Brain Targeted Intranasal Zaleplon Nano-emulsion: In

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posed of 10% Miglyol® 812, 40% Cremophor® RH40 40%Transcutol® HP and 10% water ..... solubilization, which might increase the toxicity of the sys- tem.
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RESEARCH ARTICLE

Brain Targeted Intranasal Zaleplon Nano-emulsion: In-Vitro Characterization and Assessment of Gamma Aminobutyric Acid Levels in Rabbits’ Brain and Plasma at Low and High Doses Eman Abd-Elrasheed2, Sara Nageeb El-Helaly1*, Manal M. EL-Ashmoony1,2 and Salwa Salah1 1

Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt; Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Al-Ahram Canadian University, 6th of October City, Egypt 2

ARTICLE HISTORY Received: August 27, 2017 Revised: November 03, 2017 Accepted: November 17, 2016

DOI: 10.2174/1567201814666171130121732

Abstract: Zaleplon is a pyrazolopyrimidin derivative hypnotic drug indicated for the short-term management of insomnia. Zaleplon belongs to Class II drugs, according to the biopharmaceutical classification system (BCS), showing poor solubility and high permeability. It undergoes extensive first-pass hepatic metabolism after oral absorption, with only 30% of Zaleplon being systemically available. It is available in tablet form which is unable to overcome the previous problems. The aim of this study is to enhance solubility and bioavailability via utilizing nanotechnology in the formulation of intranasal Zaleplon nano-emulsion (ZP-NE) to bypass the barriers and deliver an effective therapy to the brain. Screening studies were carried out wherein the solubility of zaleplon in various oils, surfactants(S) and co-surfactants(CoS) were estimated. Pseudo-ternary phase diagrams were constructed and various nano-emulsion formulations were prepared. These formulations were subjected to thermodynamic stability, in-vitro characterization, histopathological studies and assessment of the gamma aminobutyric acid (GABA) level in plasma and brain in rabbits compared to the market product (Sleep aid®). Stable NEs were successfully developed with a particle size range of 44.6±3.4 to 136.9±1.6 nm. A NE composed of 10% Miglyol® 812, 40% Cremophor® RH40 40%Transcutol® HP and 10% water successfully enhanced the bioavailability and brain targeting in the rabbits, showing a three to four folds increase than the marketed product.

Keywords: Zaleplon, intranasal nano-emulsion, in-vitro release, in-vivo studies, brain targeting, GABA measurement. 1. INTRODUCTION Zaleplon (ZP) is a pyrazolopyrimidine derivative, has a molecular weight of 305.34 [1]. It is a hypnotic drug indicated for the short term management of insomnia where difficulty in falling asleep is the primary complaint [2], without the risk of dependence or rebound insomnia upon discontinuation [3]. It also possesses potent anticonvulsant activity against pentylenetetrazole- and electroshock-induced convulsions [4]. The total dose of Zaleplon is 10 mg before bedtime. Elderly, debilitated patients or those with mild to moderate hepatic impairment should be given 5mg [5]. Zaleplon is a full agonist for the benzodiazepine 1 receptor located on gamma aminobutyric acid type A (GABAA) receptor ionophore complex in the brain, with lower affinity for the 2 and 3 subtypes. It selectively enhances the release of GABA neurotransmitter, which is similar in action but more selectively than benzodiazepines. *Address correspondence to this author at the Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Kasr El Aini Street, 11562, Cairo, Egypt; Tel: +2 0100 578 4049; E-mail: [email protected] 1567-2018/18 $58.00+.00

Its selectivity for the 1 receptor rather than 2 receptor, is expected to provide sedative action with reduced effects on cognition and psychomotor function. Zaleplon is rapidly and completely absorbed after oral administration. However, it undergoes extensive first pass hepatic metabolism after absorption, with only 30% of Zaleplon being systemically available [6]. Zaleplon attains peak concentration (Cmax) within 1.1 hour (tmax) approximately after administration, with terminal elimination half-life of 1 hour [7]. The ultra-short half-life gives Zaleplon a unique advantage over other hypnotics as it lacks next day residual effects on driving and other performance related skills [8, 9]. Zaleplon belongs to BCS Class II drugs with poor solubility and high permeability [10, 11]. Thus, although Zaleplon is rapidly absorbed after oral administration, its poor aqueous solubility (practically insoluble) can make its absorption, dissolution rate limited and thus delay its onset of action [12]. Intranasal drug delivery system is used for both topical and systemic therapies and is now presented as an alternative to oral and parenteral routes [13]. There are two main mechanisms for absorption through the nasal mucosa and © 2018 Bentham Science Publishers

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reaching the brain tissues; either it can be absorbed from the olfactory neural cells directly to the brain tissues or it can be absorbed from the highly vascular respiratory region into systemic circulation. Intranasal drug delivery system bypasses the first pass metabolism and can be used to achieve therapy directed to the brain tissue or cerebrospinal fluid [14]. The anatomical structure of the nose permits systemic drug delivery with a speedy onset of action due to its high permeability and high vasculature. At the same time, its nearly neutral environment can overcome limitations of the oral route and duplicate the benefits of intravenous route. Moreover, it is a non-invasive route that can be administered by the patient with a higher degree of adherence and compliance than the intravenous route [13]. Karthik et al. reported the use of lipid emulsion based delivery systems for the enhancement of Zaleplon oral bioavailability with the aid of self-nanoemulsifying systems [15]. The nano-emulsions were converted to a powder form by adsorbing them onto different adsorbents like maltodextrin, aerosil 200, pearlitol SD200, neusilin US2. A higher predicted permeability coefficient and percentage fraction oral dose was absorbed from the in-situ rat intestinal perfusion studies for self-nanoemulsifying powder formulation with respect to control. An in-vivo pharmacokinetic study (PK) was conducted in Wistar rats. The results from the PK studies revealed an enhancement of ~3 fold in the oral bioavailability upon the administration of nano-formulations compared to ZP suspension. K. M. Hosny & Z. M. Banjar developed nanoemulsiongel (NEG) composed of 15% Miglyol, 30% Labrasol as surfactant and 10% PEG200 as a co-surfactant. Carbopol 934 was used as an in-situ gelling agent, showing a particle size range of 35 to 73 nm. It successfully provided the maximum in-vitro, ex-vivo permeation and enhanced the bioavailability in the rabbits by eight folds, when compared with the marketed tablets [16]. Therefore, the aim of this study was to formulate intranasal ZP-NE to improve the solubility, dissolution and hence the bioavailability and brain targeting of the poorly water soluble ZP by formulating NE and use the benefits of intranasal delivery to bypass the first-pass effect and target the drug directly to the brain. And to conduct in-vivo pharmacokinetic studies to ensure brain targeting by assessment of the GABA level in plasma, and brain, one hour after its nasal administration in rabbits compared to the market product (Sleep aid®). 2. MATERIALS AND METHODS Zaleplon was a kind gift from Al Andalous for pharmaceutical industries (6th of October city, Egypt). Sleep aid® 5 mg tablets was purchased from October 107 Pharma S.A.E, 6 October city-Egypt, Capryol™ 90 (propylene glycol monocaprylate), Labrasol® (PEG-8 caprylic/capric glycerides), Labrafac™ Lipophile WL 1349 (caprylic/capric triglyceride), and Transcutol® HP (diethylene glycol monoethyl ether) were kind gifts from Gattefosse (SaintPriest, Lyon, France). Isopropyl Myristate (IPM) was obtained from Loba Chemie (Mumbai, India). Miglyol® 812 (caprylic/capric triglyceride) was a kind gift from Eipico for pharmaceutical industries (Cairo, Egypt). Cremophor® RH40 (polyoxyl 40 hydrogenated castor oil) was kindly provided

Abd-Elrasheed et al.

by BASF SE (Carl –Bosch – str .3867056 Ludwigsha fen Germany). Tween® 80 and Propylene glycol (Propane – 1,2 diol) (PG) were purchased from El-Nasr pharmaceutical chemicals Co. (Cairo, Egypt). Dialysis tubing cellulose membrane (Molecular weight cut off 14,000) was purchased from SERAVA Electrophoresis, Carl-Benz-Str. 7, D-69115 Heidelberg. All other chemicals and solvents were of analytical grade and used as received. 2.1. Determination of the Saturated Solubility of Zaleplon in Different Vehicles To find out the appropriate oil phase, surfactant and cosurfactant for the preparation of the NE, the solubility of Zaleplon in various oils namely; Capryol™ 90, Miglyol® 812, Labrafac™ Lipophile WL1349, and IPM; different surfactants (S) including Labrasol®, Tween® 80, Cremophor® RH40 and different co-surfactants (CoS) such as, Transcutol® HP, and PG was determined. Excess amounts of Zaleplon were added to each of oil, surfactant and co-surfactant separately in stoppered vials and shaken at 37ºC for 72 hours to reach equilibrium. The mixtures were removed from the shaker and centrifuged for 10 min at 4000 rpm to remove the excess undissolved Zaleplon. The supernatants were then filtered through 0.45μm millipore filter and the drug concentration in the filtrate was determined spectrophotometrically at max 230 nm after appropriate dilution with methanol. All experiments were performed in triplicate. 2.2. Preparation of Nano-Emulsion Systems and Construction of Pseudo-Ternary Phase Diagrams A combination of S and CoS was prepared in the form of mixture with two different ratios S/CoS 1:1 (%V/V), S/CoS 3:1 (%V/V), and they were used as one component of the pseudo-ternary phase diagram, water and oil constituting the two other components. Pseudo-ternary phase diagrams were constructed for all S, CoS and oils resulting in twelve systems as shown in (Fig. 1). Oil, S/CoS (Smix) were mixed then water was added drop wise while vortexing (2-3 min) at certain volume ratios into glass tubes and then incubated in refrigerator for 24 hours till equilibrium [17]. For each system twenty-eight samples were prepared. In case of drug loaded nano-emulsion, the drug was vortexed first with the oil for 1-2 min, then S/CoS were added and vortexed for 5 min; finally, water was added drop wise while vortexing. Only clear or slight bluish dispersions were selected and assessed for their ability to incorporate 10 mg of Zaleplon. The NE formulae that did not show any precipitation of the drug were selected for further characterization. Their compositions are shown in (Table 1). 2.3. Assessment of Thermodynamic Stability The selected Zaleplon -loaded NE formulae (F1- F6) were subjected to centrifugation for 30 min at 3500 rpm. The stable formulae that did not show precipitation of the drug were subjected to cooling/heating cycles (4 °C and 45 °C). Furthermore, those formulae which passed cooling/heating cycles formulae were subjected to freeze and thaw cycles (21 °C and +25 °C) with storage at each temperature of not less than 48 hours. The formulae were then observed for any phase separation [18].

Brain Targeted Intranasal Zaleplon Nano-emulsion

Table 1.

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Composition of Selected NE Formulae (F1-F6) and Physical Parameters Measurements.

Formula

Surfactant

Oil

F1

Cremophor® RH40(60%)

Miglyol® 812(10%)

F2

Cremophor® RH40(40%)

Miglyol® 812(10%)

F3

Cremophor® RH40(35%)

Miglyol® 812(20%)

F4

Tween® 80(40%)

Miglyol® 812(10%)

F5

Labrasol(60%)

Miglyol® 812(10%)

F6

Cremophor® RH40(60%)

Capryol™ 90

Co-surfactant

Water

Particle Size (nm)

Zeta Potential (mV)

Viscosity at MinSR** (Cp)

Viscosity at MaxSR** (Cp)

Transcutol® HP

10%

62.76±11.7

-13.25±2.19

1230±7.07

246.25±1.8

(20%)

10%

44.57±3.4

-13.40±1.1

767.50±24.7

138.37±1.2

Transcutol® HP

10%

102.10±1.3

- 5.85±0.9

1075±35.3

163.75±5.3

(40%)

10%

96±0.1

-16.05±0.1

1390±14.1

103.12±0.8

Transcutol® HP

10%

136.90±1.6

-33.20±0.1

942.50±10.6

84.75±0.3

(35%)

10%

61.50±1.4

-19.90±0.8

1065±21.2

246.25±1.7

(10%)

* values are mean ± S.D. ** MinSR : Minimum Shear Rate, MaxSR : Maximum Shear Rate.

2.4. Globule Size and Zeta-Potential Analyses The mean globule size, zeta potential, and polydispersity index of the selected NE formulae were determined by photon correlation spectroscopy using a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK). 0.1 ml of each of the prepared Zaleplon- loaded NE formulae were diluted to 100 times with double distilled water. Following dilution, the glass tubes were vortexed to ensure complete dispersion of the formulation. Measurements were performed, in triplicate, at room temperature. 2.5. Viscosity Measurement The viscosities of different NE formulae were determined using (Brookfield Spindle Digital viscometer, model LVTD.USA) and the angular velocity was increased gradually from 0.6 to 12 rpm. Viscosities were determined at minimum and maximum rate of shear over the range of speeds used after 2 min. 2.6. Drug Content 0.1 ml of each of the selected NE formulae was diluted with 140 ml of PBS solution pH 7.4, the absorbance of this solution was measured spectrophotometrically at 232 nm against a blank (0.1 ml plain NE mixed with 140 ml PBS solution pH 7.4) and the concentrations were calculated in duplicates. 2.7. PH Measurements The apparent pH of the NE formulae was measured by a pH meter Jenway model 3505 (Bibby Scientific Limited, UK).

the NE formulae after dilution was measured at (400-800) nm to determine the degree of clarity of NE formulae. 2.9. In-vitro Release Study The in vitro release study was conducted according to the method used by Avgoustakis, et al with some modifications [19]. Briefly, 0.5 ml sample of the NE containing 5 mg Zaleplon was placed within a dialysis bag composed of cellulose membrane (MWCO 14,000 g/mole; SERAVA Electrophoresis) and then the bag was securely tied from both ends using a thermo-resistant thread. The release of Zaleplon from NE formulae and from drug suspension was performed using USP dissolution tester, apparatus II (DIS 6000, Copley Scientific, UK), where the loaded bags were tied to the paddles of the dissolution tester and immersed in 250 ml freshly prepared phosphate buffer saline (PBS), pH 7.4 at 37 ± 1 °C. The solution was stirred at 50 rpm for up to 7 hrs. 3 ml samples were withdrawn at regular time intervals of (0.25, 0.5, 1, 2, 3, 4, 6, 7) hrs. The volume of dissolution medium was adjusted to 250 ml by replacing each 3ml aliquots withdrawn with 3ml of PBS to maintain sink condition. Withdrawn samples were filtered through 0.45 μm membrane filter, suitably diluted and analyzed for the drug content using UV spectrophotometer (UV-1800, Shimadzu, Japan) with the dissolution medium of plain NE as blank. All release experiments were performed in triplicate and the dialysis bags were pretreated by soaking in distilled water for 10 min and the mean drug released percentages (± S.D.) were plotted versus time. 2.10. Characterization of the Optimum Formula

2.8. Spectroscopic Characterization of Percentage Transmission A 0.1 ml of the NE formulae was diluted to 100 times with double distilled water. The percentage transmission of

An optimum formula was chosen according to the results of the in-vitro characterization studies conducted. Henceforward, further investigation of the chosen NE formula (F2) was conducted as follows.

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2.11. Transmission Electron Microscopy (TEM) Morphology and structure of the optimum formula (F2) were studied using JEOL, JEM 1230 transmission electron microscope, Tokyo, Japan. 1-2 drops of the NE was suitably diluted (1:100) with double distilled water and applied on TEM grid (400- mesh carbon coated grids), then treated with a drop of 1% phosphotungistic acid and left for 30 seconds to negatively stain the sample, negative staining used to enhance the contrast and improve the taken images [20]. The coated grid was dried and then taken on a slide and observed under the microscope. 2.12. In-vivo Studies 2.12.1. Assessment of GABA Level in Rabbits’ Plasma & Brain The efficiency of the selected NE to deliver the drug across the blood–brain barrier was evaluated on rabbits. Thirty healthy adult male albino rabbits weighing between 1.9 and 2 kg were used for the study. The protocol of the study was reviewed and approved (PI 1197) by the institutional review board; Research Ethics Committee-Faculty of Pharmacy, Cairo University (REC-FOPCU). The rabbits were housed three per cage at room temperature with free access to food and water with a 12-hrs light–dark cycle. A parallel design was conducted where the animals were divided into five groups and each group contains 6 rabbits Group 1: Intranasal Saline solution, Group 2, 3: Intranasal NE receiving low(NEL) and high doses(NEH) and Group 4, 5: Oral Sleep aid® marketed product receiving low(SL) and high doses(SH). The low and high doses were 0.625 and 0.125 mg/2kg rabbit [21]. For intranasal formulation, the conscious rabbits were held from the back in a slanted position. The formulations were administered at the openings of the nostrils. The procedure was performed gently, allowing the animals to inhale all the preparation. As for the oral formulation, the conscious rabbits were made to swallow the predetermined dose of Sleep aid® using a specialized oral applicator, then 5 ml water was administered post dose. After one hour of dose administration, rabbits from the five groups were humanely sacrificed, blood samples were collected into the heparinized tubes as an anticoagulant and then centrifuged at 6000 rpm for 15 minutes using cooling centrifuge (Beckman, Fullerton, Canada). Plasma was separated and stored at –20 °C till analysis. GABA concentration in plasma was determined by GABA Elisa Kits (Catalog No: E0900r) [22]. The brain was dissected, washed twice using normal saline, made free from adhering tissue/fluid and weighed. Aliquots of ice cold saline solution were added in a ratio 1:1. The organ was then homogenized and finally GABA concentration was determined using GABA Elisa Kits. Statistical analysis between the groups received market product (Sleep aid®) and NE formula (F2) at different doses was computed by unpaired student-t-test. 2.12.2. Estimation of Nasal Toxicity The nasal-cavity mucosa of the nostril of the sacrificed male albino rabbits in which low and high doses Zaleplon NE was administered, and compared with negative control nasal-cavity mucosa (the other nostril) of the same rabbit. Within one hour of the sacrifice of the animal, the nasal cav-

Abd-Elrasheed et al.

ity was fully exposed by a longitudinal incision through the lateral wall of the nose while avoiding the damage of the septum. Following, the mucosa was carefully removed and the pieces were washed with distilled water and preserved in 10 % formalin solution [23]. The histopathological studies were conducted according to the protocol described by Bancroft et al [24]. Briefly, the samples were dehydrated by treatment with serial dilutions of methyl alcohol, ethyl alcohol, and absolute ethyl alcohol, respectively. Specimens were cleared in xylene embedded in paraffin in the oven. The temperature of the oven was adjusted at 56 °C and the samples were kept for 24 hrs. Paraffin-beeswax tissue blocks were sectioned by a sledge microtome. The obtained tissue sections (3–4 μm thickness) were collected, de-paraffinized, stained by hematoxylin and eosin, and examined under a light microscope. 3. RESULTS AND DISCUSSION 3.1. Determination of Saturated Solubility of Zaleplon For poorly soluble drugs, the drug loading per formulation is a very critical factor in the development of nanoemulsion systems, which is dependent on the drug solubility in various formulation components (oil, surfactant, and cosurfactant). The volume of the formulation should be minimized as much as possible for adequate intranasal administration (25). Additionally, the formulation of nano-emulsion with oil of low drug solubility would require incorporation of more oil to incorporate the target drug dose, which in turn would require higher surfactant concentration to achieve oil solubilization, which might increase the toxicity of the system. Therefore, the solubilizing capacity of the oil is considered as a controlling criterion in its selection (26). Zaleplon solubility was tested in four different oils (Capryol™ 90, Miglyol® 812, Labrafac™ Lipophile WL1349, and IPM). Zaleplon showed the highest solubility in Capryol™ 90 (24.6±2.2mg/ml). Miglyol® 812 showed solubility of Zaleplon (7.3±0.2 mg/ml), while other oils; i.e. Labrafac™ Lipophile WL1349 and IPM showed very poor solubility of Zaleplon (below 2 mg/ml). Regarding the surfactants, Cremophor® RH40 showed the highest drug solubility of (43.5±4.2 mg/ml), followed by Tween® 80 (33.6±4.9 mg/ml) and Labrasol® (26.5±8.5 mg/ml). Also, for the co-surfactants, Zaleplon showed the highest solubility (43.97±4.91 mg/ml) in Transcutol® HP which was significantly higher than PG that showed solubility of (16.4±0.2 mg/ml). 3.2. Construction of Ternary Phase Diagrams As shown in Fig. (1), phase diagrams were constructed using oils/water/Surfactant-cosurfactant mixture. The dots in the phase diagram systems represent the ratios of the clear formed nano-emulsion systems. Oils from different categories were used such as medium-chain triglycerides Miglyol® 812 (caprylic/capric triglyceride) and Capryol™ 90 (propylene glycol monocaprylate) [16, 25]. Three nonionic surfactants (Tween® 80, Labrasol® and Cremophor® RH40) were chosen due to their safety for all biological tissues, compatibility with various ingredients used in the preparation of nano-emulsions plus they are not affected by pH [27]. Another factor that should be taken into consideration for the selection of the surfactants is their Hydrophile-Lipophile

Brain Targeted Intranasal Zaleplon Nano-emulsion

A: Capryol 90,Water,Tween 80:Transcutol HP 1:1

Current Drug Delivery, 2018, Vol. 15, No. 0

B: Capryol 90, Water, Tween 80:Transcutol HP 3:1

C: Capryol 90, Water, CremophorRH40 : Transcutol HP 1:1

E: Capryol 90, Water, Labrasol:Transcutol HP 1:1

F: Capryol 90, Water, Labrasol:Transcutol HP 3:1

G: Miglyol 812, Water, Labrasol:Transcutol HP 1:1

H: Miglyol 812, Water, Labrasol:Transcutol HP 3:1

I: Miglyol 812, Water, Tween 80:Transcutol HP 1:1

J: Miglyol 812, Water, Tween 80: Transcutol HP 3:1

K: Miglyol 812, Water, Cremophore RH40:Transcutol HP 1:1

D: Capryol 90, Water, Cremophor RH40:Transcutol HP 3:1

5

L: Miglyol 812, Water, Cremophore RH40:Transcutol HP 3:1

Fig. (1). Pseudo ternary phase diagram of the twelve NE systems from A to L.

Balance (HLB) values. The formation of o/w microemulsions requires a surfactant with HLB greater than 10 [28]. The HLB values of Tween® 80, Cremophor® RH40 and labrasol are 15, 14–16 and 12 respectively. On the other hand, using high HLB surfactant with a low HLB cosurfactant in proper ratio gives a successful and stable NE as reported by (Mahmoud, Saleh Al-Sawyeh et al in the formu-

lation of Simvastatin self-nanoemulsifying drug delivery system which composed of 10% polar oils (C8), 60% Cremophor® RH40 and 30% Transcutol® HP [29]. Two cosurfactants (Transcutol® HP and PG) were chosen due to their safety and low HLB values (4.2 and 4.45) respectively [27, 29]. It was found that Transcutol® HP was better than Propylene glycol, resulting in successful NE formulations.

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Similar findings were detected in the preparation of Simvastatin self- nanoemulsifying drug delivery system composed of 10% polar oils (C8), 60% Cremophor® RH40 and 30% Transcutol® HP [29]. In addition Transcutol® HP has effective solubilizing ability and permeability enhancing capabilities when present in emulsified systems [30]. 3.3. Assessment of Thermodynamic Stability All the six tested formulae (F1- F6) were thermodynamically stable and were able to withstand centrifugation, heating-cooling and freeze-thaw cycles without showing any signs of drug precipitation which comes in agreement with Mahmoud, Al-Suwayeh et al that choosing high HLB surfactant and low HLB Co-surfactant give successful and stable NE [29]. 3.4. Globule Size and Zeta-Potential Analyses From Table 1, it was observed that the prepared systems possessed globule size ranging from 44.6 to 136.9 nm. The small mean droplet size (MDS) might be attributed to the use of the proper surfactant/co-surfactant mixture which reduced the free energy of the system, giving small globule in size which comes in line with Nepal and Han et al who formulate Coenzyme Q10 (CoQ10) into self-nanoemulsifying drug delivery system (SNEDDS) composed of Witepsol® H35, Solutol® HS15 and Lauroglycol® FCC in the weight ratio of 1:0.7:4:2, respectively with mean emulsion droplet size of 32.4 nm. He found that the use of proper surfactant and/or Co-surfactant can be adsorbed around oil-water interface decreasing the interfacial tension, the emulsification time and MDS [31]. Moreover, our results revealed that increasing oil concentration from 10% (F2) to 20% (F3) resulted in a significantly larger globule size from 44.6±3.6 nm to 102.1±1.3 nm that matched with Chen et al. [32]. He found that in the preparation of Triptolide transdermal microemulsion the MDS increased from 12.7 nm into 59.8 nm upon increasing oil concentration from 1.5% into 6%. This could be attributed to the expansion of oil drop of the nanoemulsion by further addition of the oil. Also, this was consistent with Yue Yuan et al who prepared Meloxicam microemulsion for transdermal drug delivery consisting of 0.375% meloxicam, 5% IPM, 50% Tween 85/ethanol (1:1) and water. He found that upon increasing oil concentration from 5% to 10% the MDS increased from 37 to 46.9 nm and by increasing oil concentration from 10% to 15% the MDS increased from 46.9 to 61.9 nm [33]. As for the two formulae containing Cremophor® RH40, F1(62.8±11.7 nm) and F2 (44.6±3.4 nm), they showed less MDS than the corresponding Tween® 80 and Labrasol® containing formulae, F4(96±0.07 nm) and F5(136.9±1.6 nm), respectively. On the other hand, the same MDS was achieved in case of nanoemulsion containing Capryol™ 90 (propylene glycol monocaprylate), F6 (61.5±1.4 nm) and medium-chain triglyceride oil Miglyol® 812, F1(62.8±11.7 nm). It was observed that formula F2 had the smallest MDS. Poly dispersity index (PDI) values ranged from 0.222 to 0.385, these small values of PDI indicate a monodispersed population [34]. NE Formulae (F1, F2, F4, F5 and F6) showed a zeta-potential values of (-13.25, -13.4, -16.05, 33.2, and -19.9 mV), respectively, indicating higher stability

Abd-Elrasheed et al.

compared to other systems. Only one formula (F3) showed zeta-potential value of less than 10 mV and it was considered neutral. 3.5. Viscosity Measurement The viscosity of the NE formulae is of foremost importance for the performance of the NE with respect to nasal clearance and drug release. As shown in (Table 1) it was found that the NE formulae containing Cremophor® RH40 (F1, F2, F3 and F6) and Tween® 80 (F4) have higher viscosity than NE formula F5, containing Labrasol® as surfactant. In addition, upon increasing the surfactant concentration as in (F1, F6) the viscosity of the formed NE increases simultaneously. This might be attributed to the use of originally viscous constituents (example; Capryol 90, Cremophor® RH40, Tween® 80, and Transcutol). Drug content All the six formulae were found to have drug content in the range of (100 - 104.86 %) as shown in Table 1. 3.6. PH Measurement The mean pH values of all formulae ranged between 5.5 to 7.21 which is within the range of the reported pH of the physiologic nasal fluid [35]. 3.7. Spectroscopic Transmission

Characterization

of

Percentage

Percentage transmittance (%T) of the diluted NE formulae (F1- F6) was measured spectrophotometrically in the visible range (400-800 nm). F1 (98.86 %T), F2 (97.64 %T), F3 (92.65 %T), F4 (93.42 %T), F5 (93.87 %T) and F6 (94.01 %T) indicating highly clear and stable systems obtained by aqueous dilution of the prepared NE formulae. This might be attributed to the fact that the oil droplets are thought to be in a state of finer dispersions [36]. 3.8. In-vitro Drug Release The percentage release-time profiles of the nanoemulsion preconcentrates in PBS pH 7.4 are shown in (Fig. 2A). The percentage release at time 4 hrs of F1 (73.4±1.2 %), F2 (80.5±4.4 %), F3 (69.9±2.5 %), F4 (70.3±0.7 %), F5 (72.2±3.5 %) and F6 (74.7±0.8 %). Approximately 70% or more of the drug is released after 4 hours from all formulae. These release profiles in PBS pH 7.4 when compared to that of the drug suspension which release only 41.6% after 4 hrs, demonstrated significant increase in the Zaleplon release profile. The percentage release profile ranked in the following order F2>F6>F1>F5>F4>F3. A possible explanation is that it was observed that as the MDS and the viscosity decrease the % drug release increase. These suggestions are in line with the findings of E Atef et al who develop Phenytoin self-emulsifying drug delivery system (SEDDS) , [37] who stated that the amount of dissolved drug in the aqueous phase at time t is inversely proportional to the radius of the droplets and Jain, Kumar et al [38] who prepared oil/water Atrovastatin NE and he found that as droplet size and viscosity of the NE decrease the percentage release of the drug increase. Formula F2 presented the highest percentage drug release as it has the smallest MDS (44.6±3.4 nm) and its viscosity (138.4±1.2 cp). F1 & F6 showed similar release profile as

Brain Targeted Intranasal Zaleplon Nano-emulsion

the difference in the MDS between the two formulae is small (61.50±1.41 and 62.8±11.7 nm), respectively and the two formulae have the same viscosity (246.3±1.8 cp). As the MDS and viscosity of F4 (96±0.07 nm and 103.1±0.88 Cp) are smaller than F3 (102.1±1.3 nm and 163.8±5.3 cp), thus, the percentage release of F4 is higher than F3. On the other hand, F5 was observed to have higher percentage of drug released than F4 and F3, even though its MDS (136.9±1.6 nm), is higher than that of F3 and F4. This could be attributed to its lower viscosity (84.8±0.35 cp) than F3 and F4 formulae. The percentage release efficiency of the nanoemulsion preconcentrates in PBS pH 7.4 are shown in (Fig. 2B). It was found that the percentage release efficiencies of all NE formulae were higher than that of the drug suspension with the formula F2 having the higher % release efficiency of about 69.4± 2.2 %.

Current Drug Delivery, 2018, Vol. 15, No. 0

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3.10. Transmission Electron Microscopy (TEM) The morphology of the formula F2 was observed using TEM (JEM-2100, JEOL, USA). The photographs depicted in (Fig. 3) revealed that most of the globules after dilution were in the size range indicated in the particle size analysis, signifying excellent state of the nano-emulsion formed. As demonstrated, the size of the formed globules is consistent with that obtained from the globule size analysis.

Fig. (3). Transmission electron micrographs of the formulated NE (F2).

3.11. In-vivo Studies 3.11.1. Estimation of Nasal Toxicity

Fig. (2A). The percentage release-time profiles of Zaleplon from different NE formulae in phosphate buffer (pH 7.4) at 37 ± 0.5°C for 7 h.

The negative control group, administrated saline, showed no histopathological alteration and normal histological structure of the lining pseudostratified columnar epithelium with the underlying lamina propria and cartilaginous structure as recorded in (Fig. 4A1, A2). Similarly, NE formula F2 at low and high doses showed no histopathological alteration in the mucosa as recorded in Fig. 4B1, B2) for NE low dose and Fig. 4C1, C2) for NE high dose. 3.11.2. Assessment of GABA Level in Rabbits’ Plasma & Brain

Fig. (2B). The percentage release efficiency of Zaleplon from different NE formulae in phosphate buffer (pH 7.4) at 37 ± 0.5°C for 7 h.

3.9. Choice of the Optimized Formula NE formula (F2) was chosen as the best optimized system as it has the maximum in-vitro release and dissolution efficiency percentage (69.4±2.2%) than other formulae and it was successfully prepared in the nano-size (44.6±3.4 nm) which is lower than other formulae with a zeta-potential of (13.40±1.13 mv) and viscosity of (767.5±24.7 cp) at minimum shear rate and (138.4±1.2 cp) at maximum shear rate, with a neutral pH of 7.09±0.03.

Hence, the therapeutic effectiveness of ZP depends upon the availability of ZP molecules at the receptor site as it promotes sleep by enhancing the activity of the inhibitory neurotransmitter, GABA, at its receptors in the brain. The formula (F2) acts on (GABAA) receptors in the central nervous system (CNS) by binding with the 1, 2 and 2 subunits of GABAA receptors resulting in an allosteric positive modulation effect [39]. Table 2 shows the effect of intranasal saline, NE formula F2 and oral Sleep aid® on GABA level in rabbits’ plasma and brain. An increase in GABA concentration of: 18.78±1.39 and 32.48±3.19 compared to 6.10±0.54 and 10.02±0.44 ng/mL in plasma and 5.14±0.49 and 8.68±0.97 compared to 1.25±0.05and 2.37±0.09 ng/g in brain tissues were observed following oral administration of 0.625mg and 1.25 mg/2kg rabbit of F2 and Sleep aid®, respectively. The plasma and brain GABA level of groups receiving ZP-NE formula (F2) at different doses (0.625 and 1.25 mg/kg) were significantly different from those receiving the market tablet (Sleep aid®) at the same doses (at a significant level, p