Preparation and evaluation of ibuprofen-loaded microemulsion for improvement of oral bioavailability

Abstract

The purpose of the current study was to improve the solubility of ibuprofen, a poorly water-soluble drug, in a microemulsion system that is suitable for oral administration. Microemulsion was prepared using different sorts of oils, surfactants, and co-surfactants. Pseudo-ternary phase diagrams were used to evaluate the microemulsion domain. The formulations were characterized by solubility of the drug in the vehicle, droplet size, and drug release. The optimal formulation consists of 17% Labrafil M 1944CS, 28% Cremophor RH40/Transcutol P (3:1, w/w), and 55% water, with a maximum solubility of ibuprofen up to 60.3 mg/ml. The mean droplet size of microemulsion was 57 nm. The pharmacokinetic study of microemulsion was performed in rats and compared with granule formulation. The microemulsion has significantly increased the C_max and area under the curve (AUC) compared to that of the granule (p < 0.05). The relative bioavailability of ibuprofen in microemulsions was 1.9-fold higher than that of the granule. These results indicated that this novel microemulsion is a useful formulation for enhancing the oral bioavailability of ibuprofen.

Keywords::

• Ibuprofen
• microemulsion
• solubilization
• oral
• pharmacokinetics.

Introduction

Ibuprofen, a phenyl propionic acid derivative, is widely accepted as one of the best tolerated non-steroidal anti-inflammatory drugs available for the treatment of rheumatoid arthritis, osteoarthritis, and mild-to-moderate pain (Yong et al., 2004; Newa et al., 2007). However, ibuprofen shows the poor gastrointestinal absorption because of the low solubility or dissolution rate to water (Glowka, 2000; Ghorab & Adeyeye, 2001). So ibuprofen was formulated into many preparations such as inclusion complex (Ghorab & Adeyeye, 2001; Charoenchaitrakool et al., 2002), pro-drug (Bansal et al., 1994; Murtha & Ando, 1994), solid dispersion (Bodmeier & Wang, 1993; Khan & Jiabi, 1998; Greenhalgh et al., 1999), and microencapsulation (Bodmeier et al., 1992; Adeyeye & Price, 1994; 1997; Kachrimanis et al., 2000) to improve the solubility of ibuprofen. Recently, some studies reported that the solubility and oral absorption of poorly water-soluble drugs can be improved by a microemulsion system (He et al., 2003; Araya et al., 2005; Nornoo et al., 2009; Yin et al., 2009).

Microemulsion is a single optically isotropic and thermodynamically stable liquid solution with a droplet size between 10–100 nm. It consists of an oil phase, surfactant, co-surfactant, and aqueous phase. Microemulsion is a novel pharmaceutical drug delivery system for oral delivery of poorly water-soluble drugs because of it’s ability to improve drug solubilization and potential for enhancing absorption in the gastrointestinal tract (Lawrence & Rees, 2000; Kim et al., 2001; Kawakami et al., 2002).

In this study, an ibuprofen microemulsion was designed for oral administration, the effects of the excipients on the formation of microemulsion is discussed. Based on a solubility study and pseudo ternary phase diagrams, microemulsions containing ibuprofen have been developed after screening oils, surfactants, and co-surfactants. The pharmacokinetic study for the optimized formulation was also investigated.

Materials and methods

Materials

Ibuprofen as the model drug was obtained from Baike Pharmaceutical Co. Ltd. (Beijing, China). Ibuprofen granules were purchased from Tongjitang Pharmaceutical Co. Ltd. (Guizhou, China). Excipients such as Labrafac Lipophile WL1349, Labrafil M 1944 CS, Labrasol, and Transcutol P were donated by Gattefosse (Shanghai, China). Cremophor RH40 was purchased from Xietai Chemical Co. Ltd. (Shanghai, China). Tween 80, Ethanol, and Propylene Glycol were obtained from Meilin Industry and Trade Co. Ltd. (Tianjin, China). Acetonitrile and methanol were of HPLC grade and supplied from Huadong Chemical (Tianjin, China). Double-distilled water was used throughout the study. All other chemicals and solvents were analytical reagent grade.

Preparation of microemulsion

Solubility study

The solubility of ibuprofen in various oils and surfactants was determined by adding an excess amount of ibuprofen into 1 ml of each vehicle (Table 1) in the centrifugal tube. Then, the mixture was vortexed and kept for 7 days at 25°C in a shaking incubator to get to equilibrium. The equilibrated sample was centrifuged at 10,000 rpm for 30 min to remove the excess ibuprofen. The drug content in the supernatant was diluted with methanol and measured by HPLC analysis using a P3000A pump and a UV3000 UV-Vis detector with a Venusil XBP C18 column (5 μm, 250 × 4.6 mm). The mobile phase was composed of 0.02 mol/l potassium dihydrogen phosphate buffer (pH 6.5) and acetonitrile (6:4 v/v) at a flow rate of 1.0 ml/ min, and the effluent was monitored at 223 nm.

Table 1.  Solubility of ibuprofen in various vehicles at 25°C (mean ± SD, n = 3).

	Vehicle	Solubility of ibuprofen (mg/ml)
Surfactant	Tween80	250.51 ± 17.10
	Labrasol	265.65 ± 16.97
	Cremophor RH40	259.33 ± 13.78
	span80	144.70 ± 14.15
Oil	peanut oil	66.30 ± 7.90
	Labrafac Lipophile WL1349	98.86 ± 15.10
	Labrafil M 1944CS	87.64 ± 11.90
Co-surfactant	Transcutol P	389.91 ± 19.40
	Propylene glycol	162.68 ± 12.25
	Ethanol	408.39 ± 17.09

Pseudo-ternary diagrams

The area of microemulsion existence was determined with the aid of pseudo-ternary phase diagrams previously reported (Zhu et al., 2008). The weight ratio of surfactant to co-surfactant (Km) varied as 1:1, 2:1, and 3:1. At each ratio of surfactant to co-surfactant (S/CoS), the ratio of oil to the mixture of S/CoS was varied as 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, and 95:5. Distilled water was added dropwise to each oily mixture under gentle magnetic stirring at ambient temperature. Following the addition of aliquot of water phase, the mixtures were assessed visually. The points from clear to turbid and turbid to clear were investigated. Based on these diagrams, appropriate oil, surfactant, and co-surfactant were selected for the preparation of ibuprofen microemulsions.

Preparation of ibuprofen microemulsions

From the screen results of pseudo-ternary phase diagrams, ibuprofen microemulsions were prepared. In brief, an excess amount of ibuprofen was dispersed to the mixtures of oil, surfactant, and co-surfactant, an appropriate amount of water was added to the mixture drop-by-drop under magnetic stirring. The mixture was kept for 24 h at 25°C in a shaking incubator. Then undissolved ibuprofen was removed by centrifugation at 10,000 rpm for 30 min and the ibuprofen concentration in the supernatant was measured by the HPLC method mentioned above.

Characterization of microemulsions

Transmission electron microscopy

The morphology of ibuprofen microemulsions was observed using transmission electron microscopy (JEM-100SX, JEOL, Tokyo, Japan). One drop of diluted samples was negatively stained by 2% phosphotungstic acid (PTA) and placed on film-coated copper grids followed by drying before examination under the electron microscope.

Droplet size of microemulsions

The droplet size of microemulsions was measured by photon correlation spectroscopy (PCS) using a NICOMP particle sizing system (CW380, Santa Barbara, CA) at a fixed angle of 90° and at a temperature of 25°C. The droplet size analysis data were evaluated using the volume distribution.

Dilution

Each formulation containing ibuprofen was diluted 100-times with distilled water and simulated gastrointestinal fluid at 25°C and 37°C. The droplet size, appearance, and drug content were investigated.

The stability of microemulsions

The microemulsions were stored at 4°C, 25°C, 37°C, and 40°C for 6 months. The droplet size, appearance, and drug content were investigated.

In vitro diffusion studies

The in vitro diffusion studies were performed for optimal formulation using the dialysis bag method. The dialysis bag (molecular weight cut-off 10,000 Da) was soaked in ionized water for 12 h before use. Three dialyzing media were chosen: PH 6.8 PBS, 0.1 N HCl, and distilled water. The temperature was set at 37°C ± 0.5°C; 0.5 ml of microemulsion (equivalent to ~ 30 mg ibuprofen) was poured into the bag. The bags were placed in a conical flask and 300 ml release medium was added. The conical flasks were placed into a thermostatic shaker at 37°C and 50 strokes per minute. Each sample (3 ml) was withdrawn at 5, 10, 20, 30, 40, and 60 min with replacement by an equal volume of temperature-equilibrated media. The samples were filtered through 0.22 μm filters and the concentration of ibuprofen was determined by the HPLC mentioned above.

Pharmacokinetics studies

Healthy male Sprague-Dawley rats (250 ± 20 g) were supplied from the Laboratory Animal Center of Hebei University. Prior to use, the rats were kept in a temperature and humidity controlled animal observation room (25°C, 55–60% air humidity). All animal experiments complied with the requirements of the National Act of the People’s Republic of China on the use of experimental animals. Animals were fasted overnight prior to administration, but allowed free access to water. The animals were randomized to be administered orally (30 mg/kg of body weight) with microemulsion and granule, respectively. Both formulations were dispersed into saline with drug concentration 10 mg/ml prior to dosing. Blood samples were collected in tubes containing heparin at various intervals after administration. Samples were centrifuged after collection and stored at −20°C until analysis.

The plasma concentration of ibuprofen was determined by HPLC previously reported (Wang et al., 2005). Briefly, 0.2 ml plasma was mixed with 100 μl naproxen methanol solution (internal standard, 10 μg/ml) and 1.0 ml methanol. The samples were vortex-mixed for 5 min and centrifuged at 3000 rpm for 10 min. The organic portion was separated and evaporated to dryness at 40°C. The residues were then reconstituted in 100 μl of mobile phase and 20 μl was injected for analysis.

Statistical analysis

Student’s t-tests were performed to evaluate the significant differences between the two formulations. Values are reported as mean ± SD and the data were considered statistically significant at p < 0.05.

Results and discussion

Solubility study

The solubility of ibuprofen in various oils, surfactants, and co-surfactants was analyzed to screen components for microemulsions. The results were shown in Table 1. Based on the results, although Labrafac Lipophile WL1349 showed better solubility for ibuprofen than Labrafil M 1944 CS, Labrafac Lipophile WL 1349 can not form a microemulsion with Labrasol, Tween80, and Cremophor RH40. Thus, Labrafil M 1944 CS was chosen as oily phase due to its good drug solubility and emulsion-forming ability. In addition, ibuprofen had a higher solubility in Cremophor RH40, Labrasol, and Tween 80, but had less solubility in Span 80. Therefore, three surfactants (Cremophor RH40, Labrasol, and Tween 80) and three co-surfactants (Transcutol P, Propylene Glycol, and Ethanol) were chosen for further evaluation.

Pseudo ternary phase diagram study

Pseudo-ternary phase diagrams were constructed in the absence of ibuprofen to obtain appropriate concentration ranges of components in the areas of microemulsions. In this study, a total of nine phase diagrams were prepared by pairing three surfactants and three co-surfactants mentioned above. When Tween 80 or Labrasol was used as surfactant, the microemulsion area was very small regardless of what co-surfactant was used. The systems containing Cremophor RH40 as surfactant and Transcutol P as co-surfactants formed a stable and broad microemulsion area. In the case of Cremophor RH40, Ethanol as a co-surfactant also formed a broad microemulsion area, but the system was not stable when diluted 50-times with distilled water, thus only Transcutol P was chosen as a co-surfactant.

The phase diagrams containing Cremophor RH40 as a surfactant and Labrafil M 1944 CS as an oil and Transcutol P as a co-surfactant with various Km values (1:1, 2:1, and 3:1) are described in Figure 1. The addition of Transcutol P increased the area of microemulsion at all Km values over that with Cremophor RH40 alone (data not given). It was also found that the microemulsion region was increased gradually with increase in Km, reaching a maximum at Km of 3:1. Thus, the ratio of surfactant and co-surfactant was fixed at 3:1 for further studies.

Figure 1.  The pseudo-ternary phase diagrams of the oil-surfactant-water system at 1:1 (a), 2:1 (b), and 3:1 (c) weight ratios of Cremophor RH40 to Transcutol P at room temperature. E, emulsion; G, gel; L, isotropic region; shaded region, microemulsion.

Solubility of ibuprofen in microemulsions

In this study, ibuprofen was dissolved in the mixture of oil, surfactant, and co-surfactant and then miroemulsified to find an optimized formulation with higher drug-carrying capacity. Three microemulsions with the following oil:Sm:water (w/w/w) ratio were selected for experiments: F1 (8:32:60), F2 (12:30:58), and F3 (17:28:55). Among the three microemulsions, the solubility of ibuprofen was increased as the ratio of oil to S_mix (the mixture of surfactant and co-surfactant) increased (Table 2). F3 (60.3 mg/ml) produced a higher solubilizing capacity of ibuprofen than F1 (36.6 mg/ml) and F2 (50.9 mg/ml). Furthermore, the three microemulsions did not exhibit any precipitation of drug when observed for a period of 1 month. Therefore, the optimum formulation consisted of Labrafil M 1944 CS 17%, CremophorRH40 21%, and Transcutol P 7% and water 55% (w/w) exhibited the highest solubility of ibuprofen (60.3 mg/ml).

Table 2.  Compositions, droplet size, and ibuprofen content of the selected formulations (mean ± SD, n = 3).

Formulation	Oil:Sm:water	Droplet size (nm)	Drug content (mg/mL)
F1	8:32:60	53.2 ± 1.36	36.6 ± 1.66
F2	12:30:58	55.6 ± 2.81	50.9 ± 1.75
F3	17:28:55	56.9 ± 2.46	60.3 ± 1.37

Characterization of microemulsions

The physicochemical characteristics of microemulsion appear in Table 2. Morphology of the ibuprofen microemulsion was characterized using TEM (Figure 2). The average size of all microemulsions was ~ 55 nm.

Figure 2.  Transmission electron microphotography of ibuprofen-loaded microemulsions.

Microemulsions could be diluted by water in the gastrointestinal tract upon oral administration, which could lead to drug precipitation. In our study, the size range of the particle was retained even after 100-times dilution with water and simulated gastrointestinal fluid at 25°C and 37°C, and no drug crystallized out of the solution, which proves the stability of ibuprofen microemulsion in excess water and can avoid in vivo drug precipitation.

Ibuprofen microemulsion was found to be stable after 6 months of storage at 4°C, 25°C, 37°C, and 40°C. There was no significant difference in droplet size, appearance, drug content, and dilution ability during the stability study. This indicates that ibuprofen microemulsion is chemically and physically stable.

In vitro release study

In vitro release experiments were conducted to evaluate the effect of different media on the release of ibuprofen from microemulsion, and the results are shown in Figure 3. The results indicated that ibuprofen released fast in the microemulsion due to its high solubility and the cumulative amount of drug released after 30 min was above 80%.

Figure 3.  Release profiles of drug from microemulsion in PH 6.8 PBS, 0.1 N HCl, and distilled water (mean ± SD, n = 3).

Pharmacokinetic study

Figure 4 shows the blood concentration-time profiles of ibuprofen after oral administration of granule and microemulsion. The peak plasma concentration (C_max) and the time (T_max) were obtained directly from the individual plasma concentration vs time profiles. The non-compartmental pharmacokinetic parameters are presented in Table 3. The microemulsion formulation gave significantly higher AUC and C_max of ibuprofen compared with granule. The AUC increased 1.9-fold after the oral administration of microemulsion. However, T_max was not significantly different between the two formulations (Table 3).

Table 3.  Comparison of pharmacokinetic parameters between ibuprofen microemulsion and ibuprofen granule (mean ± SD, n = 5).

Group	Microemulsion	Granule
T1/2 (h)	2.14 ± 0.63	2.16 ± 0.27
Cmax (mg/L)	69.02 ± 9.88	37.14 ± 9.59
tmax (h)	0.65 ± 0.34	0.50 ± 0.31
AUC (h·mg/L)	183.19 ± 11.82	95.31 ± 15.14
MRT (h)	2.70 ± 0.24	2.77 ± 0.17

Figure 4.  The blood concentration-time profile of ibuprofen after oral administration of granule and microemulsion to rats (mean ± SD, n = 5).

The enhanced bioavailability is probably due to the combination of the following effects: (a) significantly improved solubility of ibuprofen. The solubility became 60.3 mg/mL by the optimum microemulsion, ~ 420-times that when water was obtained (Kokot & Zmidzinska, 2001). The gastrointestinal absorption can be increased by microemulsions which could keep the drug as the soluble form. (b) Significantly improved permeability of ibuprofen. Surfactants are known to increase the permeability of drugs by disturbing the cell membrane and modifying tight junctions between the cells (Jackson, 1987), the presence of a surfactant Cremophor RH40 and a co-surfactant Transcutol P, in microemulsion system may enhance permeability of intestine. (c) Reduction the activity of enteric metabolism and intestinal P-glycoprotein (P-gp) efflux pump. So the microemulsion formulation could significantly increase the oral bioavailability by the combined effects above.

Conclusion

In this study, a microemulsion system was constructed for oral delivery of ibuprofen, various formulation factors were evaluated to find an optimum microemulsion vehicle that had high drug solubility. The optimum formulation consisted of Labrafil M 1944 CS 17%, CremophorRH40 21%, Transcutol P 7%, and water 55% (w/w) exhibited the highest solubility of ibuprofen (60.3 mg/ml). The microemulsion system is physically stable at all conditions investigated for 3 months. The oral bioavailability of ibuprofen obtained from microemulsion was 1.9-fold higher than that of the granule formulation. So the employment of microemulsion was found to be a suitable carrier system for the oral administration of ibuprofen.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.