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against bacteria (e.g. E. coil, Salmonella s, S. nanoemulsion adjuvant (HBsAg- require large doses of their respective active
aureus), enveloped viruses (e.g. HIV, Herpes nanoemulsion) could be effective with fewer ingredients to surround pathogen cell walls,
simplex), fungi (e.g. Candida, administrations. Comprehensive pre-clinical which cause them to disintegrate,
Dermatophytes), and spores (e.g. anthrax). toxicology evaluation demonstrated that fundamentally "drowning" them in the
The nanoemulsion particles are HBsAg- nanoemulsion vaccine is safe and disinfectant solution. The formulation is a
thermodynamically driven to fuse with lipid- well tolerated in multiple animal models. Our broad-spectrum disinfectant cleaner that can
containing organisms. results suggest that needle-free nasal be applied to any hard surface, including
This fusion is enhanced by the immunization with HBsAg-NE could be a safe equipment, counters, walls, fixtures, and
electrostatic attraction between the cationic and effective hepatitis B vaccine, or provide floors. One product can now take the place of
charge of the emulsion and the anionic an alternative booster administration for the many reducing product inventories and
charge on the pathogen. When enough parenteral hepatitis B vaccines. This vaccine saving valuable storage space. (18-19)
nanoparticles fuse with the pathogens, they induces a Th1 associated cellular immunity
release part of the energy trapped within the and also may provide therapeutic benefit to Nanoemulsions in Cell Culture Technology
emulsion. Both the active ingredient and the patients with chronic hepatitis B infection Cell cultures are used for in vitro assays or to
energy released destabilize the pathogen who lack cellular immune responses to produce biological compounds, such as
lipid membrane, resulting in cell lysis and adequately control viral replication. Long- antibodies or recombinant proteins. To
death. In the case of spores, additional term stability of this vaccine formulation at optimize cell growth, the culture medium can
germination enhancers are incorporated into elevated temperatures suggests a direct be supplemented with a number of defined
the emulsion. Once initiation of germination advantage in the field, since potential molecules or with blood serum. The
takes place, the germinating spores become excursions from cold chain maintenance advantages of using nanoemulsions in cell
susceptible to the antimicrobial action of the could be tolerated without a loss in culture technology are better uptake of oil-
nanoemulsion. As a result, the nanoemulsion therapeutic efficacy. (16) soluble supplements in cell cultures; improve
can achieve a level of topical antimicrobial A novel technique for vaccinating against growth and vitality of cultured cells, and
activity that has only been previously a variety of infectious diseases-using an oil- allowance of toxicity studies of oil-soluble
achieved by systemic antibiotics. (12-13) based emulsion placed in the nose, rather drugs in cell cultures. (20)
than needles-has proved able to produce a
As A Mucosal Vaccine strong immune response against smallpox Nanoemulsion in Cancer Therapy and
Nanoemulsions are being used to deliver and HIV in two new studies. Developing Targeted Drug Delivery
either recombinant proteins or inactivated mucosal immunity may be very important for The effects of the formulation and particle
organisms to a mucosal surface to produce protection against HIV. In the study, the composition of gadolinium (Gd)-containing
an immune response. The first applications, nanoemulsion HIV vaccine showed that it lipid nanoemulsion (Gd-nanoLE) on the
an influenza vaccine and an HIV vaccine, can was able to induce mucosal immunity, biodistribution of Gd after its intravenous
proceed to clinical trials. The nanoemulsion cellular immunity, and neutralizing antibody (IV) injection in D1-179 melanoma-bearing
causes proteins applied to the mucosal to various isolates of HIV virus. A protein hamsters were evaluated for its application
surface to be adjuvant and it facilitates used by the team, gp120, is one of the major in cancer neutron-capture therapy.
uptake by antigen-presenting cells. binding proteins under study in other HIV Biodistribution data revealed that Brij 700
Additional research is ongoing to complete vaccine approaches. (17) and HCO-60 prolonged the retention of Gd in
the proof of concept in animal trials for other the blood and enhanced its accumulation in
vaccines including Hepatitis B and Nanoemulsion As Non-Toxic Disinfectant tumors. Upon dermal application, the drug
anthrax.(14) Mice and guinea pigs intranasally Cleaner was predominantly localized in deeper skin
immunized by the application of A breakthrough nontoxic disinfectant cleaner layers, with minimal systemic escape. This
recombinant HIV gp120 antigen mixed in for use in commercial markets that include has amounted to an absolute bioavailability
nanoemulsion demonstrated robust serum healthcare, hospitality, travel, food of 70.62%. Inhibition of P-glycoprotein efflux
anti-gp120 IgG, as well as bronchial, vaginal, processing, and military applications has by D-tocopheryl polyethyleneglycol 1000
and serum anti-gp120 IgA in mice. been developed by EnviroSystems, Inc. that succinate and labrasol would have
The serum of these animals kills tuberculosis and a wide spectrum of contributed to the enhanced peroral
demonstrated antibodies that cross-reacted viruses, bacteria and fungi in 5-10 min bioavailability of PCL. This investigation
with heterologous serotypes of gp120 and without any of the hazards posed by other provides direct evidence on the localization
had significant neutralizing activity against categories of disinfectants. The product of high-molecular-weight, lipophilic drug,
two clade-B laboratory strains of HIV needs no warning labels. It does not irritate PCL, in dermis. Further, the nanoemulsion
(HIVBaL and HIVSF162) and five primary eyes and can be absorbed through the skin, formulation has enhanced the peroral
HIV-1 isolates. The analysis of gp120-specific inhaled, or swallowed without harmful bioavailability significantly to more than
CTL proliferation, INF-g induction, and effects. The disinfectant formulation is made 70%. The developed nanoemulsion
prevalence of anti-gp120 IgG2 subclass up of nanospheres of oil droplets #106 mm formulation was safe and effective for both
antibodies indicated that nasal vaccination in that are suspended in water to create a NE peroral and dermal delivery of PCL. (22)
nanoemulsion also induced systemic, Th1- requiring only miniscule amounts of the Camptothecin is a topoisomerase-I
polarized cellular immune responses. This active ingredient, PCMX inhibitor that acts against a broad spectrum
study suggests that nanoemulsion should be (parachlorometaxylenol). The nanospheres of cancers. However, its clinical application is
evaluated as a mucosal adjuvant for carry surface charges that efficiently limited by its insolubility, instability, and
multivalent HIV vaccines. (15) Hepatitis B penetrate the surface charges on toxicity. The aim of the present study was to
virus infection remains an important global microorganisms' membranes-much like develop acoustically active nanoemulsions
health concern despite the availability of safe breaking through an electric fence. Rather for camptothecin encapsulation to
and effective prophylactic vaccines. than "drowning" cells, the formulation allows circumvent these delivery problems. The
Limitations to these vaccines include PCMX to target and penetrate cell walls. As a nanoemulsions were prepared using liquid
requirement for refrigeration and three result, PCMX is effective at concentration perfluorocarbons and coconut oil as the
immunizations thereby restricting use in the levels 1-2 orders of magnitude lower than cores of the inner phase. These
developing world. A new nasal hepatitis B those of other disinfectants; hence, there are nanoemulsions were stabilized by
vaccine composed of recombinant hepatitis B no toxic effects on people, animals, or the phospholipids and/or Pluronic F68 (PF68).
surface antigen (HBsAg) in a novel environment. Other microbial disinfectants The nanoemulsions were prepared at high
Inventi Rapid: Pharm Tech Vol. 2, Issue 1 2011pt231, CCC: $10 © Inventi Journals (P) Ltd
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REVIEW ARTICLE
drug loading of approximately 100% with a species such as free radicals and singlet osteoarthritis (OA) pain. OA is a painful
mean droplet diameter of 220-420 nm. oxygen (1O2) that are toxic to cells and condition affecting more than 30 million
Camptothecin in these systems showed tissues. (31-33) Various PDT therapies have people in the USA and is the most frequent
retarded drug release. Camptothecin in reported two different vehicles for cause of physical disability among adults,
nanoemulsions with a lower oil photosensitizers, a cremophor oil emulsion mainly elderly. Topical diclofenac is also
concentration exhibited cytotoxicity against and DPPC (dipalmitoylphosphatidylcholine) being considered as treatment for soft tissue
melanomas and ovarian cancer cells. liposomal vesicles. The reported injuries, sprains, and strains. It is estimated
Confocal laser scanning microscopy pharmacokinetic studies clearly indicate that that 20% of OA patients are not receiving
confirmed nanoemulsion uptake into cells. the former vehicle yields a significantly treatment, mainly due to gastrointestinal
Using a 1 MHz ultrasound, an increased larger selectivity of tumor targeting, mainly side effects of oral NSAIDs and
release of camptothecin from the system as a consequence of an enhanced cardiovascular risk of COX-2 inhibitors. A
with lower oil concentration could be accumulation in the malignant lesion. topical NSAID offering adequate pain relief
established, illustrating a drug-targeting Neutron Capture Therapy (NCT) is a binary targeted to the site of injury with an
effect. [23] radiation therapy modality that brings improved safety profile could become a
The scientists have investigated the together two components that when kept treatment alternative for these patients. In
nanoemulsion containing risperidone (RSP) separate had only minor effects on the cells. the USA, there are no approved topical
to accomplish the delivery of drug to the The first component is a stable isotope of NSAIDs for the treatment of OA. Pharmos' NE
brain via nose. Risperidone nanoemulsion boron or gadolinium (Gd) that can be technology consists of an efficient solvent-
(RNE) and mucoadhesive nanoemulsion concentrated in tumor cells by a suitable free topical vehicle based and drug
(RMNE) were characterized for drug content, delivery vehicle. The second is a beam of entrapment in stable, submicron particles of
pH, percentage transmittance, globule size, low-energy neutrons. Boron or Gd in or oil-in-water emulsions with a mean droplet
and zeta potential. Biodistribution of RNE, adjacent to the tumor cells disintegrates after size between 100 and 200 nm that are
RMNE, and risperidone solution (RS) in the capturing a neutron, and the high energy uniformly dispersed in an aqueous phase.
brain and blood of Swiss albino rats heavy charged particles produced through One of the unique characteristics of the
following intranasal (i.n.) and intravenous this interaction destroy only the cancer cells nanoemulsion technology is the relatively
(i.v.) administration was examined using in close proximity to it, leaving adjacent high percentage of total particle volume
optimized technetium-labeled [ (99 m) Tc- normal cells largely unaffected. (34) The occupied by the internal hydrophobic oil core
labeled] RSP formulations. Gamma success of NCT relies on the targeting of of the droplets. This provides high
scintigraphy imaging of rat brain following boron and Gd-based compounds to the solubilization capacity for lipophilic
i.v. and i.n. administrations were performed tumor mass and to achieve desirable compounds compared to other lipoidal
to ascertain the localization of drug in brain. intracellular concentrations of these agents. vehicles such as liposomes. Viscosity-
Higher drug transport efficiency (DTE%) and At the present time, there are two targets imparting agents are used for nanoemulsion
direct nose to brain drug transport (direct with NCT, namely glioblastoma (malignant thickening to produce creams with the
transport percentage, DTP%) for brain tumor) and malignant melanoma. desired semisolid consistency for application
mucoadhesive nanoemulsions indicated The perfluorochemical nanoemulsions to the skin. The skin penetrative properties
more effective and best brain targeting of (PFCE) have opened interesting of the solvent-free nanoemulsion delivery
RSP amongst the prepared nanoemulsions. opportunities in cancer therapy. It is technology and its low irritancy make this
Studies conclusively demonstrated rapid and suggested that fluorocarbon emulsions might novel topical nanovehicle a promising
larger extent of transport of RSP by RMNE find a role in photodynamic therapy, both as candidate for effective transcutaneous
(i.n.) when compared to RS (i.n.), RNE (i.n.), carriers for sensitizing dyes and to maintain delivery of lipophilic drugs. Primaquine (PQ)
and RNE (i.v.) into the rat brain. (24) tissue oxygenation in hypoxic regions of solid is one of the most widely used antimalarial
Another study reported the formulation tumors. The high solubility of oxygen in and is the only available drug till date to
of filter sterilizable emulsion formulation of fluorocarbon emulsions maintains solution combat relapsing form of malaria especially
paclitaxel using á-tocopherol as the oil phase oxygen tension, optimizing photo-oxidative in case of Plasmodium vivax and Plasmodium
and á-tocopherylpolyethyleneglycol-1000 damage. The hydrophobic anti-cancer drugs ovale. Primaquine acts specifically on the
succinate (TGPS) and poloxamer 407 as can be delivered to the tumor mass by pre-erythrocytic schizonts that are
emulsifiers. The formulation exhibited better dissolving them in a hydrophobic core of the concentrated predominantly in the liver and
efficacy and was more tolerable when emulsion. Furthermore, PFCE can be used as causes relapse after multiplication. However,
studied in B16 melanoma tumor model in an adjuvant to radiation therapy and/or application of PQ in higher doses is limited
mice. (25) chemotherapy in the treatment of solid by severe tissue toxicity including
Emulsion formulations also show tumors.(36-37) hematological and GI-related side effects that
promise in cancer chemotherapy as vehicles The preclinical studies have shown very are needed to be minimized. Lipid
for prolonging the drug release after positive effects with single dose and nanoemulsion has been widely explored for
intramuscular and intratumoral injection fractionated radiation in several rodent solid parenteral delivery of drugs. Primaquine
(W/O systems) and as a means of enhancing tumor models. Many widely used anticancer when incorporated into oral lipid
the transport of anti-cancer drugs via the drugs, including anti-tumor alkylating agents nanoemulsion having a particle size in the
lymphatic system.[26] Positively charged and doxorubicin, have shown improved range of 10-200 nm showed effective
nanoemulsions systems are expected to response by PFCE coadministration. (38) Also, antimalarial activity against Plasmodium
interact with negatively charged cell surfaces local application of toxic doses of PFCEs bergheii infection in Swiss albino mice at a
more efficiently, and this aspect of the resulted in the necrosis of cancer cells. This 25% lower dose level as compared to
positively charged nanoemulsions has been is especially promising in the treatment of conventional oral dose. Lipid nanoemulsion
explored for possibility of oligonucleotide cancers of the head and neck regions that are of primaquine exhibited improved oral
delivery to cancer cells.(27-30) Photodynamic currently difficult to treat. (39) bioavailability and was taken up
therapy (PDT) of cancer is based on the preferentially by the liver with drug
concept that certain photosensitizers can be Nanoemulsion in the Treatment of concentration higher at least by 45% as
localized in the neoplastic tissue, and Various Other Disease Conditions compared to the plain drug. (40)
subsequently, these photosensitizers can be Pharmos' (US-based company) has
activated with the appropriate wavelength developed the nanoemulsion topical Nanoemulsion Formulations for Improved
(energy) of light to generate active molecular diclofenac cream as a potential treatment for Oral Delivery of Poorly Soluble Drugs
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Nanoemulsion formulations were developed Celecoxib, a selective cyclo-oxygenase-2 biological, or diagnostic agents. Traditionally,
to enhance oral bioavailability of inhibitor, has been recommended orally for nanoemulsions have been used in clinics for
hydrophobic drugs. Paclitaxel was selected the treatment of arthritis and osteoarthritis. more than four decades as total parenteral
as a model hydrophobic drug. The oil-in- Long-term oral administration of celecoxib nutrition fluids. Although nanoemulsions are
water (o/w) nanoemulsions were made with produces serious gastrointestinal side chiefly seen as vehicles for administering
pine nut oil as the internal oil phase, egg effects. Skin permeation mechanism of aqueous insoluble drugs, they have more
lecithin as the primary emulsifier, and water celecoxib from nanoemulsion was evaluated recently received increasing attention as
as the external phase. Stearylamine and by FTIR spectral analysis, DSC thermogram, colloidal carriers for targeted delivery of
deoxycholic acid were used to impart activation energy measurement, and various anticancer drugs, photo sensitizers,
positive and negative charge to the histopathological examination. The neutron capture therapy agents, or
emulsions, respectively. optimized nanoemulsion was subjected to diagnostic agents. Because of their
The formulated nanoemulsions had a pharmacokinetic (bioavailability) studies on submicron size, they can be easily targeted to
particle size range of 90-120 nm and zeta Wistar male rats. Photomicrograph of a skin the tumor area. It is expected that further
potential ranging from +34 mV to 245 mV. sample showed the disruption of lipid research and development work will be
Following oral administration, a significantly bilayers as distinct voids and empty spaces carried out in the near future for clinical
higher concentration of paclitaxel was were visible in the epidermal region. The realization of these targeted delivery
observed in the systemic circulation when absorption of celecoxib through vehicles.
administered in the nanoemulsion relative to transdermally applied nanoemulsion and
control aqueous solution. The results of this nanoemulsion gel resulted in 3.30- and 2.97- REFERENCES AND NOTES
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[ISSN 0976-3783] Published on Web 12/03/2011, www.inventi.in
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[ISSN 0976-3783] Published on Web 12/03/2011, www.inventi.in
Research Article [Chouksey et al., 2(8): Aug., 2011]
ISSN: 0976-7126
Introduction
Nanoemulsions being colloidal nanodispersions of oil Nanoemulsions have a higher solubilization capacity
in water (or water in oil), thermodynamically stabilized than simple micellar solutions and their
by an interfacial film of surfactant(s) and co- thermodynamic stability offers advantages over
surfactant(s) have revealed tremendous potential in unstable dispersions, such as emulsions and
nanoengineering of various inorganic materials1. suspensions, because they can be manufactured with
Droplet size in thermodynamically stable little energy input (Low energy emulsification
nanoemulsions is usually 10-100 nm2. The techniques/heat or mixing) and has a long shelf life.
homogeneous systems that can be prepared over a wide The nanosized droplets leading to enormous interfacial
range of surfactant concentrations and oil to water areas associated with nanoemulsions would influence
ratios (1:4 %) are all fluids of low viscosity. the transport properties of the drug, an important factor
Nanoemulsions provide ultra low interfacial tensions in sustained and targeted drug delivery3-4. The
and large o/w interfacial areas. attraction of o/w nanoemulsion systems lies in their
ability to incorporate hydrophobic drugs into the oil
* Corresponding Author: phase thereby enhancing their solubility3.
E-mail: chouksey_rajendra@yahoo.com Nanoemulsions have been reported to make the plasma
Mob.: +91-9993815318 concentration profiles and bioavailability of drugs
more reproducible5-6. The objective of the present study
Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 2, Issue 8: Aug.: 2011, 982-988
982
Research Article [Chouksey et al., 2(8): Aug., 2011]
ISSN: 0976-7126
is to prepare nanoemulsion of atorvastatin to improve involved in these systems, as well as the limitation
its solubility and bioavailability. associated with each technique, but such knowledge is
essential for their successful commercial exploitation.
Material and Methods The rate of release of sodium salicylate from a lecithin-
Development of drug containing nanoemulsion based nanoemulsion, is dependent upon their
formulation microstructure7.
The drug stock solutions in oil mixture were prepared A complementarily of methods is generally required in
in such a way that 10 mg dose was present in each order to fully characterize these systems. At the
formulation complying the oil percentage for each macroscopic level viscosity, conductivity and dielectric
formula as selected from the phase diagram. This was methods provide useful information.
prepared by dissolving the 1000 mg of drug Phase Behavior studies
individually in the 10, 15, 20 and 25 mL of oily Phase diagram showing the nanoemulsion region,
mixture, which compiles the 10%, 15%, 20% and 25% information about of different phases as a function of
oil compositions respectively in the formulae. composition variables can be analyzed from vigorous
Screening of formulations on the basis of study of the phase diagrams.
thermodynamic stability studies Viscosity measurements
Microemulsions are thermodynamically stable systems The viscosity of the prepared nanoemulsion
and are formed at a particular concentration of oil, formulations were determined as such without dilution
surfactant: co-surfactant mixture and water, with no by Brookfield DV III ultra V6.0 RV cone and plate
phase separation, creaming or cracking. It is the rheometer (Brookfield Engineering Laboratories, Inc,
thermostability which differentiates micro emulsion Middleboro, MA) using spindle # CPE40 at 25 ±0.5°C.
from emulsions that have kinetic instability and will The software used for the viscosity calculations was
eventually phase separate3. The thermodynamic Rheocalc V2.6.
stability studies were performed on the basis of Refractive index
following tests. Refractive index of selected formulations was
Centrifugation study determined using an Abbe type refractrometer. It was
The selected formulations were centrifuged (REMI, standardized using castor oil.
India) at the 5000 rpm for 30 mins and observed for Dielectric measurements
phase separation, creaming or cracking. The They are powerful means of probing both structural
formulations which showed maximum stability (no and dynamic features of nanoemulsions systems.
creaming, cracking, phase separation) were selected Electron microscopic studies
and studied for heating-cooling cycle, freeze-thaw Morphology and structure of the nanoemulsion was
cycles and Dispersibility tests. studied using transmission electron microscopy (TEM)
Heating cooling cycles TOPCON 002B operating at 200 KV and of a 0.18 nm
It is used to see the stressed effect of heating and capable of point to point resolution. In order to perform
cooling on the nanoemulsion`s stability. In this study the TEM, the nanoemulsion formulation was diluted
the formulations were kept at 450 c and at 0 0C with distilled water (1/100). A drop of the diluted
temperature for not less then 48 h. for each temperature nanoemulsion was directly deposited on the Copper
cycle. holey film grid and observed after putting fixing agent
Freeze –thaw cycles (Accelerated ageing) and drying it in the filtered air.
This test was performed for accelerated stability testing Scattering techniques
of nanoemulsion formulations. In this study the Dynamic light scattering photon correlation
formulations were exposed at two different spectroscopy (PCS) is used to analyze the fluctuations
temperatures i.e. -210C and 210C for each temperature in the intensity of the scattering by the droplets due to
cycles not than 24 hrs. For the better estimation of Brownian motion.
accelerated stability studies three such cycles should be Interfacial tension measurement
run for each batch of formulation. The formulations The formation and properties of nanoemulsion can be
which showed the maximum stability were selected for studied by measuring the interfacial tension. Spinning-
further study. drop apparatus can be used to measure the ultra low
Characterization of the nanoemulsion interfacial tension.
Nanoemulsion has been characterized using a wide Droplet size distribution
variety of techniques. The characterization of Droplet size of the prepared nanoemulsion was
nanoemulsion is a difficult task due to their determined by using photon correlation spectroscopy,
complexity, variety of structures and components
Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 2, Issue 8: Aug: 2011, 982-988
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Research Article [Chouksey et al., 2(8): Aug., 2011]
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which analyzes the fluctuations in light scattering due
to Brownian movement of the particles8. The Comparative dissolution profile in simulated gastric
formulation (0.1 mL) was dispersed in 50 mL (500 fluid (pH 1.2)
dilution) of distilled water in a volumetric flask and
gently mixed by inverting the flask and measurement 120
done using a Zetasizer (Nano ZS-90, UK). Light 100
% Drug release
scattering was monitored at 25°C at a 90° angle. 80
Refractive index/ Isotropicity 60
Refractive index of nanoemulsions formulations was 40
determined using an Abbe type refractrometer. It 20
basically gives an idea about the isotropicity of the 0
formulations. The isotropic nature of microemulsions 0 20 40 60 80
and their optical clarity makes their study by Time (min)
spectroscopic techniques straightforward, particularly
in comparison to conventional macroemulsions. Self-nanoemulsifying pellets Tablet Avas Tablet Atorlip
Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 2, Issue 8: Aug: 2011, 982-988
984
Research Article [Chouksey et al., 2(8): Aug., 2011]
ISSN: 0976-7126
GM1 that showed the highest release profile of drug in Tubes were stored at room temperature, 25 ± 2°C and
in vitro studies was subjected to in vivo studies. 6 relative humidity (55 ± 5%) for 30 minutes. The clotted
mg/kg9 body weight of the Atorvastatin Ca was used in blood was then centrifuged at 5000 rpm for 30 min.
the study.Six tablets with label claim of 10 mg The serum was separated and stored at -21 °C until
(Atorlip®) were taken, crushed in mortar, and mixed drug analysis was carried out using RP-HPLC method.
with 50 ml of double distilled water (1.2 mg/mL). Each The collected serum was extracted with ethyl-acetate (5
rat in this group was given 1.0 ml using oral feeding ml) by using Sodium phosphate buffer (pH 7, 1 ml) as
needle. Same dose of drug suspension and a protein-precipitating agent for analysis and
nanoemulsion was administered. The blood sampling centrifuged at 5000 rpm for 5 min and dried at room
was carried out for around 48 hours and 10 to 12 temperature. After evaporating the ethyl acetate solid
samples of blood were taken from each animal in the residue was reconstituted using 100 µl mobile phase
group. Blood was withdrawn from the juglar vein after and then analyzed by RP-HPLC. The samples were
anesthesia at 0 (pre-dose), 0.25, 0.5, 0.75, 1.0, 1.25, injected and the area was noted down in triplicate. The
1.5, 6, 12, 24, 36 and 48 h in micro centrifuge tubes. mean areas obtained by HPLC are given in the Table 2.
Table 2: Working formula for nanoemulsion (GM1)
Oil :Sefsol 218+ Oleic acid , Surfactant : Tween 20, Cosurfactant: Carbitol
Volume of components for Nanoemulsion Formulation Code
Oil (ml) Surfactant (ml) Co – surfactant (ml) Aqueous phase Atorvastatin (mg)
(ml)
0.10 0.19 0.19 0.52 1.2 GM1
pharmacokinetic (PK) parameters (tmax, Cmax, AUC0→t,
The formulations were given orally using oral feeding AUMC0→t and MRT 0→t) were calculated individually
needle. Dose for the rats was calculated based on the for each subject in the group and the values were
weight of the rats (6 mg/ kg). expressed as mean ±SD. The comparative in-vivo
Pharmacokinetic and statistical analysis bioavailability profiles of nanoemulsion formulation
Pharmacokinetic parameters were calculated by using GM1, API suspension and Tablet suspension are
non-compartmental analysis also called as Model shown in the Table 4.
independent analysis using WinNonLin version 4.0
(Pharsight Corp., Mountain View, CA). All
Table 4: Comparative observation table for in-vivo bioavailability studies in rat animal model (n=6).
Sampling time SNEDDS Tablet suspension API suspension
(Hours) (Mean Conc. ± SD) (Mean Conc. ± SD) (Mean Conc. ± SD)
0 0 0 0
0.25 24719.36 ± 142.54 4747.476 ± 63.76 3636.91 ± 57.56
0.5 35582.73 ± 345.54 5491.985 ± 36.87 4307.00 ± 24.87
0.75 37687.34 ± 733.16 5815.137 ± 62.97 4456.82 ± 54.89
1 41090.76 ± 531.78 6343.85 ± 64.86 4959.85 ± 86.36
1.25 43603.77 ± 632.63 6750.599 ± 145.35 5271.45 ± 75.37
1.5 37508.95 ± 456.56 5801.549 ± 48.25 4345.85 ± 86.74
6 24312.26 ± 731.84 3761.141 ± 65.36 2887.23 ± 75.36
12 12093.38 ± 234.47 1870.322 ± 124.12 1437.17 ± 44.66
24 6370.94 ± 32.47 987.3089 ± 32.85 765.24 ±75.05
36 3851.75 ± 42.74 599.0169 ± 24.86 548.16 ± 36.87
48 2811.38 ± 57.38 436.0885 ± 14.60 338.09 ± 23.21
Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 2, Issue 8: Aug: 2011, 982-988
985
Research Article [Chouksey et al., 2(8): Aug., 2011]
ISSN: 0976-7126
Tablet Suspension 2.24 ± 1.74 6750.599 ± 145.35 35432.4 ± 1143.53 765.86 ±32.7
API drug 2.52 ± 1.93 5271.45 ± 75.37 68232.5 ± 6734.46 893.57 ±84.2
Suspension
a
time of peak concentration; b peak of maximum concentration;e area under the concentration time profile curve until
last observation; fRelative bioavailability of formulations
Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 2, Issue 8: Aug: 2011, 982-988
987
Research Article [Chouksey et al., 2(8): Aug., 2011]
ISSN: 0976-7126
as to that of and API drug suspension was 559.86% delivery systems. Adv. Drug Deliv. Rev., 45(1):
respectively. 89-121.
Therefore, based on higher drug release, optimum 4. Eccleston J. (1994). Microemulsions. In:
globule size, minimum polydispersity, lower viscosity, Encyclopedia of Pharmaceutical Technology
lower surfactant concentration, higher solubility as well (Swarbrick J.,Boylan J.C. Eds.), Marcel
as higher bioavailability without variable absorption Dekker New York. 9: 375-421.
has been optimized. The composition of optimized 5. Kawakami K., Yoshikawa T., Moroto Y.,
formulation (GM1) was Sefsol 218 (5% v/v), Oleic Kanaoka E., Takahashi K., Nishihara Y. and
acid (5% v/v), Tween-20 (19% v/v), Carbitol (19%v/v) Masuda K. (2002). Microemulsion formulation
and distilled water (52% v/v) as oil, surfactant, for enhanced absorption of poorly soluble
cosurfactant and aqueous phase respectively, drugs II. J. Control. Release, 81: 75-82.
containing 10 mg of AT Calcium. 6. Constantinides P. P. (1995). Lipid
The in vivo studies revealed significantly greater extent microemulsions for improving drug dissolution
of absorption than the conventional tablet suspension and oral absorption and biopharmaceutical
and API drug suspension formulation. The absorption aspects. Pharm. Res., 12(11): 1561-1572.
of AT Calcium from Tween 20 nanoemulsion resulted 7. Khoshnevis P., Mortazavi S. A., Lawrence M.
in 3.56 fold increase in bioavailability as compared to J. and Aboofazeli R. (1997). In-vitro release of
conventional Tablets suspension and 5.59 fold to that sodium salicylate from water-in-oil
of API drug suspension. Furthermore, this result phospholipid microemulsions. J. Pharm.
attributed that the presence of surfactants in Pharmacol., 49 (S4): 47.
nanoemulsion system might have caused changes in 8. Attwood D., Mallon C. and Taylor C. J.
membrane permeability of the GI tract and the (1992). Phase studies of oil-in water
inhibition of an apically polarized efflux system, which phospholipid microemulsions. Int. J. Pharm.
could lead to enhancement of the oral absorption. 84: R5–R8.
9. Shen H. R. and Zhong M. K. (2006).
Acknowledgements Preparation and evaluation of self
Author is thankful to Lupin Pharmaceuticals Pvt. Ltd. microemulsifying drug delivery system
for providing drug sample. We are also thankful to (SMEDDS) containing atorvastatin. Pharmacy
Medical Superintendent, G. R. Medical College, and Pharmacology., 58: 1183-1191.
Gwalior for kind cooperation, guidance and help 10. Shafiq S., Faiyaz S., Sushma T., Farhan J. A.,
during my tenure of my research work. I am also Khar R. K. and Mushir A. (2007).
thankful to Dean Research, Gyan Vihar University, Development and bioavailability assessment of
Jaipur for all the support and guidance. ramipril nanoemulsion formulation. Eur. J.
Pharm. Biopharm., 66(2): 227-243.
References
1. Date A. A. and Patravale V. B. (2004). Current
strategies for engineering drug nanoparticles.
Curr. Opin. Colloid Interface Sci., 9: 222- 235.
2. Sintov A. C. and Shaprio L. (2004). New
microemulsions vehicle facilitates
percutaneous penetration invitro and cutaneous
drug bioavailability in-vivo. J. Control
Release., 95: 173-183.
3. Lawrence M. J. and Reece G. D. (2000).
Microemulsion-based media as novel drug
Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 2, Issue 8: Aug: 2011, 982-988
988
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 3, Issue 3, 2011
Research Article
PREPARATION AND EVALUATION OF THE SELF EMULSIFYING DRUG DELIVERY SYSTEM
CONTAINING ATORVASTATIN HMGCOA INHIBITER
RAJENDRA CHOUKSEY1*, HARISH PANDEY2, A. K. JAIN3, HIMESH SONI4, G. K. SARAOGI4
1 Gyan Bihar University, Jaipur (Rajasthan), 2 Sai Satya Sai College of Pharmacy, Sehore (M.P.), 3 GR Medical College Gwalior (M.P.),
4 Lakshmi Narain College of Pharmacy, Bhopal (M.P.) India Email: himeshsoni@rediffmail.com
Received: 28 March 2011, Revised and Accepted: 26 April 2011
ABSTRACT
As a consequence of modern drug discovery techniques, there has been a steady increase in the number of new pharmacologically active lipophilic
compounds that are poorly water‐soluble. It is a great challenge for pharmaceutical scientists to convert those molecules into orally administered
formulations with sufficient bioavailability. Among the approaches to improve the oral bioavailability of these molecules, the use of self‐emulsified
drug delivery systems (SEDDS) has been shown to be reasonably successful in improving the oral bioavailability of poorly water‐soluble and
lipophilic drugs. In the present study we formulate self‐emulsified drug delivery systems (SEDDS) through the nanoemulsion for the effective
delivery of Atorvastatin which is a HMG‐COA Inhibiter.
Keywords: Atorvastatin, Self emulsifying drug delivery systems, Oral drug delivery, Bioavailability.
INTRODUCTION catabolism of LDL, its also reduces LDL production and the number
of LDL particles. The absolute bioavailability of atorvastatin (parent
The oral route is one the most commonly used method for drug) is approximately 14% and the systemic availability of HMG‐
administration of drugs and drug delivery. The only disadvantages CoA reductase inhibitory activity is approximately 30% this low
of this method are sluggish onset time, the possibility of erratic systemic availability is ascribed to presystemic clearance in
absorption, degradation of some specific drugs by digestive gastrointestinal mucosa and/or hepatic first‐pass metabolism.
enzymes. The poorly water‐soluble drugs such as HIV protease
inhibitors, glycoprotein inhibitors and anticancer drugs have The food decreases the rate and extent of drug absorption by
problems to produce and retain a good solubility in gastrointestinal approximately 25% and also responsible for reduction in C max and
tract. The drug delivery industry scientists are used a wide range of AUC, LDL‐C level. A blood/plasma ratio of approximately 0.25
methods to improve the dissolution rate of poorly water‐soluble indicates poor drug penetration into red blood cells. Based on
drugs, including formulations containing nanoparticles, a solid observations in rats, atorvastatin calcium is likely to be secreted in
solution formulation or self emulsifying drug delivery system human milk. So it is aimed to formulate a SEDDS containing a
(SEDDS), and stable amorphous form of the drug 1‐5. SEDDS is an lipophilic drug, atorvastatin and to explore the potential of this
isotropic mixture of oil, surfactant and/or co‐surfactant can be used carrier for improvement of the oral bioavailability of
for formulations to improve the absorption of drugs in atrovastatin.11
gastrointestinal tract and solve the solubility problems. SEDDS can
produce fine oil/water emulsion after dilution in gastrointestinal MATERIALS AND METHODS
fluids and provide large interfacial area for drug partitioning
Materials
between oil and water phases and so increase in solubility rate and
extent of absorption 6‐10. Atorvastatin, a 3‐hydroxy‐3‐methylglutaryl Atorvastatin was obtained from Lupin Laboratory, Aurangabad as a
coenzyme A (HMG‐CoA) reductase inhibitor (a statin), is a lipid gift sample.
regulating drug with actions on plasma lipids similar to those of
simvastatin. It is used to reduce LDL‐cholesterol, apolipoprotein B, Formulation of self emulsifying drug delivery system (SEDDS)
and triglycerides, and to increase HDL‐cholesterol in the treatment
of hyperlipidaemias , including hypercholesterolaemias and The drug stock solutions in oil mixture were prepared in such a way
combined (mixed) hyperlipidaemia (type IIa or IIb that 10 mg dose is present in each formulation complying the oil
hyperlipoproteinaemias), hypertriglyceridaemia (type IV), and percentage for each formulae selected from the phase diagram. This
dysbetalipoproteinaemia (type III). Atorvastatin lowers plasma was prepared by dissolving the 1000 mg of drug individually in the
cholesterol and lipoprotein levels by inhibiting HMG‐CoA reductase 10, 15, 20 and 25 mL of oily mixture, which complies the 10%, 15%,
and cholesterol synthesis in the liver and by increasing the number 20% and 25% oil compositions respectively in the formulae. The
of hepatic LDL receptors on the cell‐surface to enhance uptake and drug stock is shown in Table no 1.
Table 1: Preparation of drug stock for each formula selected in phase diagram
S.NO Oil percentage in formulations Amount of drug Volume of oil Final concentration
(mg) (mL) (mg/μL)
1 10% 1000 10 10 mg/100 μL
2 15% 1000 15 10 mg/150 μL
3 20% 1000 20 10 mg/200 μL
4 25% 1000 25 10 mg/250 μL
Construction of pseudoternary phase diagrams 15‐16 were chosen in increasing concentration of cosurfactant with
respect to surfactant and increasing concentration of surfactant with
Surfactant and co‐surfactant (Smix) in each group were mixed in respect to cosurfactant for detailed study of the phase diagrams for
different volume ratios (1:0, 1:1, 1:2, 1:3, 2:1, 3:1, 4:1) and the stock the nanoemulsions formation.
of 100 mL of each groups was prepared (Table 2). These Smix ratios
Chouksey et al.
Int J Pharm Pharm Sci, Vol 3, Issue 3, 2011, 147152
Table 2: Different volumes of surfactant and cosurfactant taken to make a stock Smix ratio
S.No Volume of Surfactant Volume of Cosurfactant Ratio of Smix
(mL) (mL)
1 100 0 1:0
2 50 50 1:1
3 33.3 66.7 0.5:1 or 1:2
4 25 75 1:3
5 66.7 33.3 2:1 or 1:0.5
6 75 25 3:1
7 80 20 4:1
For each phase diagram, oil and specific Smix ratio was mixed diagrams, the oil phase (oleic acid: Sefsol, 1:1) was mixed with
thoroughly in different volume ratios from 1:9 to 9:1 in different different ratio of surfactant and cosurfactant (Tween 20 and
small glass test tubes. Sixteen different combinations of oil and each Carbitol® respectively) and mixture was titrated with distilled water
Smix, 1:9, 1:8, 1:7, 1:6, 1:5, 2:8 (1:4), 1:3.5, 1:3, 3:7 (1:2.3), 1:2, 4:6 until it turned turbid. Examine each and every point I detailed and
(1:1.5), 5:5 (1:1), 6:4 (1:0.7), 7:3 (1:0.43), 8:2(1:0.25), 9:1 (1:0.1) note it down. Pseudo ternary phase diagrams were drawn by using
were made so that maximum ratios were covered for the study to data obtained in aqueous titration method as shown in Figure.1 (A‐
delineate the boundaries of phases precisely formed in the phase G). The amount of water added to give water concentration in the
diagrams. range of 5‐95% of total volume at 5% intervals. After every 5%
addition of the water to the oil and Smix mixture, visual
For the determination of existence zone of microemulsion, observations were made as shown in Figure 1. The ratio of
pseudoternary phase diagrams were constructed using water surfactant and co surfactant (Tween 20 and Carbitol ®) were used for
titration method [17‐19]. To construct pseudoternary phase the titration
A B
Water Water
S:CoS-2:1
Ratio-1:2
S:CoS-3:1 S:CoS-1:3
Oil Smix
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Int J Pharm Pharm Sci, Vol 3, Issue 3, 2011, 147152
F G
Water
Water
Ratio-1:1 Ratio-1:0
Oil Smix
Oil Smix
Fig. 1: AG Phase diagram of Smix containing the surfactant and co surfactant in ratio 1:0, 1:1, 1:2, 1:3, 2:1 3:1and 4:1 respectively
Self emulsifying capacity Heating cooling cycles
For evaluation of self‐emulsification properties of formulations, 1 ml It is used to see the stressed effect of heating and cooling on the
of each formulation was added to 0.1N hydrochloric acid (50 mL) nanoemulsion`s stability. In this study the formulations were kept at
under continuous stirring (60 rpm) at 37°C and then spreadability, 450 c and at 0 0C temperature for not less then 48 hrs for each
tendency to emulsifying and progress of emulsion droplets were temperature cycle.
observed. Formulation were categorized as clear, non clear, stable or
unstable. In the other hand, refractive index of different formulations Freeeze –thaw cycles (Accelerated ageing)
were measured and compared with 0.1N hydrochloric acid.
This test was performed for accelerated stability testing of
Solubility determination in the various oils, surfactants and co nanoemulsion formulations. In this study the formulations were
surfactants exposed at two different temperatures i.e ‐21 0C and 210C for each
temperature cycles not than 24 hrs. For the better estimation of
2 ml of different oils was taken in small vials separately and excess
accelerated stability studies three such cycles should be run for each
amount of the drug was added to each vial. The vials were tightly
batch of formulation .The formulations which showed the maximum
stopper and were continuously stirred for 72 hrs in mechanical
stability were selected for further study.
shaker at 250C and after that, oils were centrifuged. The supernatant
was separated and dissolved in methanol and solubility was Dispersibility tests
quantified by UV‐Spectroscopy (Shimadzu 1701, Japan) method at
247 nm after appropriate dilution with methanol. The efficiency of self‐emulsification of oral nanoemulsion was
assessed using a standard USP XXII dissolution apparatus 2. [20].
Screening of formulations on the basis of thermodynamic One ml of each formulation was added to 500 mL of distilled water
stability studies
and in 0.1N HCl respectively at 37 ± 0.5 oC. A standard stainless
The thermodynamic stability studies were performed on the basis of steel dissolution paddle rotating at 50 rpm provided gentle
following tests. agitation. The in vitro performance of the formulations was
visually assessed using the following grading system (Table 5).
Centrifugation study Those formulations that passed the thermodynamic stability and
The selected formulations were centrifuged (REMI, India) at the also dispersibility test in Grade A were taken for the further
5000 rpm for 30 mins and observed for phase separation, creaming studies. Further from each Smix Group one formulation is selected,
or cracking. The formulations which showed maximum stability (no having the least Smix concentration irrespective of Smix ratio
creaming, cracking, phase seperation) were selected and studied for used, but passing dispersibility test in Grade A in distilled water as
heating‐cooling cycle, freeze‐thaw cycles and Dispersibility tests. well as in 0.1N HCl.
Table 3: Observation table of dispersibility study
S.No. Observation Grade
1 Rapidly forming (within 1 min) nanoemulsion, having a clear or slight bluish A
2 Rapidly forming, slightly less clear microemulsion, in bluish colour B
3 Fine milky emulsion that formed within 2 minutes C
4 Dull, grayish white emulsion having slightly oily appearance that is slow to emulsify (longer than 2 min). D
5 Formulation, exhibiting either poor or minimal emulsification with large oil globules present on the surface. E
Viscosity Table 4: Observation table of viscosity measurements
The viscosity of the prepared nanoemulsion formulations were Code Zero time After 120 sec
determined as such without dilution by Brookfield DV III ultra V6.0 Mean viscosity ± SD (cps) Mean viscosity ± SD (cps)
RV cone and plate rheometer (Brookfield Engineering Laboratories, GM1 27.51 ± 1.01 27.21 ± 0.93
Inc, Middleboro, MA) using spindle # CPE40 at 25 ±1.0°C. The GM2 21.32 ± 1.1 20.85 ± 0.64
software used for the viscosity calculations was Rheocalc V2.6. The GM3 10.3 ± 0.91 10.02 ± 0.63
results are shown in Table 6.4. GM4 28.22 ± 0.91 27.76 ± 1.75
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Int J Pharm Pharm Sci, Vol 3, Issue 3, 2011, 147152
Droplet size analysis (Particle size distribution) 200 KV and of a 0.18 nm capable of point to point resolution.
Combination of bright field imaging at increasing magnification and
Droplet size of the prepared nanoemulsion was determined by using of diffraction modes was used to reveal the form and size of the
photon correlation spectroscopy, which analyzes the fluctuations in nanoemulsion. In order to perform the TEM an observation, the
light scattering due to Brownian movement of the particles . The nanoemulsion formulation was diluted with distilled water (1/100).
formulation (0.1 mL) was dispersed in 50 mL (500 dilution) of A drop of the diluted nanoemulsion was directly deposited on the
distilled water in a volumetric flask and gently mixed by inverting Copper holey film grid and observed after putting fixing agent and
the flask and measurement done using a Zetasizer (Nano ZS‐90, UK). drying it in the filtered air.
Light scattering was monitored at 25°C at a 90° angle.
Table 5: Droplet size of the various emulsion formulations
S. No Formulation Code Particle Size
1 GM1 42.8±5.2 nm
2 GM2 195±10.5 nm
3 GM3 107±9.7 nm
4 GM4 81.6±6.1 nm
In vitro drug release study
In vitro release test was performed using Dialysis bag method and
release study was carried out in 250 ml of distilled water, 1 ml of
emulsion formulation (Single dose containing 10 mg of AT Calcium)
was placed in treated dialysis bag (MWCO 12,000 g/mole; Sigma,
USA) and 1 mL samples was withdrawn at regular time intervals (0,
0.5, 1, 1.5, 2,2.5,3,3.5, 4,4.5,5.5, 6,6.5,7, 8,9, 10, 12, 22 and 24 h) and
same amount of distilled water was replaced.[21‐22]. The
withdrawn 1 ml samples were diluted with 3 ml methanol and
analyzed for the drug content by using developed UV‐spectroscopy
at 247 nm. The same method was used for the suspension
Fig. 2: TEM of formulation GM1
containing 10 mg of AT Calcium in 1 ml distilled water. The release
Transmission electron microscopy of drug from different selected nano emulsion formulations was
compared with drug suspension and finally selected formulation
Morphology and structure of the nanoemulsion were studied using was used for the further study i.e. for preparation of solid self‐nano
transmission electron microscopy (TEM) TOPCON 002B operating at emulsifying drug delivery system (SSNEDDS).
Comparative dissolution profile of different formulations
120
100
% Cumulative release
80
60
40
20
0
0 5 10 15 20 25 30
Time (Hrs)
GM1 GM2
GM3 GM4
GM5 API suspension
Fig. 3: Invitro Drug Release of AT nano emulsion by dialysis bag method in distilled water
RESULTS AND DISCUSSION although the free energy required to form an emulsion is very low,
the formation is thermodynamically spontaneous.
The SEDDS of the drug Atorvastatin were prepared by emulsification
method. Constructing a phase diagram is one of the primary steps The relationship between the phase behaviour of a mixture and its
and makes a backbone for the SEEDS, particularly when the aim is to composition can be selected with the aid of a phase diagram. Sefsol
accurately delineate a phase boundary. Observations are made 218 (oil) and oleic acid (oil), Tween 20, Tween 80 (surfactants) and
carefully to separate metastable systems from phase boundary, Carbitol (co surfactants), were used to study the phase diagrams in
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Int J Pharm Pharm Sci, Vol 3, Issue 3, 2011, 147152
detail. The systems were observed for visual clarity and flowability is seen using TEM. Some equally distributed droplet sizes are
caracteristics. Those which did not show a change in the miniscus measured using TEM, as it is capable of point‐to‐point resolution.
after tilting to an angle of 90° were classified as nanogels a The droplet size is in agreement with the results obtained from
metastable system were not selected. After taking observation, droplet size analysis.
pseudoternary phase diagrams were constructed based on the
observations marked during titration. Phase diagrams were Dissolution study was performed by dialysis bag method in distilled
constructed separately for each ratio of Smix prepared so that o/w water for the final selection of formulation for further development
nanoemulsion regions could be identified. In the phase diagrams of solid self‐nanoemulsifying dosage forms. The dissolution study
(Figure. 6.1, A‐G) only o/w nanoemulsion region is shown. After was performed for 24 hrs. Drug dissolution from formulation GM1
building the backbone of the nanoemulsion delivery system, was very fast as 99.65±1.29% of drug released in 6.5 hrs; while
different formulations were selected at different point from the formulations GM2, GM3, GM4 and GM5 showed comparatively slow
phase diagram justifying the drug dose. release i.e. 82.36±2.12, 93.97±0.56, 72.69±0.81 and 61.06±1.31 in
6.5 hrs. In contrast to this drug released from API suspension was
Vigorous studies were done for the phase diagram construction, found to be very low i.e. 13.77±0.98% in 6.4 hrs. This result was
phase boundry were plotted on the ternary phase diagram. After attributed to the fact that formulation GM1 is having comparatively
building the backbone of the nanoemulsion delivery system, smaller size (42.8 nm) of oil droplets and hence the larger surface
different formulations were selected at different point from the area for dissolution as justified by droplet size distribution and by
pseudophase diagram which justified the drug dose considering the TEM photograph. The another possible reason was the oil
drug solubility in the oils phase. As per saturation solubility studies concentration in formulation GM1 (10%) as compared to the GM2
of AT Calcium in oily mixture, Sefsol 218: Oleic acid (1:1), around (15%), although having the same concentrations of the Smix, which
130 mg of drug can be solubilized per mL.The concentration 10% was not sufficient to emulsify the increased amount of oil in GM2.
(100 μL) of oil in 1 mL formulation is just able to solubilize 10 mg of
AT Calcium. Therefore 10% was selected as the least oil CONCLUSION
concentration to be taken for one mL formulation from the phase
diagram. The selected formulations shown were screened on the The formulation was found to be the optimized formulation on the
basis of the thermodynamic stability studies. base of results of pseudo ternary phase diagram, in vitro drug
release, droplet size and other parameters. The present study was
Nanoemulsions are thermodynamically stable systems and are clearly indicated that the usefulness of SEDDS in the improvement of
formed at a particular concentration of oil, surfactant and water, the dissolution rate and there by oral bioavailability of poorly water
with no phase separation, creaming or cracking. It is the soluble drugs like ATV without incompatibility between the
thermostability which differentiates nano or micro emulsion from ingredients.
emulsions that have kinetic stability and will eventually phase
separate. Thus, the selected formulations were subjected to different ACKNOWLEDGEMENT
thermodynamic stability by using heating cooling cycle, The authors are thankful to Suresh Gyan vihar University, Jaipur for
centrifugation and freeze thaw cycle stress tests. Those providing necessary support to this project and also thankful to
formulations, which survived thermodynamic stability tests, were Lupin Lab for the gift sample of atorvastatin.
taken for dispersibility test. Those formulations, which survived
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5. http://www.microfluidicscorp.com.
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152
Bulletin of Pharmaceutical Research 2011;1(2):xx-xx
An Official Publication of Association of Pharmacy Professionals
ISSN: 2249-6041 (Print)
RESEARCH ARTICLE
*E-mails: chouksey_rajendra@yahoo.com
Tel.: +91-9993815318
Received: June 30, 2011 / Revised: July 22, 2011 / Accepted: July 25, 2011
Atorvastatin is one of the most important hypolipidemic drug available today and circumventing the
major problem of its poor bioavailability remains a bigger challenge to pharmaceutical scientists.
The objective of the present study was to carry out pharmacokinetic studies of nanoemulsion
formulation of atorvastatin (AT) in order to assess the in vivo performance of developed formulation.
Optimized formulation was selected for in vivo study on the basis of higher drug release, optimum
globule size, minimum polydispersity value, lower viscosity, and overall lower surfactant and co-
surfactant concentration and exhibited better bioavailability as compared to pure drug.
Key words: Nanoemulsion, Atorvastatin, In vivo studies, Bioavailability.
INTRODUCTION
Nanoemulsions being colloidal nanodispersions drug, an important factor in sustained and
of oil in water (or water in oil), targeted drug delivery (Swarbrick and Boylan,
thermodynamically stabilized by an interfacial 1994). The attraction of o/w nanoemulsion
film of surfactant(s) and co-surfactant(s) have systems lies in their ability to incorporate
revealed tremendous potential in hydrophobic drugs into the oil phase thereby
nanoengineering of various inorganic materials enhancing their solubility. Nanoemulsions have
(Date and Patravale, 2004). Droplet size in been reported to make the plasma concentration
thermodynamically stable nanoemulsions is profiles and bioavailability of drugs more
usually 10-100 nm (Sintov and Shapiro, 2004). reproducible (Kawakami et al 2002;
The homogeneous systems that can be prepared Constantinides, 1995; Lawrence and Rees,
over a wide range of surfactant concentrations 2000).
and oil to water ratios (1:4) are all fluids of low The design of effective formulation for drugs has
viscosity. Nanoemulsion provides ultra low long been a major challenge, because drug
interfacial tension and large o/w interfacial efficacy can be severely limited by instability or
areas. Nanoemulsions have a higher poor solubility in the vehicle. Although solid
solubilization capacity than simple micellar dispersion is one of the most widely used
solutions and their thermodynamic stability technique for solubility enhancement (Pabreja
offers advantages over unstable dispersions, and Dua, 2011; Dahiya and Kaushik, 2010),
such as emulsions and suspensions, because nanoemulsions being a versatile technology have
they can be manufactured with little energy the greater potential to increase the
input (low energy emulsification bioavailability of drug in many ways. They act as
techniques/heat or mixing) and has a long shelf supersolvents for poorly soluble drugs.
life. The nanosized droplets leading to enormous Nanoemulsions have a higher solubilization
interfacial areas associated with nanoemulsions capacity than simple micellar solutions and their
would influence the transport properties of the thermodynamic stability offers advantages over
1
Chouksey et al Bull. Pharm. Res. 2011;1(2)
unstable dispersions, such as emulsions and observed for phase separation, creaming or
suspensions, because they can be manufactured cracking. The formulations which showed
with little energy input (heat or mixing) and has maximum stability (no creaming, cracking, phase
a long shelf life thus scale up technology is easy. separation) were selected and studied for
The nanosized droplets leading to enormous heating-cooling cycle, freeze-thaw cycles and
interfacial areas associated with nanoemulsions dispersibility tests.
would influence the transport properties of the
drug, an important factor in sustained and Accelerated ageing studies
targeted drug delivery. The droplet size of This test was performed using freeze-thaw
nanoemulsion is between 14 to 100 nm, and can cycles for accelerated stability testing of
be sterilized by filtration. The use of nanoemulsion formulations after ageing. In this
nanoemulsion as drug delivery systems can study the formulations were exposed at two
improve the efficacy of drug, allowing the total different temperatures i.e. -21°C and 21°C, for
dose to be reduced and thus minimizing side each temperature cycles was not for more than
effects. Atorvastatin, a synthetic cholesterol- 24 h. For the better estimation of accelerated
lowering agent, is a HMG-CoA (3-hydroxy-3- stability studies, three such cycles run for each
methylglutaryl-coenzyme A) reductase inhibitor. batch of formulation. The formulations which
The calcium salt of atorvastatin is used in the showed the maximum stability were selected for
treatment of primary hypercholesterolemia and further study.
dyslipidemia. Atorvastatin is highly lipophillic in
nature (log P (octanol/water), 6.36), freely Viscosity measurements
soluble in methanol and soluble in The viscosity of the prepared nanoemulsion
dimethylsulphoxide (DMSO) and dimethyl formulations were determined as such without
formamide. It is soluble in aqueous medium with dilution by Brookfield DV III ultra V6.0 RV cone
a pH of less than 4.0. It is very slightly soluble in and plate rheometer (Brookfield Engineering
water, phosphate buffer (7.4) and acetonitrile; Laboratories, Inc, Middleboro, MA) using spindle
while it is slightly soluble in ethanol with # CPE40 at 25 ±0.5°C. The software used for the
absolute bioavailability of mere 14%. viscosity calculations was Rheocalc V2.6.
Atorvastatin is drug of choice among
hypolipidemic drug available today and Droplet size distribution
circumventing the major problem of its poor Droplet size of the prepared nanoemulsion was
bioavailability remains a bigger challenge of determined by using photon correlation
pharmaceutical scientists. With this background spectroscopy, which analyzes the fluctuations in
in mind, the objective of the present study was to light scattering due to Brownian movement of
develop and characterize an optimal stable the particles (Attwood et al 1992). The
nanoemulsion formulation of atorvastatin with formulation (0.1 ml) was dispersed in 50 ml
an aim to increase its bioavailability. The in vitro (500 dilution) of distilled water in a volumetric
assessment of nanoemulsion formulation of flask and gently mixed by inverting the flask and
atorvastatin has been published. measurement done using a Zetasizer (Nano ZS-
90, UK). Light scattering was monitored at 25°C
MATERIALS AND METHODS at a 90° angle.
Development of nanoemulsion formulation
The drug stock solutions in oil mixture were In-vitro drug release performance
prepared in such a way that 10 mg dose was The study was performed by using dialysis bag
present in each formulation complying the oil method (Shafiq et al 2007). The dialysis
percentage for each formula as selected from the membrane used in the study was Cellulose
phase diagram. This was prepared by dissolving membrane (Sigma, USA) with a molecular weight
the 1000 mg of drug individually in the 10, 15, cut off of 12000 g/mol. In vitro release test was
20 and 25 ml of oily mixture, which compiles the performed in 250 ml of distilled water, which
10%, 15%, 20% and 25% oil compositions was based on USP XXIV method at 100 rpm and
respectively in the formulae. 37±0.5°C. One ml of nanoemulsion formulation
(single dose containing 10 mg of AT Calcium)
Evaluation of nanoemulsion was placed in treated dialysis bag and 1 ml
The selected formulations were centrifuged samples were withdrawn at regular time
(REMI, India) at the 5000 rpm for 30 min and intervals (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5.5, 6,
2
Chouksey et al Bull. Pharm. Res. 2011;1(2)
6.5, 7, 8, 9, 10, 12, 22 and 24 h) and same Pharmacokinetic and statistical analysis
amount of distilled water was replaced. The Pharmacokinetic parameters were calculated by
withdrawn 1 ml samples were diluted with 3 ml using non-compartmental analysis using
methanol and analyzed for the drug content by WinNonLin version 4.0 (Pharsight Corp.,
using developed RP-HPLC method at 247 nm. Mountain View, CA). All pharmacokinetic (PK)
The same method was used for the suspension parameters (tmax, Cmax, AUC0→t,) were calculated
containing 10 mg of AT Calcium in 1 ml distilled individually for each subject in the group and the
water. values were expressed as mean±SD.
rate and extent of absorption as compared to v/v), Carbitol (19% v/v) and distilled water
marketed formulations. The composition of (52% v/v) as oil, surfactant, co-surfactant and
optimized formulation (GM1) was safsol 218 aqueous phase respectively, containing 10 mg of
(5% v/v), Oleic acid (5% v/v), Tween-20 (19% AT Calcium.
50000
40000
Concentration (ng/ml)
30000
20000
10000
0
0 0.5 1 1.5 12
36
Table 1. Comparative observation table for in vivo bioavailability studies in rat animal model*
Sampling time GM1 Tablet suspension API suspension
(h) (Mean±SD) (Mean±SD) (Mean±SD)
0 0 0 0
0.25 24719.36±142.54 4747.476±63.76 3636.91±57.56
0.5 35582.73±345.54 5491.985±36.87 4307.00±24.87
0.75 37687.34±733.16 5815.137±62.97 4456.82±54.89
1 41090.76±531.78 6343.85±64.86 4959.85±86.36
1.25 43603.77±632.63 6750.599±145.35 5271.45±75.37
1.5 37508.95±456.56 5801.549±48.25 4345.85±86.74
6 24312.26±731.84 3761.141±65.36 2887.23±75.36
12 12093.38±234.47 1870.322±124.12 1437.17±44.66
24 6370.94±32.47 987.3089±32.85 765.24±75.05
36 3851.75±42.74 599.0169±24.86 548.16±36.87
48 2811.38±57.38 436.0885±14.60 338.09±23.21
*(n=6)
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