Etodolac (ET) (poorly soluble drug) nanosuspensions were prepared by both pH shift method and antisolvent techniques in order to increase its dissolution rate. Various stabilizers were used, namely, Tween 20 and 80, HPMC, PVP K44, PVA, PEG 400, NaCMC, and
Formulation of a poor water soluble drug has always been a challenging problem to pharmaceutical industry [
Nanosuspensions are colloidal dispersions and biphasic system consisting of drug particles dispersed in an aqueous medium in which the diameter of the suspended particles is less than 1
According to Noyes–Whitney and Ostwald–Freundlich principles, the particle size in the nanometer range can lead to increased dissolution velocity and saturation solubility for a nanosuspension, which is usually accompanied by an increase in bioavailability [
Nanosuspensions can be prepared by two methods, namely, “bottom up technology” and “top down technology” [
In bottom up technology, the nanoprecipitation method presents numerous advantages, as being a straightforward technique and being rapid and easy to perform. The drug is dissolved in a solvent, which is then added to nonsolvent that causes precipitation of the fine drug particles [
Etodolac (ET) is a nonsteroidal anti-inflammatory (NSAI) drug prescribed for the treatment of acute pain, osteoarthritis, and rheumatoid arthritis at an oral dose of 200 mg twice daily [
ET is administered as a racemate. Both enantiomers are stable and there is no evidence of
Depending on the biopharmaceutical classification system [
In the present study, nanosuspensions of ET were prepared by two different methods, namely, pH-shift method and antisolvent method. Different stabilizers were utilized for their steric stabilization character such as polyvinylpyrrolidone (PVP), hydroxypropyl methylcellulose (HPMC), polyvinyl alcohol (PVA), beta-cyclodextrin (
ET of 99.2% purity, (AMSA-S.P.A, Italy), was kindly supplied by (Alamryia Pharmaceuticals, Alexandria, Egypt), hydroxypropyl methylcellulose (HPMC) (viscosity 6 cps) by (Colorcon, U.K.), sodium carboxymethyl cellulose (Na CMC) by (Elnasr Pharmaceutical Chemicals Co., Egypt), polyvinyl alcohol (PVA) by (Alfa Aesar, Germany),
Preliminary studies were carried out to determine the most promising drug to stabilizer ratio (in terms of weight) that produces nanosuspensions with least aggregation. Different ratios of drug to stabilizer (9 : 1 to 1 : 1) were examined.
Calculated amount of the drug was weighed and dissolved in an appropriate volume of 0.1 N NaOH (22.5 mg/mL) containing the calculated amount of the stabilizer used. The drug to polymer ratio (3 : 1) was kept constant in all formulations. The solution’s pH was reduced from 12 to 5.5 within 5 min by the addition of 0.1 N HCl [
Drug content, percentage yield, particle Size (P.S.), and size distribution of nanosuspensions prepared by pH shift method.
Formula code | Stabilizer | Drug content (%) |
% Yield |
P.S. in nm | PdI* |
---|---|---|---|---|---|
F1 | Crude drug | — | — | 3676 ± 42.6 | 0.52 |
F2 | Nanosuspension without stabilizer | 100 | 99.46 | 7129 ± 63.2 | 1.00 |
F3 | Na CMC | 98.7 | 92.02 | 827 ± 5.4 | 0.49 |
F4 | HPMC | 89.5 | 90.91 | 1025 ± 11.14 | 0.56 |
F5 | PVP K44 | 85.3 | 93.75 | 1702 ± 97.2 | 0.72 |
F6 | PVA | 90.0 | 96.77 | 2000 ± 50.7 | 0.85 |
F7 | PEG 400 | 92.6 | 95.54 | 790 ± 3.3 | 0.51 |
F8 | Tween 20 | 95.7 | 90.91 | 618 ± 4.5 | 0.49 |
F9 | Tween 80 | 92.5 | 90.36 | 393 ± 2.05 | 0.34 |
F10 |
|
98.2 | 96.77 | 866 ± 5.9 | 0.53 |
PdI*: polydispersibility index.
Calculated amount of the drug was weighed and dissolved in 5 mL ethyl alcohol. The organic solution of the drug was added at once to 10 mL of water containing the calculated amount of water soluble stabilizer under stirring at 1200 rpm using magnetic stirrer at room temperature. The drug to polymer ratio (3 : 1) was kept constant for all formulations. By adding the organic solution of the drug to water, nanosuspensions were formed spontaneously due to the poor solubility of the drug in water. The same procedure was carried out to prepare drug nanosuspension without stabilizer for particle size comparison study and denoted as F2*. Eight stabilized formulations were prepared by antisolvent method and denoted as F11 : F18 (Table
Drug content, percentage yield, particle Size (P.S.), and size distribution of nanosuspensions prepared by antisolvent method.
Formula code | Stabilizer | Drug content (%) |
% Yield |
P.S. in nm | PdI* |
---|---|---|---|---|---|
F1 | Crude drug | — | — | 3676 ± 42.6 | 0.52 |
F2 | Nanosuspension without stabilizer | 100 | 99.74 | 4234 ± 67.4 | 1.00 |
F11 | Na CMC | 92 | 85.77 | 464 ± 7.6 | 0.83 |
F12 | HPMC | 96.8 | 98.33 | 603 ± 5.9 | 0.78 |
F13 | PVP K45 | 80.0 | 87.92 | 290 ± 6.8 | 0.85 |
F14 | PVA | 85.5 | 91.93 | 468 ± 9.1 | 0.85 |
F15 | PEG 400 | 90.0 | 92.86 | 474 ± 6.2 | 0.78 |
F16 | Tween 20 | 98.0 | 93.09 | 119 ± 4.6 | 0.79 |
F17 | Tween 80 | 95.0 | 92.80 | 160 ± 3.9 | 0.54 |
F18 |
|
95.0 | 93.61 | 517 ± 7.4 | 0.78 |
PdI*: polydispersibility index.
The prepared nanosuspensions were cooled rapidly to −70°C and stored overnight. Freeze-drying of the samples was performed with a Telstar freeze-dryer (Terrassa, Spain) at a temperature of −50°C with a pressure below 1 mbar; then the vials were removed after 48 hr of drying. The samples were stored in well-closed vials at −20°C for further investigations [
FTIR spectra were performed using Perkin-Elmer FTIR series (model-1615) spectrophotometer to find out any possible drug-stabilizer interactions. Samples of ET and of each stabilizer used (PVP, HPMC, PVA,
Morphologies of raw ET and nanosized ET were examined using a scanning electron microscope (JEOL JSM-7001F, Japan) operated at an accelerating voltage of 1 KV and a secondary detector. Freshly prepared ET nanosuspensions and dispersion of raw ET were deposited on a glass slide following evaporation of solvent.
Particle size and size distribution of the prepared nanosuspensions were measured using a dynamic light scattering particle size analyzer, model Zetasizer Nano-ZS; model number ZEN 3500, Malvern (UK). Raw data were collected over 5 minutes at 25°C and at an angle of 90 degrees and further processed using the ZPW388 software program. Nanosuspensions were diluted 50-fold in deionized water before measurement.
ET content in lyophilized nanosuspensions’ samples was analyzed by dissolving 10 mg of each sample separately in 5 mL ethanol. The samples were vortexed for 1 min at room temperature and filtered (0.45
To evaluate the physical appearance, nanosuspension samples were observed for agglomeration and color change for two weeks at room temperature after preparation.
Lyophilized samples equivalent to 50 mg of ET were used to study the drug release rate using USP dissolution apparatus II (Erweka, Germany). Lyophilized powder was dispersed in 500 mL of either water or phosphate buffer pH 6.8 at
Male Albino Wister rats were provided by the laboratory Animal Center, King Saud University, Riyadh, Saudi Arabia. The study was performed in accordance with the guidelines of the local institutional animal ethics committee.
Bioavailability of two preparations (F1 & F17) was assessed in rats. Twelve rats, weighing 0.25–0.3 kg, were divided into two groups randomly (six animals each) and given ET orally with gastric catheter at a single dose of 20 mg/kg after fast overnight but were allowed free access to water. Orbital sinus blood (approximately 2 mL) was collected into tubes containing 2.6 mmol sodium edetate before and at 15 and 30 min and 1, 3, 5, 12, 24, and 48 hr after drug administration. Plasma was separated by centrifugation for 15 min at 10000 rpm at 4°C using 3–30 k centrifuge (Sigma, Germany). The specimens were stored at −70°C until analysis [
Plasma samples were analyzed for ET using reported HPLC method with some modifications [
HPLC analysis was carried out on a Waters Breeze system (Waters Corporation, Milford, MA, USA) equipped with 1525 binary pump with on-line degasser, 717+ autosampler, 5CH thermostated column compartment, and 2487 UV dual wavelength absorbance detector. The chromatographic separations were performed on Symmetry C18 column (3.5
The mean best-fit linear regression equation was used to estimate the concentrations of ET at different time. All pharmacokinetic parameters were calculated using the pharmacokinetic software WinNonlin (version 5.2, Pharsight Corp., Mountain View, CA, USA) using noncompartmental method. The maximum plasma concentration (
The elimination rate constant (
All the results were expressed as mean values ± standard deviation (SD). One-way analysis of variance (ANOVA) with Turkey’s multiple comparisons post hoc was used to test for significance, at a 5% significance level. Statistical difference dealing (
Manufacturing of nanosuspensions involves the generation of a large number of small particles with enormous surface area. This significantly increases the Gibb’s free energy of the system and, due to the high interfacial tension, these systems are thermodynamically unstable. Accordingly, nanoparticles will tend to minimize their total energy by undergoing agglomeration [
The process of agglomeration depends on the activation energy, which is influenced by the addition of stabilizers to the system (such as, surfactants and polymers). These stabilizers reduce the interfacial tension between the particles and the dispersion medium and act as wetting agents. The second requirement is to provide a barrier between the drug particles to prevent agglomeration by electrostatic attraction (ionic surfactants) or steric stabilization (nonionic surfactants and polymers) [
It is well known that an appropriate stabilizer is very important to control particle growth during the production of uniform nanoparticles. Many reports have shown that if preliminary particles can be arrested efficiently by appropriate stabilizers, the nanosuspensions system can be maintained for a longer time [
The adsorption properties of stabilizers can be affected by the nature of stabilizer and drug surface, for example, molecular weight is an important factor for polymeric stabilizers. The chain length should be high enough, so that polymers chains have an optimum length to overcome the Van der Waals forces of attraction. Furthermore, another important factor is the size of the polymer [
Electrolytes are present in the gastrointestinal tract and the contact of the nanocrystals with these electrolytes cannot be avoided. Electrostatic stabilization is reduced in its efficiency in an electrolyte containing environment. To compensate for this it is ideal to use steric stabilizers, which are less impaired in their effect by electrolytes [
The adsorption layer of the stabilizer shifts the plain of shear, at which the zeta potential is measured, to a larger distance from the particle surface. Consequently the measured zeta potential is lower. In such cases zeta potentials of about 20 mV are still sufficient to fully stabilize the system [
Based on the above consideration various surfactants and polymers which exhibit steric stabilization characteristics were preferably used in this study such as Tween 20, Tween 80, HPMC, PVP K30, PVA, PEG 400, Na CMC, and
In conclusion, a preliminary study revealed that drug to stabilizer ratio of 3 : 1 was of choice in terms of performance and particle size.
FTIR spectroscopy Figures
Fourier transformed infrared (FTIR) spectra of pure drug, drug nanosuspension, pure stabilizers, and all nanosuspensions formulations.
IR spectra of ET nanosuspension stabilized with
IR spectra of ET nanosuspension stabilized with Tween 80
IR spectra of ET nanosuspension stabilized with Na CMC
IR spectra of ET nanosuspension stabilized with Tween 20
IR spectra of ET nanosuspension stabilized with PEG 400
IR spectra of ET nanosuspension stabilized with PVP K44
IR spectra of ET nanosuspension stabilized with HPMC
IR spectra of ET nanosuspension stabilized with PVA
Pure drug and nanoparticles surface morphology and shape were analyzed by SEM, representative examples are shown in Figure
Photomicrographs of SEM of pure ET powder (a), pH shift method using PVP as a stabilizer (b), antisolvent method using PVP as a stabilizer (c), and pH shift method using Tween 80 as a stabilizer (d).
Visual examination of all prepared nanosuspensions was done on a basis of color change and agglomeration. There was no color change after lyophilization of nanosuspensions. Sedimentation of PVP, HPMC and PVA contained nanosuspension prepared by pH shift method occurred within 1–3 h. Large particle sizes (agglomerates) were observed. When we evaluated feasibility of nanosuspension production, poor performance was seen when the stabilizer was HPMC, increasing drug-to-stabilizer ratio did not result in stabilization of nanosuspension. It was noticed that when drug-to-stabilizer ratio was shifted from 3 : 1 to 2 : 1 for HPMC, more viscosity was imparted to the system compared to other stabilizers. More viscosity of HPMC was inherent disadvantage as it would slow down the process for nanosuspension [
Other stabilizers showed good stabilization as the nanosuspension did not sediment during the time of visual observation. Nanosuspensions prepared by antisolvent method using the same stabilizers were stable at room temperature for at least 4 days. These results may be attributed to the particle size and molecular weight (MW) of the stabilizer itself, in addition to the method of preparation [
During the preparation process there was no any drug loss step involved, so theoretically the formulation was considered as being 100% drug content. Table
The mean particle size and the span of particle size distribution (polydispersity index, PI) are two important characteristic parameters because they affect the saturation solubility, dissolution rate, physical stability, even bioavailability of nanosuspensions. Particle size has primary impact on saturation solubility and ripening [
Particle size measurements of pure ET and its lyophilized nanosuspensions are listed in Tables
It was found that in absence of stabilizer the drug particle size increased from 3.676
In solvent change method, the drug is firstly dissolved in a solvent. Then this solution is mixed with a miscible antisolvent in presence of surfactants or polymers. Rapid addition of a drug solution to the antisolvent (usually water) leads to sudden supersaturation of drug in the mixed solution, and generation of ultrafine crystalline or amorphous drug solids. This process involves two phases; nuclei formation and crystal growth. When preparing a stable suspension with a minimum particle size, a high nucleation rate but low growth rate is necessary. The major challenge is to avoid crystal growth due to Ostwald ripening being caused by different saturation solubility’s in the vicinity of the differently sized particles [
Particle size ranged from 119 nm for Tween 20 to 603 nm for HPMC, which seemed to be affected by relative viscosity of the polymeric dispersion in the presence of stabilizers and followed the trend: Tween 20 > Tween 80 > PVP > Na CMC > PVA > PEG 400 >
It has been reported that particle-size distribution of solid particles in nanosuspensions is usually less than one micron with an average particle size ranging between 200–600 nm [
Tween 20 or 80 as stabilizers were adsorbed on the surface of the hydrophobic drug ET, a mechanical barrier was formed against crystal growth and agglomeration. The stabilizer occupied the adsorption sites on the surface of freshly formed drug crystals during process of crystallization. As a result, it inhibits subsequent growth by inhibiting the incorporation of drug molecules from solution into crystal lattices, as already observed for other drugs such as Felodipine and Folic acid [
HPMC is a large molecule, nonionic stabilizer and provides stability via steric stabilization. HPMC is a polymer containing a number of methoxyl and hydroxypropyl groups, and the hydrophobic parts have good affinity for drug particles and can be adsorbed onto the drug particle surface providing an effective steric barrier against growth. In addition, hydrogen bonds can be formed between the drug molecule and HPMC [
It can be seen from Table
As mentioned before, nanosuspensions prepared by pH shift method showed larger particle size than those prepared by antisolvent method (Tables
The dissolution profiles of the drug release from the different nanosuspension formulations were illustrated in Figures
Dissolution parameters of ET nanosuspensions prepared by pH shift or antisolvent method in both distilled water and phosphate buffer (PB) pH 6.8.
Formula code | pH shift method | Antisolvent method | |||||||
---|---|---|---|---|---|---|---|---|---|
Distilled water | PB pH 6.8 | Formula code | Distilled water | PB pH 6.8 | |||||
|
|
|
|
|
|
|
| ||
F1 | 17.80 | 9.27 | 82.40 | 13.73 | F1 | 17.8 | 9.27 | 82.40 | 13.73 |
F3 | 78.90 | 15.29 | 98.08 | 32.70 | F11 | 24.30 | 4.66 | 99.00 | 49.50 |
F4 | 79.90 | 26.63 | 77.65 | 17.26 | F12 | 84.50 | 15.09 | 99.00 | 49.50 |
F5 | 39.10 | 7.56 | 96.65 | 46.33 | F13 | 97.50 | 49.75 | 96.00 | 32.32 |
F6 | 75.23 | 12.71 | 83.70 | 15.67 | F14 | 57.60 | 9.80 | 93.60 | 20.80 |
F7 | 82.70 | 14.06 | 90.05 | 15.47 | F15 | 93.40 | 46.70 | 91.60 | 48.21 |
F8 | 52.00 | 9.24 | 67.90 | 11.85 | F16 | 95.20 | 47.60 | 91.20 | 31.93 |
F9 | 77.65 | 12.94 | 97.60 | 32.50 | F17 | 95.40 | 48.28 | 83.40 | 28.37 |
F10 | 96.00 | 48.00 | 98.08 | 31.80 | F18 | 107.7 | 4.39 | 90.50 | 31.16 |
F1 = Crude drug.
DR10 min: % drug released after 10 min; DE: % dissolution efficiency after 10 min.
(a) Dissolution profiles of ET nanosuspensions prepared by the pH shift method in distilled water.
(a) Dissolution profiles of ET nanosuspensions prepared by pH shift method in phosphate buffer pH 6.8.
(a) Dissolution profiles of ET nanosuspensions prepared by antisolvent method in distilled water.
(a) Dissolution profiles of ET nanosuspensions prepared by antisolvent method in phosphate buffer pH 6.8.
The dissolution profiles of lyophilized ET nano-solid suspensions prepared by the pH shift method, in comparison with crude drug as a reference are shown in Figures
It was reported that the saturation solubility increases with decreasing particle size. However, this effect is only pronounced for particle below approximately 2
The increase in the dissolution rate of ET from
The percentage of drug dissolved ranged from 96 (
Using phosphate buffer pH 6.8 as dissolution medium, the crude drug showed a significant increase in its dissolution rate under sink condition. Dissolution rate study of the drug in distilled water showed that about 17.8% of drug dissolved in water within 10 minutes. Changing the dissolution medium to buffer pH 6.8 the percentage of dissolved drug increased to 82.4 at the same time (
Nanosuspensions of
Dealing with the antisolvent method Figures
On the other hand, the effect of stabilizer used on dissolution rate in buffer pH 6.8 of different formulations can be arranged in the following order; Na CMC = HPMC > PVP > PVA > PEG = Tween 20 >
Since nanoparticles prepared by antisolvent method using Tween 80 as a stabilizer showed a promising results concerning the particle size and dissolution rate, it was selected for further
In the present investigation, the pharmacokinetic parameters of a nanosuspension of ET (F17) were compared to that of pure ET suspension (F1) after administration by oral gastric catheter in rats. Figure
Pharmacokinetic parameters after oral administration of ET formulations.
Pharmacokinetic parameter | Formulation A |
Formulation B |
---|---|---|
|
36.5 ± 2.27 | 55.07 ± 9.88 (63.417) |
|
38.16 ± 2.26 | 58.20 ± 9.55 |
|
2.38 ± 0.30 | 3.78 ± 0.13 |
|
4.0 | 3.0 |
|
10.93 ± 0.62 | 11.60 ± 0.97 |
|
0.063 ± 0.004 | 0.06 ± 0.01 |
MRT (h) | 11.51 ± 0.77 | 12.92 ± 0.27 |
% absorbed in 1 h | 7.79 ± 0.26 | 21.20 ± 0.69 |
Clearance rate (mg/mL) | 132 | 86 |
Mean plasma concentration-time profiles for F1 and F17 formulations after oral administration of a single dose of 20 mg/kg. Each data point is the mean ± SD from six experiments (
In order to demonstrate a faster absorption of ET nanosuspension (F17), particularly during the first hour after administration, the percentage of ET absorbed after 1 h was estimated (Table
Figure
Percentage ET unabsorbed versus time for the two formulations F1 (Pure ET suspension) and F17 (ET nanosuspension).
The improved oral bioavailability of ET could be explained by the combination of the following effects: firstly, the drug molecules were absorbed rapidly from gastrointestinal wall due to the significantly improved dissolution rate by the reduced particle size with increased surface area and reduced diffusion layer thickness [
ET nanosuspensions were prepared by pH shift or antisolvent method in presence of different stabilizers with drug to stabilizer ratio of 3 : 1. Solvent precipitation method succeeded in producing particles in the nanosized range. The dissolution of nanosized ET was significantly enhanced compared with the crude pure drug. The results showed that the particle size minimization produced by pH shift method was not the major determining factor in the dissolution improvement. Rather, the type of stabilizer used in the formulations was of greater importance. The results also demonstrated that nanoprecipitation can thus be a simple and effective approach to produce submicron particles of poorly water-soluble drugs. The
The authors declare that there is no conflict of interests regarding the publication of this paper.
This research project was supported by a grant from the “Research Center of the Female Scientific and Medical Colleges,” Deanship of Scientific Research, King Saud University.