int j pharm sci nanotech vol 9; issue 1 international journal...

3102 Int J Pharm Sci Nanotech Vol 9; Issue 1 January February 2016

Research Paper

Biopharmaceutical and Preclinical Studies of Zaleplon as Semisolid Dispersions with Self-emulsifying Lipid Surfactants for Oral Delivery

Narendar Dudhipala1†, Arjun Narala1†, Dinesh Suram1†, and Karthik Yadav Janga1,2,* 1Department of Pharmaceutics, University College of Pharmaceutical Sciences Kakatiya University, Warangal, India, and 2Department of Pharmaceutics, Kakatiya Institute of Pharmaceutical Sciences, Warangal, India.

Received August 14, 2015; accepted October 16, 2015

ABSTRACT

The objective of this present study is to develop a semisolid dispersion (SSD) of zaleplon with the aid of self-emulsifying lipid based amphiphilic carriers (TPGS E or Gelucire 44/14) addressing the poor solubility of this drug. A linear relationship between the solubility of drug with respect to increase in the concentration of lipid surfactant in aqueous medium resulting in A

L type phase diagram was

observed from phase solubility studies. Fusion method was employed to obtain semisolid dispersions (SSD) of zaleplon which showed high content uniformity of drug. The absence of chemical interactions between the pure drug, excipients and formulations were conferred by Fourier transmission infrared spectroscopic examinations. The photographic images from polarized optical microscopic studies revealed the change in crystalline form of drug to

amorphous or molecular state. The superior dissolution parameters of zaleplon from SSD over pure crystalline drug interpreted from in vitro dissolution studies envisage the ability of these lipid surfactants as solubility enhancers. Further, the caliber of TPGS E or Gelucire 44/14 in encouraging the GI absorption of drug was evident with the higher human effective permeability coefficient and fraction oral dose of drug absorbed from SSD in situ intestinal permeation study. In conclusion, in vivo studies in Wister rats demonstrated an improvement in the oral bioavailability of zaleplon from SSD over control pure drug suspension suggesting the competence of Gelucire 44/14 and TPGS E as conscientious carriers to augment the dissolution rate limited bioavailability of this active.

KEYWORDS: Zaleplon; Self-emulsifying lipid surfactants; In situ intestinal permeation; bioavailability; polarized optical microscopy.

Introduction

Solubility is one of the major constraints in dosage form development of BCS class II and IV pharmaceutical actives. Most of the new and existing chemical entities were of these classes displaying low dissolution rate and/or poor permeation across biological membranes restraining their oral bioavailability. Numerous conven-tional techniques like aqueous soluble inclusion complexes with cyclodextrins; drug derivatization by strong or weak electrolytes; reduction in particle size with the aid of stabilizers and solid solution of drug in suitable vehicle to manipulate from solid crystalline state to amorphous or molecular level were widely investigated by formulation scientists to improve dissolution rate of lipophilic or hydrophobic drugs (Jasna et al., 2012; Swati et al., 2012; Köllmer et al., 2013; Vandana et al., 2014). However, the applicability of these strategies were confined due to various limitations such as the remnant of unwanted organic solvents from solvent evaporation technique, gastric irritation by cyclodextrins, and aggregate formation of micronized particles resulting in reduced surface area for dissolution (Chaumeil, 1998;

Kapsi and Ayres, 2001; Veiga et al., 2001). The dispersion of drug in inert hydrophilic carrierssuch as high-molecular-weight polyethylene glycols (PEGs) and polyvinylpyrrolidones are reported to have poor intrinsic solubilizing property (Saharan et al., 2009; Serajuddin, 1999).

In recent past, the lipid surfactants have attracted many researchers as potential carriers with greater solubilizing feature and permeation enhancing propensity to augment the dissolution and absorptionof poor aqueous soluble and/or permeable drugs respectively (Eedara et al., 2014). Gelucire 44/14, a saturated polyglycolized glyceride with blend of mono-, di-, and triglycerides and mono- and di-fatty acid esters of PEG, and vitamin E TPGS (d-a-tocopheryl PEG 1000 succinate) are two such lipid surfactants which demonstrated the greater solubility dependent systemic exposure of therapeutic moieties(Gattefossé, 1983; Karatas et al., 2005; Sandrien et al., 2008). The hydrophilic-liphophilic balance (HLB) value of Gelucire 44/14 is 14 and TPGS E is 13 which indicate their superior hydrophilic characteristics. Additionally, TPGS E exhibits amphiphilic nature with the hydrophilic head

International Journal of Pharmaceutical Sciences and Nanotechnology

Volume 9Issue 1January – February 2016

MS ID: IJPSN-8-14-15-DUDHIPALA

3102

Dudhipala et al: Biopharmaceutical and Preclinical Studies of Zaleplon as Semisolid Dispersions with Self-emulsifying... 3103

and lipophilic tailportions attributing to the least micelle concentration (i.e., 0.02%w/w) (Sundeep and Emilio, 2004). The self-emulsifying behavior of these lipid surfactants along with good wettability and dispersibility offer higher surface area of drug in the form of fine emulsion resulting in improved dissolution rate and progress in drug absorption. Additionally, lipid-based formulations are established to ensue improved oral bioavailability due to postprandial “food effect”(Charman et al., 1997; Sunesen et al., 2005).

Zaleplona pyrrazolopyrimidine hypnotic drug prescribed for the management of Insomnia and in pentylenetetrazole/electroshock-induced convulsions as an effective anticonvulsant since it interacts with GABA receptor (Winkler et al., 2014; Melissa et al., 2015). The oral bioavailability of this drug was poor (~30%) owing to dissolution rate restricted gastric absorption. However, previous reports statedan augment in solubility of zaleplon via solid dispersions with PEGs and complexation with cyclodextrins (Waghmare et al., 2008; Popescu et al., 2015). In our previous investigations, lipid vehicles had showed great prominence in improving the dissolution and bioavailability of zaleplon (Janga et al., 2012; Janga et al., 2013).

The main objective of this study is to develop semi-solid dispersions of zaleplon with the aid of lipid surfactants (Gelucire 44/14 and TPGS E). Initially, solubility studies were performed to assess the solubilizing character of the amphiphilic carriers. Semi-solid dispersions were obtained by fusion method and further characterized for drug content. Drug excipient compatibility was assessed by FT-IR examination. Optical polarized microscopic studies were done to scrutinize the transformation of crystalline nature of drug in the formulation. Further, in vitro dissolution studies were performed to evaluate the improvement in the dissolution rate of drug. In situ intestinal permeation studies and pharmacokinetic studies were conducted in male wistar rats to elucidate the potential of lipid surfactants as suitable carriers in improving the solubility and bioavailability of zaleplon.

Materials and Methods

Materials and chemicals

Zaleplon(ZL) was a kind gift sample from Symed laboratories, Hyderabad, India. Gelucire 44/14(G) (Lauroyl macrogol-32 glycerides EP) was generously donated byGatteffose, Saint-Priest Cedex, France. TPGS (D-alpha-tocopheryl polyethylene glycol 1000 succinate) was obtained from BASF corp., North America. All other chemicals used were of analytical grade and solvents were of HPLC grade. Freshly collected double distilled water was used throughout the study.

Biopharmaceutical Methods

Phase solubility studies

To assess the role of lipid surfactants on the solubility of zaleplon, phase solubility studies were performed as

reported method published by Higuchi and Connors (Higuchi and Connors, 1965). In brief, an excess of drugwas added to 5ml aqueous solutions containing 5, 10, 15, 20, 25 and 30% (w/v) of Gelucire 44/14 and TPGS-E. The capped vials were placed on rotary shaker and agitated at 37 oC for 48 h. The resulting samples were filtered by passing through a 0.45 μm membrane filter (Millipore, USA). The filtrates were suitably diluted and analyzed to determine the ZL concentration by the HPLC. The experiment was carried out in triplicates. Gibbs free energy of transfer , o

trG which demonstrates

the solubilzation of zaleplon, as calculated from the following equation.

o s2.303 RT log S /S otrG …..(1)

Where So/Ss is the ratio of molar solubility of zaleplon in aqueous solutions of carriers to that in pure water. The value of gas constant (R) is 8.31 J K−1 mol−1 and T is temperature in degree Kelvin.

Preparation of semisolid dispersions

The semi solid dispersions (SSDs) were prepared by fusion method using drug and lipid carrier (TPGS or Gelucire 44/14) at various weight ratios (1:1, 1:2, 1:3, 1:4) and were coded as ZST1, ZST2, ZST3, ZST4 and ZSG1, ZSG2, ZSG3, ZSG4 respectively. The required amounts of lipid surfactant was taken in china dish and melted on mantle heater at 50-60 oC followed by the addition of zaleplon. The resultant mixtures were thoroughly mixed for 5 min and allowed to cool. Finally, the semisolid dispersions were transferred into screw capped vials and stored in refrigerator until further characterization. The respective physical mixtures of ZL were prepared by blending the appropriate amounts of drug and carrier at weight ratio of 1:4 respectively.

Drug content

The drug content in the semi solid dispersions were assessed by taking accurately weighed samples of SSD (equivalent to 10 mg of zaleplon) in a conical flask with 100 mL 0.1 N hydrochloric acid and agitated by placing on rotary shaker for 1h. Further, these samples were centrifuged and supernatant was filtered through a 0.45 μm membrane filter (Millipore, USA). The filtrates were suitably diluted and quantified for drug using HPLC.

In vitro dissolution studies

To understand the effectiveness of lipid surfactants in improving the dissolution characteristics of zaleplon, in vitro dissolution study of pure drug, semi-solid dispersions and physical mixtures (ZPT4 and ZPG4) were executed using USP type II (paddle) apparatus (Electrolab, TD L8, Mumbai, India) in simulated gastric fluid (pH 1.2) without enzyme as dissolution medium. A volume of 500 mL dissolution medium was maintained at a temperature 37 ± 0.5°C with paddle speed set at 50 rpm throughout the experiment. About 5 mL samples was collected at prefixed time intervals and replenished with fresh dissolution medium to ensure the maintenance of constant volume. The clear solutions


obtained after passing the samples through 0.45 μm membrane filter (Millipore, USA) were analyzed for drug by HPLC to interpret the dissolution parameters.

Dissolution parameters

The cumulative amount of drug released at the end of 60 minutes of study (Q60) was interpolated from the graph. Dissolution efficiency (DE) is expressed as a percentage of the area of the rectangle described by 100% dissolution in the same timeafter calculating it from the area under the dissolution curve quantified by trapezoidal rule (Malladi et al., 2010). DE is measured by the following equation(2).

tydt

DEy to

100

= ×100% …..(2)

Equation 3 and 4 represents the mean dissolution time (MDT), the mean time taken by the drug to get dissolved and mean dissolution rate (MDR), the average dissolution velocity of the drug from SSD under in vitro dissolution conditions respectively (Hiba et al., 2010):

nj jnj

tMDT =1

=1

= j

j

MM

…..(3)

/ njMDR =1= jM t

n …..(4)

Where j is the sample number, n is the number of dissolution sample times, t or tj is the midpoint of the jth time period (easily calculated with [t + (t−1)]/2 and Mj is the further amount of drug dissolved between tj and t−1.

The relative dissolution rate (RDR) was determined by dividing the dissolution efficiency ofsemi solid dispersions with respective to pure drug (Valleri et al., 2004).

Optical polarized microscopy

To verify the crystalline properties of the drug in semisolid dispersion the optical birefringence of drug, carriers and mixtures (optimized solid dispersions, physical mixtures) was examined under an optical microscope with uncrossed polarizers (Nikon Eclipse E600 Pol, Nikon Instech Co., Japan). The test samples for polarizing microscopy studies were prepared in rectangular glass capillaries (gap thickness 100 μm and width 1 mm, length 5 cm). Both ends were sealed with epoxy resin.

Fourier transform infrared (FT-IR) spectroscopy

To predict the chemical compatibility between pure drug and excipients, Fourier transform infrared (FT-IR) spectra of pure drug, TPGS E or Gelucire 44/14, Physical mixtures and optimized semi solid dispersions were examined with aid of FT-IR spectrophotometer (Thermo Scientific, USA) by the conventional KBr pellet method. The scanning range and resolution of spectra was set at 4000–500 cm-1and 4 cm-1 respectively.

In situ intestinal absorption study

The in situ single-pass perfusion studies were performed using established methods reported (Zakeri

et al., 2007). The study was conducted with the prior approval of Institutional Animal Ethical Committee (IAEC), St. Peter’s institute of pharmaceutical sciences. Euthanasia and disposal of carcass was in accordance with specified the guidelines by IAEC. Male Wister rats weighing between 180-200 gm used in the study were obtained from Mahaveera Enterprises (146-CPCSEA no: 199; Hyderabad, India). The animals were housed in separate cages in a clean room and maintained under controlled condition of temperature with free access to food and water.

The rats fasted overnight with free access to water were anesthetized by intraperitoneal injection of thiopental sodium (60 mg/kg body weight) and placed on a thermostatic surface to maintain body temperature. An incision was made through a midline portion of anesthetized rat to expose the abdominal contents. The lower part of the small intestine segment used for perfusion was exposed and semi-circular incisions were made on both ends and cannulated with PE tubing followed by ligation with silk suture. After cannulation the surgical area was covered with cotton soaked in physiological saline (37°C). The intestine segment was flushed with phosphate buffered saline (PBS) (pH 7.4 at 37°C) and stabilized by perfusing the blank PBS for 15 min. The perfusates prepared by dispersing optimized semi solid dispersions (ZST4 and ZSG4) equivalent to 3 mg of zaleplon and/or pure drug (3mg) containing phenol red (7.5 μg/mL) in PBS were passed at a steady flow rate of 0.2 mL/min (NE-1600, New Era Syringe Pumps, USA) for 90 min. The perfusate was collected for every 15 min and at the end of the perfusion the circumference and length of the perfused intestine was measured. The samples were stored at -20°C until further analysis by HPLC. Control (drug dispersed in 1% w/v PEG 400) containing the same amount of the zaleplon was included in the study for comparison. Each experiment was performed in triplicate. Prior to analysis, the perfusate samples were allowed to thaw, deproteinized with methanol, centrifuged and the drug content in the supernatant was quantified for zaleplon by HPLC.

Data analysis

The absorption rate constant (Ka) was calculated from the slope of the remaining amount of drug vs. time plot. The effective permeability coefficient was determined using the following equation

in in outQ C / C Aeff (rat)P =[ .ln ( ] / …..(5)

Where Qin is the rate of perfusion (0.2 mL/min), A is the surface area within the intestinal segment that is assumed to be the area of a cylinder (2πrL) with the length (L) (measured at the end of the experiment) and radius (r) of 0.18 cm. Cin and Cout are the inlet and fluid-transport-corrected outlet solution concentrations respectively. The enhancement ratio (ER) was calculated byusing the following equation: ER = Peff(rat) of optimized semisolid dispersion/Peff(rat) of control. The data from the rat permeability studies were extrapolated to predict the


fraction dose (Fa) absorbed and effective permeability coefficient in human by fitting the data into equations 7 and 8 respectively (Zakeri et al., 2007).

Peff rate –(38450)× ( )Fa =1 – …..(6)

eff human eff ratP P( ) ( ) = 11.304 × – 0.00038

Preclinical Pharmacokinetic Study

Study Protocol

Male albino wistar rats (180-200 g) used in the study had free access to food and water. Prior to the study the protocol has been approved by Institutional Animal Ethical Committee, Vaagdevi Institute of Pharmaceutical Sciences, Warangal, India. Before dosing, the animals were kept for overnight fasting and were divided into three groups containing six in each and were randomly administered with each treatment. Control group received an oral suspension of zaleplon (drug dispersed in 0.5% w/v of sodium carboxymethyl cellulose) and the test groups were treated with ZST4 and ZSG4 respectively, at a dose of 10 mg/kg body weight. At predetermined time intervals, blood samples (500 μL) were collected from retro orbital plexus into micro-centrifuge tubes. The blood was allowed to clot and the serum was separated by centrifugation at 10,000 rpm for 10 min in a micro-centrifuge (Remi equipments, India) and stored at –20 °C until analysis.

Sample analysis

Zaleplon was quantitatively determined in serum by HPLC using 55:45 (v/v) acetonitrile and water respectively as mobile phase at a flow rate of 1.0 mL/min equipped with LC-10 AT solvent delivery unit (Shimadzu, Japan). An octadecylsilane (C18) reverse phase stainless steel analytical column (250 × 4.6 mm) with 5 μm particle size was employed for chromatographic separation (Lichrospher, Merck, Germany). The column eluent was monitored at a wavelength of 232 nm using an SPD-10 AVP ultraviolet detector and the sensitivity was set at 0.005 AUFS at ambient temperature. The serum samples were processed as described earlier in reports (Zhang et al., 2006). Briefly, 200 μL of serum sample was treated with 100 μL of methanol, 100 μL of internal standard (1 μg/mL of 2-naphthol in methanol) and 100 μL of 2.0 M sodium hydroxide solution and vortexed for 3 min. The mixture was extracted with 3 mL of ethyl acetate followed by centrifugation and the separated organic layer was dried under vacuum. The residue was reconstituted with 100 μL of mobile phase and an aliquot of 20 μL was injected onto the HPLC. The concentration vs. peak area ratio plot was linear (r2 > 0.990) over the concentration range of interest and the zaleplon content in samples was quantified using this plot.

Pharmacokinetic parameters

The peak concentration (Cmax) and its time (tmax) were obtained directly from the serum concentration vs. time profile. The area under the curve (AUC0-t) was calculated by using trapezoidal rule method. The AUCt-∞ was

determined by dividing the serum concentration at last time point with elimination rate constant (k). The relative bioavailability (F) was estimated by dividing the AUC0-∞ of optimized semisolid dispersion formulations with control oral suspension of drug.

Statistical analysis

The data obtained was subjected to student ‘t’ test and one way analysis of variance (ANOVA) and the significance of difference between formulations was calculated by student-Newman-Keuls (compare all pairs) with InstatGraphpad prism software (version 4.00; GraphPad Software, San Diego California). The level of statistical significance was chosen at P<0.05.

Stability studies

The formulations stored in glass vials were covered with aluminum foil and kept in refrigerator (4 ± 2°C) for a period of 90 days. At definite time intervals samples were withdrawn and evaluated for % cumulative drug release by in vitro dissolution studies.

Results and Discussion

Phase solubility studies

The solubility of zaleplon in distilled water was found to be 0.0568±0.004 mg/mL, which indicates its poor aqueous solubility. We could establish a linear relationship between the solubility of drug and amount of carrier, as the concentration of carrier increased the solubility of Zaleplon increased and demonstrated AL type phase diagram (Table 1). Good wetting properties of Gelucire 44/14 and TPGS-E ensured their choice as potential carriers to improve the solubility of poorly water soluble drugs (Damian et al., 2000; Sang and Jin, 2003). Thus the enhanced solubility of this therapeutic moiety may be due to its high wettability in the presence of Gelucire 44/14 and TPGS-E. The values of Gibbs free energy change ( G ) specifies the process of transfer of drug from pure crystalline phase to aqueous phase with carrier. The obtained values of G were negative at all the concentrations of carrier ensuring the spontaneous solubilization of zaleplon. Further, fall in the G values with respectto the increased lipid carrier concentrations demonstrates that the reaction became more positive with additional weight fraction of carrier in pure water (Table 1)(Higuchi and Connors, 1965).

Drug content

The drug content in all the semisolid dispersions was found to be in the range of 95-105% indicating the high content uniformity of solid dispersions.

In vitro dissolution studies

The enhanced in vitro dissolution release profiles of zaleplon from semi solid dispersions and respective physical mixtures indicates the efficiency of self-emulsifying lipid surfactants in augmenting solubility of drug are represented in Figure 1. Dissolution parameters extrapolated from in vitro dissolution profile of zaleplon from semisolid dispersions were found to be increased in

3106

comparis(Table 2semisolid50.1- 84.5to physicpure druimprovemsemisolidof these wetting moleculesThe mea1.59 ± 0substanti(0.96 ± 0.pure drudissolutio

TABLE 1

Solubility a

%Carri

05

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Fig. 1. Diss

TABLE 2

Compositiosimulated g

FormulaControlZST-1 ZST-2 ZST-3

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ment in drug rd dispersions a

amphiphilic and micellars (Yan et al., an dissolution0.18 and 1.52ially higher .24), ZPG4 (1ug (0.48 ± 0on rate (MDR

and thermodyna

TP

ier (w/v) Concezalepl

0 0.0965 5.1

10 11.015 15.420 23.725 31.530 35.R2

f Graph ge of three determi

solution profiles

on and Dissolutiogastric fluid (pH

ation Z : C l --

1:1 1:2 1:3

f physical mixent amount after 60 mi

ignificantly h(ZPT4 (57.0%6.1%) (p<0.

release from lattributes to surfactants,

r solubilizatio2007; Repka n rate of Z2 ± 0.24 res

when com.00 ± 0.12) (p0.11) (p<0.01R) and low m

mic parameters

PGS-E entration of lon (mg/mL)

65 ± 0.004 15 ± 1.24 03 ± 0.75 42 ± 1.64 78 ± 1.32 58 ± 0.54 14 ± 0.24 0.990

AL nations±S.D

of zaleplon from

on parameters ofH 1.2) (mean±SD;

Q60 31.2 ± 6.1 252.4 ± 5.4* 462.7 ± 6.5* 577.5 ± 6.3* 6

xtures and pudrug releas

n (Q60) was higher when c%), ZPG4 (57..01) (Table lipid surfactathe emulsifyileading to t

on of lipophiand McGinitST4 and ZSpectively, wh

mpared withhysical mixtu). The highe

mean dissolut

of zaleplon in w

otrG (J/mol)

0 -3977.93-4739.69-5079.64-5508.05-5791.78-5898.62

m semi solid dispe

f zaleplon from s; n=3).

DE 27.3 ± 1.1 2846.3 ± 4.1* 2656.4 ± 4.5* 2168.3 ± 4.8* 21

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(MadpspthpadT(7foentrm

water-carrier syst

% Carrier (w/v)

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0 05

1015202530

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MDT 8.4 ± 1.57 0.4.4 ± 1.31* 0.82.7 ± 1.86* 1.12.7 ± 1.15* 1.33

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MDT) valuesattributes to dissolution mepresence of Tpontaneous fhese self-emu

physiological addressing thedrugs (Table 2The dissolutio76.2 ± 3.7) anolds over thnhancement

natural crystaransformation

molecular stat

tem.

Gelucire 44/14 Concentration of aleplon (mg/mL)

0.0965 ± 0.0042.82 ± 0.044.92 ± 0.307.46 ± 0.05

12.48 ± 0.8414.76 ± 0.7615.93 ± 0.31

0.981AL

TPGS-E and B) G

sions with GELU

MDR R48 ± 0.112 ± 0.21* 1.692 ± 0.24* 2.063 ± 0.27* 2.50

TABLE 2

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otrG (J/mol)

0-3375.56-3932.23-4348.55-4863.22-5031.04-5107.34

Gelucire 44/14 (M

UCIRE 44/14 or

RDR --

9 ± 0.10*6 ± 0.21*0 ± 0.24*

Contd…

Issue 1 Janua

n from SSD nt of drug iigh dissolutio

Gelucire 44/14fine micella

surfactants umplifies theeous solubilité, 1983, Sang(DE) of zale7 ± 3.6) was ure drug (27f zaleplon froof pure drug

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Mean±SD; n=3).

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Formulation Z : C Q60 DE MDT MDR RDR ZST-4 1:4 84.5 ± 6.8* 76.2 ± 3.7* 18.5 ± 1.10* 1.59 ± 0.18* 2.79 ± 0.28*ZSG-1 1:1 50.1 ± 5.1* 44.5 ± 2.7* 26.4 ± 0.70* 0.79 ± 0.23* 1.63 ± 0.11*ZSG-2 1:2 60.3 ± 4.6* 53.9 ± 1.9* 24.1 ± 0.45* 1.01 ± 0.12* 1.97 ± 0.15*ZSG-3 1:3 72.3 ± 4.8* 64.5 ± 2.6* 23.7 ± 0.98* 1.26 ± 0.17* 2.37 ± 0.28*ZSG-4 1:4 80.1 ± 5.7* 73.7 ± 3.6* 19.9 ± 0.30* 1.52 ± 0.24* 2.70 ± 0.10*ZPT-4 1:4 66.8 ± 5.1* 57.0 ± 3.1* 27.7 ± 0.94* 0.96 ± 0.25* 2.08 ± 0.26*ZPG-4 1:4 64.5 ± 5.4* 57.6 ± 4.2* 25.2 ± 0.43* 1.00 ± 0.12* 2.11 ± 0.31*-Q60 indicates percent drug release in 60 minutes respectively. -DE, MDT, MDR and RDR indicate dissolution efficiency, mean dissolution time, mean dissolution rate and relative dissolution rate respectively -Z : C- indicates zaleplon : carrier -ZST- semisolid dispersion of zaleplon with TPGS-E; ZSG- semisolid dispersion of zaleplon with GELUCIRE 44/14, ZPT- physical mixture of zaleplon and TPGS-E, ZPG-physical mixture of zaleplon and GELUCIRE 44/14 -* indicates p<0.01 vs Control

Polarized Optical Microscopy

The polarized optical microscopic studies were performed to observe the crystallinity of drug in the lipid surfactant based semisolid dispersions, respective physical mixtures and pure drug. Fig 2 A2 & B2 shows the optical birefringence with interference colors indicating the crystallinity of drug. TPGS-E and Gelucire 44/14 showed no evidence of interference colors in their optical birefringence ensuring the transformation of crystalline to amorphous/molecular state (Fig 2A1&B1). In converse, the typical interference colors of drug in the optical birefringence of physical mixtures make sure that the drug remained in crystalline nature (Fig 2A3&B3).

Further, the transformation in crystalline nature of drug to amorphous or molecular form would lead to enhancement in the solubility of drug which was evident from in vitro dissolution studies ensuingin improved dissolution characteristics of drug from ZST4 and ZSG4 formulation compared to respective physical mixtures and pure drug. The amphiphilic lipid carriers used in the study i.e., Gelucire and TPGS has a tendency to form micellar dispersion holding drug in the core of formed micelle up on contact with aqueous media (Zhang et al., 2008).

Fourier transmission Infra-red spectroscopic studies.

The FT-IR spectra of zaleplon, TPGS-E, Gelucire 44/14, ZPT4, ZPG4, ZST4 and ZSG4 are shown in Figure 3. The pure drug zaleplon exhibited characteristic peaks at 3089 cm–1 (C-H aromatic), 2934 cm–1 (C-H aliphatic), 1614 cm–1 (C=N), 1223 cm-1 (C-N), 1577 cm–1

(C=C aromatic) and intense absorption peaks at 2232 and 1651 cm–1 corresponding to cyanide and amide carbonyl group respectively whereas the peaks at 684 and 801 cm–1 can be assigned to the aromatic stretching of the m-substituted phenyl group (Fig. 3). The physical mixtures (ZPT4 and ZPG4) showed typical zaleplon peaks at 1223, 1577, 1651, 1614 and 2232 cm–1. ZST4 and ZSG4 showed the disappearance of peaks at 3089, 1614, 1577 and 1223 cm–1 and the drop in intensity of peaks at 2934, 2232 and 1651 cm–1 indicate physical interaction. However the absence of extra peaks suggests that there was no possible chemical interaction between the drug and formulation ingredients.

In situ perfusion studies

In situ single pass perfusion technique/method was used to gain an insight on the potential of semisolid

dispersion with lipid surfactants for improved absorption of drug across GI barrier. Further, the data interpreted was extrapolated so as to obtain the predictive effective permeability coefficient and fraction oral dose absorbed in humans and shown in Table 3. The obtained Peffrat values for control, ZST4 and ZSG4 formulations were 5.5 ± 0.58, 24.5 ± 3.96 and 20.2 ± 4.05 × 10-6 cm/sec respectively. The significant enhancement in Peff(rat) for zaleplon from ZST4 and ZSG4 formulations (p<0.001) with respect to control reveals that these lipid surfactants obviate the barrier properties of the gastrointestinal tract thus favoring the drug absorption through gastric mucosa (Sundeep and Emilio, 2004; Manda et al., 2011). The Peff(human) and fraction oral dose absorbed in human (Fa) values were predicted by correlating with the obtained rat Peff values(Zakeri et al., 2007). The predicted Peff(human) of zaleplon for control, ZST4 and ZSG4 formulations were 5.3 ± 0.86, 31.2 ± 4.26 and 25.7 ± 2.54 × 10-4 cm/sec respectively. The significant improvement in Peff(human) of zaleplon from self-emulsifying semisolid dispersions (ZST4 and ZSG4) (p<0.001) with respect to control can be attributed to the increased solubility of the drug and enhanced effective surface area for absorption owing to dispersion of drug asmicelle form (Eedara et al., 2014; Sang and Jin, 2003; Manda et al., 2014). The absorption rate constant (Ka) indicative of rate of absorption was also significantly higher for ZST4 and ZSG4 formulations compared to control (p<0.001). The ER values of ZST4 and ZSG4 formulations were 3.67 ± 1.2 and 4.45 ± 1.6 which was above 1 indicating an enhanced permeation of zaleplon through GI barrier. Obviously the higher Fa values 61.0 ± 9.58% and 54.0 ± 8.54 of zaleplon from ZST4 and ZSG4 formulations respectively with respect to control (19.1 ± 3.5%) confers their potential as a carrier for improved absorption of zaleplon (Table 3). Literature evidence suggests that TPGS-E and Gelucire 44/14, amphiphilic carriers, offer the advantage of spontaneously solubilization of lipophilic drugs upon contact with an aqueous medium to form a fine dispersion, which inturn impede the function of P-gp and facilitate the absorption (Sandrien et al., 2008; Festo et al., 2000; Dintaman and Silverman, 1999). Our results are also in support to these findings and in our case the zaleplon absorption was increased by 3 times with ZST4 and ZSG4 formulations containing TPGS-E and Gelucire 44/14with respect to the control.


Fig. 2. Polarized optical microscopic images of A) and B) TPGS-E and Gelucire 44/14, 1) zaleplon 2) Physical mixtures ((2A) ZPT4& (2B) ZPG4) 3) Formulations ((3A) ZST4& (3B) ZSG4).

Fig. 3. FT-IR Spectra of A1) TPGS-E, B1) Gelucire 44/14, A2 and B2) zaleplon A3) ZPT4, B3) ZPG4, A4) ZST4 and B4) ZSG4)

TABLE 3

In situ parameters of zaleplon in rats following oral administration of optimized semisolid dispersion formulations (ZST-4 and ZSG-4) and control oral suspension (mean±SD, n=3).

Formulation Peff(rat) (cm/sec) ×10-6 Peff(human) (cm/sec) ×10-4 Fa (%) Ka(h-1) ER

Control 5.5 ± 0.58 5.3 ± 0.86 19.1 ± 3.5 0.018 ± 0.003 - ZSG-4 20.2 ± 4.051b 25.7 ± 3.241b 54.0 ± 8.541b 0.109 ± 0.0761b 3.67 ± 1.2 ZST-4 24.5 ± 3.961b 31.2 ± 4.261b2a 61.0 ± 9.521b 0.136 ± 0.0951b 4.45 ± 1.61b

-Peff(rat), Peff(human), Fa (%) , Ka and ER represent effective permeability coefficient in rat, predicted effective permeability coefficient in human, % fraction oral dose absorbed in human, absorption rate constant, and enhancement ratio respectively. -ZST-4: semisolid dispersion of zaleplon with TPGS-E 1:4 ratio; ZSG-4: semisolid dispersion of zaleplon with GELUCIRE 44/14 1:4 ratio. -1, 2 indicates control and ZSG-4 formulation respectively -a and b indicates significant difference at p<0.05and p<0.001 respectively


Pharmacokinetic study

Pharmacokinetic studies were conducted to draw out the conclusion in assessing the suitability of self-emulsifying solid dispersions in enhancing the zaleplon oral delivery. The mean serum concentration vs. time profiles of zaleplon following peroral administration of ZST4 and ZSG4 formulations in comparison to control was shown in Fig. 4 and the relevant pharmacokinetic parameters were represented in Table 4. It is evident from fig. 4 that peak serum concentration of zaleplon when treated with ZST4 and ZSG4 formulations was significantly higher compared to treatment with control (p<0.001). On the other hand the time to reach peak serum concentration (Tmax) remained constant which suggests that the immediate solubilization of zaleplon from ZST4 and ZSG4 formulations due to spontaneous emulsification. The slower elimination rate of zaleplon from ZST4 and ZSG4 formulations with respect to control resulted in higher biological half-life and mean residence time. The AUC values which indicate the extent of absorption was 2971 ± 214.6 and 2483 ± 244.3 ng h mL-1 following oral administration of ZST4 and ZSG4 respectively and was significantly higher compared to control (923.9 ± 106.2 ng h mL-1) (p<0.001). The relative bioavailability (F) of zaleplon following oral administration of (ZST4, ZSG4) was also significantly higher compared to control (p<0.001). Overall, it is apparent from the results that the rate and extent of absorption of zaleplon has been markedly improved from ZST4 and ZSG4 formulations to control.

The remarkable improvement in the bioavailability of zaleplon from ZST4 and ZSG4 formulations can be attributable to many factors which either in combination or alone contribute for favoured magnitude of absorption. One of the key factors for hike in bioavailability of zaleplon is because of the better presentation of the drug at the site of administration. The reasons include the tremendous increase in the effective surface area by the formation of fine micelle and further, enhanced diffusion of micelle through the aqueous filled channels present within the cell membrane structure. Additionally, these self-emulsifying amphiphilic surfactants can alter the barrier properties of GI membrane either by fluidization of membrane or by modulating the P-gp activity by inhibiting the efflux function, hence, promoting the partitioning of the drugs into the bilayer facilitating absorption (Sandrien et al., 2008; Manda et al., 2011; Festo et al., 2000; Dintaman and Silverman, 1999). Thus, these results surmise the suitability of self-emulsifying lipid surfactants as potential carriers in enhancing the dissolution rate limited oral bioavailability of BCS class II drug, zaleplon.

Stability studies

The stability of the ZST4 and ZSG4 formulations was ascertained by monitoring the dissolution profiles upon storage at refrigerated temperature for a period of 90 days. The formulations were stable without any change in color. The dissolution profiles were similar and we could not notice any significant change in the dissolution characteristics of zaleplon up on storage at 4oC±2oC for a period of 90 days (Fig. 5A & 5B).

Fig. 4. Pharmacokinetic profiles of zaleplon in rat serum following oral administration of ZSG4, ZST4 formulations and control suspension (Mean±SD; n=6)

3110

TABLE 4

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Address correspondence to: Karthik Yadav Janga, Department of Pharmaceutics, Kakatiya Institute of Pharmaceutical Sciences, Warangal, India. E-mail: [email protected]; [email protected] †Authors have contributed equally in the present work

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