-Bridge C18 column, 4.6 x 150 mm, 5 µm, connected to a C18 guard column (Waters Assoc., Milford, USA); isocratic mobile phase of methanol-water (75:25, v/v) adjusted to pH 3.0 with orthophosphoric acid; flow rate of 1.0 ml/min, injection volume of 20 µL and wavelength of 222 nm.

Ex vivo permeation study
For each formulation, an ex vivo test using Franztype diffusion cell were carried out for the purpose of evaluating the permeation of IBU from cream formulations into the skin. Phosphate buffer (pH 7.4) was used as receptor solution and excised skin from pig ear was used as biological membrane. Pig ear skin is a very attractive model for ex vivo percutaneous absorption studies since it mimics human skin (Yuan, Capomacchia, 2005).
The pig ear was obtained from local slaughterhouse and the skin was excised from the dorsal part of the pig's ear using a scalpel. The hair and excess fat were removed and only skin cuts in the range of 1.5 -2.0 cm 2 were used in the experiments (Lucca et al., 2015). The receptor compartment was filled with 7.5 mL of phosphate buffer pH 7.4. Cells were maintained at 37°C ± 0.5 in a thermostatic bath under agitation (900 rpm) (Santis et al., 2012). Cream samples (200 mg) were applied on the membrane by using an automatic pipette for semisolid preparations. The diffusion area of each donor compartment corresponded to approximately 1.00 cm 2 .
After applying samples, an aliquot of 1 mL of receptor solution was sampled each 90 min for analysis of IBU content, and the same volume was replaced with fresh buffer to maintain sink conditions. After that, IBU levels were analyzed by high-performance liquid chromatography (HPLC). The permeation of IBU in the receptor medium was monitored for 450 min (Herkenne et al., 2007).
To calculate the amount of IBU permeated (Q) at time t, dilutions obtained after the first collection were considered, and the following equation (1) was used according to Sato et al. (2007): (1) Where: C measured = concentration of the sample at time t V r = volume of receptor solution of the diffusion cell C a = concent ration of the sample removed V a = volume of the sample removed Rodrigo José Lupatini Nogueira, Richard Michael Grazul, Ademar Alves Da Silva Filho, Jorge Willian Leandro Nascimento A permeation curve of IBU, expressed in µg cm -2 of permeated drug versus time (min), was then generated. Values obtained by HPLC were used to calculate kinetic parameters (kinetic model), latency time (lag time), steady-state flux (J) and permeability coefficient (Kp) of IBU through the membrane (Santis et al., 2012;Stahl, Wohlert, Kietzmann, 2011).
To determine the kinetics of permeation, Higuchi, First-order and Zero-order model kinetics were calculated. The closest linear correlation coefficient (R 2 ) to 1 corresponded to the membrane permeation kinetics of IBU (Sato et al., 2007).

Penetration in the epidermis and dermis
To determine IBU penetration in the dermis and epidermis, a validated method by Santis et al. (2012) was used. After the permeation test, the system was dismantled and the skin was removed from the diffusion cell, and cleaned. Epidermis was separated from the dermis using a surgical scalpel, and both were subjected to the extraction process of IBU by adding 1 mL of mobile phase (methanolwater 75:25 v/v). Material was vortexed three times for 30 sec. Samples were then centrifuged at 6400 rpm for 10 min. Supernatant was collected and samples were analyzed by HPLC (Zamarioli et al., 2015). As a result, a graph representing dermal and epidermal penetration of IBU was generated. Results were used to evaluate the ability of copaiba oil as a permeation enhancer of IBU through different skin structures (Santis et al., 2012).

Statistical analysis
Results are presented as mean ± standard deviation. Statistical significance was determined by Student's t test for two group comparisons, and oneway ANOVA followed by the Newman-Keuls test for multiple comparisons through GraphPad Prism software. Statistical differences were considered significant when p-values were less than 0.05.

Preliminary stability
As shown in Table II, all formulations tested in two stages of analysis were stable regarding organoleptic characteristics of color, odor, uniformity, brightness and absence of precipitates compared with the commercial reference product.
Concerning the pH determination, our results showed no significant differences between times t 0 and t 12 for the three samples. It indicates that neither acidic nor alkaline decomposition products were generated during the preliminary stability test.
We also found that samples were stable at t 0 and t 12 in rotation, with no precipitates, coalescence and/or phase separation following a stability test under mechanical stress by centrifugation. These results show that samples exhibit physical stability even after increasing the force of gravity and mobility of the droplets.

Ex vivo permeation and penetration study
The drug permeation through pig ear skin, between donor and receiver compartment, in vertical diffusion cells, were tested for all the samples. The highest amounts of IBU permeated after 450 minutes were observed from those samples containing copaiba oil, as shown on Figures 1 and 2. When we evaluate the ability of copaiba oil as an IBU permeation enhancer through the pig ear skin membrane, was observed a higher distribution of this drug in dermis when compared to epidermis (Figure 3). FIGURE 1 -Release of ibuprofen (IBU) over time -cumulative permeation curves of IBU from cream formulations containing 5 or 10% of copaiba oil in comparison to a control and commercial product. It represents the average of total IBU amount transferred by a diffusion area using pig ear skin as the membrane model monitored for 450 minutes (n=3). FIGURE 2 -Cutaneous permeation of ibuprofen (IBU) through pig skin at 450 min in vertical diffusion cells. *p < 0.05; **p < 0.01 in comparison with Commercial product; a p < 0.05 and b p < 0.01 in comparison with control (CO) (n=3).
The effects of copaiba oil on IBU release through permeation at 450 min were further substantiated by permeation flux (J, µg cm -2 h -1 ) of IBU through pig ear skin (Table III). Based on the latency period, it was possible to define the kinetics of the permeation of three mathematical models (Higuchi, first order or zero order), aiming to determine the order of the process. The linear correlation coefficient (R 2 ) for each model was determined (Table IV).

DISCUSSION
Several studies reported the use of the GC-MS technique for qualitative and quantitative analysis of copaiba oil. This chromatographic technique was effective in this study since all identified terpenes by GC-MS in copaiba oil were known from other copaiba oil samples and match those already reported in the literature (Dias, et al., 2014;Custodio, Veiga-Junior, 2012). β-caryophyllene was the main detected compound, which is in agreement with previous chromatographic studies that have shown β-caryophyllene as the major compound found in copaiba oil samples (Leandro et al., 2012).
Stability studies performed to evaluate IBU content in IBCO5, IBCO10 and commercial product, at t 0 and t 12 after heat/cool stress, are in accordance with the specification range set by the British Pharmacopoeia, 2014 (95 to 105%). These data demonstrated that even after a period of heat/cool stress, samples were able to maintain their chemical stability, showing that the formulations meet the pharmacopeial specification.
Based on the quantification of IBU obtained from HPLC, we observed drug permeation through pig ear skin between donor and receiver compartment in vertical diffusion cells. This pattern was seen in all samples tested (Figure 1). It has been previously shown that several commercial formulations containing IBU 5% in gel increased drug permeation through porcine skin for 8 h (Herkenne et al., 2007).
IBCO5 and IBCO10 samples only differ from others by having the copaiba oil in their compositions. The highest amounts of IBU permeated after 450 minutes were observed on those samples containing that oil. It thus indicates that since the copaiba oil contains a significant terpene composition which may play an important role in promoting cutaneous permeation of IBU through porcine skin (Figure 1). Interestingly, some studies found that the permeation enhancement capacities of the crude natural oils were significantly higher than its main terpene components (Lan et al., 2014;Monti et al., 2002).
Cutaneous permeation of IBU through pig skin shows a remarkable release of the drug at 450 min in the presence of copaiba oil (IBCO5 and IBCO10) (Figure 1). Both IBCO5 and IBCO10 showed significant differences (p<0.01 and p<0.05, respectively) when compared with control or commercial product ( Figure 2). Interestingly, IBCO5 induced a two-fold increase in IBU release in comparison with the commercial product. On the other hand, higher amount of copaiba oil (10%) (IBCO10) did not induce further release of IBU than the formulation with 5% (IBCO5). In addition, the commercial product showed no difference in comparison with control ( Figure 2). Therefore, despite the fact that the copaiba oil demonstrates permeation enhancement activity of IBU, our data suggests that copaiba oil is not more efficient when its concentration is doubled.
IBCO5 and IBCO10 showed significantly higher steady-state flux (J) of IBU through pig ear skin (p<0.01 and p<0.05, respectively) than control or commercial product. Moreover, copaiba oil at 10% (IBCO10) did not increase permeation flux when compared with a lower concentration (Table III). A similar result was found by Oliveira et al. (2010) when they compared the skin permeation efficacy of kojic acid using two different concentrations of copaiba oil (25% and 50%) in vertical Franz diffusion cells. There wasn't a statistically significant difference between these two concentrations when the steady-state flux was evaluated. This study concluded that both concentrations could be used as skin permeation enhancers for hydrophilic drugs.
Analyzing the lag or latency time, which is the time that the drug takes to start its permeation, a shorter time (90 min) was found for IBCO5, IBCO10 and the commercial product than the control (120 min). These differences in lag time can be explained by the absence of copaiba oil as skin permeation enhancer in the control, and copaiba oil was used for IBCO5 and IBCO10 and the commercial product contains nerolidol, an amphiphilic sesquiterpene also considered a drug penetration enhancer due its capacity for the disruption of the highly organized lipid packing in the stratum corneum (Chen et al., 2016).
According to our results, all samples followed a zero order model as a permeation kinetic profile due to their higher coefficient. This model indicates that the steadystate flux (J) is constant for the interval of calculated time (after lag time) and independent of the drug concentration used. This kinetic model is typical for formulations with infinite doses (Sato et al., 2007;Ruela et al., 2016).
Following permeation experiments, the amount of IBU was determined in different skin layers after their separation as described in Section 2.5. Very significant differences were observed in all formulations after 450 min, including IBCO5 (p < 0.01) and IBCO10 (p <0.05). Importantly, at least a two-fold amount of IBU was found in the porcine dermis in comparison with epidermis ( Figure 3). The amount of IBU accordingly increased with the depth of the skin at a certain time mainly in the presence of copaiba oil. This finding supports the hypothesis of a unique role played by copaiba oil as a skin permeation enhancer of IBU.

CONCLUSION
Our physical and chemical studies indicate the feasibility of developing stable IBU formulation with copaiba oil as permeation enhancer. In addition, permeation and penetration data suggests that copaiba oil at 5% and 10% incorporated into an emulsified base not only markedly improved drug skin permeation but also showed penetration enhancing properties of IBU in the dermis. As a result, copaiba oil increased the depth of IBU penetration in skin layers. Altogether, our study demonstrates that copaiba oil is an attractive tool to be explored as a potent cutaneous enhancer of drugs such as IBU.