1. INTRODUCTION:
The poorly water soluble drugs are the challenging candidates for the formulation. Hence, the selection of suitable formulation is necessary to develop the solubility as well as the bioavailability of such drugs.
The solubility of weakly water soluble drugs can be enhanced via different methods11. Mainly there are three approaches through which we can improve the solubility of the drug and it includes pharmaceutical approach, pharmacokinetic approach, and biological approach. When we compare these three approaches, the pharmacokinetic approach is having many drawbacks like adverse effects, time consumption, high cost etc. Because of this, the pharmaceutical approach is generally preferred. In this approach they will be modifying the formulation or manufacturing process and it includes co-solvency, particle size reduction, hydrotrophy, solid dispersions, micellar solubilization, complexation10, pH adjustment and lipid based drug delivery system. But, all these methods are having drawbacks in one or the other way like change in drug character or agglomeration. Methods like micronization and nanosuspension leads to a thermodynamically unstable product2. Thus, researchers support the lipid –based formulations since this will be helpful in overcoming the formulation challenges3. The lipid- based drug delivery system is having a significant growth in the past 15 years and it can also deal with low soluble chemical entities.
Lipid based formulation is a helpful tool in increasing the solubility of BCS Class II and IV drugs. In this formulation, the drug is incorporated into the oil, surfactant and co-surfactant system to result in nanoemulsion. LBDDS varies from oily solution to a complex mixture comprising of oil, surfactant, co-surfactant and co-solvent. Then later it forms a self-dispersing system which leads to the formation of self-emulsifying drug delivery system (SEDDS)/Self-emulsifying nanoemulsifying drug delivery system (SNEDDS)7,9. They are oil-in-water (o/w) emulsions having a droplet diameter of about 50 to 1000nm. The particles can be present either in oil or in water based on which it can be sorted either as oil-in water or water-in- oil system.
Nanoemulsions are of two types; oil/water (oil droplets will be dispersed in aqueous phase) or water/oil (water droplets dispersed in oil phase)19. They are more stable compared to microemulsions. Nanoemulsions are non-toxic as well as non-irritant. They also have high surface energy and do not show creaming, sedimentation and flocculation18. It can be prepared by different techniques. The techniques include high pressure homogenization, micro fluidization, phase inversion temperature technique, solvent displacement method, phase inversion composition technique15 and spontaneous emulsification (Aqueous phase titration method). Though, many literatures supports the nature of the nanoemulsions as thermodynamically stable still stringent and broad study is required to study the same. Hence, the present wok aims at formulation and accelerated stability study to determine the shelf life and the effect of added ingredients on the stability.
Stability of a product is the ability of the product to retain its originality when kept in a specific container. It should be able to maintain its physical, chemical, microbiological, therapeutic and toxicological conditions. Pharmaceutical goods are likely to encounter their requirement on behalf of detecting purity, value and potency during their distinct storage phase at exact storage circumstance. When it comes to the liquid dosage form like suspension, emulsion, microemulsion and nanoemulsion, the stability of the product plays an important role. It has been said that the nanoemulsions are capable of increasing the chemical as well as physical stability with decreasing the adverse effects. Stability of the product according to WHO (World Health Oraganization) depends on different environmental factors like temperature, humidity, light and the physical and chemical nature of the drug and excipients. Researchers have carried out accelerated stability studies based on the ICH (International Conference on Harmonization) guidelines. And they have found out the shelf life of the optimized formulation3. Since real time stability studies requires more time, the accelerated stability studies will be helpful tool for the researchers to identify the shelf life as well as the stability of the product in addition it also requires less time.
2. MATERIALS AND METHODS:
Artesunate was obtained from Mylan Laboratories, Hyderabad. Capryol 90, Lauroglycol 90, Labrafac PG, Labrafac Lipophile WL 1349, Isopropyl Myristate, Oleic acid, Arachis oil and Sesame oil was obtained from Gatteffosse India Pvt. Ltd., Mumbai. Potassium dihydrogen ortho-phosphateand KBr (IR grade) was obtained from Qualigen Fine Chemicals, Mumbai. Acetonitrile (HPLC grade) was bought from Merck chemicals India Pvt. Ltd. Mumbai.
2.1 Screening of oil, surfactant and co-surfactant:
2.1.1 Screening of oil: Solubility study of drug in oil4:
With the aid of shake flask method, the solubility of artesunate was determined6. To 1ml of chosen vehicle an additional amount of drug was added (capryol 90, Lauroglycol 90, isopropyl myristate, labrafac lipophile WL 1349, labrafac PG, Oleic acid, arachis oil, sesame oil) in a volumetric flask, and blended via a vortex mixer in order to mix the drug and vehicle properly. After this, vials were next kept in an isothermal shaker at 25±10°C for 72 h to attain equilibrium. Samples which have been equilibrated were detached and centrifuged at 3000rpm for 15min. The supernatant was taken and with the help of 0.45μm membrane filter, it was filtered. UV spectroscopy with 210nm was used to determine the strength of the drug.
2.1.2 Screening of Surfactant and Co Surfactant4
Based on the capability to emulsify the respective oil phase, the surfactant and co surfactants were examined. Through blending the oil, surfactant and water (for clear emulsion), the emulsifying capability was established.
2.2 Purity of the drug by DSC and melting point:
A pierced aluminium crucible bearing a capacity of 40µL was taken and the sample was filled into it and evaluated with the help of TA-Instruments software with 20-250ºC of temperature range and a heating rate of 20º C/min with a stream of nitrogen. Later the thermogram was recorded.
2.3 Compatibility studies of drug, lipid, surfactant, co surfactant and formulation by FTIR:
With the aid of FTIR, the compatibility of drug, lipid, Surfactant, Cosurfactant and formulation was analysed. A physical blend of drug, Lipid and surfactants (both separately and mixture) was made and mixed with anhydrous potassium bromide (KBr) in 1:4 ratio. Using mortar and pestle 100mg of this blend was broken up into fine powder followed by compression to develop a transparent KBr pellet by means of a hydraulic press at 15tons pressure. Each KBr pellet was examined using FTIR spectrophotometer (Shimadzu, Japan) at 4mm/s at a resolution of 2cm over a wave number zone from 4000 to 400cm-1. The IR spectrum of the physical blend (1:4 ratios) was evaluated with that of pure drug, lipid and surfactant and IR peak comparison was performed to identify some emergence or loss of peaks. Even for the optimized batch of drug loaded NE formulation, FTIR was carried out.
2.4 Formulation of nanoemulsion (NE):
Capryol 90 for artesunate was chosen for NE formulation on basis of solubility study. Using simple spontaneous emulsification approach NE was formulated. NE formulation was carried out by dissolving specific quantity of drug in oil and then again blending it with surfactant and co-surfactant mixture (SCoS)17, i.e., cremophor EL and ethanol.
2.5 Formulation development and optimization of artesunate nanoemulsion20,21:
2.5.1 Spontaneous emulsification method (Aqueous titration method):
Spontaneous emulsification process (titration technique) was used to prepare nanoemulsions. They can be made just via combining oil, water, surfactant, and cosurfactant, in the exact fraction, by gentle agitation16.
2.5.2 Selection of NE formulation:
Mixture of oil and SCoS from the pseudoternary phase diagrams were chosen and proceeded with subsequent assessment tests for stability.
2.5.3 Evaluation of NE:
2.5.3.1 Thermodynamic stability studies4:
The final product were subjected to the following stability analysis
a) Heating and Cooling Cycle:
Cooling phase was made in refrigerator at 4°C and heating phase was made in Hot air oven for 48 h at 45°C. Next, centrifugation test was carried out for those samples which were steady at all these temperatures.
b) Centrifugation:
At 3500 rpm, centrifugation study for the selected formulations was done for 30 min. Next for the freeze thaw cycle the formulation which didn’t show any phase separation was considered.
c) Freeze Thaw Cycle:
It was conducted in Deep freezer with a temperature ranging between -21°C and +25°C where the formulation was kept in stock for not < 48 h at every temperature. The formulations which passed the thermodynamic stability tests were taken into account for further examination.
2.5.3.2 Globule size, zeta potential and polydispersity index12:
Dynamic Light Scattering (DLS) technique was helpful in determining the mean droplet size and PDI of the NE. The instrument employed was Malvern Zetasizer Nano, Series ZEN1002 (Malvern, UK) in cuvette DTS0012 with a 532nm green laser and a scattering angle of 173°C 13.
2.5.3.3 Refractive index, % transmittance, viscosity, conductivity:
Abbe-type refractometer (Macro Scientific Works, Delhi) was used to determine the drug loaded formulations .Visible spectroscopy using Shimadzu UV-Visible spectrophotometer was used to determine the percentage transmittance of NE formulations. Brookfield DVE viscometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA) was employed to identify the viscosity of the formulation. About 0.5 g of sample was taken for analysis without dilution by using spindle no. 63 at different rpm at 25±0.5°C. An electro-conductometer (Conductivity meter 305, Systronic) was used to determine the electro-conductivity of the resulting system. Tested NEs were prepared with a 0.01 N aqueous solution of NaCl as a substitute of doubled distilled water for the conductivity determination. The trail were carried out in triplicate at 25±1°C.
2.6 Drug loading:
With the help of the optimized report, drug was dispersed in the relevant oil and blended with SCoS to obtain self-emulsifying concentrate. Overall quantity of the drug was presumed to be there in the oil phase, this because of the fact that the solubility of the drug was well beyond the dose.
2.7 In vitro release by USP apparatus I6:
Using USP dissolution apparatus Type I, the quantitative in vitro release test 7was carried out in 250 mL simulated intestinal fluid with pH 6.8 at 50 rpm with the temperature range of 37±0.5°C. In capsule size 2, the optimized SNEDDS formulation enclosing single dose of Artesunate was packed. Solutions were removed at standard time period (0, 0.5, 1, 1.5, and 2, h) and an aliquot quantity of dissolution media was restored. The discharge of drug from SNEDDS formulation was evaluated with that of conventional tablet formulation and with the help of UV-Spectrophotometer (with 210 nm) the samples were analyzed.
2.8 Stability studies as per ICH guidelines5:
As stated by international conference on harmonization (ICH) guidelines, accelerated stability examination were executed on optimized ART nano emulsion. At accelerated temperature of 30, 40, 50 and 60°C with ambient humidity, 3 sets of optimized formulation were taken in glass vials and set aside. At standard intervals of 0, 1, 2 and 3 months, the samples were removed. By using stability-indicating HPLC method with a wavelength of 210 nm, the drug content of the samples was determined. As controls (100% drug), the zero time samples were employed. Examination was conducted at every specific time interval by removing 20μl of each formulation and diluting with mobile phase KH2PO4: ACN (50:50). The quantity of drug decomposed and the quantity left over at each time interval was analyzed. Graphical approach was employed to establish the order of degradation. At every temperature, the degradation rate constant (K) were established. Arrhenius plot was created with log K and 1/T to find out the shelf-life of optimized nanoemulsion formulation. The degradation rate constant at 25°C (K25) was established by extrapolating the value of 25°C from Arrhenius plot. The shelf-life (T0.9) for the formulation was decided by using the formula:
T0.9 = 0.1052
K25
3. RESULT AND DISCUSSION:
3.1 Screening of oil, surfactant and co surfactant:
3.1.1 Solubility studies of drug in different oils:
Solubility is a vital principle in formulation of SNEDDS, as the drug residues in liquid form solubilized in the oil phase. Therefore, the oil phase in which the drug illustrated utmost solubility was chosen for the purpose. From table 1 it was clear that Capryol 90 showed greatest solubility of artesunate 142 ± 0.54 mg/mL. Thus, for the formulation of SNEDDS, capryol 90 was chosen. The drug shows an enhanced solubility due to more affinity towards the oil.
Table 1: Solubility studies
|
OIL |
ARTESUNATE (mg/mL) |
|
Oleic acid |
5 ± 0.24 |
|
Labrafac PG |
25 ± 0.14 |
|
Lauroglycol 90 |
12 ± 0.20 |
|
Capryol 90 |
142 ± 0.54 |
|
Labrafac Lipophile WL 1349 |
5 ± 0.87 |
|
Isopropyl Myristate |
18 ± 0.47 |
|
Arachis oil |
7 ± 0.12 |
|
Sesame oil |
11 ± 0.21 |
3.1.2 Screening of surfactants and co-surfactants:
A clear and consistent o/w emulsion for capryol 90 was established using the SCoS mixture of Cremophor EL and ethanol. Therefore, for the formulation, the cremophor EL and Ethanol was chosen. A clear and a consistent emulsion of capryol 90 were generated with the help of SCoS mix.
3.2 Purity of the drug by DSC and melting point:
The melting behavior of the drug substance was supported by the literature reports. The Merck Index states that Artesunate melts at about 135-137oC. The melting point of Artesunate was detected to be 136.33oC as shown in the Table 2; it confirms that the sample is pure without any impurities.
Table 2: Melting point studies
|
Substance |
Melting point |
Observed Melting points |
Average Melting Point |
||
|
01 |
02 |
03 |
|||
|
Artesunate |
135-137°C |
136oC |
137oC |
136oC |
136.33oC |
3.3 Compatibility studies of the drug, lipid, surfactant, co surfactant and formulation by FTIR:
The results obtained from FTIR clearly indicated that there was no contact among the drug and excipients.
1)2873 cm-1 to CH3 stretching vibration 2) 3000-3500 cm-1 to OH stretching vibration 3)1747 cm-1 to bending vibration 4)1158 cm-1 to >C-O stretching vibration, these are the major peaks in artesunate spectra. The functional groups with consequent peaks of pure artesunate and in physical mixture of artesunate were shown in Table 3. As evident from the results, there were no interactions between artesunate and selected oil+SCos and as all the peaks were present in the physical mixture. Therefore, pure drug of artesunate and physical mixture of artesunate are compatible with each other.
Table 3: FT-IR studies showing functional group, pure drug artesunate and its
Mixture (Wave numbers)
|
Functional group |
Pure drug (Wave number) |
Mixture (Wave number) |
Interference |
|
CH3, stretching |
2873 cm-1 |
2924 cm-1 |
No Shifting |
|
OH stretching |
3000-3500 cm-1 |
3187 cm-1 |
No Shifting |
|
N-H, bending |
1747 cm-1 |
1718 cm-1 |
No Shifting |
|
>C-O, stretching |
1158 cm-1 |
1287 cm-1 |
No Shifting |
3.4 Pseudo ternary phase diagrams:
Capryol 90:
In SCoS ratio 1:1 when surfactant and co-surfactants in equivalent ratio was employed only a lesser region of NE was produced with oil solubilisation up to 70% with 30% of SCoS. However, while co surfactant was twice than surfactant (SCoS 1:2) there was a minor raise in NE part yet the oil solubilisation improved up to 74% with 28% of SCoS. This may be recognized to the truth that the adding up of co-surfactant might direct to better penetration of the oil phase in the hydrophobic region of the surfactant monomer there by further declining the interfacial tension, which directs to rise in the fluidity of the interface therefore escalating the entropy of the system. Once the Concentration of Cosurfactant was increased to two fold (SCoS 1:2) NE area improved significantly with 74% oil solubilized by means of 28% SCoS. There was no distinction in NE area and the oil solubulisation stayed constant for SCoS 1:3.
Oil phase: Capryol 90; SCoS: Cremophor EL: Ethanol
Table 4: Visual observations during aqueous phase titration for phase diagram construction using SCoS 1:1 to 1:3
|
SCoS |
Oil: SCoS |
||||||||
|
F1 |
F2 |
F3 |
F4 |
F5 |
F6 |
F7 |
F8 |
F9 |
|
|
1:1 ratio |
1:1 |
1:2 |
1:3 |
1:4 |
1:5 |
1:6 |
1:7 |
1:8 |
1:9 |
|
E |
E |
E |
E |
NE |
NE |
NE |
NE |
NE |
|
|
1:2 ratio
|
1:1 |
1:2 |
1:3 |
1:4 |
1:5 |
1:6 |
1:7 |
1:8 |
1:9 |
|
M |
E |
E |
EG |
E |
M |
E |
NE |
NE |
|
|
1:3 ratio |
1:1 |
1:2 |
1:3 |
1:4 |
1:5 |
1:6 |
1:7 |
1:8 |
1:9 |
|
E |
E |
M |
EG |
EG |
E |
EG |
E |
M |
|
NE is Nanoemulsion, E is Emulsion EG is Emulsion gel, M is Milky
SCoS 1:1
SCoS 1:2
SCoS 1:3
Fig 1: Pseudo-ternary phase diagrams with Capryol 90, indicating o/w nanoemulsion region at different SCoS ratios from 1:1 to 1:3.
3.5 Formulation Selection:
Various concentrations of oil which could solubilize single dose of drug were chosen at 5% intervals (10%, 15%, 20%, 25% and 30%) from every phase diagram. Therefore, more number of formulations could be selected covering the NE area of the phase diagram. For every percentage of oil selected, only those formulations were selected from the phase diagram which employed less concentration of SCoS for the development of NE and proceeded with stability studies.
3.6 Evaluations of NE:
3.6.1 Thermodynamic stability studies:
Thermodynamic stability studies were helpful in differentiating the NE formulations from those of kinetically stable to the one which undergoes phase separation. This denotes that the formulations enclose satisfactory measure of SCoS concentration necessary for NE formulation, which reduces the energy needed for NE formation. Stability of the NE depends on this energy (deceased). Due to poor dispersibility, the formulation undergoes phase separation with infinite dilutions on entering into the GI tract. Whereas, those formulations that has passed the dispersibility studies, were remained as a NE upon the dispersion into the GIT (aqueous environment). There will be slow desorption of the surfactant situated at the globule interface for the oral NE which when undergoes the progression of dilution by the GI fluids.
Those formulations which were examined for globule size, zeta potential, % intensity and PDI studies have passed thermodynamic stability tests. Through the visual observation of clarity and transparency, the refractive index and viscosity studies were determined and confirmed. The formulations that were clear and free from turbidity were believed as a stable one, while others were believed as unstable formulation. Table 5 indicates the thermodynamic stability studies.