3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, more commonly known as the statins, are the most commonly prescribed lipid-modifying drugs [1,2]. Competitive inhibition of HMG-CoA reductase by the statins decreases hepatocyte cholesterol synthesis, which results in an increased extraction of LDL-C from the blood and decreased circulating of the LDL-C concentration [3,4].
Pitavastatin (PT), (+)-monocalcium bis (3R,5S,6E)-7-(2- cyclopropyl-4-[4-fluorophenyl]-3-quinolyl-3,5-dihydroxy-6- heptanoate), is a potent synthetic inhibitor of HMG-CoA reductase and was developed for the treatment of hypercholesterolaemia. It can reduce plasma levels of LDL cholesterol by 40% in hypercholesterolaemic patients [5,6]. In humans, PT is only minimally metabolized by the cytochrome P450 2C9 isoenzyme. The major metabolic pathway of PT involves its initial glucuronidation by uridine diphosphateglucuronosyltransferase and then spontaneous lactonization by the elimination of the glucuronide moiety. Moreover, the lactone form can be reversibly converted to the parent drug . PT is excreted predominantly into the bile and thereby enters the enterohepatic circulation. Very little parent drug is excreted into the urine [7,8].
Microemulsions (MEs) have recently attracted much attention in pharmaceutical research areas . High thermodynamic and kinetic stability, low viscosity and optical transparency make them very attractive as a pharmaceutical application form to improve the solubility, the dissolution and the oral absorption of poorly water-soluble drugs . Further advantages of using MEs as drug delivery systems include a better drug solubilization and the protection against enzymatic hydrolysis, as well as the potential for an enhanced absorption due to a surfactant-induced improvement in the permeability. In addition, MEs represent an interesting and potentially quite powerful alternative carrier system for drug delivery because of their high solubilization capacity, transparency, ease of preparation, and high diffusion and absorption rates, compared to solvents without the surfactant system [11,12]. MEs are forming spontaneously and are composed of surfactant (S), co-surfactant (CoS), oil and water with a particle size of less than 100 nm in diameter. The combination of surfactants with oils to form MEs offers an advantage with a low free energy and a large surface area, which were considered to be responsible for transporting drugs to gastrointestinal membrane for absorption [13,14].
PT was provided by Basel Drug Company (Istanbul, Turkey). Diphenhydramine was purchased from Sigma Chemical Co (St Louis, MO). Lutrol F 127 was purchased from BASF (Ludwigshafen, Germany). Span 80, oleic acid, and isopropyl alcohol were obtained from Sigma- Aldrich (St Louis, MO). Cell culture reagents and supplies were obtained from Gibco Invitrogen (Grand Island, NY).
Preparation of the microemulsions
In order to find out the concentration range of the components for the existing range of the ME, pseudo-ternary phase diagrams were constructed using the water titration method at an ambient temperature. Oleic acid was selected as the oil phase. The effects of the Ss (mixtures of span 80 and lutrol F 127 at w/w ratios of 9.5:0.5 with a hydrophilic-lipophilic balance (HLB) of 5.53 and of the CoS (isopropyl alcohol) on the pseudoternary phase diagram were systematically observed at room temperature. For each phase diagram at a specific S/ CoS weight ratio, the ratios of oil to the mixture of S/CoS were varied as, 1:1, 2:1, 3:1, 4:1 and 5:1 (w/w). Lutrol F 127 were melted at 50°C–60°C and blended with span 80 to make the S mixture. Afterwards, the oil phase and the S mixture were mixed. These mixtures were titrated, drop-by-drop, with distilled water while being gently stired at 25 ± 1°C until. The appearances from clear to turbid and turbid to clear were investigated, respectively. After being equilibrated, the mixtures were assessed by visual characterization (Figures 1A-E) [15,16].
After the identification of ME region in the phase diagrams, the ME formulation was selected with the desired component ratios. The blank and the PT containing ME were prepared by the same way. The PTincorporating ME was prepared by dissolving the drug powder into the ME system. The final concentration of PT in ME system was 1 mg/mL.
Characterization of the microemulsions
The ME was analyzed for various properties. The average droplet size and the polydispersity index (PDI) of the ME in the presence and absence of PT were measured by photon correlation spectroscopy (Nano ZS, Malvern Instruments, UK). The viscosities of the MEs in the presence and absence of PT were detected at 25.0 ± 1°C using a Brookfield digital viscometer-III rheometer V 3.3 HB (Middleboro, MA) (Spindle: SC4–21). The refractive index and the electric conductivity of the ME in the presence and absence of PT were measured using a refractometer (Atago RX-7000 CX, Japan) at 25 ± 1°C and monitored quantitatively by using a conductometer and its conductometer probe (Jenway 4071, UK) at 25 ± 1°C, respectively.
Stability of the microemulsion
To evaluate the stability of the optimized ME formulation of PT, the formulation was added into sealed glass vials which were stored at 25 ± 1°C and 40 ± 1°C in climate cabin for 6 months. The clarity and droplet size were investigated at predetermined intervals (n=6).
Cytotoxicity studies of the pitavastatin solution
The Caco-2 and MCF-7 cells were obtained from American Type Culture Collection (ATCC) and used for the cytotoxicity tests. The experimental cells were counted in a hemocytometer (Reichert Co., USA) using the trypan blue exclusion method. The cells were plated on 96-well flat-bottomed plates, with each well at a density of 1×104 cells, and incubated for 24 h at 37°C in the CO2 incubator. After the cells were attached on 96-well flat-bottomed plates, 100 μL of PT solutions in Hank’s Balanced Salt Solution (HBSS) at different concentrations (100 to 2000 μg/mL) were directly added to plates. The cells were incubated with the PT solution for the measurement duration of 1, 2 and 3 days, at 37°C in the CO2 incubator. The cytotoxicity following the above-mentioned treatments was evaluated by 3-[4, 5-dimethylthiazol- 2-yl]-3, 5-diphenyltetrazolium bromide dye (MTT) assay. Briefly, at the end of the incubation period the culture medium was aspirated and cells were washed with phosphate buffer solution (PBS) (pH=7.4). Then cells were incubated with 100 μl of the MTT solution (0.5 mg/ mL) in Dulbecco Modified Eagle Medium (DMEM) without Fetal Bovine Serum (FBS) for 4 h at 37°C. The MTT solution with culture medium was removed without a formation of the formazan crystals. 100 μl of dimethyl sulfoxide (DMSO) was added in order to dissolve the formed formazan crystals. After solubilizing the crystals, the absorbance was measured with the ELISA microplate reader (Thermo vario scan-FHA multiplate reader) at a wavelength of 570 nm. The results of the experiments were evaluated due to the cell viability (%). All experiments were performed in triplicate.
Cell viability (%) = T/C×100 (Equation 1)
Cytotoxicity studies of the pitavastatin microemulsion
The Caco-2 and the MCF-7 cells were also used for the cytotoxicity tests of the microemulsions. The cells were plated on 96-well flatbottomed plates, with each well at a density of 1×104 cells, and incubated for 24 h at 37°C in the CO2 incubator. After the cells were attached on 96-well flat-bottomed plates, ME in the absence and presence of PT at a desired concentration (1000 μg/ml) were directly added to the cell culture medium of 100 μl. The cells were incubated with the ME for the measurement duration of 1, 2 and 3 days, at 37°C in the CO2 incubator. The cytotoxicity following the above-mentioned treatments was evaluated by the MTT assay.
HPLC analysis of pitavastatin
The HPLC analysis method was modified from previous studies . The samples were analyzed by using a HPLC system (The HPLC system was equipped with a Agilent 1100 series, a reverse phase ACE 5 C18 column (150 mm × 4.6 mm, 5 μm, ACT, Scotland)) that has a UV spectroscopic detector. The mixture of methanol, distilled water and formic acid solutions (75:25:0.05 v/v/v) was used as the mobile phase at a flow rate of 0.8 ml/min. The injection volume was 20 μl. The wavelength of the DAD detector was set as 254 nm. All samples were filtrated through membrane filter (0.2 μm Nylon, Millipore Millex-GN) before injection. The peak area correlated linearly with PT concentration in the range of 5–250 μg/ml with the lowest detection limit at 0.5 μg/ml, and the average correlation coefficient was 0.999.
Statistical data analysis
Statistical analysis was performed using one way analysis of variance (ANOVA) to evaluate differences between the ME and the solution, both containing PT. The data were considered as statistically significant at p<0.05.
Construction of the pseudo ternary phase diagrams
Investigation of the phase behavior of these systems demonstrated that our approach was suitable for determining the water phase, the oil phase, the S concentration, and the CoS concentration at which the transparent ME system was formed. The construction of a phase diagram makes it easy to identify the concentration range of the components in the MEs. Figure 1 shows the phase diagrams constructed to determine the optimum S/CoS concentration ratio for the formulation of a w/o ME consisting of span 80, lutrol F 127, oleic acid, isopropyl alcohol, and water. The S/CoS and S mixture (span 80/ lutrol F 127) ratios were found to be 1:1 and 9.5:0.5 for the optimized ME. As shown in figure 1A, the area of w/o ME becomes enlarged and is highest at an S/CoS ratio of 1:1. The exact composition according to oil, S, CoS, and aqueous phases is shown in table 1.
|Formulation||Oil (%)||S 1(%)||S 2 (%)||CoS (%)||Water (%)|
Table 1: The contents of the optimized ME formulation (S1- Lutrol F 127, S2- Span 80, CoS- isopropyl alcohol)
Characterization of the microemulsion
The characteristic parameters of the ME were shown in table 2. The particle size analysis showed that the mean droplet size of the ME in the presence and absence of PT was between 39.05 ± 0.87 and 33.29 ± 1.76 nm and the PDIs were 0.044 for both MEs, with and without PT. When PT was loaded to the ME system, the formulation showed no change in its transparency, but the mean droplet size of ME slightly decreased from 39 nm to 33 nm (Table 2). The possible reason might be that loading the ME with PT affects the density of the formulation and thus reflects the particle size. When PT was dissolved and dispersed into the emulsifying membrane layer (composed of S and CoS) and oil phase, the chemical groups in PT could react with the other groups of the S, the CoS and the oil phase by forming hydrogen bonds. This may be because of the decreased surface tension due to the presence of the S and the CoS . The PDI value is describing the width of the particle size distribution of the formulation. All PDI values were smaller than 0.2, which shows a narrow distribution of the particle size.
|Electrical conductivity (µS/cm)||Viscosity (cP)||Particle size (nm)||PDI||Refractive index||PT Content (mg/mL)|
|ME||1.1 ± 0.03||104.7 ± 0.15||39.05 ± 0.87||0.044 ± 0.01||1.412 ± 0.12||-|
|ME-PT||1.1 ± 0.02||103.8 ± 0.25||33.29 ± 1.76||0.044 ± 0.02||1.415 ± 0.21||1 ± 0.17|
Table 2: Characterization of the microemulsions (ME is a blank microemulsion, ME-PT is a microemulsion with PT).
It was described in the literature, that a CoS free viscous ME was showing a pseudo-plastic behavior. The less viscous CoS containing preparations had Newtonian flow. Therefore, incorporating the CoS into the MEs resulted in a significant viscosity reduction of the formulations, along with a flow change to a simple Newtonian behavior [16,20]. In our study, the viscosity of the MEs (with and without) were nearly close to each other, namely 104.7 ± 0.15 cP (blank ME) and 103.8 ± 0.25 cP (PT loaded ME) (p>0.05) (Table 2). The refractive indexes of both ME formulations ranged from 1.412 ± 0.12 (blank ME) to 1.415 ± 0.21 (PT loaded ME). When PT was loaded into ME, refractive index of ME was increased (Table 2). The electrical conductivity was about 1.1 μs/cm for both ME formulations (Table 2).
Stability of the microemulsion
After storage of the PT loaded ME at 25 ± 1°C and 40 ± 1°C for 6 months, the ME was still clear and transparent without any phase separation. The droplet size did not changed significantly (p>0.05) (Table 3). It was suggested that the PT incorporated ME was stable during these storage time of 6 months.
|Droplet size||Droplet size|
|0||33.29 ± 0.95||33.29 ± 0.95|
|3||33.72 ± 0.61||34.01 ± 0.55|
|6||33.34 ± 0.44||34.72 ± 0.85|
Table 3: Stability test results of the PT loaded microemulsion (ME-PT).
Cytotoxicity studies of the pitavastatin solution
In this study, the effect of PT at different concentrations on Caco-2 cells and MCF-7 cells line has been showed by the MTT test. The cell viabilities (Figure 2) after the application of the PT solutions were found to be between 98.37 ± 5.86% and 99.99 ± 5.75% for Caco–2 cells after 72 h at different concentrations. This demonstrates that the PT solution was not toxic on Caco-2 cells (Figure 2A). In addition, after exposure to PT at different concentrations for 72 h, the cytotoxic effect on MCF-7 was also investigated by using the MTT assay. The cell viabilities after application of the PT solutions were found between 98.04 ± 6.53% and 98.74 ± 9.41% for the MCF-7 cells (Figure 2B). Evaluation of the results revealed that PT was not cytotoxic for these cell lines during the test period (Figure 2B).
Cytotoxicity studies of the pitavastatin loaded microemulsion
The cytotoxicity results of PT loaded ME was shown in figures 3A and 3B, expressed as the cell viability %. The concentration of PT (1000 μg/mL) in the MEs caused no cytotoxicity on the Caco-2 cell line (Figure 3A) and on the MCF-7 cell line (Figure 3B). When the ME with and without PT was applied to the cell culture medium, the samples did not show any cytotoxicity on the MCF-7 cells line during the test period. Furthermore, the ME formulation containing PT did not affect the MCF-7 cells line growth and survival during the direct and continuous exposure to the cell for the test period. The cell viability of MCF-7 was found to be between 99.62 ± 2.27% and 98.91 ± 2.49% for both MEs during 72 h. According to the in vitro cytotoxicity studies, it was concluded that there was no cytotoxic effect on Caco-2 cell and MCF-7 for both ME formulations with and without PT (ME and MEPT).
• Recently, many studies have been performed by using ME systems for hyperlipidemic therapy and there are only a few studies about formulations which have been developed for the application of PT. In this study, a new w/o ME system for the oral delivery of PT was developed through the construction of pseudo-ternary phase diagrams and optimization with a simple method. According to the physicochemical characterization and the in vitro cytotoxicity studies, it can be concluded that PT may be incorporated into the ME formulation which is a new drug carrier system.
• The ME formulations (ME and ME-PT) were not cytotoxic for Caco-2 and MCF-7 cell lines.
These results indicate that, this ME is a promising formulation for the alternative oral drug delivery of PT with hyperlipidaemia as the indication.