Spray dried self-nanoemulsifying drug delivery systems for sertraline HCl: Pharmacokinetic study in healthy volunteers

Purpose: The aim of this study is to improvelow oral bioavailability of sertraline HCl by formulation and characterization ofsolid self-nanoemulsifying drug delivery system [SNEDDS] using spray drying technique. Methods: Solubility of sertraline HCl in different vehicles was determined, and ternary phase diagrams were constructed.Various formulations were prepared and characterized by morphological characterization, differential scanning calorimetry and droplet size analysis. The formulations were evaluated for in vitro release profi le in comparison to the marketed product [Lustral® tablets]. The in vivo study was performed on healthy human volunteers for pharmacokinetic analysis of the optimized formulations. Results: In vitro release data showed signifi cant improvement of dissolution rate of sertraline HCl in form of liquid SNEDDS compared to the plain drug. Optimized liquid SNEDDS were chosen for the preparation of solid SNEDDS by spray drying technique. High dissolution effi ciency values of solid SNEDDS indicated the increase in dissolution characteristics of sertraline HCL in solid SNEDDS. F6 SNEDDS, comprising Capmul® 20%, Cremophor® 53.4%, Transcutol® 26.6% showed highervalues for AUC[0-72 h], AUC [0-∞] and AUMC[0-72h] compared to Lustral® tablets. Conclusion: The prepared formulation reveals the potentiality of incorporating sertraline HCl in a SNEDDS formulation to improve the biological performance of the drug. Research Article Spray dried self-nanoemulsifying drug delivery systems for sertraline HCl: Pharmacokinetic study in healthy volunteers Hussein O. Ammar1, Mahmoud M. Ghorab2, Dina M. Mostafa3, and Amira M. Ghoneim1* 1Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences and Pharmaceutical Industries, Future University in Egypt 2Department of Pharmaceutics, Faculty of Pharmacy, Cairo University, Egypt 3Department of Pharmaceutical Technology, National Research Centre, Cairo, Egypt Received: 09 June, 2018 Accepted: 25 June, 2018 Published: 27 June, 2018 *Corresponding author: Hussein O. Ammar, Future university in Egypt, 90th St., Fifth Settlement, New Cairo, Cairo Egypt, Tel: 01224472851; E-mail:


Introduction
Depression is the most familiar mental disorder that affects a large number of individuals in all countries. However, depression is under diagnosed and often under cured [1].
Recent research believes that depressive episodes may intensify severity of following episodes and may escalate theurgency for more health care resources, if left untreated.
Sertraline HCl -a selective serotonin re-uptake inhibitor -is indicated for the treatment of depression and anxiety disorders, panic disorder and post-traumatic stress disorder [2]. It is considered appropriate for the treatment of depressive symptoms in elderly patients, including those suffering from Alzheimer's disease, as it has minimal anticholinergic activity and is essentially devoid of cardiovascular effects [2].
However, sertralineHCl is practically insoluble in water and undergoes extensive fi rst pass metabolism, resulting in poor bioavailability [40-45%]. Therefore, this required the drug to be taken in high doses in order to maintain adequate plasma levels [3]. Various problems are associated with its oral delivery such as gastrointestinal disturbances such as dry mouth, diarrhea, decreased appetite, nausea, impotence and insomnia [4]. In order to overcome the solubility problems, several formulation techniques can be approached. Among these techniques are self-nanoemulsifying drug delivery systems.
Nanoemulsions are isotropic systems, thermodynamically stable and the diameter of the droplets are within the range of 10-100 nm [5]. Self-nanoemulsifying drug delivery system is a pre-mixture of drug, oil, surfactant and cosurfactant that can be used to deliver drugs that are oil soluble. After gentle shaking and gastric juice dilution in stomach, it can form nanoemulsion spontaneously [6]. It is a suitable drug delivery system for oil-soluble drugs because it can be self-emulsifi ed study in healthy volunteers. Int J Pharm Sci Dev Res 4(1): 009-019. DOI: http://doi.org/10.17352/ijpsdr.000017 to nanoemulsion readily and steadily under mild condition in GI tract, providing large surface area for absorption and hence enhancing the bioavailability and lessening the irritation caused by the direct contact of the drug with GI wall. The premixture can be stored for a very long period in capsules because of the high thermodynamic stability [7].
SNEDDS have the ability to decrease the slow and inadequate release of a drug, promote the formation of the solubilized phase and the extent of the transportation through intestinal lymphatic system, hence augmenting drug absorption from the GI tract [8].
Usually, the SNEDDS are prepared as liquid dosage forms. These liquid SNEDDS have to be administered in soft gelatin capsules. The major problems associated with soft gelatin capsules are the high production costs, poor stability, inadequate drug loading and low portability [9]. This shows the importance of the formulation of the solid SNEDDS which aims to merge the qualities of the conventional SNEDDS with those of solid dosage forms.
Several solidifi cation techniques are used to prepare solid SNEDDS. Examples of these techniques are: adsorption to solid carrier, melt extrusion, spray drying, nanoparticle technology, etc. One of these techniques is adsorption to solid carrier which is considered one of the most uncomplicated and economical technique, which gives stable free fl owing solid SNEDDS powder. It can be easily fi lled in hard gelatin capsule and easily disperses upon GI fl uid contact [10].
Many recent studies have discovered that the solidifi cation technique and selection of several solid carriers could negatively affect the size of the droplet of the solid SNEDDS and the concentrations of excipients therein [11]. However, there remains a lack of investigations on the effect of different types of solid carriers in solid SNEDDS formulations. It is also of great importance that the solidifi cation procedure should conserve small droplet size for enhancing bioavailability of the formulated drug.
In this study, we have chosen to investigate the effect of spray-drying on the preparation of solid SNEDDS. It is a simple and inexpensive method commonly used for microencapsulation of drug components. In this study, we also elucidate the feasibility of dispensing sertraline HCl in liquid and solid SNEDDS for enhancing its bioavailability. The investigations include preparation, dissolution, characterization and pharmacokinetic evaluation of selected formulations.

Solubility study
The solubility of sertraline HCl in assorted oils, surfactants and cosurfactants was determined by adding an excess amount of drug to 2 ml of selected vehicles in 5 ml stoppered vials, and mixed using a vortex mixer. The vials were then kept at 25 ± 1.0 °C in an isothermal shaker for 72 h to attain equilibrium. The samples were removed from the shaker and centrifuged at 3,000 rpm for 15 min. The supernatant was taken and fi ltered through a 0.45 μm membrane fi lter. The concentration of the drug was determined spectrophotometrically at 273.4 nm.

Construction of ternary phase diagrams
According to the solubility studies of drug, Capmul®, PGML® and Captex® were selected as oil phase. Tween 80® and Cremophore EL® were used as surfactants and Transcutol®was used as cosurfactant. Oil, surfactant and cosurfactant were arranged in variuos combination for phase studies [12]. Surfactants and cosurfactant [Smix] were blended in different ratios [1:1, 2:1, 3:1 and 4:1]. Mixture of oil and Smix was prepared at ratios [w/w] of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7; 2:8, 1:9 and 0:10 in vials. A visual examination was made directly for clarity, and precipitation of drug and excipients [13]. An aliquot [0.2 ml] of the formulation was added to 300 ml of double distilled water in a glass beaker at 37 °C, and the contents were mixed mildly with a magnetic stirrer at 100 rpm. The outcome emulsions were stored for 48 h at ambient temperature and observed for clarity, drug precipitation and coalescence of droplets. Emulsions showing phase separation, cracking or coalescence of oil droplets were judged as unstable emulsions. Phase diagram was designed to pinpoint the selfemulsifying region using Triplot v1-4 software.

Preparation of liquid SNEDDS of sertraline HCl
A group of liquid SNEDDS formulations were prepared using oil, surfactant and cosurfactant. Sertraline HCl [25 mg] was added to the mixture, and then the ingredients were mixed by mild stirring and vortex mixing, and heated at 37˚C. The sample was stored at room temperature until further experimentaion [14].

Characterization of liquid SNEDDS
Droplet size, particle size distribution and zeta potential analysis: One gram of SNEDDS formulation containing 25 mg of sertraline HCl was diluted to 100 mL with distilled water in a fl ask and was blended gently by inversion. The droplet size, zeta potential and the polydispersity index [PDI] of the

Characterization of solid SNEDDS
Visual observation: In order to assess the emulsifi cation properties, one gram of each formulation was introduced into 100 ml of distilled water in a beaker at room temperature and the content was mixed manually. The tendency to form a transparent or unclouded emulsion was evaluated as good, and when the formation was poor or milky in appearance, it was judged as bad. The prepared nanoemulsions were checked for 24 hrs for any sign of drug precipitation [16].
Robustness to dilution: Solid SNEDDS were diluted to 10, 100, 1000 times with different dissolution media, viz. water, 0.1 N HCl, and buffer with different pH values [1.2, 4.5, 6.8]. The diluted nanoemulsions were stored for 24 hr and observed for any sign of phase separation or drug precipitation [17].

Morphological analysis:
The outer microscopic structures of solid SNEDDS formulations were examined using a scanning electron microscope with an image analysis system [ImageInsideVer 2.32]. The powders were fi xed to a brass grid using double-sided adhesive tape, stained by 1% aqueous solution of phosphotungestic acid and observed after drying.

Solid state characterization by DSC:
The thermal characteristics of sertraline HCl powder, PVP, lactose, physical mixtures and solid SNEDDS formulations were inspected using a differential scanning calorimeter [LabX XRD-6000, Shimadzu X-Ray Diffractometer, Japan]. About 2 mg of the samples were placed in sealed aluminum pans before heating under a nitrogen fl ow [25 ml/min] at a heating rate of 10 °C/ min from 50 °C to 200 °C.
Assessment of droplet size, polydispersity index and zeta potential: One gram of solid SNEDDS formulation containing 25 mg of sertraline HCl was diluted to 100 mL with distilled water in a fl ask and was mixed gently. The droplet size, zeta potential and the polydispersity index [PDI] of the solid SNEDDS formulations were determined by photon correlation spectroscopy that analyze the fl uctuation in light scattering due to the Brownian motion of the droplets as function of time. Light scattering was monitored at 25 °C at a 90 0 angle [16].
In vitro drug release study of solid SNEDDS: One gram of SNEDDS formulations containing 25 mg of sertraline HCl was put in hard gelatin capsule [0 size]. Optimized formulation release was compared with that of Lustral ® tablets [25 mg] to evaluate the release enhancement by solid SNEDDS formulations.

In vivo study
Protocol: The design of this study composed of "An open label, randomized, three treatments, three periods, three sequences, single dose, crossover, balanced, comparative evaluation of relative bioavailability of test [two sertraline HCl-loaded solid SNEDDS formulations at a dose of 25 mg] and reference formulation [Lustral ® , half of white capsular shaped 50mg sertraline hydrochloride fi lm-coated tablets] in 6 healthy human subjects under fasting conditions". This study was approved by the research ethics committee for experimental and clinical studies at Faculty of Pharmacy, Cairo University.
Each individual orally administered a single dose of test and reference formulation with 240 mL of water after recommended wash out period of 14 days. Drinking water was restricted [at least] from 1 h before dosing and up to 2 h after dosing, supine position was restrained for four hours after dosing. Blood samples were collected in microcentrifuge tubes containing EDTA as an anticoagulant before [0.

Pharmacokinetic analysis
Noncompartmental analysis was performed by using WinNonlin ® software [Pharsight Co., Mountain View, CA, USA] to calculate the pharmacokinetic parameters. Also, the area under the plasma concentration-time curve [AUC] was calculated using the linear trapezoidal method.

Results and Discussion
Solubility study Solubility
When Capmul® is incorporated as the oil phase with Cremophor®; surfactant, the nanoemulsion area is much larger than that with Tween 80®. Cremophor®is a good emulsifi er for the drug used in the formulation; it may produceminimization in the surface tension and fl uidizes the interfacial surfactant fi lm which can broaden the range of existence of microemulsion system [20]. Also Capmul®, which is a medium chain monoglyceride, is expected to raise the interfacial fl uidity of surfactant boundaries in the micelles because of the entrapment of Capmul®in the surfactant with the high HLB value, enhancing the emulsifi cation process upon dilution with aqueous medium [21].
The present study indicates that Cremophor® has better ability to emulsify PGML® than Tween 80®. Although, HLB values of both surfactants used in the study are greater than 10, there is considerable difference in their ability to emulsify oils. Results obtained indicate that apart from HLB value, other factors such as the structure and the relative length of hydrophobic chains of surfactants infl uence nanoemulsifi cation. These results are in conformation with results reported in literature [17]. The systems I, II, IV and V were selected based on the ability of the prepared ternary systems to form nanoemulsion containing the highest oil content [ Figures 1-4

Droplet size, particle size distribution and zeta potential analysis
The droplet size of the nanoemulsion is a crucial factor in self-emulsifi cation performance because it determines the rate and extent of the release of the drug leading to enhancement of absorption [9]. From the results, it can be concluded that in most systems, the formula containing Smix ratio 1:1 or 2:1 shows the least globule size compared to the formulae containing Smix ratios 3:1 and 4:1. The reason may be due to higher surfactant concentration that results in more rapid maturation of the droplets. The charge of oil droplets of SNEDDS is another property that should be assessed for increased absorption [23]. The charge of the oil droplets in SNEDDS is negative due to the presence of free fatty acids. The results of droplet size determination and zeta potential of the different SNEDDS are shown in table 2.

Morphological characterization
Transmission electron microscopy is the most important   Combination of bright fi eld imaging at increasing magnifi cation and of diffraction modes was used to reveal the form and size of the nanoemulsion. The globules of the nanoemulsion appear as dark and the surroundings were bright. Globules were seen as uniform in size and spherical in shape which indicates the good state of nanoemulsion.

In vitro drug release study
The In vitro dissolution studies were carried out in order to ensure the quick release of the drug in the dissolution medium and they act as a critical quality control tool for the dosage forms. Moreover, In vitro dissolution studies also give an idea about the self-nanoemulsifi cation effi ciency of the developed system [25]. The results are illustrated in fi gure 6.     Robustness to dilution: Diluted nanoemulsions with various dissolution media, must stay robust to all dilutions, and should not separate or precipitate even after 24 h of storage [29]. Diluted SNEDDS did not show any precipitation or phase separation on storage in different dilution media. This shows that all media were robust to dilution.   Citation: Ammar  Comparison between the mean particle size of formulations using lactose and PVP reveals that formulations prepared with PVP show signifi cantly smaller [P <0.05] particle size than formulations prepared with lactose.    In general, zeta potential can be positive or negative in the range of −30 to +30 mV and the measured zeta potential of all solid SNEDDS lie within this range.

In vitro drug release study of solid SNEDDS
In the self-emulsifying systems, the required free energy to form an emulsion is low, therefore allowing formation of an interface spontaneously between the oil droplets and water.
It is suggested that the oil/surfactant/cosurfactant and water phases swelleffectively, decreasing the oil droplet size and eventually increasing the release rate [39]. Comparing the release effi ciency of solid SNEDDS with Lustral ® tablets (89.23 %) reveals that F14 and F19 prepared from both lactose and PVP show higher [P <0.05] release effi ciency than Lustral ® tablets; this could be due to the increased effective surface area and alteration in the native crystalline form of the drug.
Overall, SNEDDS prepared by PVP show higher [P <0.05] release effi ciency than those prepared by lactose. This also agrees with the particle size results which show that SNEDDS prepared with PVP have smaller particle size than those prepared with lactose.
On the basis of these results, F6 prepared with lactose and F14 prepared with PVP were chosen for further investigation. F6 was chosen because it shows the smallest particle size and the lowest PDI in all SNEDDS prepared by lactose, also the release effi ciency is comparable to that of Lustral ® tablets. Although F2, F6 and F10, prepared with PVP, show smaller particle size than F14, the latter was chosen because it shows higher [P <0.05] release effi ciency than Lustral ® tablets and also lower PDI than other solid SNEDDS.

In vivo study
The LC       The improved bioavailability of SNEDDS may be also due to its lymphatic transport through transcellular pathway [42]. It is also reported that the long-chain oils enhance lipoprotein synthesis and consequent lymphatic absorption [43]. The single layer of epithelial cell of the intestine is considered the main rate-limiting barrier for drug absorption/diffusion. The enhanced permeability could be due to high content of surfactants in SNEDDS which caused disturbing of the cell membrane. Surfactant likewise exhibited a reversible effect on the opening of tight junction; it may interact with the polar head groups found in the lipid bilayers, altering hydrogen bonding and ionic forces between these groups. Surfactant may also insert itself between the lipophilic tails of the bilayers, causing disruption of the lipid-packing arrangement [44,45]. Surfactants can also weaken the interfacial surface tension and augment the penetration of sertraline HCl.
Also, the presence of a surfactant in the nanoemulsion system has caused changes in membrane permeability by the inhibition of an apically polarised effl ux system, which might cause enhancement of the oral absorption [45]. Not only is the bioavailability of sertraline HCl improved, but also SNEDDS form emulsion in GI fl uid under mild agitation caused by GI motility. This lipid emulsions have advantages in terms of high drug loading capacity and reduction in irritation or toxicity of the incorporated drug. [46] . Moreover, the emulsion droplets lead to a more uniform and rapid distribution of drug in the GI tract. The mucosal irritation is minimized due to the contact between the drug and the gut wall [47].

Discussion
In this study, the liquid SNEDDS of sertraline HCl were prepared then the optimized formulations were spray dried using lactose and PVP as solid carriers. The solid SNEDDS consisted of smooth granular particles, DSC analysis suggested that the drug in the solid SNEDDS is in the molecularly state or in the amorphous state. In vitro release study test showedthat the solid SNEDDS had a faster release ratethan the Lustral® tablets. In vivo study in healthy volunteers showed that solid SNEDDS gave signifi cant increase inthe bioavailability of sertraline HCl compared to the Lustral® tablets.
From all the afore-mentioned results, it is evident that we can throw a strong beam of light on the potentiality of incorporating sertraline HCl in a SNEDDS formulation to improve the biological performance of the drug, via enhancing the bioavailability of the drug as well as minimizing its side effects.

Confl ict of interest
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (Research Ethics Committee for experimental and clinical sciences at Faculty of Pharmacy Cairo University) and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from all patients for being included in the study.