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Current Drug Delivery, 2012, 9, 182-196
Development and Characterization of Self-Nanoemulsifying Drug Delivery Systems (SNEDDS) of Atorvastatin Calcium Shiva Kumar Mantri, Shailaja Pashikanti and K.V. Ramana Murthy* University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam -530 003, Andhra Pradesh, India Abstract: The main aim of the present investigation is to develop and characterize the self-nanoemulsifying drug delivery systems (SNEDDS) of atorvastatin calcium (ATV) for improving the dissolution thereby oral bioavailability and to minimize the gastric degradation. Naturally occurring different vegetable oils, various surfactants and co-surfactants were studied for ATV solubility to identify the components of SNEDDS. Ternary phase diagrams comprising surfactant, cosurfactant and oil were plotted. In the ternary phase diagrams the area of self-nanoemulsifying region was marked for the compositions that are giving dispersion with a globule size 200 nm. Effect of drug loading on the phase behavior of selected system was studied. A series of SNEDDS were prepared by selecting from the nanoemulsifying area of 2.5% ATV system. Prepared SNEDDS were evaluated for visual observations, turbidity, effect of pH of the dispersion media on globule size and zeta potential, robustness to dilution and in vitro dissolution study and optimized. FT-IR and DSC were studied for interaction between drug and excipients if any. Forced degradation and accelerated stability studies were conducted for optimized SNEDDS. ATVF 04 and 11 were selected as optimized SNEDDS due to their smaller mean globule size (75.2 and 85.8 nm respectively), lower turbidity values, faster drug release and higher DE values among the other SNEDDS. The optimized ATV SNEDDS were not affected by the pH of dissolution medium. FT-IR study revealed no interaction between drug and excipients used. Forced degradation studies indicated the stability of ATV in the gastric environment. Accelerated stability studies showed no significant changes in the mean globule size, zeta potential, drug content and drug release before and after storage of optimized SNEDDS.
Keywords: Self-nanoemulsifying drug delivery systems, atorvastatin calcium, vegetable oils, ternary phase diagrams, globule size, zeta potential, turbidity, nanoemulsifying area, drug release, oral bioavailability, forced degradation.. 1. INTRODUCTION Atorvastatin (ATV), a synthetic cholesterol-lowering agent, is an inhibitor of HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase, which catalyses the conversion of HMG-CoA to mevalonate, an early and rate limiting step in sterol biosynthesis [1]. Atorvastatin is a monocarboxylic acid with a pKa of 4.46 and is commonly used as atorvastatin calcium. Atorvastatin calcium is [R-(R',R')]-2(4- fluorophenyl)- beta, delta- dihydroxy- 5- (1- methylethyl)- 3- phenyl- 4- [(phenylamino) carbonyl]- lH- pyrrole-1- heptanoic acid, calcium salt (2:1) trihydrate. It is a white to off-white crystalline powder; very slightly soluble in water and pH 7.4 buffer (insoluble in aqueous solutions of pH 4 or below). The solubility in aqueous solution of pH 2.1 is about 20.4 g/mL, while the solubility in aqueous solution of pH 6.0 is about 1.23 mg/mL [2]. Atorvastatin is rapidly absorbed after oral administration, maximum plasma concentrations occur within 1 to 2 hours. The absolute bioavailability of atorvastatin is approximately 14%, which is attributed to low solubility, presystemic clearance in gastrointestinal mucosa and/or hepatic first-pass metabolism [3,4]. The log P of ATV is 5.7 [5]. An approach, which will increase drug solubility and inhibit first-pass metabolism is highly desirable for improving *Address correspondence to this author at the University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam -530 003, Andhra Pradesh, India; Tel: +91 891 284 4931; Cell: 91 944 141 5779; E-mail:
[email protected] 1-/12 $58.00+.00
the therapeutic performance of ATV. Several approaches are available to improve the solubility, thereby the dissolution and bioavailability of poorly water soluble drugs, they include micronisation, salt formation, solid dispersions, inclusion complexation with cyclodextrins etc. The use of formulations (i.e. lipid based drug delivery systems) containing natural and/or synthetic lipids as a potential strategy for improving the oral bioavailability of poorly water soluble, lipophilic drug candidates has received increasing interest in recent years [6-8]. For poorly water soluble compounds, lipids are believed to assist absorption by reducing the inherent limitations of slow and incomplete dissolution and by facilitating the formation of colloidal species within the intestine that are capable of maintaining otherwise poorly water soluble drugs in solution. Importantly, the formation of these solubilizing species does not necessarily arise directly from the administered lipid, but more frequently results from the intraluminal processing of these lipids (via digestion and dispersion) prior to absorption [9,10]. The co-administration of drugs with lipids can also influence the drug absorption pathway. Whilst most orally administered drugs gain access to the systemic circulation via the portal blood, some highly lipophilic drugs are transported to the systemic circulation via the intestinal lymphatics, thereby avoiding hepatic first-pass metabolism [11,12]. In addition, lipids can delay gastric transit and enhance passive intestinal permeability [13]. More recently, certain lipids and lipidic excipients have been suggested to improve drug absorption through mitigation of presystemic drug metabo© 2012 Bentham Science Publishers
Development and Characterization of Self-Nanoemulsifying Drug Delivery Systems (SNEDDS)
lism associated with gut membrane-bound cytochrome P-450 enzymes or via inhibition of the P-glycoprotein efflux transporter [14,15]. These formulations have also proven useful in preparing stable formulations of moisture sensitive drugs [16]. Lipid based drug delivery systems, particularly selfnanoemulsifying drug delivery systems (SNEDDS) [17-20] have received great attention recently for its potential in improving oral bioavailability for the delivery of poorly water soluble drugs due to their high solvent capacity, ease of dispersion and formation of very fine droplet size. SNEDDS are isotropic mixtures of oil, surfactant, co-surfactant/co-solvent and drug that form fine oil-in-water nanoemulsion when introduced into aqueous phases under gentle agitation. The aim of our present investigation was to develop SNEDDS of ATV for improving its dissolution rate thereby oral bioavailability and to conduct forced degradation and stability studies of the optimized SNEDDS. 2. MATERIALS AND METHODS
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laboratory. A gradient HPLC (Shimadzu, Class VP series) with two LC-10AT VP pumps and variable wavelength programmable Photo Diode Array (PDA) detector, SPD-M10A VP was used. The HPLC system was equipped with the Shimadzu LC Solution software (Version 1.12). Samples were chromatographed on a reversed phase C18 column (GeminiTM 5μ, 250 x 4.6 mm). Acetonitrile and ammonium acetate (20 mM, adjusted to pH 4 with glacial acetic acid) in the ratio of 70:30 v/v were used as mobile phase. The mobile phase components were filtered before use through a 0.45 μm membrane filter and pumped from the respective solvent reservoirs at a flow rate of 1.0 mL/min. Eluents were monitored using UV detection at a wavelength of 242 nm. The volume of injection port was 20 μL. Calibration curve was constructed for atorvastatin in the range of 1-10 μg/mL and a good linear relationship was observed between the concentration of atorvastatin and peak area with a strong correlation coefficient (r = 0. 9998). The regression equation (y = 80275x - 1272.6) was used for estimating the atorvastatin samples.
2.1. Materials
2.3. Solubility of Atorvastatin in Vehicles
Atorvastatin is a gift sample from M/s. Dr. Reddy’s Laboratories Ltd., Hyderabad, India. Methanol (HPLC Grade), Acetonitrile (HPLC Grade), Ammonium acetate (HPLC Grade), Glacial acetic acid (ExcellaR Grade) and Hydrochloric acid (ExcellaR Grade) were purchased from Qualigens Fine Chemicals, Mumbai, India. Vegetable oils (arachis oil, sesame oil, olive oil, sunflower oil and soybean oil) were purchased from local market. Capmul MCM C8 and Capmul PG 8 are gift sample from M/s. Abitech Corporation, WI. Cremophor EL and Cremophor RH 40 are products of BASF, Germany and obtained as gift samples from M/s. Alkem Lab Ltd., Mumbai, India. Labrafil M 1994CS, Labrafil M2125CS, Labrasol, Lauroglycol 90, Plurol oleique CC497, Peceol and Transcutol P are the products of Gattifossé, France and obtained as gift samples from Alkem Laboratories Ltd. Triple distilled water was prepared in our laboratory. Atocor 10 is a commercial formulation (B.No. V 70305) and it is a product of M/s. Dr. Reddy’s Laboratories Ltd., Hyderabad, India.
Naturally occurring different vegetable oils, various surfactants and co-surfactants were studied for ATV solubility in order to identify the components for construction of ternary phase diagrams. An excess amount of ATV (approximately 500 mg) was placed in screw-capped glass vials containing 2 g of vehicle (i.e., oil or surfactant or co-surfactant). Glass vials were sealed with caps and vortexed for 10 min using a cyclomixer in order to facilitate proper mixing of ATV with the vehicles. Then vials were shaken reciprocally using a mechanical rotary shaker for 48 hrs at 25° C and allowed for another 24 hrs to attain equilibrium conditions with out shaking at the same temperature. The vials were centrifuged at 3000 rpm for 10 min using a centrifuge to obtain a clear supernatant liquid. Supernatant (100 mg) which was pre-filtered through a 0.45 μm membrane filter and extracted for ATV with methanol and diluted suitably. The extracted samples were filtered through a 0.45 μm membrane filter and analyzed for ATV using HPLC method. The amount of ATV dissolved in various vehicles was calculated.
UV/Visible Double Beam Spectrophotometer (S165, M/s. Elico Ltd, India) was used for analysis of atorvastatin in in vitro samples. A gradient High Performance Liquid Chromatography (HPLC) (Class VP series, M/s. Shimadzu Corporation, Japan) was used for analysis of atorvastatin in plasma samples. The Nephelo Turbidity Meter (131, M/s. Systronics, India) was used for the determination of turbidity of the dispersed formulations. Zeta Sizer (Nano-ZS, M/s. Malvern Instruments, UK) was used for determination of globule size of the dispersed formulations. USP XXIV Dissolution Rate Test Apparatus (Disso 2000, M/s. Labindia, India) was used for performing the dissolution studies. Fourier Transformed-Infrared Spectrometer (Nicolet Nexus 470, Thermo Nicolet Corp., USA) was used for obtaining FTIR spectra. 2.2. Analysis of Atorvastatin by HPLC In the present investigation, samples of atorvastatin were estimated by a validated HPLC method developed in our
2.4. Construction of Pseudo-Ternary Phase Diagrams Ternary phase diagrams comprising surfactant, cosurfactant and oil were plotted, each of them, representing an apex of the triangle [18, 21]. Olive oil as oil phase and Cremophor EL and Labrasol as surfactants and Transcutol P and Capmul MCM C8 as co-surfactants were selected (based on the solubility studies). Ternary phase diagrams were constructed for four systems, Cremophor EL-Transcutol P-Olive oil, Cremophor EL-Capmul MCM C8-Olive oil, LabrasolTranscutol P-Olive oil and Labrasol-Capmul MCM C8-Olive oil. The surfactant concentration was varied from 0 to 75% w/w, oil concentration was varied from 25 to 70% w/w and co-surfactant concentration was varied from 0 to 30% w/w. The total composition of the system was maintained equal to 100% w/w. Seventy compositions of the three components of each system were prepared and identified in the ternary phase diagram. 25 mg of each formulation was dispersed
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into 50 mL of distilled water and the globule size of the resulting dispersions was determined using Zeta sizer (NanoZS, M/s. Malvern Instruments, UK). In the ternary phase diagrams the area of self-nanoemulsifying region was marked for the compositions that are giving dispersion with a globule size 200 nm.
scribed above. About 400 mg of the formulation (equivalent to 10 mg of the ATV) was filled in size ‘1’ hard gelatin capsules, sealed and stored at ambient temperature (25° C) until used. These SNEDDS were evaluated for visual observations, turbidity, effect of pH of the dispersion media on globule size and zeta potential, robustness to dilution and in vitro dissolution study and were optimized.
2.5. Effect of ATV Loading 2.6.1. Visual Observations
The drug loading has considerable influence on the globule size and phase behavior of the spontaneously emulsifying systems [18, 22, 23]. In the view of this, effect of ATV loading on the globule size, phase behaviour and area of nanoemulsion formation was studied on Cremophor EL-Capmul MCM C8-Olive oil system which gave more area of nanoemulsification region among the other.
To assess the self-emulsification properties, ATV SNEDDS (25 mg) was introduced into 50 mL of distilled water in a glass Erlenmeyer flask at 37° C and the contents were gently stirred manually. After equilibrium, time of selfemulsification, dispersibility and appearance were observed [21, 24] and rated according to grading system (Table 2).
The seventy compositions of Cremophor EL-Capmul MCM C8-Olive oil were incorporated with 2.5, 5 and 10% w/w of ATV (i.e. 210 formulations). Required amount of ATV was added to the screw capped glass vials containing required amount of surfactant and co-surfactant. Drug was solubilized using a vortex mixer or by heating at 40° C in a water-bath wherever necessary. Finally required amount of oil was added to the vials and vortex mixed for 2 min for proper mixing. The mean globule size of the resulting dispersions up on diluting 25 mg of the formulations with 50 mL distilled water was measured using Zeta sizer. The area of nanoemulsification region was identified as described above by constructing pseudo-ternary phase diagrams.
2.6.2. Turbidity Measurement Turbidity of the prepared dispersions was measured using Nephelo Turbidity Meter using 30 mL of the dispersion. The Nephelo Turbidity Meter was carefully calibrated with formazin standard solution before the measurements. 2.6.3. Effect of pH of the Dispersion Media on Globule Size and Zeta Potential Effect of pH of dispersion media (viz. distilled water, 0.1N HCl, pH 4.5 acetate buffer and pH 6.8 phosphate buffer) on the mean globule size and zeta potential of the nanoemulsions was studied for the selected ATV SNEDDS. 25 mg of the formulation was dispersed in 50 mL of selected dispersion media and the globule size and zeta potential was measured using Zeta sizer.
2.6. Preparation and Evaluation of ATV SNEDDS A series of SNEDDS (F 01-14, the composition was shown in Table 1) which showed desired size (200 nm) were selected from 2.5% ATV system and prepared as deTable 1.
Composition (% w/w) of ATV SNEDDS
Formulation
ATV
Olive Oil
Cremophor EL
Capmul MCM C8
ATVF 01
2.5
24.4
73.1
0.0
ATVF 02
2.5
24.4
68.3
4.9
ATVF 03
2.5
24.4
63.4
9.8
ATVF 04
2.5
24.4
58.5
14.6
ATVF 05
2.5
24.4
53.6
19.5
ATVF 06
2.5
24.4
48.8
24.4
ATVF 07
2.5
24.4
43.9
29.3
ATVF 08
2.5
29.3
68.3
0.0
ATVF 09
2.5
29.3
63.4
4.9
ATVF 10
2.5
29.3
58.5
9.8
ATVF 11
2.5
29.3
53.6
14.6
ATVF 12
2.5
29.3
48.8
19.5
ATVF 13
2.5
29.3
43.9
24.4
ATVF 14
2.5
29.3
39.0
29.3
Development and Characterization of Self-Nanoemulsifying Drug Delivery Systems (SNEDDS)
Table 2.
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Grading System for Visual Observations of Self-Emulsifying Formulations
Grade
Dispersibility
Appearance
Self-Emulsification Time
A
Rapid emulsification
Clear or slightly bluish
