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Intranasal in situ gel loaded with saquinavir mesylate nanosized microemulsion: Preparation, characterization, and in vivo evaluation
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IJP 14306 1–7 International Journal of Pharmaceutics xxx (2014) exxx–exxx
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
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Intranasal in situ gel loaded with saquinavir mesylate nanosized microemulsion: Preparation, characterization, and in vivo evaluation
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Khaled Mohamed Hosny a,b, * , Ali Habiballah Hassan c
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a
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Beni Suef University, Beni Suef, Egypt Department of Pharmaceutics, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia c Department of Orthodontics, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 June 2014 Received in revised form 26 August 2014 Accepted 28 August 2014 Available online xxx
Saquinavir mesylate (SM) is a protease inhibitor with activity against human immunodeficiency virus type 1 (HIV-1) and is available in tablet form, which has three major problems. First, the drug undergoes extensive first pass metabolism. Second, the drug has a poor aqueous solubility. And third, it has low GIT permeability and absorption. These constrains lead to decrease oral bioavailability (4% only) and administration of large doses which increase the incidence of occurrence of the side effects. The aim of this research was to utilize nanotechnology to formulate (SM) into a nasal in situ nanosized microemulsion gel (NEG) to provide a solution for the previously mentioned problems. The solubility of (SM) in various oils, surfactants, and cosurfactants was estimated. Pseudo-ternary phase diagrams were developed and various nanosized microemulsion (NE) were prepared, and subjected to characterization, stability study, and droplet size measurements. Gellan gum was used as an in situ gelling agent. The gel strength, critical ionic concentration, gelation characteristics, in vitro release, and ex vivo nasal permeation were determined. The pharmacokinetic study was carried out in rabbits. Stable NEs were successfully developed with a droplet size range of 25–61 nm. A NEG composed of 17.5% Labrafac PG, 33% Labrasol, and 11% Transcutol HP successfully provided the maximum in vitro and ex vivo permeation, and enhanced the bioavailability in the rabbits by 12-fold when compared with the marketed tablets. It can be concluded that the nasal NEG is a promising novel formula for (SM) that has higher nasal tissue permeability and enhanced systemic bioavailability. ã 2014 Published by Elsevier B.V.
Keywords: Saquinavir mesylate Nanosized microemulsion Intranasal Human immunodeficiency virus (HIV-1) In situ gel
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1. Introduction
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Saquinavir is a protease inhibitor with activity against human immunodeficiency virus type-1 (HIV-1). Protease inhibitors block the part of HIV called protease. HIV-1 protease is an enzyme required for the proteolytic cleavage of the viral polyprotein precursors into the individual functional proteins found in infectious HIV-1. Saquinavir binds to the protease active site and inhibits the activity of the enzyme. This inhibition prevents cleavage of the viral polyproteins resulting in the formation of immature non-infectious viral particles (Forestier et al., 2001) However, the drug undergoes extensive first pass hepatic metabolism after oral absorption (Li and Chan, 1999). Additionally, the drug has a poor GIT permeability; therefore, the absorption of
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* Corresponding author at: Department of Pharmaceutics, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia. Tel.: +966 592722634; fax: +966 26951696. E-mail address: [email protected] (K.M. Hosny).
(SM) is diffusion rate limited, resulting in low oral bioavailability (about 4%) (Richard, 1998). Administration of a drug through the nasal cavity has many advantages. The large surface area of the nasal cavity and the relatively high blood flow, thereby achieving a rapid absorption and avoidance of hepatic first-pass elimination are attractive features, as well as the rapid onset of action, and ease of administration; all of these advantages have made the development of nasal administrated compounds attractive to formulation scientists (Kublik and Vidgren, 1998). However, the mucociliary clearance is the primary barrier for the delivery of drugs by this route. One possible solution for this problem is to prolong the residence time of the drug in the nasal cavity. Recently, lipid-based formulations have been shown to be a promising strategy for improving the bioavailability of poorly tissue permeable drugs (Monteiro et al., 2012; Mahajan, 2014) This strategy includes the incorporation of drugs into inert lipid vehicles, such as oils or surfactant dispersions, liposomes, lipid nanocarriers, microemulsions, and nanoemulsion (NE) (Khaled and Zainy, 2013).
http://dx.doi.org/10.1016/j.ijpharm.2014.08.064 0378-5173/ ã 2014 Published by Elsevier B.V.
Please cite this article in press as: Hosny, K.M., Hassan, A.H., Intranasal in situ gel loaded with saquinavir mesylate nanosized microemulsion: Preparation, characterization, and in vivo evaluation. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.08.064
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NEs are comprised of isotropic mixtures of oils, surfactants and cosurfactants, which undergo spontaneous emulsification in aqueous fluids. NE mixtures with nanometric droplet sizes between 20 and 200 nm offer several advantages, including a high solvent capacity, small particle size, and excellent stability. In addition, small droplets can enhance the drug’s permeation across the biological membranes, thereby increasing bioavailability (Elshafeey et al., 2009). In situ gel forming systems are liquid aqueous solutions before administration but turn to gel under physiological conditions. There are several possible mechanisms that lead to in situ gel formation, such as pH change, ionic cross-linkage, and temperature modulation (Hosny, 2009). Gellan gum is a linear, anionic polysaccharide secreted by the bacterium Pseudomonas elodea. Deacetylated gellan gum (DGG) is approved in the USA and EU as a gelling, stabilizing, and suspending agent in food products. It can form strong clear gels at physiological ion concentrations and has been widely investigated for use as an in situ gelling agent, which increases the residence time of the drug (Jansson et al., 2005). The objective of this work was to utilize nanotechnology to formulate (SM) into a nasal in situ nanosized microemulsion gel (NEG) in order to provide a solution to the three major problems associated with the marketed (SM) tablets.
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2. Experimental methods
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2.1. Materials
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Pure saquinavir mesylate powder was obtained from Roche (Cairo, Egypt). Labrafac PG, Labrafil M 1944, Labrafact, Labrasol, and Transcutol HP were samples from Gattefosse (Saint-Priest, France). Sefsol, safflower oil, and avocado oil were a kind gift from Nikko Chemicals Co., Ltd. (Tokyo, Japan). Linoleic acid and isopropyl myristate were purchased from Acros Organics (NJ, USA). Isopropanol, and propylene glycol were obtained from TEDIA Company, Inc. (OH, USA). Triacetin, castor oil, oleic acid, Tween 20, Tween 80, Span 20, Span 80, PEG 200, PEG 400, and ethanol were obtained from Sigma–Aldrich (St. Louis, USA). Marketed tablets of saquinavir (Invirase, Roche, Cairo, Egypt). All other reagents and chemicals were of analytical grade.
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3. Methods
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3.1. Solubility studies
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The solubility of (SM) in various oils (castor oil, Labrafac PG, Labrafil M1944, isopropyl myristate, sefsol, oleic acid, linoleic acid, safflower oil, Labrafac, avocado oil, and triacetin), surfactants (Span 20, Span 80, Tween 20, Tween 80, and Labrasol) and cosurfactants (PEG 200, PEG 400, Transcutol HP, ethanol, propylene glycol, and isopropanol) was determined by dissolving an excess amount of (SM) in 2 mL of each solution separately. The mixtures were shaken at 25 0.5 C for 48 h in a water bath. After reaching equilibrium, the mixtures were centrifuged at 3000 rpm for 15 min. The supernatant was diluted with isopropanol, and the concentration of (SM) was quantified spectrophotometrically at 239 nm.
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3.2. Construction of pseudo-ternary phase diagrams
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Based on the solubility studies, the selected oil (Labrafac PG), surfactant (Labrasol), and cosurfactant (Transcutol HP) were used to prepare the NE. The surfactant and cosurfactant (Smix) were mixed at different mass ratios (1:1, 2:1, 3:1, 1:2, and 1:3). Different combinations of oil and Smix were made so that the maximum ratios were covered in order to precisely delineate the boundaries
of phases formed in the phase diagrams. The pseudo ternary phase diagrams of the oil, Smix and water were developed using the aqueous titration method.
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3.3. Visual assessment and emulsification ability of the ternary mixtures
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The efficiency of the prepared NE was visually assessed for its ability to emulsify spontaneously and for the clarity of the final emulsion.
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3.4. Spectroscopic characterization of optical clarity
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The optical clarity of the aqueous dispersions of the NE formulations was measured spectroscopically. Briefly, 0.5 mL of each NE was diluted with 9.5 mL distilled water. The absorbance of each solution was measured at 638 nm.
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3.5. NE droplet size analysis
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To test the droplet size of the NE, 100 mg of each formulation was diluted with 10 mL distilled water. The mean droplet size of the resulting dispersion was determined by dynamic light scattering using a Zetatrac machine from Microtrac Inc. (PA, USA).
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3.6. Conductivity
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The conductivity was determined using the Zetatrac machine from Microtrac Inc. (PA, USA).
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3.7. Thermodynamic stability studies
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The NE formulations were centrifuged at 3000 rpm for 30 min. The formulations that did not show any phase separations were tested with heating and cooling cycles. Three freeze-thaw cycles (20 C and +25 C) were performed on these formulations. The NEs that passed the dispersion stability tests were selected for the formulation of in situ gels.
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3.8. Formulation of in situ gel
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The formulations shown in Table 1 were prepared by sprinkling the cationic induced in situ gel polymer (DGG) over 6 mL distilled water, at 80 1 C; this solution was stirred at 50 rpm until the polymer dissolved, then allowed to cool overnight (Narayana et al., 2009). A separate 4 mL NE containing 500 mg (SM) was then added to the polymeric solution and mixed. The final NEGs contained 0.5% (DGG) were stored overnight in the refrigerator prior to further evaluation.
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3.9. Evaluation of in situ gels
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3.9.1. Critical ionic concentration (CIC) for DGG phase transition (CIC) for phase transition is an important parameter for ion-activated in situ gels. The CIC was determined by mixing 1 mL of 0.5% DGG solution and various amounts of artificial nasal fluid (NF), which consisted of 150 32 mM Na+, 41 18 mM K+, and 4 2 mM Ca2+; in bottles placed in a water bath at 32 C. After 20 s, the bottles were turned over. If the gels adhered to the bottom instead of flowing or sliding down the side, the formulation showed gel formation and was considered “+”. The minimum NF concentration in the mixture that could induce gel formation was estimated as the CIC.
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Please cite this article in press as: Hosny, K.M., Hassan, A.H., Intranasal in situ gel loaded with saquinavir mesylate nanosized microemulsion: Preparation, characterization, and in vivo evaluation. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.08.064
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Table 1 UV absorbance, mean droplet size SD, and conductivity for different nanosized microemulsion formulations.
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NE
Smix ratio
Labrafac PG (%)
Smix (%)
Water (%)
UV absorbance at 638 nm
Mean droplet size (nm)
Conductivity ms/cm
NE-1 NE-2 NE-3 NE-4 NE-5 NE-6 NE-7 NE-8 NE-9 NE-10
1:1 1:1 2:1 2:1 2:1 2:1 3:1 3:1 3:1 3:1
5 9.5 5 10 12.5 15 10 15 17.5 20
50 55 40 45 47.5 50 35 40 44 45
45 35.5 55 45 40 35 55 45 38.5 35
0.0035 0.0049 0.0021 0.0041 0.0056 0.0079 0.0024 0.0063 0.0071 0.0076
33 3.1 42 3.7 25 2.9 37 2.6 48 4.1 58 3.5 31 2.6 55 3.9 59 2.7 61 4.1
0.158 0.154 0.161 0.150 0.154 0.145 0.156 0.152 0.140 0.137
3.9.2. Expansion coefficient (S%) of the in situ gels When the solution transforms into a gel, its volume may increase. The nasal cavity is small; thus, gel expansion may cause discomfort. Therefore, investigating the S% of the in situ gels is necessary (Zheng et al., 2011). The S% was determined by mixing 1 mL of 0.5% DGG solution with 0.25 mL NF in a graduated test tube and placed in a water bath at 32 C with a final total liquid volume, VI, of 1.25 mL. The volume of the gel after transition, VG, was determined by adding 2.0 mL of NF and recording the total volume VT. Thus, VG = VT 2, and the expansion coefficient (S%) = (VG VI)/ VI 100%. 3.9.3. Rheological properties of the in situ gel The apparent single viscosity values were measured using the Brookfield digital viscometer. The viscosity of the NEGs was measured at 10 rpm after 30 s before and after gelation. 3.9.4. Measurement of gel strength A sample of the formulation (5 g) was gelled by neutralization with NF. A 3.5 g weight was then placed on top of the gel. The gel strength, which is an indication of the viscosity of the nasal in situ gel at physiological conditions, was determined by the time, in seconds, it took the weight to penetrate 3 cm into the gel. 3.9.5. In vitro release study A 5 mL sample of the NEG was placed within a dialysis bag composed of cellulose membrane and immersed into a vessel containing 100 mL PBS, pH 6 at 37 1 C; the solution was stirred at 50 rpm for up to 12 h. Samples of the NEG (2 mL) were taken at different time intervals, and the (SM) content was determined. 3.9.6. Ex vivo drug permeation studies Fresh nasal tissue was carefully removed from the nasal cavities of goats obtained from the local slaughterhouse. Tissue samples were mounted on Franz diffusion cells with a permeation area of 1.76 cm2. PBS (7 mL, pH 6) was placed in the receptor chamber. After a pre-incubation time of 20 min, 1 g of neutralized NEG-8 or NEG-9 and 1 mL 5% (SM) suspension (composed of the pure drug suspended in distilled water) were placed in the donor chamber. At predetermined time intervals, 0.5 mL samples were withdrawn over a period of 12 h, and the (SM) content was measured using HPLC (Campanero et al., 2001). Results were expressed as the amount that permeated the receptor chamber, and the percentage of permeation was calculated using the following equation: SP (1) 100 Percentage permeated ¼ ST where Sp and ST represent the amount of (SM) that permeated into the receptor chamber and the initial amount of (SM) in the donor chamber, respectively.
3.9.7. In vivo drug absorption study
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3.9.7.1. Selection of animals. The in vivo drug absorption study was conducted according to the institutional guidelines of the Animal Ethics Committee of the Faculty of Pharmacy at King Abdulaziz University. Twelve albino male rabbits, weighing between 2 and 2.5 kg, were used in the in vivo study. Based on the results of the in vitro and ex vivo studies, NEG-9 was selected for the in vivo study. The rabbits were kept in fasting condition for 24 h before the experiment commenced, and the rabbits were grouped into two groups, each containing 6 rabbits. Group I was administered the (SM) tablet (reference) at a dose of 10 mg/kg. Group II was administered intranasal NEG-9 at a dose of 10 mg/kg. Blood samples (2 mL) were collected before the administration of the drug and again 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4.5, 6, 9, 12, and 24 h after the administration of the drug. Samples were stored at 20 C until analysis.
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3.9.7.2. Pharmacokinetic analysis. The Cmax and Tmax were calculated using the plasma concentration-time curve in the WinNonlinTM Nonlinear Estimation Program. One way analysis of variance (ANOVA) was employed to assess the Na+ the Tmax, Cmax, AUC0–1, t1/2, and MRT data of (SM) from the tested NEG9 formulation and the reference tablet at a level (p 0.05) using the SPSS program. The AUC0–24 was calculated using the linear trapezoidal rule (Yuan et al., 2008). The AUC0–1 and the relative bioavailability (BAR) of the nasal NEG-9 were also calculated by using the following formula: AUNEG DoseTablet (2) Relative bioavailability ðBAR Þ ¼ AUCTablet DoseNEG
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4. Results
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4.1. Solubility studies
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Fig. 1 shows the solubility of (SM) in different oils, surfactants, and cosurfactants. The solubilisation of (SM) was highest in Labrafac PG (25.35 3.44 mg/mL) when compared with the other oils. Of the surfactants screened, Labrasol showed a superior solubilising potential (62.18 2.75 mg/mL). Additionally, Transcutol HP was selected as a co-surfactant due to its efficient solubilising effect (46.82 2.26 mg/mL).
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4.2. Pseudo-ternary phase diagram
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Five ternary phase systems were constructed, in which the (Smix) was prepared at different mass ratios (1:1, 2:1, 3:1, 1:2, and 1:3) (Fig. 2). In the Smix (1:1), it was observed that the maximum concentration of oil that could be solubilised was 9.5% using 55% of
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Please cite this article in press as: Hosny, K.M., Hassan, A.H., Intranasal in situ gel loaded with saquinavir mesylate nanosized microemulsion: Preparation, characterization, and in vivo evaluation. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.08.064
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Fig. 1. Graphical representation of solubility of (SM) in various oils, surfactants and cosurfactants.
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the Smix. However, when the concentration of Transcutol HP was increased with respect to Labrasol (1:2 or 1:3), the NE area was decreased when compared to the 1:1 Smix ratio. As the surfactant concentration was increased in the Smix (2:1 or 3:1), higher NE regions were observed.
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4.3. Characterization of the NE
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Table 1 shows all the NEs that had droplets in the 25–61 nm nanosize range. Among the NEs, the mean UV absorbance at 638 nm varied between 0.0021 and 0.0079, and the droplet size increased as the concentration of oil increased; however, the droplet size decreased as the concentration of the surfactant in the Smix ratio was increased (Smix 3:1). The conductivity measurements (0.137–0.161 ms/cm) indicated the NEs were of the oil-in-water type.
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4.4. Thermodynamic stability of the NEs
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The stress tests, including centrifugation and freeze thaw cycles, showed that all of the formulations were physically stable. The concentrations of (SM) in the NEs remained almost constant, and no degradation was observed.
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4.5. Formulation and evaluation of the in situ gel
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Each of the NEGs had a clear appearance. The results of the gelation studies, CIC, S%, and the viscosities of the NEGs in solution and gel form are given in Table 2. The gel strength values between 25 and 50 s were considered sufficient, and the gels that exhibited gel strength within this range were selected for in vitro release. Fig. 3 shows the release profile of (SM) from the selected NEGs (6, 8, and 9), as well as from the in situ gel prepared from an aqueous (SM) suspension. NEG-8 (85%) and NEG-9 (82%) showed the highest cumulative percentage of the drug released after 12 h.
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4.6. Ex vivo drug permeation studies
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Fig. 4 shows the nasal permeation profile of (SM) while in a 5% aqueous suspension, as well as when contained in NEG-8 and NEG-9. The extent to which the drug permeated the receptor chamber from the NEGs and from the aqueous suspension after 12 h was significantly different. The cumulative percentage of (SM) that permeated from the NEG-8 was 64%, while the cumulative percentage that permeated from the NEG-9 was 69%. However, in the case of the (SM) aqueous suspension, the cumulative percentage that permeated was only 11.2%. In other words, the NEG-9 in situ gel showed a 6.5-fold higher permeation percentage than the aqueous suspension.
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4.7. In vivo pharmacokinetics study
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There was a statistically significant difference in the Tmax, Cmax, AUC0–1, t1/2, and MRT data between the (SM) from the NEG-9 and the marketed tablet (Table 3 and Fig. 5), at a level (p 0.05). In the case of the (SM) table, the MRT was 6.5 h, whereas, the MRT for the NEG-9 was 11.25 h. The mean AUC0–1 for the marketed tablet was significantly different than the AUC0–1 for the NEG-9. These results confirmed that the formulation of (SM) into an NEG increased its bioavailability by greater than 12-fold.
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5. Discussion
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NEs are thermodynamically stable systems and are formed when a particular concentration of oil, Smix and water are combined. In the case of Smix (1:1), the maximum concentration of Labrafac PG oil that could be solubilised in the phase diagram was 9.5% using 55% of the Smix. As the Labrasol concentration increased in the Smix (2:1) and (3:1), a higher NE region was
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Fig. 2. Pseudo-ternary phase diagrams indicating oil/water nanosized microemulsion region at different Smix ratios.
Please cite this article in press as: Hosny, K.M., Hassan, A.H., Intranasal in situ gel loaded with saquinavir mesylate nanosized microemulsion: Preparation, characterization, and in vivo evaluation. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.08.064
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Table 2 CIC%, S%, gelling capacity, gel strength, and viscosities SD in solution and gel form for different prepared in situ NEGs. Formulation
CIC (%)
S%
Gelling capacity
Viscosity before gellation (cp)
Viscosity after gellation (cp)
Gel strength (second)
NEG-1 NEG-2 NEG-3 NEG-4 NEG-5 NEG-6 NEG-7 NEG-8 NEG-9 NEG-10
0.18 0.15 0.19 0.18 0.17 0.16 0.20 0.19 0.16 0.15
2.8 2.4 2.9 2.8 2.7 2.5 3.0 2.9 2.6 2.2
+ ++ + ++ +++ +++ ++ +++ +++ ++++
1.87 0.21 1.95 0.26 1.91 0.19 2.18 0.29 2.47 0.31 2.71 0.34 2.05 0.21 2.61 0.33 3.01 0.41 3.38 0.37
19.43 1.21 23.47 1.72 21.03 1.25 25.55 2.08 28.27 2.87 34.99 3.01 27.11 2.16 31.03 3.01 33.16 3.02 37.44 3.51
16.43 20.74 17.14 21.75 24.02 45.93 22.35 37.88 41.96 59.42
Fig. 3. (SM) release profile from different NEG formulations (NEG-6, 8, and 9) and from control in situ gel. 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304
observed, and about 20% of the oil could be solubilised. This may be due to a further reduction in the interfacial tension, thereby increasing the fluidity of the interface, this could result in greater penetration of the oil phase in the hydrophobic region of the Labrasol monomers (Yuan et al., 2008). The presence of Transcutol HP as cosurfactant decreases the ending stress of the interface and makes the interfacial film sufficiently flexible to exhibit different curvatures that are required to form NEs over a wide range (Kawakami et al., 2002). However, when the concentration of Transcutol HP was increased with respect to Labrasol (Smix ratio 1:2 and 1:3), the NE area decreased as compared to the Smix ratio (1:1); this could be due to the formation of a smaller amount of micelles (Lawrence and Rees, 2000). Transcutol HP is a polar co-solvent that can highly incorporate into water, and the relatively low Transcutol HP
content in the NE system, such as with NE-7, decreases the hydrophilicity of the Smix, and therefore the amount of oil could be solubilise in the o/w NE was decreased (about 10% only). The NE formulations were then evaluated for their physical stability. There was no sign of phase separation or turbidity observed for the ten formulations (NE1-10) during their preparation, indicating the formulations were physically stable. All of the formulations passed stability stress tests, including centrifugation and freeze thaw cycles. All the tested NEs had droplets within the nanosize range (between 25 and 61 nm). The droplet size increased as the concentration of oil in the formulations increased. The droplet size decreased as the concentration of Labrasol in the Smix increased (ratio 3:1). The addition of a surfactant to the NE systems may cause the interfacial film to condense and stabilize, resulting in smaller droplet diameters, whereas the addition of the Transcutol HP cosurfactant may cause the film to expand (Djekic and Primorac, 2008). The conductivity measurements (0.137–0.161 ms/cm) indicated that the NEs were of the o/w type, and an increase in the Labrafac PG concentration in the NE resulted in a decrease in conductivity. In situ nasal nanosized microemulsion gels (NEG) were prepared successfully using 0.5% deacetylated gellan gum as an ionic induced gelling agent. All of the NEG formulas had a clear appearance. Results from the viscosity tests for the NEGs in solution and in gel form indicated that there was a marked increase in viscosity values (19.43–37.44 cp) in the presence of NF when compared to the NEGs in solution (1.87–3.38 cp); this change was due solely to the gel conversion, which indicated the in situ nature of all the prepared gels. When the DGG solutions transformed into gels, no obvious volume expansion was observed. The expansion coefficient was only about 3%. This slight expansion may not cause discomfort to patients when the in situ gel is administered into the nasal cavity (Zheng et al., 2011). When developing an NEG, the gel strength is an important factor to consider; the ideal gel strength allows for the easy nasal administration of the gel but does not let the gel leak from the nose. It is very important that the nasal gel formulation must have suitable gel strength. A gel strength less than 25 s may not have sufficient structural integrity and may erode rapidly, however, gels Table 3 Pharmacokinetic parameters of (SM) after administration of 10 mg/kg intranasal in situ NEG-9 and 10 mg/kg of marketed tablet (n = 6). The mean difference is significant at the 0.05 level.
Fig. 4. Percentage of cumulative (SM) permeation for the various NEG (8 and 9), and for (SM) aqueous suspension through nasal tissue.
Pharmacokinetic parameters
NEG-9
(SM) marketed tablet
Tmax (hr) Cmax (mg/ml) AUC0 t (mg hr/ml) K (hr1) MRT (hr) AUC0–1 (mg hr/ml)
5.3 0.4 87.13 1.32 293.51 4.34 0.477 0.036 11.25 0.51 404.49 6.43
4.2 0.5 22.25 0.62 25.24 2.11 0.348 0.031 6.51 0.32 34.22 3.43
Please cite this article in press as: Hosny, K.M., Hassan, A.H., Intranasal in situ gel loaded with saquinavir mesylate nanosized microemulsion: Preparation, characterization, and in vivo evaluation. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.08.064
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Fig. 5. Plasma-concentration time curve of (SM) after administration of 10 mg/kg intranasal in situ NEG-9 and 10 mg/kg of marketed Tablet (n = 6). The mean difference is significant at the 0.05 level.
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with a strength greater than 50 s may be too stiff and cause discomfort (Tanaji et al., 2008). For this reason, the NEGs-1,2,3,4,5,7, and 10 were excluded from further evaluations. The in vitro release profiles for NEGs-6, 8, and 9 exhibited inflection points; the initial release rate of the drug was very rapid due to incomplete gel formation, but the release slowed down after the gel was completely formed and remained at a steady state. The results showed that NEG-8 and NEG-9 showed the highest cumulative percentage of the drug released, 85% and 82%, respectively, after 12 h. This could have been due, in part, to these NEGs having the smallest droplet size and the lowest viscosity of all the prepared NEGs. The ex vivo permeation results showed that the NEG system significantly increased the nasal permeation of (SM). NEG-9 showed the highest release of the drug when compared with the other formulation. This formulation showed a 6.5-fold higher permeation percentage than the (SM) aqueous suspension, these characteristics make NEG-9 an excellent carrier for the nasal administration of (SM). Also, an applied NE is expected to penetrate and to exist intact in the nasal tissue, thereby altering both the lipid and polar permeation pathways (Thacrodi and Panduranga, 1994). Additionally, the hydrophilic domain of the NEs can hydrate the external surface of the nasal tissue to a greater extent and play an important role in the tissue uptake of the drugs. When the aqueous fluid of the NEs enters the polar pathway, it increases the interlamellar volume of the tissue lipid bilayer, resulting in the disruption of its interfacial structure (Sanjula et al., 2007). The pharmacokinetic study results revealed that intranasal application of (SM) can significantly modify its pharmacokinetic profile and can increase its bioavailability by more than 12-fold in comparison with the marketed oral tablet formulation. This was due to the fact that (SM) is a drug with poor aqueous solubility and GIT permeability, and the preparation of this drug as a nasal NE enhanced its solubility and diffusion which leads to enhancing the bioavailability by more than 12-folds. Also, the pharmacokinetic of drugs upon delivery in nanosized microemulsion formulation are dictated by the properties of the nanosized microemulsions rather than by the physicochemical properties of the drug molecules. Additionally, the presence of Transcutol HP in the NE formula cause steric hindrance which help in reducing the tissue uptake by evading the RES, this will increase the residence time of nanosized microemulsions in the blood circulation Furthermore, gellan gum in situ NEG has a better mucoadhesive property, thereby ensuring intimate contact between the NE and the nasal tissue, and prolongs the retention of the formulation at the site of administration, which enhances permeation (Cao et al., 2009). In addition, the enhancement in the bioavailability of the drug by more than
12-fold could be attributed to the decreased amount of hepatic first pass metabolism that is associated with the oral administration of the (SM) marketed tablets.
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6. Conclusion
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The formulation of (SM) as a nasal in situ nanosized microemulsion gel, which is a novel drug delivery system, provided the maximum in vitro and ex vivo nasal permeation. The bioavailability of (SM) was enhanced by more than 12-folds in relation to the marketed tablet. The improved nasal NEG formula was composed of 17.5% Labrafac PG, 33% Labrasol, and 11% Transcutol HP, and was gelled with 0.5% Deacetylated Gellan gum. The use of this formula could eliminate the major drawbacks of the conventionally used tablet. Of course, it will not obviate the need for further clinical evaluation for this novel formula, which may inform clinicians of other important data.
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Declaration of interest
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The authors reported that there is no any conflict of interest.
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References
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Campanero, M.A., Escolar, M., Arangoa, M.A., et al., 2001. Development of a chromatographic method for the determination of saquinavir in plasma samples of HIV patients. Biomed. Chrom. 16, 7–12. Cao, L., Ren, W., Zhang, Z., et al., 2009. In situ gel based on gellan gum as new carrier for nasal administration of mometasone furoate. Int. J. Pharm. 365, 109–115. Djekic, L., Primorac, M., 2008. The influence of cosurfactants and oils on the formation of pharmaceutical nanosized microemulsions based on PEG-8 caprylic/capric glycerides. Int. J. Pharm. 352, 231–239. Elshafeey, A.H., Kamel, A.O., Fathallah, M.M., 2009. Utility of nanosized nanosized microemulsion for transdermal delivery of tolterodine tartrate: ex-vivo permeation and in-vivo pharmacokinetic studies. Pharm. Res. 226, 2446–2453. Forestier, F., de Renty, P., Peytavin, G., Dohin, E., Farinotti, R., Mandelbrot, L., 2001. Maternal-fetal transfer of saquinavir studied in the ex vivo placental perfusion model. Am. J. Obstet. Gynecol. 185, 178–181. Hosny, K.M., 2009. Preparation and evaluation of thermosensitive liposomal hydrogel for enhanced transcorneal permeation of ofloxacin. AAPS PharmSciTech 10, 1336–1342. Jansson, B., Hagerstrom, H., Fransen, N., Edsman, K., Bjork, E., 2005. The influence of gellan gum on the transfer of fluorescein dextran across rat nasal epithelium in vivo. Eur. J. Pharm. Biopharm. 59, 557–564. Kawakami, K., Yoshikawa, T., Moroto, Y., et al., 2002. Microemulsion formulation for enhanced absorption of poorly soluble drugs I. Prescription design. J. Control. Rel. 81, 65–74. Khaled, H., Zainy, B., 2013. The formulation of a nasal nanoemulsion zaleplon in situ gel for the treatment of insomnia. Expert Opin. Drug Deliv. 10, 1033–1041. Kublik, H., Vidgren, M., 1998. Nasal delivery systems and their effect on deposition and absorption. Adv. Drug Delv. Rev. 29, 157–177. Lawrence, M.J., Rees, G.D., 2000. Microemulsion-based media as novel drug delivery systems. Adv. Drug Deliv. Rev. 45, 89–121. Li, X., Chan, W.K., 1999. Transport, metabolism and elimination mechanism of anti-HIV agents. Adv. Drug Deliv. Rev. 39, 81–103.
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Mahajan, H.S., 2014. Nanoemulsion-based intranasal drug delivery system of saquinavir mesylate for brain targeting. Drug Deliv. 21, 148–154. Monteiro, L.M., Lione, V.F., do Carmo, F.A., et al., 2012. Development and in silico bioavailability studies of dapsone oral nanoemulsion systems. Int. J. Nanomed. 7, 5175–5182. Narayana, R.C., Harish, N.M., Gulzar, A.M., Prabhakara, P., Singh, A.K., Subrahmanyam, E.V., 2009. Formulation and in vitro evaluation of in situ gels containing secnidazole for vaginitis. Yakugaku Zasshi 129, 569–574. Richard, B.K., 1998. The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease inhibitors. J. Clin. Invest. 101, 289–294. Sanjula, B., Faiyaz, S., Alka, A., et al., 2007. Design, development and evaluation of novel nanoemulsion formulations for transdermal potential of celecoxib. Acta Pharm. 57, 315–332.
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Tanaji, N., Rahul, T., Nitin, J., et al., 2008. Formulation and evaluation of pH induced in-situ nasal gel of salbutamol sulphate. Int. J. Pharm. Sci. Nanotech. 12, 177–183. Thacrodi, D., Panduranga, K., 1994. Transdermal absorption of nifedipine from microemulsions of lipophilic skin penetration enhancers. Int. J. Pharm. 111, 235–240. Yuan, J.S., Ansari, M., Samaan, M., Acosta, E.J., 2008. Linker-based lecithin microemulsions for transdermal delivery of lidocaine. Int. J. Pharm. 349, 130–143. Zheng, C., Xiangrong, S., Feng, S., et al., [129_TD$DIFF][130_TD$DIFF]2011. Formulation and evaluation of in situ gelling systems for intranasal administration of gastrodin. AAPS PharmSciTech 12, 1102–1109.
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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
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Intranasal in situ gel loaded with saquinavir mesylate nanosized microemulsion: Preparation, characterization, and in vivo evaluation
3 Q1
Khaled Mohamed Hosny a,b, * , Ali Habiballah Hassan c
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a
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Beni Suef University, Beni Suef, Egypt Department of Pharmaceutics, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia c Department of Orthodontics, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 June 2014 Received in revised form 26 August 2014 Accepted 28 August 2014 Available online xxx
Saquinavir mesylate (SM) is a protease inhibitor with activity against human immunodeficiency virus type 1 (HIV-1) and is available in tablet form, which has three major problems. First, the drug undergoes extensive first pass metabolism. Second, the drug has a poor aqueous solubility. And third, it has low GIT permeability and absorption. These constrains lead to decrease oral bioavailability (4% only) and administration of large doses which increase the incidence of occurrence of the side effects. The aim of this research was to utilize nanotechnology to formulate (SM) into a nasal in situ nanosized microemulsion gel (NEG) to provide a solution for the previously mentioned problems. The solubility of (SM) in various oils, surfactants, and cosurfactants was estimated. Pseudo-ternary phase diagrams were developed and various nanosized microemulsion (NE) were prepared, and subjected to characterization, stability study, and droplet size measurements. Gellan gum was used as an in situ gelling agent. The gel strength, critical ionic concentration, gelation characteristics, in vitro release, and ex vivo nasal permeation were determined. The pharmacokinetic study was carried out in rabbits. Stable NEs were successfully developed with a droplet size range of 25–61 nm. A NEG composed of 17.5% Labrafac PG, 33% Labrasol, and 11% Transcutol HP successfully provided the maximum in vitro and ex vivo permeation, and enhanced the bioavailability in the rabbits by 12-fold when compared with the marketed tablets. It can be concluded that the nasal NEG is a promising novel formula for (SM) that has higher nasal tissue permeability and enhanced systemic bioavailability. ã 2014 Published by Elsevier B.V.
Keywords: Saquinavir mesylate Nanosized microemulsion Intranasal Human immunodeficiency virus (HIV-1) In situ gel
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1. Introduction
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Saquinavir is a protease inhibitor with activity against human immunodeficiency virus type-1 (HIV-1). Protease inhibitors block the part of HIV called protease. HIV-1 protease is an enzyme required for the proteolytic cleavage of the viral polyprotein precursors into the individual functional proteins found in infectious HIV-1. Saquinavir binds to the protease active site and inhibits the activity of the enzyme. This inhibition prevents cleavage of the viral polyproteins resulting in the formation of immature non-infectious viral particles (Forestier et al., 2001) However, the drug undergoes extensive first pass hepatic metabolism after oral absorption (Li and Chan, 1999). Additionally, the drug has a poor GIT permeability; therefore, the absorption of
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* Corresponding author at: Department of Pharmaceutics, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia. Tel.: +966 592722634; fax: +966 26951696. E-mail address: [email protected] (K.M. Hosny).
(SM) is diffusion rate limited, resulting in low oral bioavailability (about 4%) (Richard, 1998). Administration of a drug through the nasal cavity has many advantages. The large surface area of the nasal cavity and the relatively high blood flow, thereby achieving a rapid absorption and avoidance of hepatic first-pass elimination are attractive features, as well as the rapid onset of action, and ease of administration; all of these advantages have made the development of nasal administrated compounds attractive to formulation scientists (Kublik and Vidgren, 1998). However, the mucociliary clearance is the primary barrier for the delivery of drugs by this route. One possible solution for this problem is to prolong the residence time of the drug in the nasal cavity. Recently, lipid-based formulations have been shown to be a promising strategy for improving the bioavailability of poorly tissue permeable drugs (Monteiro et al., 2012; Mahajan, 2014) This strategy includes the incorporation of drugs into inert lipid vehicles, such as oils or surfactant dispersions, liposomes, lipid nanocarriers, microemulsions, and nanoemulsion (NE) (Khaled and Zainy, 2013).
http://dx.doi.org/10.1016/j.ijpharm.2014.08.064 0378-5173/ ã 2014 Published by Elsevier B.V.
Please cite this article in press as: Hosny, K.M., Hassan, A.H., Intranasal in situ gel loaded with saquinavir mesylate nanosized microemulsion: Preparation, characterization, and in vivo evaluation. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.08.064
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NEs are comprised of isotropic mixtures of oils, surfactants and cosurfactants, which undergo spontaneous emulsification in aqueous fluids. NE mixtures with nanometric droplet sizes between 20 and 200 nm offer several advantages, including a high solvent capacity, small particle size, and excellent stability. In addition, small droplets can enhance the drug’s permeation across the biological membranes, thereby increasing bioavailability (Elshafeey et al., 2009). In situ gel forming systems are liquid aqueous solutions before administration but turn to gel under physiological conditions. There are several possible mechanisms that lead to in situ gel formation, such as pH change, ionic cross-linkage, and temperature modulation (Hosny, 2009). Gellan gum is a linear, anionic polysaccharide secreted by the bacterium Pseudomonas elodea. Deacetylated gellan gum (DGG) is approved in the USA and EU as a gelling, stabilizing, and suspending agent in food products. It can form strong clear gels at physiological ion concentrations and has been widely investigated for use as an in situ gelling agent, which increases the residence time of the drug (Jansson et al., 2005). The objective of this work was to utilize nanotechnology to formulate (SM) into a nasal in situ nanosized microemulsion gel (NEG) in order to provide a solution to the three major problems associated with the marketed (SM) tablets.
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2. Experimental methods
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2.1. Materials
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Pure saquinavir mesylate powder was obtained from Roche (Cairo, Egypt). Labrafac PG, Labrafil M 1944, Labrafact, Labrasol, and Transcutol HP were samples from Gattefosse (Saint-Priest, France). Sefsol, safflower oil, and avocado oil were a kind gift from Nikko Chemicals Co., Ltd. (Tokyo, Japan). Linoleic acid and isopropyl myristate were purchased from Acros Organics (NJ, USA). Isopropanol, and propylene glycol were obtained from TEDIA Company, Inc. (OH, USA). Triacetin, castor oil, oleic acid, Tween 20, Tween 80, Span 20, Span 80, PEG 200, PEG 400, and ethanol were obtained from Sigma–Aldrich (St. Louis, USA). Marketed tablets of saquinavir (Invirase, Roche, Cairo, Egypt). All other reagents and chemicals were of analytical grade.
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3. Methods
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3.1. Solubility studies
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The solubility of (SM) in various oils (castor oil, Labrafac PG, Labrafil M1944, isopropyl myristate, sefsol, oleic acid, linoleic acid, safflower oil, Labrafac, avocado oil, and triacetin), surfactants (Span 20, Span 80, Tween 20, Tween 80, and Labrasol) and cosurfactants (PEG 200, PEG 400, Transcutol HP, ethanol, propylene glycol, and isopropanol) was determined by dissolving an excess amount of (SM) in 2 mL of each solution separately. The mixtures were shaken at 25 0.5 C for 48 h in a water bath. After reaching equilibrium, the mixtures were centrifuged at 3000 rpm for 15 min. The supernatant was diluted with isopropanol, and the concentration of (SM) was quantified spectrophotometrically at 239 nm.
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3.2. Construction of pseudo-ternary phase diagrams
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Based on the solubility studies, the selected oil (Labrafac PG), surfactant (Labrasol), and cosurfactant (Transcutol HP) were used to prepare the NE. The surfactant and cosurfactant (Smix) were mixed at different mass ratios (1:1, 2:1, 3:1, 1:2, and 1:3). Different combinations of oil and Smix were made so that the maximum ratios were covered in order to precisely delineate the boundaries
of phases formed in the phase diagrams. The pseudo ternary phase diagrams of the oil, Smix and water were developed using the aqueous titration method.
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3.3. Visual assessment and emulsification ability of the ternary mixtures
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The efficiency of the prepared NE was visually assessed for its ability to emulsify spontaneously and for the clarity of the final emulsion.
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3.4. Spectroscopic characterization of optical clarity
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The optical clarity of the aqueous dispersions of the NE formulations was measured spectroscopically. Briefly, 0.5 mL of each NE was diluted with 9.5 mL distilled water. The absorbance of each solution was measured at 638 nm.
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3.5. NE droplet size analysis
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To test the droplet size of the NE, 100 mg of each formulation was diluted with 10 mL distilled water. The mean droplet size of the resulting dispersion was determined by dynamic light scattering using a Zetatrac machine from Microtrac Inc. (PA, USA).
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3.6. Conductivity
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The conductivity was determined using the Zetatrac machine from Microtrac Inc. (PA, USA).
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3.7. Thermodynamic stability studies
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The NE formulations were centrifuged at 3000 rpm for 30 min. The formulations that did not show any phase separations were tested with heating and cooling cycles. Three freeze-thaw cycles (20 C and +25 C) were performed on these formulations. The NEs that passed the dispersion stability tests were selected for the formulation of in situ gels.
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3.8. Formulation of in situ gel
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The formulations shown in Table 1 were prepared by sprinkling the cationic induced in situ gel polymer (DGG) over 6 mL distilled water, at 80 1 C; this solution was stirred at 50 rpm until the polymer dissolved, then allowed to cool overnight (Narayana et al., 2009). A separate 4 mL NE containing 500 mg (SM) was then added to the polymeric solution and mixed. The final NEGs contained 0.5% (DGG) were stored overnight in the refrigerator prior to further evaluation.
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3.9. Evaluation of in situ gels
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3.9.1. Critical ionic concentration (CIC) for DGG phase transition (CIC) for phase transition is an important parameter for ion-activated in situ gels. The CIC was determined by mixing 1 mL of 0.5% DGG solution and various amounts of artificial nasal fluid (NF), which consisted of 150 32 mM Na+, 41 18 mM K+, and 4 2 mM Ca2+; in bottles placed in a water bath at 32 C. After 20 s, the bottles were turned over. If the gels adhered to the bottom instead of flowing or sliding down the side, the formulation showed gel formation and was considered “+”. The minimum NF concentration in the mixture that could induce gel formation was estimated as the CIC.
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Table 1 UV absorbance, mean droplet size SD, and conductivity for different nanosized microemulsion formulations.
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NE
Smix ratio
Labrafac PG (%)
Smix (%)
Water (%)
UV absorbance at 638 nm
Mean droplet size (nm)
Conductivity ms/cm
NE-1 NE-2 NE-3 NE-4 NE-5 NE-6 NE-7 NE-8 NE-9 NE-10
1:1 1:1 2:1 2:1 2:1 2:1 3:1 3:1 3:1 3:1
5 9.5 5 10 12.5 15 10 15 17.5 20
50 55 40 45 47.5 50 35 40 44 45
45 35.5 55 45 40 35 55 45 38.5 35
0.0035 0.0049 0.0021 0.0041 0.0056 0.0079 0.0024 0.0063 0.0071 0.0076
33 3.1 42 3.7 25 2.9 37 2.6 48 4.1 58 3.5 31 2.6 55 3.9 59 2.7 61 4.1
0.158 0.154 0.161 0.150 0.154 0.145 0.156 0.152 0.140 0.137
3.9.2. Expansion coefficient (S%) of the in situ gels When the solution transforms into a gel, its volume may increase. The nasal cavity is small; thus, gel expansion may cause discomfort. Therefore, investigating the S% of the in situ gels is necessary (Zheng et al., 2011). The S% was determined by mixing 1 mL of 0.5% DGG solution with 0.25 mL NF in a graduated test tube and placed in a water bath at 32 C with a final total liquid volume, VI, of 1.25 mL. The volume of the gel after transition, VG, was determined by adding 2.0 mL of NF and recording the total volume VT. Thus, VG = VT 2, and the expansion coefficient (S%) = (VG VI)/ VI 100%. 3.9.3. Rheological properties of the in situ gel The apparent single viscosity values were measured using the Brookfield digital viscometer. The viscosity of the NEGs was measured at 10 rpm after 30 s before and after gelation. 3.9.4. Measurement of gel strength A sample of the formulation (5 g) was gelled by neutralization with NF. A 3.5 g weight was then placed on top of the gel. The gel strength, which is an indication of the viscosity of the nasal in situ gel at physiological conditions, was determined by the time, in seconds, it took the weight to penetrate 3 cm into the gel. 3.9.5. In vitro release study A 5 mL sample of the NEG was placed within a dialysis bag composed of cellulose membrane and immersed into a vessel containing 100 mL PBS, pH 6 at 37 1 C; the solution was stirred at 50 rpm for up to 12 h. Samples of the NEG (2 mL) were taken at different time intervals, and the (SM) content was determined. 3.9.6. Ex vivo drug permeation studies Fresh nasal tissue was carefully removed from the nasal cavities of goats obtained from the local slaughterhouse. Tissue samples were mounted on Franz diffusion cells with a permeation area of 1.76 cm2. PBS (7 mL, pH 6) was placed in the receptor chamber. After a pre-incubation time of 20 min, 1 g of neutralized NEG-8 or NEG-9 and 1 mL 5% (SM) suspension (composed of the pure drug suspended in distilled water) were placed in the donor chamber. At predetermined time intervals, 0.5 mL samples were withdrawn over a period of 12 h, and the (SM) content was measured using HPLC (Campanero et al., 2001). Results were expressed as the amount that permeated the receptor chamber, and the percentage of permeation was calculated using the following equation: SP (1) 100 Percentage permeated ¼ ST where Sp and ST represent the amount of (SM) that permeated into the receptor chamber and the initial amount of (SM) in the donor chamber, respectively.
3.9.7. In vivo drug absorption study
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3.9.7.1. Selection of animals. The in vivo drug absorption study was conducted according to the institutional guidelines of the Animal Ethics Committee of the Faculty of Pharmacy at King Abdulaziz University. Twelve albino male rabbits, weighing between 2 and 2.5 kg, were used in the in vivo study. Based on the results of the in vitro and ex vivo studies, NEG-9 was selected for the in vivo study. The rabbits were kept in fasting condition for 24 h before the experiment commenced, and the rabbits were grouped into two groups, each containing 6 rabbits. Group I was administered the (SM) tablet (reference) at a dose of 10 mg/kg. Group II was administered intranasal NEG-9 at a dose of 10 mg/kg. Blood samples (2 mL) were collected before the administration of the drug and again 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4.5, 6, 9, 12, and 24 h after the administration of the drug. Samples were stored at 20 C until analysis.
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3.9.7.2. Pharmacokinetic analysis. The Cmax and Tmax were calculated using the plasma concentration-time curve in the WinNonlinTM Nonlinear Estimation Program. One way analysis of variance (ANOVA) was employed to assess the Na+ the Tmax, Cmax, AUC0–1, t1/2, and MRT data of (SM) from the tested NEG9 formulation and the reference tablet at a level (p 0.05) using the SPSS program. The AUC0–24 was calculated using the linear trapezoidal rule (Yuan et al., 2008). The AUC0–1 and the relative bioavailability (BAR) of the nasal NEG-9 were also calculated by using the following formula: AUNEG DoseTablet (2) Relative bioavailability ðBAR Þ ¼ AUCTablet DoseNEG
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4. Results
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4.1. Solubility studies
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Fig. 1 shows the solubility of (SM) in different oils, surfactants, and cosurfactants. The solubilisation of (SM) was highest in Labrafac PG (25.35 3.44 mg/mL) when compared with the other oils. Of the surfactants screened, Labrasol showed a superior solubilising potential (62.18 2.75 mg/mL). Additionally, Transcutol HP was selected as a co-surfactant due to its efficient solubilising effect (46.82 2.26 mg/mL).
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4.2. Pseudo-ternary phase diagram
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Five ternary phase systems were constructed, in which the (Smix) was prepared at different mass ratios (1:1, 2:1, 3:1, 1:2, and 1:3) (Fig. 2). In the Smix (1:1), it was observed that the maximum concentration of oil that could be solubilised was 9.5% using 55% of
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Fig. 1. Graphical representation of solubility of (SM) in various oils, surfactants and cosurfactants.
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the Smix. However, when the concentration of Transcutol HP was increased with respect to Labrasol (1:2 or 1:3), the NE area was decreased when compared to the 1:1 Smix ratio. As the surfactant concentration was increased in the Smix (2:1 or 3:1), higher NE regions were observed.
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4.3. Characterization of the NE
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Table 1 shows all the NEs that had droplets in the 25–61 nm nanosize range. Among the NEs, the mean UV absorbance at 638 nm varied between 0.0021 and 0.0079, and the droplet size increased as the concentration of oil increased; however, the droplet size decreased as the concentration of the surfactant in the Smix ratio was increased (Smix 3:1). The conductivity measurements (0.137–0.161 ms/cm) indicated the NEs were of the oil-in-water type.
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4.4. Thermodynamic stability of the NEs
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The stress tests, including centrifugation and freeze thaw cycles, showed that all of the formulations were physically stable. The concentrations of (SM) in the NEs remained almost constant, and no degradation was observed.
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4.5. Formulation and evaluation of the in situ gel
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Each of the NEGs had a clear appearance. The results of the gelation studies, CIC, S%, and the viscosities of the NEGs in solution and gel form are given in Table 2. The gel strength values between 25 and 50 s were considered sufficient, and the gels that exhibited gel strength within this range were selected for in vitro release. Fig. 3 shows the release profile of (SM) from the selected NEGs (6, 8, and 9), as well as from the in situ gel prepared from an aqueous (SM) suspension. NEG-8 (85%) and NEG-9 (82%) showed the highest cumulative percentage of the drug released after 12 h.
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4.6. Ex vivo drug permeation studies
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Fig. 4 shows the nasal permeation profile of (SM) while in a 5% aqueous suspension, as well as when contained in NEG-8 and NEG-9. The extent to which the drug permeated the receptor chamber from the NEGs and from the aqueous suspension after 12 h was significantly different. The cumulative percentage of (SM) that permeated from the NEG-8 was 64%, while the cumulative percentage that permeated from the NEG-9 was 69%. However, in the case of the (SM) aqueous suspension, the cumulative percentage that permeated was only 11.2%. In other words, the NEG-9 in situ gel showed a 6.5-fold higher permeation percentage than the aqueous suspension.
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4.7. In vivo pharmacokinetics study
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There was a statistically significant difference in the Tmax, Cmax, AUC0–1, t1/2, and MRT data between the (SM) from the NEG-9 and the marketed tablet (Table 3 and Fig. 5), at a level (p 0.05). In the case of the (SM) table, the MRT was 6.5 h, whereas, the MRT for the NEG-9 was 11.25 h. The mean AUC0–1 for the marketed tablet was significantly different than the AUC0–1 for the NEG-9. These results confirmed that the formulation of (SM) into an NEG increased its bioavailability by greater than 12-fold.
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5. Discussion
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NEs are thermodynamically stable systems and are formed when a particular concentration of oil, Smix and water are combined. In the case of Smix (1:1), the maximum concentration of Labrafac PG oil that could be solubilised in the phase diagram was 9.5% using 55% of the Smix. As the Labrasol concentration increased in the Smix (2:1) and (3:1), a higher NE region was
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Fig. 2. Pseudo-ternary phase diagrams indicating oil/water nanosized microemulsion region at different Smix ratios.
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Table 2 CIC%, S%, gelling capacity, gel strength, and viscosities SD in solution and gel form for different prepared in situ NEGs. Formulation
CIC (%)
S%
Gelling capacity
Viscosity before gellation (cp)
Viscosity after gellation (cp)
Gel strength (second)
NEG-1 NEG-2 NEG-3 NEG-4 NEG-5 NEG-6 NEG-7 NEG-8 NEG-9 NEG-10
0.18 0.15 0.19 0.18 0.17 0.16 0.20 0.19 0.16 0.15
2.8 2.4 2.9 2.8 2.7 2.5 3.0 2.9 2.6 2.2
+ ++ + ++ +++ +++ ++ +++ +++ ++++
1.87 0.21 1.95 0.26 1.91 0.19 2.18 0.29 2.47 0.31 2.71 0.34 2.05 0.21 2.61 0.33 3.01 0.41 3.38 0.37
19.43 1.21 23.47 1.72 21.03 1.25 25.55 2.08 28.27 2.87 34.99 3.01 27.11 2.16 31.03 3.01 33.16 3.02 37.44 3.51
16.43 20.74 17.14 21.75 24.02 45.93 22.35 37.88 41.96 59.42
Fig. 3. (SM) release profile from different NEG formulations (NEG-6, 8, and 9) and from control in situ gel. 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304
observed, and about 20% of the oil could be solubilised. This may be due to a further reduction in the interfacial tension, thereby increasing the fluidity of the interface, this could result in greater penetration of the oil phase in the hydrophobic region of the Labrasol monomers (Yuan et al., 2008). The presence of Transcutol HP as cosurfactant decreases the ending stress of the interface and makes the interfacial film sufficiently flexible to exhibit different curvatures that are required to form NEs over a wide range (Kawakami et al., 2002). However, when the concentration of Transcutol HP was increased with respect to Labrasol (Smix ratio 1:2 and 1:3), the NE area decreased as compared to the Smix ratio (1:1); this could be due to the formation of a smaller amount of micelles (Lawrence and Rees, 2000). Transcutol HP is a polar co-solvent that can highly incorporate into water, and the relatively low Transcutol HP
content in the NE system, such as with NE-7, decreases the hydrophilicity of the Smix, and therefore the amount of oil could be solubilise in the o/w NE was decreased (about 10% only). The NE formulations were then evaluated for their physical stability. There was no sign of phase separation or turbidity observed for the ten formulations (NE1-10) during their preparation, indicating the formulations were physically stable. All of the formulations passed stability stress tests, including centrifugation and freeze thaw cycles. All the tested NEs had droplets within the nanosize range (between 25 and 61 nm). The droplet size increased as the concentration of oil in the formulations increased. The droplet size decreased as the concentration of Labrasol in the Smix increased (ratio 3:1). The addition of a surfactant to the NE systems may cause the interfacial film to condense and stabilize, resulting in smaller droplet diameters, whereas the addition of the Transcutol HP cosurfactant may cause the film to expand (Djekic and Primorac, 2008). The conductivity measurements (0.137–0.161 ms/cm) indicated that the NEs were of the o/w type, and an increase in the Labrafac PG concentration in the NE resulted in a decrease in conductivity. In situ nasal nanosized microemulsion gels (NEG) were prepared successfully using 0.5% deacetylated gellan gum as an ionic induced gelling agent. All of the NEG formulas had a clear appearance. Results from the viscosity tests for the NEGs in solution and in gel form indicated that there was a marked increase in viscosity values (19.43–37.44 cp) in the presence of NF when compared to the NEGs in solution (1.87–3.38 cp); this change was due solely to the gel conversion, which indicated the in situ nature of all the prepared gels. When the DGG solutions transformed into gels, no obvious volume expansion was observed. The expansion coefficient was only about 3%. This slight expansion may not cause discomfort to patients when the in situ gel is administered into the nasal cavity (Zheng et al., 2011). When developing an NEG, the gel strength is an important factor to consider; the ideal gel strength allows for the easy nasal administration of the gel but does not let the gel leak from the nose. It is very important that the nasal gel formulation must have suitable gel strength. A gel strength less than 25 s may not have sufficient structural integrity and may erode rapidly, however, gels Table 3 Pharmacokinetic parameters of (SM) after administration of 10 mg/kg intranasal in situ NEG-9 and 10 mg/kg of marketed tablet (n = 6). The mean difference is significant at the 0.05 level.
Fig. 4. Percentage of cumulative (SM) permeation for the various NEG (8 and 9), and for (SM) aqueous suspension through nasal tissue.
Pharmacokinetic parameters
NEG-9
(SM) marketed tablet
Tmax (hr) Cmax (mg/ml) AUC0 t (mg hr/ml) K (hr1) MRT (hr) AUC0–1 (mg hr/ml)
5.3 0.4 87.13 1.32 293.51 4.34 0.477 0.036 11.25 0.51 404.49 6.43
4.2 0.5 22.25 0.62 25.24 2.11 0.348 0.031 6.51 0.32 34.22 3.43
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Fig. 5. Plasma-concentration time curve of (SM) after administration of 10 mg/kg intranasal in situ NEG-9 and 10 mg/kg of marketed Tablet (n = 6). The mean difference is significant at the 0.05 level.
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with a strength greater than 50 s may be too stiff and cause discomfort (Tanaji et al., 2008). For this reason, the NEGs-1,2,3,4,5,7, and 10 were excluded from further evaluations. The in vitro release profiles for NEGs-6, 8, and 9 exhibited inflection points; the initial release rate of the drug was very rapid due to incomplete gel formation, but the release slowed down after the gel was completely formed and remained at a steady state. The results showed that NEG-8 and NEG-9 showed the highest cumulative percentage of the drug released, 85% and 82%, respectively, after 12 h. This could have been due, in part, to these NEGs having the smallest droplet size and the lowest viscosity of all the prepared NEGs. The ex vivo permeation results showed that the NEG system significantly increased the nasal permeation of (SM). NEG-9 showed the highest release of the drug when compared with the other formulation. This formulation showed a 6.5-fold higher permeation percentage than the (SM) aqueous suspension, these characteristics make NEG-9 an excellent carrier for the nasal administration of (SM). Also, an applied NE is expected to penetrate and to exist intact in the nasal tissue, thereby altering both the lipid and polar permeation pathways (Thacrodi and Panduranga, 1994). Additionally, the hydrophilic domain of the NEs can hydrate the external surface of the nasal tissue to a greater extent and play an important role in the tissue uptake of the drugs. When the aqueous fluid of the NEs enters the polar pathway, it increases the interlamellar volume of the tissue lipid bilayer, resulting in the disruption of its interfacial structure (Sanjula et al., 2007). The pharmacokinetic study results revealed that intranasal application of (SM) can significantly modify its pharmacokinetic profile and can increase its bioavailability by more than 12-fold in comparison with the marketed oral tablet formulation. This was due to the fact that (SM) is a drug with poor aqueous solubility and GIT permeability, and the preparation of this drug as a nasal NE enhanced its solubility and diffusion which leads to enhancing the bioavailability by more than 12-folds. Also, the pharmacokinetic of drugs upon delivery in nanosized microemulsion formulation are dictated by the properties of the nanosized microemulsions rather than by the physicochemical properties of the drug molecules. Additionally, the presence of Transcutol HP in the NE formula cause steric hindrance which help in reducing the tissue uptake by evading the RES, this will increase the residence time of nanosized microemulsions in the blood circulation Furthermore, gellan gum in situ NEG has a better mucoadhesive property, thereby ensuring intimate contact between the NE and the nasal tissue, and prolongs the retention of the formulation at the site of administration, which enhances permeation (Cao et al., 2009). In addition, the enhancement in the bioavailability of the drug by more than
12-fold could be attributed to the decreased amount of hepatic first pass metabolism that is associated with the oral administration of the (SM) marketed tablets.
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6. Conclusion
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The formulation of (SM) as a nasal in situ nanosized microemulsion gel, which is a novel drug delivery system, provided the maximum in vitro and ex vivo nasal permeation. The bioavailability of (SM) was enhanced by more than 12-folds in relation to the marketed tablet. The improved nasal NEG formula was composed of 17.5% Labrafac PG, 33% Labrasol, and 11% Transcutol HP, and was gelled with 0.5% Deacetylated Gellan gum. The use of this formula could eliminate the major drawbacks of the conventionally used tablet. Of course, it will not obviate the need for further clinical evaluation for this novel formula, which may inform clinicians of other important data.
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Declaration of interest
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The authors reported that there is no any conflict of interest.
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