International Journal of Pharmaceutics 462 (2014) 129–134

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Pharmaceutical Nanotechnology

Anti HIV nanoemulsion formulation: Optimization and in vitro–in vivo evaluation Sabna Kotta a , Abdul Wadood Khan a , Shahid H. Ansari b , Rakesh Kumar Sharma c , Javed Ali a,∗ a

Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India Department of Pharmacognosy & Phytochemistry, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India c Division of CBRN Defence, Institute of Nuclear Medicine and Allied Sciences, Brig SK Mazumdar Marg, New Delhi 110054, India b

a r t i c l e

i n f o

Article history: Received 30 October 2013 Received in revised form 13 December 2013 Accepted 18 December 2013 Available online 25 December 2013 Keywords: Nanoemulsion Efavirenz Condensation method Phase inversion composition

a b s t r a c t The objective of the present work is to develop a dose adjustable nanotechnology based liquid formulation of efavirenz with improved bioavailability for HIV therapy. Nanoemulsion of efavirenz was developed using phase inversion composition method with the help of ternary phase diagram. Globule size of the o/w nanoemulsion was studied with the help of dynamic light scattering and further confirmed with TEM analysis. Optimized formulations were subjected for in vitro dissolution studies and in vivo studies were done in rats to calculate pharmacokinetics parameters and compared with efavirenz suspension. TEM results revealed that the globule size of optimized formulation was less than 30 nm. In vitro release profile showed more than 80% release within 6 h which was highly significant (p > 0.05) and pharmacokinetic studies also proved a promising in vivo absorption profile when compared to the efavirenz suspension. The developed nanoemulsion proved to be an effective dose adjustable formulation of efavirenz for pediatric HIV therapy. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Acquired immunodeficiency syndrome (AIDS) is a set of symptoms and infections resulting from the specific damage to the immune system caused by infection with the human immunodeficiency virus (HIV) which is a retrovirus of the lenitivirus family. HIV is a retrovirus that mainly infects vital components of the human immune system such as CD4+ T cells, macrophages and dendritic cells. It directly and indirectly destroys CD4+ T cells. CD4+ T cells are required for the proper functioning of the immune system. Forty million people infected with human immunodeficiency virus type 1 (HIV-1) globally. AIDS has claimed over 25 million lives since its discovery in 1981. Prevalence of the disease is extremely high in resource-constrained countries therefore development of scalable and cost-effective anti retroviral (ARV) medicines is a crucial access to enable patients to receive the appropriate medication. Most of the current clinical therapies have bioavailability issues either due to poor solubility or due to extreme first pass metabolism. Poor drug availability in the cellular and anatomical reservoirs is affected by expression of efflux transporters (e.g., P-glycoprotein), presence of

∗ Corresponding author. Tel.: +91 811312247; fax: +91 126059633. E-mail addresses: [email protected], [email protected] (J. Ali). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.12.038

drug metabolizing enzymes (e.g., cytochrome P-450), poor permeability properties, non-targeted distribution, and rapid clearance. The reduced bioavailability and short residence of anti retroviral agents have profound impact on the clinical management of the disease. The overall consequence is that upon discontinuation of therapy or when drug resistance develops, HIV is able to re-seed the systemic circulation and continues to propagate the infection (Clarke et al., 2000; Kulkovsky and Bray, 2006). Therefore there is a need for a delivery system to overcome the solubility and bioavailability issues. Efavirenz is HIV-1 specific and a first-choice anti retroviral (ARV) in adult and pediatric pharmacotherapy. Efavirenz inhibits the activity of viral RNA-directed DNA polymerase (i.e., reverse transcriptase). It is highly lipophilic, non-nucleoside reverse transcriptase inhibitor (NNRTI) which falls under class II according to Biopharmaceutical classification system with an oral bioavailability of about 40%. The low solubility in water and lower absorption in gastric fluid is the reason for low bioavailability (Madhavi et al., 2011; WHO Public Assessment Reports). Vedha Hari et al. (2012) formulated efavirenz nanoparticles using methacrylate polymers (Eudragit E100) by emulsion solvent evaporation method and succeeded in attaining high bioavailability with low dose of efavirenz. Similarly Jain et al. (2013) prepared surface stabilized nanoparticles for oral bioavailability enhancement

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of efavirenz and found that a 2.02-folds increase in peak plasma concentration and 2.29-fold increase in AUC(0–infinity). But dose adjustable liquid formulation is desired particularly for pediatric patients and hence present work deals with development of a nanoemulsion formulation made using nanotechnology showing more than 2.5-fold increases in bioavailability when compared with drug suspension. The use of nanotechnology platforms for delivery of drugs is revolutionizing medicine in many areas of disease treatment (Farokhzad and Langer, 2009). The utility of lipid based oral formulations to augment the absorption of poorly water soluble, lipophilic drugs has been renowned for many years. More recently, experience with the effective development of self-emulsifying or nanoemulsion based lipidic formulations for cyclosporin (sandimmun Neoral−, Novartis Pharmaceutical Ltd., Surrey, UK), and subsequently for ritonavir (Norvir® , Abbott Laboratories, IL, USA), and saquinavir (Fortovase® , Roche Pharmaceuticals, NJ, USA) has stimulated a significant increase in the application of lipid based formulations for the improved oral delivery of poorly water soluble drugs. In fact, the most popular approach is the incorporation of the active lipophilic component into inert lipid vehicles such as oils, surfactant dispersions, nanoemulsions, self-emulsifying formulations, self microemulsifying formulations, emulsions, liposomes and self nanoemulsifying systems. Among these approaches, nanoemulsions can improve the bioavailability by increasing the solubility of hydrophobic drugs very effectively (Parveen et al., 2011; Bali et al., 2010; Vyas et al., 2002). Nanoemulsions are stable isotropic systems of two immiscible liquids and are prepared by means of appropriate surfactants with a droplet diameter approximately less than 100 nm. In order to enhance delivery of efavirenz to systemic circulation, in the present study, we have formulated nanoemulsions with a droplet size of less than 30 nm with propylene glycol monocaprylate as oil phase. To examine oral bioavailability optimized nanoemulsion administered orally to adult female albino Wistar rats. Control preparation of efavirenz was made as aqueous suspension with 0.5% sodium carboxy methyl cellulose for comparison purpose. 2. Materials and methods Efavirenz was gifted by Lupin Ltd. (Pune, India). Capryol 90 (propylene glycol monocaprylate) and Transcutol® HP (diethylene glycol monoethyl ether) were gifted by Gattefosse (Saint Priest, Cedex, France). Tween 20 (poly-oxyethylene sorbitan monolaurate) was purchased from Merck (Schuchardh, Hokenbrunn, Germany). Water was obtained from Milli-Q-water purification system (Millipore, MA, USA). All other chemicals and reagents were of analytical grade and procured from Merck (Mumbai, India) and S.D. Fine Chem. (Mumbai, India). 3. Preparation of the nanoemulsions Based on the results of solubility studies, propylene glycol monocaprylate having an HLB value of 6 was used as the oil phase for the development of nanoemulsion. Drug was incorporated in the oil phase and was used for the formulation of nanoemulsion. Nanoemulsions were prepared by phase inversion composition method. Geucire 44/14 was used as surfactant and Transcutol® HP is selected as the cosurfactant. Surfactant and cosurfactant were mixed (Smix ) in 1:0, 2:1, 3:1 and 4:1 ratio. Double distilled water was used as the aqueous phase for the formulation. The oil and Smix was thoroughly mixed with the help of a vortex mixer. Nanoemulsions were prepared by stepwise addition of water to oil/surfactant mixtures at 25 ◦ C. The region of formation of O/W nano-emulsions in

the water/surfactant/oil system was assessed visually. The amount of aqueous phase was incremented to provide concentration of aqueous phase above 50% of total volume. After each addition of aqueous phase, physical state of the mixture was marked whether it is transparent or opaque (Parveen et al., 2011; Bali et al., 2010). 4. Preparation of efavirenz aqueous suspension For the comparison of bioavailability efavirenz aqueous suspension was prepared with 0.5% sodium carboxy methyl cellulose as a suspending agent. 5. Accelerated physical stability studies Stability of nanoemulsions was studied using heating–cooling cycles, centrifugation, and freeze–thaw cycle stress tests. Heating–cooling cycles between 45 ◦ C temperature and room temperature (25 ± 2 ◦ C) with storage time of 24 h at each temperature (six cycles each) followed by centrifugation (5000 rpm for 30 min) and then freeze–thaw cycles at −20 ◦ C in a deep freezer (Vest frost, Hyderabad, India) and room temperature (25 ± 2 ◦ C) for 24 h were carried out six times (six cycles each) (Bali et al., 2010, 2011). The nanoemulsions that were stable were considered for further studies (Ballesteros and Frutos, 2003; Bali et al., 2011). 6. Characterization of the nanoemulsions 6.1. Globule size analysis The mean volume diameter of the nanoemulsion was determined by photon correlation spectroscopy (PCS), also known as dynamic light scattering (DLS), using Malvern Zetasizer 1000 HS (Malvern Instruments, Worcestershire, UK). The formulation was diluted with distilled water and sonicated in a bath sonicator for 10 min prior to analysis. Light scattering was monitored at 25 ◦ C in triplicate at a scattering angle of 90◦ (Brusewitza et al., 2007). 6.2. Effect on dilution on physicochemical stability of nanoemulsion Since our investigation pursues the development of oral liquid formulations, in order to determine their ability to withstand dilution in the gastric environment, efavirenz nanoemulsion were diluted (1/100) in stomach-mimicking conditions (HCl 0.1 N, pH 1.5), in a standard USP XXII dissolution apparatus 2 (Veego, Mumbai, India). A standard stainless steel dissolution paddle rotating at 50 rpm provided gentle agitation at 37 ± 0.5 ◦ C and then observed transparency by measuring percentage transmittance spectrophotometrically using Shimadzu UV-Vis spectrophotometer (Chiappetta et al., 2009). 6.3. Transmission electron microscopy (TEM) The morphology of the oil droplets in the nanoemulsion formulations was visualized with TEM analysis. TEM analysis was also significant in order to visualize any precipitation of the drug upon addition of the aqueous phase. The nanoemulsion was diluted 100 times and a drop was applied to 300-mesh copper grid. The grid was left for 1 min. The grid was inverted and a drop of phosphotungstic acid (PTA) was applied to the grid for 10 s. Excess of PTA was removed by absorbing on a filter paper and the grid was analyzed using Morgagni 268D (FEI Company, OR, USA) operated at 60–80 kV at 1550× magnification. 6.4. Viscosity determination The viscosity of the formulations (0.5 ml) was determined without dilution with Brookfield DV III ultra V6.0 RV Cone and Plate

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Rheometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA) using spindle #CPE40 at 25 ± 0.5 ◦ C. The software used for the viscosity calculations was Rheocalc V2.6. For each sample, continuous variation of shear rate (80–400 s−1 ) was applied and the resulting shear stress was measured (Ammar et al., 2009).

significantly lower than the maximal recommended (3.5 ml), and therefore we assume that the blood loss during our experimental protocol did not affect the pharmacokinetic properties of efavirenz.

6.5. Refractive index

HPLC method was used for the analysis of efavirenz for solubility studies, in vitro release as well as for in vivo evaluation. The software used in the system was Class VP, version 5.032. C18 reverse phase column (250 mm × 4.6 mm, particle size 5 ␮m; Merck, Darmstadt, Germany) with a PDA detector (Waters 2998) at 247 nm was used for analysis. Mobile phase was 0.1 M formic acid and acetonitrile (45:55, v/v) with a flow rate of 1 mL min−1 and retention time was 9 ± 0.4 min with a run time of 15 min. The method was validated as per ICH guidelines Samples for solubility studies and in vitro release studies were directly injected after proper dilution and filtration. Plasma samples (100 ␮l) were transferred to 1.5 ml centrifuge tubes (Eppendorff AG, Hamburg, Germany) and the plasma was precipitated with 200 ␮l of IS solution. 1 ml of ethyl acetate was added to the resulting mixture and vortexed for 50 s and centrifuged at 10,000 × g for 10 min. The organic layer transferred to a sample vial, evaporated with a stream of nitrogen for 15 min at 40 ◦ C, reconstituted with 0.5 ml of mobile phase, vortexed and analyzed (Mogatle and Kanfer, 2009; Sailaja et al., 2007). The method was validated for the bioanalytical method as per ICH guidelines.

The refractive index of the system was measured by an Abbe refractometer (Bausch and Lomb Optical Company, Rochester, NY) by placing one drop of the formulation on the slide in triplicate at 25 ◦ C. 7. In vitro drug release Dissolution studies were performed to determine the extent of drug release from the formulations. In vitro release studies were performed in 900 mL of distilled water containing 2% sodium lauryl sulphate (SLS) using dissolution apparatus type II (Veego Scientific, Mumbai, India) at 50 rpm and 37 ± 0.5 ◦ C (USP, 2006). Nanoemulsion formulation (5 mL) was placed in treated dialysis bag (MWCO 12,000 g/mol, Sigma Aldrich, USA). Samples (5 ml) were withdrawn at regular time intervals at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6 and 8 h. An aliquot amount of dissolution medium was replaced. The samples were analyzed for the drug content using HPLC (Shimadzu LC-10AT VP, Kyoto, Japan) having a UV detector at 247 nm. 8. In vivo evaluation of the optimized nanoemulsion 8.1. Animal protocol for oral and intravenous administration Approval for experimental protocol to carry out in vivo study was obtained from Jamia Hamdard, Institutional Animal Ethics Committee, New Delhi and their guidelines were adhered for the complete study (Registration No. 841/CPCSEA, 2011). The animals used for in vivo experiments were adult Wistar albino rats (150–200 g) obtained from Central Animal House of Hamdard University, New Delhi, India. The animals were housed in a climatecontrolled environment with full access to food and water. Prior to any experimentation, the animals were allowed to acclimate for at least 48 h. Experiments were performed in rats fasted for 12 h (n = 6 for each group). For the oral absorption study of efavirenz aqueous suspension (control) and the nanoemulsions formulations, the animals were randomly divided into three groups each containing 6 animals to receive control (placebo nanoemulsion), standard (efavirenz suspension) and optimized efavirenz nanoemulsion. Drug formulations were orally administered by gavage, where a stomach tube is inserted into the esophagus of conscious rats. Each conscious animal was administered with dose which is equivalent to the human dose of 200 mg of efavirenz nanoemulsion or control aqueous suspension as per the body weight. So each rat was administered with 18 mg/kg of efavirenz nanoemulsion or control aqueous suspension as per the body weight which was calculated on the basis of comparison of surface area of animal to the surface are of an adult human factored with metabolic rate (Reagan-Shaw and Ahmad, 2008). The rats were anesthetized using ether and blood samples withdrawn at 0 (pre-dose), 1, 2, 4, 6, 8 and 24 h from the tail vein of rat in to EDTA tubes. The collected blood was centrifuged at 5000 rpm for 20 min to separate plasma. After extraction drug analysis was carried out using HPLC (Shimadzu LC-10AT VP, Kyoto, Japan) at 247 nm. It is significant to mention that blood sampling could alter pharmacokinetic and pharmacodynamic behavior of drugs due to fluid loss. In this experimental protocol, total blood volume extracted was approximately 700 ␮l during 24 h. This volume is

8.2. Chromatographic method for efavirenz analysis

8.3. Non-compartmental pharmacokinetic analysis Non-compartmental pharmacokinetic analysis of efavirenz following oral administration of nanoemulsion and suspension was performed and compared. Pharmacokinetic parameters such as the maximum plasma concentration (Cmax ), time to reach maximum concentration (Tmax ), half-life (t1/2 ), area-under-the-curve from zero to 24 h (AUC0→24 ), area-under-the-curve from zero to infinity (AUC0→∞) were calculated. Since the same dose was administered the relative bioavailability values of efavirenz in the nanoemulsion formulations were determined relative to the aqueous suspension. 8.4. Data analysis All the values are reported as mean ± SEM from at least four independent experiments. The statistical differences between the groups were tested using Student’s t-test and, with more than two groups, ANOVA was used to compare results. Experimental results were considered statistically significant at 95% confidence (i.e., p < 0.05). 9. Results Efavirenz nanoemulsions were prepared using Propylene glycol monocaprylate as oil phase, Geucire 44/14 as surfactant and Transcutol HP® as cosurfactant. Since the drug exhibited highest solubility in Capryol 90 among the oils used for solubility studies, it was selected as the oil phase for the formulation of nanoemulsion. Surfactant and cosurfactant were selected based on the miscibility with the selected oil phase. Complete miscibility with the oil phase is necessary to obtain a clear and transparent nanoemulsion. The obtained nanoemulsions were clearly identified by checking the transparency of the formulation visually. The selected formulations were subjected to accelerated physical stability testing. Results are shown in Table 1. Based on the observation accelerated physical stability testing, six different formulations were selected in which the Smix was minimum. Table 2 shows the % composition of oil, Smix and water of the six selected nanoemulsions those passed the accelerated physical stability test. Results of dilution test confirmed the high physical stability of the efavirenz nanoemulsion

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Table 1 Observation for thermodynamic stability study and dispersibility test for formulations prepared by high energy method. Formulation 1:0

2:1

1:9 1:8 1:7 1:6 1:5 1:4

3:1

1:9 1:8 1:7 1:6 1:5 1:4

4:1



Heating–cooling cycle. √ √ √ √ √ √ √

1:9 1:8 1:7 1:6 1:5 1:4 1:3.5

1:9 1:8 1:7 1:6 1:5

Centrifugation √ √ √ √

Freeze–thaw cycle. √ √ √ √

Dispersibility √ √ √ √

Inference √ √ √ √

× ×

× × ×

× × ×

× × × × × × √ √ √ √

× × × × × × √ √ √ √

× × × × × × √ √ √ √

√ √ √ √

× × × × × × √ √ √ √

× × √ √ √ √ √

× × √ √ √

× × √ √ √

√ √ √

× × √ √ √

× ×

× ×

× ×

indicates passed and × indicates failed batches.

Table 2 Percentage composition of oil, Smix and water of the selected formulations. Formulation

Geucire %

Transcutol %

Water %

Oil: Smix

F1 F2 F3 F4 F5 F6

9.534 11.44 10.725 12.87 13.728 17.16

4.767 5.72 3.575 4.29 3.432 0

82.84 80.02 82.84 79.98 79.98 79.98

1:5 1:6 1:5 1:6 1:6 1:6

Table 3 Mean globule size and polydispersity index. Formulation

Mean globule size (nm) ± SEM (n = 3)

F1 F2 F3 F4 F5 F6

54.610 28.833 65.483 27.007 26.427 49.430

± ± ± ± ± ±

1.927 2.084 1.978 2.435 1.960 3.346

Polydispersity index (PDI) ± SEM (n = 3) 0.160 0.417 0.153 0.252 0.117 0.185

± ± ± ± ± ±

Fig. 1. TEM image of optimized formulation (F5).

0.0038 0.0034 0.0040 0.0064 0.0034 0.0046

under dilution, proved that they could be diluted in GI fluids still maintaining the nanosized character without drug precipitation. Thus it is anticipated that absorption will be enhanced. The optimized nanoemulsion formulations were characterized for percentage transmittance viscosity, refractive index, droplet size and size distribution. All selected formulation showed a value of percentage transmittance closer to 100%, which indicated that all of the formulations were clear and transparent. Viscosity studies led to the conclusion that the selected nanoemulsions had a very low viscosity. The value of refractive index for the optimized formulations was found to be close to 1.42, the refractive index of water. This suggested the isotropic nature of the selected formulations. From the particle size distribution studies (Table 3), it was concluded that there was a very significant difference (p < 0.001) between the particle size. Formulations F2, F4 and F5 showed the small particle size and least polydispersity index of 28.833 ± 2.084 and 0.417 ± 0.0034, 27.007 ± 2.435 and 0.252 ± 0.0064 and 26.427 ± 1.960 nm and 0.117 ± 0.0034

Fig. 2. In vitro drug release profile.

respectively. TEM photographs (Fig. 1) of the formulations showed nanosized droplets which were in agreement with the results obtained using dynamic light scattering. The formulation F5 showed the highest drug release (90.02 ± 3.2) among the formulations selected (Fig. 2). Therefore, the formulation F5 containing Capryol 90 (2.86%, v/v), Gelucire (13.728%, v/v), Transcutol® HP (3.432%, v/v) and double distilled water (79.98%, v/v) showed the

S. Kotta et al. / International Journal of Pharmaceutics 462 (2014) 129–134

Fig. 3. Plasma concentration time profile. Table 4 Pharmacokinetic formulations.

parameters

(mean ± SEM,

n = 6)

of

different

efavirenz

Parameter

Efz nanoemulsion (F5)

Efz suspension

tmax (h) Cmax (␮g/mL) AUC0→24h (␮g h/mL) AUC0→∞ (␮g h/mL) T1/2 (h)

4 3.12 ± 0.0596 43.53 ± 3.134 101.59 ± 3.584 36.59 ± 2.26

4 1.9 ± 0.066 20.65 ± 3.238 39.17 ± 4.189 23.78.0 ± 2.77

highest drug release least droplet size (26.427 ± 1.960 nm), minimum polydispersity index (0.117 ± 0.0034) and lowest viscosity (30.60 ± 1.98 cP) was selected for the in vivo studies. The plasma concentration time profile of efavirenz in adult albino Wistar rats following oral administration of the nanoemulsion formulations (F5) was compared with the plasma concentration time profile (Fig. 3) obtained following administration of efavirenz suspension. All the pharmacokinetic parameters were calculated and compared statistically and a significant difference was observed (Table 4). The Cmax of nanoemulsion formulation F5 was extremely significant (p < 0.0001) in comparison to that of drug suspension. AUC0→24h of F5 was also found to be extremely significant (p < 0.001) in comparison to that of drug suspension. The value of tmax for the formulation F5 and drug suspension was found to be 4 h. The in vivo studies conducted to compare bioavailability of efavirenz from nanoemulsion formulations demonstrated significantly (p < 0.001) greater extent of absorption than that from drug suspension. The absorption of efavirenz from nanoemulsion F5 resulted in 2.6-fold increase in bioavailability as compared to the drug suspension. 10. Discussion Poor water solubility and consequent limited absorption is a major limitation with many drugs despite their good therapeutic efficacy. Nanoemulsion provides an opportunity for the improvement in the in vitro and in vivo performance of poorly water soluble drugs. And thus serve as an ideal carrier for the delivery of drugs belonging to BCS classes II and IV. The use nanoemulsion for the delivery of efavirenz could improve its solubility and permeability through the mucous membranes significantly. The current study was performed to prove the role of nanoemulsion to enhance the bioavailability of efavirenz, a first choice antiretroviral agent for adult and pediatric pharmacotherapy of AIDS. Pharmaceutical acceptability of excipients and the non-toxicity issues of the components used make the formulation of nanoemulsion are actually significant. There is huge restriction in selection of the components which are acceptable for oral formulations. It is reported that large amounts of surfactants, generally ionic

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surfactants, cause irritation, therefore for oral drug delivery, nonionic surfactants are preferred in a small concentration as possible (Constantinides, 1995). Therefore, selection of formulations was based on the criterion of their being a minimum concentration of Smix used in the formulation. So the selected formulations contains minimum amount of surfactant mixture to emulsify the oil which contain the desired dose of drug. Emulsions that have water as a continuous phase and oil as a dispersed phase are called ‘direct’, ‘water-based’, or ‘O/W’ emulsions. For direct emulsions, the surfactant is generally soluble in the aqueous phase and provides more stability of water films. But, emulsions that have oil as a continuous phase are called ‘inverse’, ‘oil-based’, or ‘W/O’ emulsions, here the surfactant is generally soluble in the oil phase and provides more stability of oil films (Mason et al., 2006). In the present study we have formulated and optimized a direct nanoemulsion of efavirenz. Excipients chosen to formulate oral nanoemulsion in the present study were of definite regulatory status. Efavirenz possess the highest solubility in propylene glycol monocaprylate (PGM) (749.88 ± 21.89 mg/mL); hence, PGM was selected as oil phase for nanoemulsion formulation. PGM caprylate is a mixture of the propylene glycol monoesters and diesters of fatty acids composed predominately of caprylic acid. It is medium-chain (6–12 carbons) fatty acid esters of propylene glycol (Gattefosse, 2013; Chemicalland21, 2013). Medium chain triglycerides (MCT) are commonly used in nanoemulsion formulation. The majority of them does not require micelle-containing bile salts or chylomicron formation, but are directly absorbed into the liver via the portal vein rather than through the thoracic duct lymph system that is the conventional route for the absorption of triglycerides containing long-chain fatty acids. MCT possess higher ester content per gram than long chain triglycerides (LCT) so drugs have higher solubility in MCT than LCT (Bath et al., 1996). The selection of surfactant and co-surfactant in the study was chosen based on their emulsification efficiency. Safety is the main decisive factor in choosing a surfactant. Non-ionic surfactants are comparatively less toxic and less affected by pH and ionic strength than ionic surfactants. The emulsification ability is much more related to the hydrophilic–lipophilic balance (HLB) value of the surfactant. Surfactants with HLB value >10 are greatly superior at providing fine, uniform nanosclae droplets (Mac Gregor et al., 1997). The right blend of low and high hydrophilic lipophilic balance (HLB) surfactants leads to the formation of stable nanoemulsion formulations (Craig et al., 1995). The surfactant and cosurfactant used the study are Gelucire 44/14 and Transcutol HP with HLB value of 14 and 4.2, respectively. Gelucire44/14 is EP Lauroyl macrogol-32 glycerides EP is a non-ionic water dispersible surfactant composed of well-characterized PEG-esters, a small glyceride fraction and free PEG and Transcutol HP is highly purified diethylene glycol monoethyl ether EP/NF. Surfactants increase the permeability by interfering with the lipid bilayer of the epithelial cell membrane. Cosurfactants can decrease interfacial tension between oil and water in nanoemulsion, adjust the flexibility of interfacial membrane and reduce the required amount of surfactant sometimes. Slow addition of the aqueous phase to the oil surfactant mixture with the help of a vortex mixture results in the formation of fine oilin-water nanoemulsion. Since the free energy required to form an emulsion is very low, the formation is thermodynamically spontaneous (Kim and Ku, 2000; Craig et al., 1995). Surfactants form a layer around the emulsion droplets and reduce the interfacial energy as well as provide a mechanical barrier to coalescence. The nanoemulsions which were stable after heating–cooling cycles selected for the centrifugation studies. The centrifugation

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test showed that the tested nanoemulsions had good physical stability. Through, turbidity was observed in freeze–thaw cycle stress test when the nanoemulsions were stored at −21 ◦ C. This may be due to the coagulation of the internal phase at low temperature; however, these nanoemulsions were easily recovered by storing at ambient temperature. Despite the dilution and acidification in the gastric environment, efavirenz nanoemulsion is not expected to dissociate precipitate drug substantially in their transit through the stomach. The values of percentage transmittance indicated that the selected formulations are clear and transparent. Optimum viscosity is a vital factor in the formulation of liquid dosage forms. The refractive indices of the nanoemulsion samples taken from three different regions of the sample are almost same as indicated by low standard deviations confirms the isotropic nature of nanoemulsion. The droplet size of all the selected formulations was noted to be less than 70 nm. Droplet size of the F2, F4 and F5 was significantly less than F1, F3 and F6 (p < 0.001). This is because of slightly higher amount of surfactants, i.e., 17% instead of 13% as in the case of F1, F3 and F6. Dissolution profile of all nanoemulsion formulations was significantly higher (p < 0.001) than efavirenz suspension. Among these F5 exhibited the highest drug release in 2% SLS in distilled water. Thus, an improvement in absorption and consequently increased bioavailability due to enhanced dissolution could be anticipated. In vivo testing of the optimized nanoemulsion F5 was done in healthy female albino Wistar rats. The mean efavirenz plasma concentration–time profile following oral administration of F5 and drug suspension are shown in Fig. 2, and comparative results of pharmacokinetic parameters are presented in Table 4. Pharmacokinetic analysis of efavirenz plasma levels after oral administration demonstrated that efavirenz nanoemulsion greatly improved the bioavailability of the drug; both the AUC and Cmax of efavirenz nanoemulsion (F5) were significantly higher than the drug suspension. The tmax parameter was similar (4 h) for all the tested formulations suggested that nanoemulsion increased the amount of efavirenz absorbed without changing the rate of absorption. An additional advantage of nanoemulsion compared with the suspension is the reduction in intersubject variability. Both the AUC0→24h and Cmax for nanoemulsion showed significantly lower variability with regards to the suspension. The mean AUC0→24h and AUC0→∞ of efavirenz nanoemulsion were 2.87 times higher than the drug suspension of efavirenz; thus, bioavailability-enhancing capacity of efavirenz nanoemulsion could be successfully proven. Acknowledgements The authors are grateful to the Defense Research Development Organization (DRDO), Government of India for providing a fellowship as financial assistance to Sabna Kotta. A portion of this work has been presented as a poster in FIP world Congress-2013, Dublin, Ireland.

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Anti HIV nanoemulsion formulation: optimization and in vitro-in vivo evaluation.

The objective of the present work is to develop a dose adjustable nanotechnology based liquid formulation of efavirenz with improved bioavailability f...
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