Improved oral bioavailability of fexofenadine

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bioavailability from the prepared dispersions as compared to pure drug. Keywords. Bioavailability, dissolution, fexofenadine hydrochloride, Gelucire 44/14, ...
http://informahealthcare.com/ddi ISSN: 0363-9045 (print), 1520-5762 (electronic) Drug Dev Ind Pharm, 2014; 40(8): 1030–1043 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2013.801984

RESEARCH ARTICLE

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Improved oral bioavailability of fexofenadine hydrochloride using lipid surfactants: ex vivo, in situ and in vivo studies Basanth Babu Eedara, Prabhakar Reddy Veerareddy, Raju Jukanti, and Suresh Bandari Department of Pharmaceutics, St. Peter’s Institute of Pharmaceutical Sciences, Vidyanagar, Hanamkonda, Warangal, India

Abstract

Keywords

The aim of the present study was to improve the dissolution, permeability and therefore oral bioavailability of the fexofenadine hydrochloride (FEX), by preparing lipid surfactant based dispersions using self-emulsifying carriers, i.e. Gelucire 44/14 (GLC) and d-a-tocopheryl polyethylene glycol 1000 succinate (Vitamin E TPGS or TPGS). The reprecipitation studies were conducted using these carriers to evaluate inhibition of reprecipitation by maintaining super saturation state. The aqueous solubility of the FEX was increased linearly with increasing GLC, TPGS concentrations as verified by the phase solubility studies. The dispersions of FEX were prepared in different drug/GLC (GD) and drug/TPGS (TD) ratios by melt method and evaluated. The prepared dispersions showed improved dissolution rate in distilled water as dissolution media and highest dissolution rate was achieved with dispersions prepared using TPGS. The solid state characterization was carried by differential scanning calorimetry and scanning electron microscopy indicated reduced crystallinity of the drug. Fourier transform infrared spectroscopy revealed the compatibility of drug with carriers. The ex vivo permeation studies conducted using intestinal gut sac technique, resulted in reduced efflux of the drug by inhibiting intestinal P-glycoprotein from the dispersions. The in situ perfusion studies and in vivo pharmacokinetic studies in male wistar rats showed improved absorption and oral bioavailability from the prepared dispersions as compared to pure drug.

Bioavailability, dissolution, fexofenadine hydrochloride, Gelucire 44/14, permeation, vitamin E TPGS

Introduction Oral drug administration is preferred generally over the other routes of administration to treat chronic diseases.1 However, many potential orally active drug candidates are suffering with low oral bioavailability caused by low solubility, low permeability and high first pass metabolism or combinations thereof.2 Among the various factors that affect oral bioavailability, efflux pumps at the site of absorption also poses an obstacle for substrate bioavailability. Particularly, P-glycoproteins (P-gp) of ABC super family efflux transporters at intestine level significantly limit the oral bioavailability of drugs. Fexofenadine hydrochloride (FEX; Figure 1A), a safer orally active metabolite of terfenadine, is second generation H1 receptor antagonist widely prescribed for the treatment of seasonal allergic rhinitis.3 It has low oral bioavailability approximately 33% in humans4 because of its very slightly soluble nature in water, low intestinal permeability and also the intestinal P-gp limits its oral absorption as it is a substrate for P-glycoprotein.5 Earlier reports suggest that certain excipients such as nonionic surfactants are commonly used in pharmaceutical formulations to improve oral bioavailability of P-gp substrate drugs by enhanced

Address for correspondence: Basanth Babu Eedara, Department of Pharmaceutics, St. Peter’s Institute of Pharmaceutical Sciences, Hanamkonda 506001, Warangal, Andhra Pradesh, India. Tel: +918702567303. Fax: +918702567304. E-mail: [email protected]

History Received 10 December 2012 Revised 25 April 2013 Accepted 30 April 2013 Published online 5 June 2013

dissolution and by inhibiting P-glycoprotein mediated efflux at intestine level.6,7 Some of the nonionic surfactants with P-gp inhibitory activity are tweens, spans, cremophors (EL and RH40), pluronic block copolymers, Gelucire 44/14 and vitamin E TPGS.8–12 Gelucire 44/14 (GLC) is a solid waxy excipient prepared by poly glycolysis of hydrogenated palm kernel oil with poly ethylene glycol 1500 (PEG 1500). Its composition13 is a mixture of mono- and di-fatty acid esters of PEG 1500 (72%), mono-, diand triglycerides (20%), free PEG 1500 (8%) with a hydrophilic lipophilic balance (HLB) value of 14. The fatty acid distribution14 of Gelucire 44/14 includes caprylic acid (4–10%), capric acid (3–9%), lauric acid (40–50%), myristic acid (14–24%), palmitic acid (4–14%), stearic acid (5–15%). TPGS (Figure 1B; HLB ¼ 13) is nonionic water soluble derivative of natural vitamin E, prepared by the esterification of d-a-tocopheryl acid succinate with polyethylene glycol 1000. Both GLC and TPGS are amphiphilic in nature and reported as solubility and absorption enhancers with P-glycoprotein inhibitory activity.15,16 Thus, in this study dispersions of FEX were prepared using GLC and TPGS as carriers by melt method and the effect of these excipients on the solubility, permeability and oral bioavailability of FEX were evaluated. Subsequently, prepared dispersions were characterized by differential scanning calorimetry, scanning electron microscopy, fourier transform infrared spectroscopy and dissolution studies. In order to understand the effect of these excipients on the permeability and bioavailability of FEX,

DOI: 10.3109/03639045.2013.801984

Improved oral bioavailability of fexofenadine hydrochloride

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apparatus (Electrolab, Mumbai, India) at the paddle rotation speed of 50 rpm at 37  0.5  C. A sample of 5 mL of FEX in ethanol (100 mg/mL) was added to each test media and visually observed for the effect of these carriers on the inhibition of reprecipitation for a period of 24 h.

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Phase solubility studies

Figure 1. Molecular structures of fexofenadine hydrochloride (FEX) (A) and vitamin E TPGS (B).

ex vivo permeation studies using intestinal sac method in both absorptive direction (Mucosal to serosal), secretory direction (Serosal to mucosal), in situ single pass intestinal perfusion (SPIP) studies and in vivo pharmacokinetic studies were conducted in rats.

Materials and methods Materials FEX was a kind gift sample from Ami Life Sciences Pvt. Ltd, Vadodara, Gujarat, India. GelucireÕ 44/14 was obtained as a gift sample from Gattefosse (France). Vitamin E TPGS was obtained from Isochem (France) as a generous gift sample. All other chemicals used were of analytical grade and solvents were of high performance liquid chromatography (HPLC) grade. Fresh deionized double distilled water was used throughout the study. Animals and legal prerequisites All the animal experiments, ex vivo intestinal permeability, in situ permeability and in vivo pharmacokinetic studies were performed with the prior review and approval of the Institutional Animal Ethical Committee, St. Peter’s Institute of Pharmaceutical Sciences, Hanamkonda. Healthy male wistar rats (250–300 g) were purchased from Mahaveera Enterprises (146-CPCSEA no:199; Hyderabad, India), acclimatized for at least 1 week in separate cages in a clean room and maintained under controlled conditions of room temperature with free access to food and water. The rats were fasted overnight before the experiment with free access to water. HPLC analysis The FEX concentration in all the samples was analyzed using an isocratic Shimadzu (Kyoto, Japan) HPLC equipped with LC-10 AT solvent delivery unit, SPD-10 AVP UV Spectrophotometric detector with Lichrospher C18 column (5 mm, 4.6  250 mm). The system was operated at ambient temperature using a mobile phase consisting of mixture of potassium dihydrogen orthophosphate buffer (0.05 M containing 0.5% v/v of triethylamine; pH 4.2 adjusted with orthophosphoric acid), acetonitrile and methanol (50:38:12 v/v/v) at a flow rate of 1 mL/min. The UV detector wavelength was set at 220 nm. The sample injection volumes of 20 mL were used and the sensitivity was set at 0.005 absorbance units full scale (AUFS). Reprecipitation study Reprecipitation studies were carried out in 300 mL of distilled water without carriers and with GLC, TPGS (300 mg in 300 mL) as test media by cosolvent method17 using USP (XXIV) type II

As reported by the Higuchi and Connors,18 phase solubility studies were carried out by placing an excess amount of FEX to 10 mL aqueous solutions containing different concentrations of carriers i.e. GLC and TPGS (5%, 10%, 15%, 20%, 25% and 30% w/v) in a 25 mL glass vials. These vials were covered with cellophane tape to avoid solvent loss by evaporation and then shaken for 48 h at 37  C in a thermostatically controlled water bath. The samples were filtered through 0.45 mm membrane filter and the filtrate obtained was diluted suitably with methanol and FEX concentration was measured using HPLC analysis. Three determinations were performed for each sample. The phase solubility diagrams were plotted by taking carrier concentration against amount of FEX solubilized in aqueous solutions. Gibbs free energy of transfer (DG  tr) represents information about whether the system encouraging or discouraging the solubility of drug in aqueous medium. Gibbs free energy of transfer, DG  tr, of FEX from plain distilled water to aqueous solution of the carriers was calculated according to the Equation (1). DGotr ¼ 2:303RTLog

S0 SS

ð1Þ

where S0 and Ss are the molar solubility of FEX in aqueous solution of the carrier and in the plain water, respectively. R is the general gas constant (R ¼ 8.31 J K1 mol1) while T is temperature in degree kelvin. From the phase solubility diagrams, the values of apparent stability constant, Ks, between drug carrier combinations was calculated using the following Equation (2). Ks ¼

Slope ð1  SlopeÞ So

ð2Þ

where So is the solubility of drug in the absence of carrier. Preparation of dispersions and physical mixtures Dispersions of FEX were prepared in 1:1, 1:2 and 1:4 (FEX: GLC/TPGS) weight ratios by melt method. Accurately weighed quantity of carrier (GLC/TPGS) in a porcelain dish was melted at 40–50  C in a water bath. To this molten carrier accurately weighed drug was added and mixed for 10 min with continuous stirring on water bath to obtain the uniform mixture. This mixture was solidified at 4  C and evaluated after equilibration at room temperature for overnight. Similarly physical mixtures (PMs) were prepared by mixing the drug and carriers with a spatula for 5 minutes and evaluated. Drug content determination Drug content uniformity of dispersions and physical mixtures was determined by taking formulation equivalent to 10 mg of FEX and dissolved in 100 mL of methanol. An aliquot of sample was centrifuged at 10 000 rpm for 15 min and followed by separation of supernatant. After dilution into the validated range with methanol, the supernatant samples were analyzed for FEX concentration using HPLC. Each determination was performed in triplicate.

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Solid state characterization Solid state characterization of pure FEX, carriers (GLC, TPGS), optimized dispersions and their respective physical mixtures was performed by differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and fourier transform infrared spectroscopy (FTIR).

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DSC thermograms were recorded using differential scanning calorimeter (TA-60WSI, Shimadzu, Kyoto, Japan). Under nitrogen atmosphere of flow rate 80 mL/min, 4 mg of the samples sealed in a flat bottomed aluminum pan (Shimadzu DSC-60, Kyoto, Japan) were heated at a scanning rate of 10  C/min from 30 to 300  C. The heat of fusion of pure crystalline drug, PMs and optimized dispersions was calculated from the peak area of the melting endotherm. The degree of crystallinity (Dcrys) of drug in PMs and dispersions was calculated by using the Equation (3).19 Dcrys

The initial dissolution rate (IDR) was calculated over the first 15 min of dissolution using the following Equation (5). IDR ¼ %dissolved=min

Differential scanning calorimetry

DHFD ¼f  100 DHF

dissolution in the same time. DE at 15 min was calculated by using the following Equation (4).20 Rt y  dt  100% ð4Þ D:E: ¼ 0 y100  t

ð3Þ

where ‘f ’ is the fraction of drug present in the formulation, DHFD is the heat of fusion of the drug (FEX) in formulations (dispersions/PMs) and DHF is the heat of fusion of the pure drug (FEX). Scanning electron microscopy JSM-840 scanning electron microscope (Jeol Corporation, Tokyo, Japan) was used to study the surface morphology of samples, previously mounted on a brass stub using double sided adhesive tape and coated with platinum (approximately 3–5 nm) in vacuum in order to make them electrically conductive. The micrographs at different magnifications were recorded by working at an excitation voltage of 15 Kv. Fourier transform infrared spectroscopy FTIR spectroscopy of the samples, formulated in KBr tablets (10 mm discs) containing 2 mg of drug mixed with 200 mg of IR grade dry potassium bromide, was carried out using FTIR spectrophotometer (Spectrum GX-FTIR, Perkin Elmer, Waltham, MA). The spectrum was recorded over spectral region from 4000–400 cm1 using a resolution of 1 cm1, at room temperature. In vitro dissolution studies Dissolution studies of pure FEX, dispersions and physical mixtures were performed by using USP (XXIV) type II apparatus (Electrolab, Mumbai, India) at the paddle rotation speed of 50 rpm in 900 mL distilled water as dissolution media at 37  0.5  C. A sample equivalent to 30 mg of FEX of the prepared systems was filled into size 3 hard gelatin capsules and placed into the dissolution medium. Aliquots of 5 mL were collected at predetermined time intervals over a period of 2 h and were replaced with the same volume of fresh dissolution medium. The samples were subsequently filtered using 0.45 mm membrane filter and the filtrate obtained was suitably diluted and analyzed by HPLC. All experiments were performed in triplicate. Treatment of dissolution data The dissolution efficiency (DE) is defined as the area under the dissolution curve up to a certain time (t), expressed as a percentage of the area of the rectangle described by 100%

ð5Þ

21

Mean dissolution time (MDT) is an alternative parameter that explains the dissolution rate, i.e. MDT is the mean time for the drug to dissolve under in vitro dissolution conditions. Mean dissolution time (MDT) and mean dissolution rates (MDR) were calculated using the Equations (6) and (7).21 Pn j¼1 Dt DMj MDTin vitro ¼ Pn ð6Þ j¼1 DMj Pn MDR ¼

j¼1

DMj =Dt n

ð7Þ

where n is the number of dissolution sample times, j is dissolution sample number, Dt is the time at midpoint between tj and tj1 (easily calculated with (tj þ tj1)/2) and DMj is the additional amount of drug dissolved between tj and tj1. The relative dissolution rate (RDR) was ratio between amount of drug dissolved from each formulation and that dissolved from the pure drug at 120 min.22 The time taken to release 50% (t50%) of drug during dissolution was also calculated by the interpolation of plotted dissolution profiles. Ex vivo intestinal permeability studies Intestinal permeability of the FEX from pure drug and 1:4 ratios of GD, TD were evaluated by applying the intestinal gut sac technique. The rats were anesthetized by intraperitonial bolus administration of thiopental sodium (50 mg/kg). A segment of jejunum (20 cm) was excised by making a midline incision of 2 cm in the abdominal cavity. Immediately after dissection the rats were sacrificed with a cardiac injection of saturated solution of KCl. Then intestinal segments were immediately flushed with warmed (37  C), pH 7.4, pregassed oxygenated phosphate buffered saline (PBS) to remove lumen contents. The transport of FEX in absorptive direction, i.e. from mucosal to serosal (M/S) direction was studied by using non everted intestinal sacs immediately after washing, whereas the transport in secretory direction i.e. from serosal to mucosal (S/M) direction was studied by using the intestinal sacs everted gently on a glass rod after washing. These segments were tied at one end with a braided silk suture, filled with the 1 mL of drug suspension (5 mg/mL), prepared by dispersing pure FEX or dispersions in 0.2% w/v sodium carboxy methyl cellulose and then tied at the other end. Each tissue was placed individually in a beaker containing 50 mL of pH 7.4 PBS in a 37  C shaking water bath (60 cycles/min). At the appropriate time points, aliquots of 2 mL were withdrawn and were stored at 20  C until analysis by HPLC. When the experiment was completed, the length of the intestinal segment was measured without stretching. Each experiment was performed in triplicate. Ex vivo permeation data analysis The cumulative amount of drug permeated during the study was determined and plotted against the time to calculate the apparent permeability (Papp, cm/s) using the following Equation (8). Papp ¼

F AC0

ð8Þ

Improved oral bioavailability of fexofenadine hydrochloride

DOI: 10.3109/03639045.2013.801984

where ‘‘F’’ is the flux (F, mg/min) of the drug through the intestinal sac was determined from the slope of linear portion of cumulative amount permeated versus time profiles, A is the surface area of the intestinal sac assumed to be the area of a cylinder (2rL) with the length (L; measured at the end of experiment) and radius (r) of 0.18 cm,23 C0 (mg/mL) is the initial drug concentration. Efflux ratio was calculated using the following Equation (9), to assess the magnitude of efflux1.

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Efflux ratio ¼

Papp SM Papp MS

ð9Þ

where PappSM is the average apparent permeability coefficient from serosal to mucosal side and PappMS is the average apparent permeability coefficient from mucosal to serosal side. In situ permeability studies 24,25

to assess the in SPIP studies were carried as reported earlier situ intestinal absorption of FEX from dispersions. The male wistar rats (250–300 g) were fasted overnight before the experiment with free access to water and anesthetized by thiopental sodium (50 mg/kg) via intraperitoneal administration and placed on a heating pad to maintain body temperature at 37  C. Upon verification of the loss of pain reflex, a midline abdominal incision was made and a 10 cm section of the proximal rat jejunum was located gently with plastic tubing rinsed with saline (37  C) to clear the segment and attached to the perfusion assembly which consisted of a syringe pump (Harvard Apparatus PHD 2000 pump, Holliston, MA) and a 30 mL syringe connected to it. Care was taken to handle the intestine gently and to minimize the surgery in order to maintain an intact blood supply. The outlet tubing was placed 10–15 cm aboral to the first opening. The entire surgical area was then covered with wet cotton to reduce evaporation. Blank perfusion buffer (PBS, pH 7.4 at 37  C) was infused for 15 min by a syringe pump followed by perfusion of compounds (pure drug and 1:4 ratios of GD, TD dispersed in perfusion buffer) at a flow rate of 0.2 mL/min for 90 min. Sampling was made every 15 min for a 90 min perfusion period with perfusion solution after 20 min equilibration. Equilibration of 20 min prior to sampling was found to be sufficient for both washout and to reach an initial steady state.26,27 Water flux was quantified based on direct measurement of the volume at the outlet.7,28 All samples including perfusion samples collected at various time points, original drug solution and inlet solutions collected at the exit of the syringe were stored at 20  C until analysis by HPLC. When the experiment was completed, the length of the intestinal segment was measured without stretching and the animal was euthanatized with a cardiac injection of saturated solution of KCl and disposal of carcass in accordance of the guidelines. At the end of permeation study, the intestinal sacs were excised, washed gently with PBS (pH 7.4) and immediately placed in 10% formaldehyde saline solution for 24 h and embedded in paraffin wax. Then the paraffin embedded sections were stained with hematoxylin/eosin for histological evaluation. Permeability calculations The intestinal effective permeability (Peff, cm/s) was calculated based on outlet perfusate steady state concentrations achieved after approximate 30 min. The steady state intestinal effective permeability (Peff, cm/s) was calculated using Equation (10). Peff ¼

½Qin lnðCoutðcorrÞ =Cin Þ0  A

ð10Þ

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where Qin is the flow rate of perfusate (0.2 mL/min), A is the surface area of the intestinal segment assumed to be the area of a cylinder (2rL) with the length (L; measured at the end of experiment) and radius (r) of 0.18 cm,23 Cin and Cout are the inlet and fluid transport corrected outlet solution concentrations, respectively. The latter was corrected with water flux on the basis of the ratio of volume of perfusion solution collected and infused for each sampling point (5 min).7 The Cout(corr) was calculated by using the Equation (11).29 CoutðcorrÞ ¼ Cout Qout =Qin

ð11Þ

where Qout and Qin are the measured flows (mL/min) of intestinal perfusates exiting and entering the intestine for the specified time interval, respectively. The absorption rate constant was calculated by using the Equation (12).7 Ka ¼ PeffðhumanÞ 

A V

ð12Þ

where Peff (human) is the predicted human effective intestinal permeability calculated by using the Equation (13). PeffðhumanÞ ¼ PeffðratÞ  3:253 þ 0:032  104

ð13Þ

and A/V is the intestinal area to volume ratio was taken as 10 cm. Pharmacokinetic study Male Wistar rats were fasted overnight with free access to water prior to oral administration and were divided into three groups each containing six animals. The pure FEX and the 1:4 ratios of GD, TD were suspended in water containing 0.2% w/v sodium carboxy methyl cellulose and immediately administered orally at a dose equivalent to 10 mg/kg body weight with an oral gavage via the esophagus into stomach. At predetermined time intervals, blood samples (500 mL) were collected from retro orbital plexus under diethyl ether anesthesia, into micro centrifuge tubes and allowed to clot and centrifuged at 6000 rpm for 5 min in a microcentrifuge (Remi equipments, India) to separate the serum. The serum obtained was stored at 20  C until HPLC analysis. Serum treatment and analysis The serum samples were stored at 20  C in a freezer until analysis and allowed to thaw at room temperature before processing. The serum samples were treated as described earlier in reports.30 Briefly, 100 mL of serum sample was treated with 50 mL of internal standard working solution (5 mg mL1 carbamazepine), 13 mM formic acid solution and vortexed for 30 s. The mixture was extracted with 5 mL of dichloromethane: ethyl acetate: diethyl ether (30:40:30, v/v/v) followed by vortexing for 60 s, centrifugation for 5 min at 5500 rpm at 4  C and the separated organic layer was dried under vacuum. The dried residue was reconstituted with 200 mL of mobile phase, vortexed for 10 s and an aliquot of 20 mL was injected onto the HPLC. The calibration curve from the standard samples was linear (r240.992) over the concentration range of interest. The limit of detection (LOD) and quantification (LOQ) were 15 and 30 ng/mL, respectively. Pharmacokinetic parameters Various pharmacokinetic parameters were obtained from the serum concentration versus time profile. AUC0–t: area under concentration–time curve from 0 to t was calculated by using trapezoidal rule method; AUC0–1: area under concentration–time curve from t to infinity was determined by dividing the serum

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concentration at last time point with elimination rate constant (K); Cmax: peak concentration, T1/2: elimination half-life and Tmax: time to reach peak concentration were obtained directly from the serum concentration vs. time profile; the relative bioavailability (RA) was calculated by dividing the AUC0–1 of dispersions with pure drug.

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Statistical analysis All the results were expressed as mean with standard deviation (mean  S.D). The data obtained were subjected to one way analysis of variance (ANOVA) and the significance of difference between formulations was calculated by student–Newman–Keuls (compare all pairs) using Instat Graphpad prism software (version 4.00; GraphPad Software, San Diego, CA). The differences were considered to be significant at p50.05.

Results and discussion Re-precipitation study As shown in Figure 2(A), the test media without any carrier became turbid immediately after addition of drug solution and remained turbid for more than 24 h; whereas the test media with carriers remained clear (Figure 2B and C) without any sign of precipitation throughout the period of study. These results suggest the solubility enhancing property of these carriers by inhibiting reprecipitation of drug. As these carriers are surfactant in nature the hydrogen bonding and hydrophobic interactions between drug and carrier may inhibit the reprecipitation of drug by improving the solubility. Phase solubility studies The influence of GLC and TPGS on solubility of FEX in distilled water at 37  C is presented in Figure 3. FEX aqueous solubility was observed to be 1.45  0.15 mg/mL. Phase solubility parameters showed an increase in drug solubility with GLC (3.95  0.19 to 11.34  0.31 mg/mL at 5 to 30 % w/v GLC concentrations) and TPGS (4.29  0.28 to 18.25  0.34 mg/mL at 5 to 30 % w/v TPGS concentrations), with r2 values 0.9862 and 0.9932. The phase solubility diagrams followed an AL-type system as the computed slopes are less than unity,18 where a linear increase in FEX solubility was observed with increase in carrier concentrations. These results are in accordance with the established formation of soluble complex between water soluble carriers and poorly soluble drugs.31 The values of the slopes for the linear curves enlisted in Table 1 indicating the relative solubilizing efficiency of GLC and TPGS. However TPGS has higher solubilising capacity than GLC. This can be further confirmed from the 12.6 fold increased solubility of FEX in presence of TPGS to 8 fold increase with GLC at same concentrations studied. The Gibbs free energy of transfer (DG  tr) gives an indication of the process of transfer of FEX from pure water to the aqueous solutions of carriers. The negative DG  tr values indicate improved dissolution.32 DG  tr values were all negative for both the carriers at various concentrations, indicating the spontaneous nature of FEX solubilisation and also the values decreased with increasing concentrations of carriers, thereby demonstrating that dissolution became more favourable as the concentration of carriers was increased. The DG  tr values were least for TPGS illustrating that the process of FEX transfer from water to its aqueous solution was the most favourable amongst the carriers studied. In addition, the apparent stability constant (Ks) of FEX with GLC and TPGS were 115.1 and 210.9 mL/g, respectively. The values of apparent stability constant, Ks, obtained for GLC and TPGS binary solution revealed the formation of stable

Figure 2. Photo images of dispersion states of fexofenadine hydrochloride (FEX) in each test solutions (A) without any carrier (B) with Gelucire 44/14 (C) with vitamin E TPGS at 15 min.

FEX-Carrier dispersion. The values of the stability constant in the range of 100 to 1000 mL/g are considered to be ideal.33 Actually smaller values of (KS) indicate too weak interaction, whereas larger values are symptomatic of an incomplete release of drug from the complex. Drug content determination The drug content of the prepared dispersions and physical mixtures was found to be in the range of 97.3–101.5% indicating the application of the present method for the preparation of simple dispersion with high content uniformity. Solid state characterization Differential scanning calorimetry The DSC thermograms of the FEX, GLC, TPGS, 1:4 ratios of dispersions (GD and TD) and respective PMs were shown in

Improved oral bioavailability of fexofenadine hydrochloride

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DOI: 10.3109/03639045.2013.801984

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Figure 3. Phase solubility diagrams for fexofenadine hydrochloride (FEX) in the presence of (A) Gelucire 44/14 and (B) Vitamin E TPGS in distilled water at 37  0.5  C (n ¼ 3).

Table 1. Phase solubility and thermodynamic parameters of fexofenadine hydrochloride in different concentrations of Gelucire 44/14 and vitamin E TPGS water systems (n ¼ 3). Gelucire 44/14 Concentration of carrier (% w/v) 0 5 10 15 20 25 30 Stability constant (mL/g) R2 value Slope Type of curve

Vitamin E TPGS

Solubility of FEX (mg/mL)

DG tr (J/mole)

Solubility of FEX (mg/mL)

DG tr (J/mole)

1.45  0.15 3.95  0.19 5.42  0.21 6.13  0.13 7.75  0.25 9.79  0.28 11.34  0.31 115.1 0.9862 0.3122 AL

– 2.58 3.40 3.72 4.32 4.93 5.30

1.45  0.15 4.29  0.28 6.30  0.25 8.75  0.18 12.05  0.21 14.35  0.19 18.25  0.34 210.9 0.9932 0.5449 AL

– 2.80 3.79 4.64 5.46 5.91 6.53

Figure 4. The pure FEX, GLC, TPGS (Figure 4A, B and E) were characterized by a single, sharp melting endotherm peaks at 199.31  C, 41.72  C and 35.54  C with an enthalpy of fusion (DH) of 51.39 J/g, 34.76 J/g and 12.84 J/g, respectively. The DSC curve of FEX revealed a typical behavior of crystalline anhydrous substance. The 1:4 ratio of FEX-GLC physical mixture (Figure 4C) showed both the endothermic peaks of drug and GLC at approximately 199.53  C and 45.26  C with an enthalpy of fusion (DH) of 21.95 J/g for drug. The 1:4 ratio of FEX-TPGS (Figure 4F) physical mixture also showed the endothermic peaks of both drug and TPGS at approximately 198.08  C and 38.80  C with an enthalpy of fusion (DH) of 3.63 J/g for drug. The degree of crystallinity of drug in the physical mixtures with GLC and TPGS was found to be 10.67% and 1.76%, indicating that a part of the FEX in the PMs is present in its crystalline form. Whereas the 1:4 ratio of dispersions (GD, TD; Figure 4D and G) did not show any melting endotherm of FEX. This may be attributed due to amorphous form of the FEX in the dispersions or the dissolution of crystalline drug into the molten carrier. Scanning electron microscopy The scanning electron photomicrographs of pure FEX, GLC, TPGS, 1:4 ratios of dispersions (GD, TD) and respective PMs were shown in Figure 5. Pure FEX (Figure 5A) existed as blunt smooth crystalline particles with irregular size and shape.



Pure GLC (Figure 5B) and TPGS (Figure 5C) have been presented as waxy lipid with smooth surface. In both the physical mixtures (Figure 5D and E), one can observe the presence of drug crystals in the smooth waxy carriers, whereas the dispersions of GLC (Figure 5F) and TPGS (Figure 5G) appeared as uniform, homogenous and the surface was similar to that of pure GLC and TPGS as smooth waxy surface, indicating that drug was adsorbed and homogeneously dispersed into carriers at the molecular level. Fourier transform infrared spectroscopy The FTIR spectra of pure FEX, GLC, TPGS, 1:4 ratios of dispersions (GD, TD) and respective PMs were shown in Figure 6. The prominent peaks of FEX were observed at 3296 cm1 (O–H alcohol/phenol stretching), 2935 cm1 (O–H carboxylic acid stretching), 1705 cm1 (C¼O carboxylic acid stretching), 1278 cm1 (C–N stretching), 744 cm1and 702 cm1(C–H aromatic out of plane bending vibration) and pure GLC and TPGS peaks at 1734 cm1 (C¼O stretching of ester) and 1111 cm1(C–O stretching of alcohol (primary or secondary)) were observed in both PMs and dispersions compared with pure forms. Retention of characteristic drug peaks with additive carrier peaks without any significant change in the position of drug peaks in the physical mixtures and dispersions clearly suggests no possible interaction between drug and carriers and confirms the stability of FEX in prepared dispersions and PMs.

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Figure 4. Differential scanning calorimetry thermograms of (A) Pure fexofenadine hydrochloride (FEX), (B) Gelucire 44/14 (GLC), (C) Physical mixture of FEX and GLC at 1:4 ratio, (D) Dispersion of FEX and GLC at 1:4 ratio (GD 1:4), (E) Vitamin E TPGS (TPGS), (F) Physical mixture of FEX and TPGS at 1:4 ratio and (G) Dispersion of FEX and TPGS at 1:4 ratio (TD 1:4).

In vitro dissolution study Dissolution of pure FEX, PMs and dispersions (GD, TD) were carried out in distilled water as dissolution media and their dissolution curves are shown in Figure 7. All the dispersions and PMs showed an enhanced dissolution when compared with the pure FEX. The dissolution rate of the drug from dispersions was increasing with increasing the ratio of the carriers (GLC and TPGS). The dispersions prepared with TPGS showed higher dissolution rate as compared to dispersions prepared with GLC. Table 2, shows the Q15 (percentage drug dissolved in 15 min), DE15 (DE at 15 min), MDT (mean dissolution time), MDR (mean dissolution rate), IDR (initial dissolution rate), RDR (relative dissolution rate) and t50% (the time needed to dissolve 50% of drug) for FEX/GLC dispersions. As shown in Table 2, Q15, DE15 were increasing with increasing carrier ratios, highest dissolution rate was observed with GD (1:4 ratio), i.e. Q15 ¼ 79.0% and the DE was improved around 4.7 fold compared to pure drug. The MDT of FEX was decreased from 45.18 min (for pure FEX) to 10.65 min (GD 1:4 ratio) and similarly the time taken to release 50% of FEX (t50%) was also decreased as the concentration of the carrier was increasing. Whereas the MDR, RDR, IDR values were increasing as the concentration of the carrier was increasing with highest dissolution rate for GD 1:4 ratio. Similarly, Table 3 shows all the dissolution parameters for FEX/TPGS dispersions. As shown in Table 3 highest dissolution rate was shown by TD (1:4 ratio) i.e. Q15 ¼ 91.9% and also DE15 of TD (1:4) was improved around 5.4 fold compared to pure drug. The MDT of the FEX was also decreasing to 9.74 min for 1:4

ratios TD. In dispersions, MDR, RDR and IDR of FEX also increase with increasing concentrations of TPGS. The time taken to release 50% of FEX (T50%) from TD (1:4 ratio) and respective PM was found to be 7 minutes and 38 minutes, respectively. It can be concluded that the dissolution profiles of the dispersions showed significantly higher dissolution profiles compared to pure drug. This enhanced dissolution with these carriers may be due to several factors like reduced crystallinity, particle size reduction and improved wettability. As the hydrophilic soluble carrier dissolves, the insoluble drug gets exposed to dissolution medium in the form of fine particles for faster dissolution. Ex vivo intestinal permeability study The apparent permeability (Papp, cm/s) of the FEX from pure drug and dispersions (1:4 ratios of GD, TD) in both the absorptive (Mucosal to serosal) and secretory (Serosal to mucosal) directions was determined by applying the intestinal gut sac technique and represented in Figure 8, Table 4. As shown in the Figure 8 the permeability of the FEX from the pure drug was higher in the secretory direction compared to absorptive transport. The permeability of FEX from GD, TD significantly (p50.05, p50.01) decreased in the secretory direction compared to pure drug. Whereas in the absorptive direction the permeability of FEX from GD, TD significantly (p50.05, p50.001) increased compared to pure drug. The efflux ratios of the FEX from the pure drug and dispersions (GD, TD) were found to be 2.69, 1.10 and 0.49 indicating decreased efflux of the FEX from the dispersions.

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Figure 5. Scanning electron photomicrographs of (A) Pure fexofenadine hydrochloride (FEX), (B) Gelucire 44/14 (GLC), (C) Vitamin E TPGS (TPGS), (D) Physical mixture of FEX and GLC at 1:4 ratio, (E) Physical mixture of FEX and TPGS at 1:4 ratio, (F) Dispersion of FEX and GLC at 1:4 ratio (GD 1:4) and (G) Dispersion of FEX and TPGS at 1:4 ratio (TD 1:4).

These results suggest the P-gp inhibitory activity of these excipients decreased the efflux of drug by inhibiting the intestinal P-gp function. It has been reported that the surfactants with HLB value of 10–17 shows the improved drug absorption by inhibiting the efflux transporters at the intestine level.12 As these excipients have an HLB value within the range of 10–17 they improved absorption by inhibiting efflux transporters. It has been also reported that both TPGS and GLC reduces the efflux of rhodamine 123 (P-gp substrate) in Caco-2 cells by inhibiting the P-glycoprotein as they alters the membrane fluidity.15,34 Similarly Hong-Mei et al.35 reported the improved bioavailability of FEX by formulating micro emulsions for intranasal delivery. He also reported that this enhancement in bioavailability might be due to the P-gp inhibitory activity of labrasol and PEG 400.

In situ permeability studies The transport of FEX across proximal rat jejunum from dispersions prepared with drug alone and 1:4 ratios of dispersions (GD, TD) was shown in Figure 9. The effective intestinal permeability values were determined based on reaching the steady state concentration of drug in the perfusate, which was confirmed by plotting (Cout/Cin)0 verse time. The inset of Figure 9 shows the steady state of drug when the (Cout/Cin)0 of the drug remains constant. The effective intestinal permeability coefficient in rats (Peff(rat)), predicted human effective intestinal permeability coefficient (Peff(human)), absorption rate constant (Ka) and enhancement ratio were calculated and represented in Table 5. The effective permeability coefficient (Peff(rat)), for GD and TD was found to be 19.7  0.39 (106), 37.7  0.68 (106) was significantly higher (p50.001) than the pure drug

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Figure 6. Fourier transform infrared spectroscopy of (A) Pure fexofenadine hydrochloride (FEX), (B) Gelucire 44/14 (GLC), (C) Physical mixture of FEX and GLC at 1:4 ratio, (D) Dispersion of FEX and GLC at 1:4 ratio (GD 1:4), (E) Vitamin E TPGS, (F) Physical mixture of FEX and TPGS at 1:4 ratio and (G) Dispersion of FEX and TPGS at 1:4 ratio (TD 1:4).

Figure 7. (A) In vitro dissolution profiles of pure fexofenadine hydrochloride (FEX), physical mixture of FEX and Gelucire 44/14 (GLC) at 1:4 ratio (GPM 1:4) and dispersions (GD) at different FEX/GLC ratios. (B) In vitro dissolution profiles of FEX, physical mixture of FEX and vitamin E TPGS (TPGS) at 1:4 ratio (TPM 1:4) and dispersions (TD) at different FEX/TPGS ratios. Each point represents the mean  SD. (n ¼ 3).

7.04  0.56(106) alone. The estimated human effective permeability coefficient and absorption rate constant (Ka) values indicates significantly higher rate of absorption of drug from dispersions than pure drug. The enhancement ratio for GD, TD was found to be 2.8 and 5.4 which is greater than 1 indicating enhanced permeation of drug from the dispersions. Improved dissolution rate by these amphiphilic carriers provides

high concentration of drug at the absorption site as well as high permeation of the drug, led to the higher absorption rate. The histological micrographs of the intestines after the permeation studies with pure drug (Figure 10B) and dispersions prepared (1:4 ratios of GD, TD; Figure 10C and D) shows the lack of changes in the intestinal structure with all the layers of the intestine as compared to intestine without treatment (Figure 10A).

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Table 2. Summary of dissolution parameters for dispersions and physical mixtures prepared using Gelucire 44/14 (n ¼ 3). Parameters

Q15*

DE15y

MDTz

MDRz

IDRz

RDRz

FEX GPM (1:4)ô GD (1:1)ô GD (1:2)ô GD (1:4)ô

16.92  1.2 23.20  0.7 35.00  1.6 64.94  1.1 79.00  1.7

8.46 11.6 17.5 32.47 39.50

45.18 40.57 39.86 19.81 10.65

0.44 0.75 0.96 1.57 1.87

1.13 1.55 2.33 4.33 5.27

– 2.18 2.37 2.56 2.67

t50%

(min)

z

4120 46 36 11 9

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FEX, GPM, GD represents pure fexofenadine hydrochloride, Gelucire 44/14 physical mixture and dispersions. *Q15: percent drug released at 15 min. yDE15: Dissolution efficiency at 15 min. zMDT, MDR, IDR, RDR and t50% (min): mean dissolution time, mean dissolution rate, initial dissolution rate, relative dissolution rate and time taken to release 50% of the drug, respectively. ôNumbers represent the weight/weight ratios of FEX/Gelucire 44/14. Table 3. Summary of dissolution parameters for dispersions and physical mixtures prepared using vitamin E TPGS (n ¼ 3). Parameters

Q15*

DE15y

MDTz

MDRz

IDRz

RDRz

FEX TPM (1:4)ô TD (1:1)ô TD (1:2)ô TD (1:4)ô

16.92  1.2 27.20  1.1 40.97  1.5 72.79  2.1 91.90  1.7

8.46 13.60 20.48 36.39 45.95

45.18 37.44 33.32 16.44 9.74

0.44 0.83 1.08 1.68 2.08

1.13 1.81 2.73 4.85 6.13

– 2.21 2.34 2.52 2.77

t50%

(min)

z

4120 38 30 9 7

FEX, TPM, TD represents pure fexofenadine hydrochloride, vitamin E TPGS physical mixture and dispersions. *Q15: percent drug released at 15 min. yDE15: Dissolution efficiency at 15 min. zMDT, MDR, IDR, RDR and t50% (min): mean dissolution time, mean dissolution rate, initial dissolution rate, relative dissolution rate and time taken to release 50% of the drug, respectively. ôNumbers represent the weight/weight ratio of FEX/vitamin E TPGS.

Figure 8. Apparent permeability coefficients (Papp) values of pure fexofenadine hydrochloride (FEX), dispersion of FEX and Gelucire 44/14 at 1:4 ratio (GD 1:4) and dispersion of FEX and vitamin E TPGS at 1:4 ratio (TD 1:4) using rat intestine in both absorptive and secretory directions (Mucosal to serosal and vice versa) (mean  SD; n ¼ 3).

Histological studies revealed there is no disruption of the intestinal epithelium with the concentration of carriers used in the formulation of dispersions (GD, TD at 1:4 ratios). Pharmacokinetic study Figure 11 shows the mean serum concentration versus time profiles of FEX (10 mg/kg) following single oral administration of

dispersions (1:4 ratios of GD, TD) in comparison with the pure drug in rats and the corresponding pharmacokinetic parameters are derived and given in Table 6. Both the dispersions have showed improved pharmacokinetic parameters relative to the absorption compared to pure drug. These results shows higher Cmax (mean peak concentration) for the dispersions compared to pure drug, whereas the Tmax (time to reach the peak concentration) remained constant for both dispersions and pure drug.

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Table 4. Ex vivo parameters of fexofenadine hydrochloride from dispersions across rat intestine in both absorptive and secretory directions (M to S and S to M; mean  SD; n ¼ 3). Papp(105 cm/s) Formulation

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FEX GD (1:4)x TD (1:4)jj

M to S

S to M

Efflux ratio

7.33  2.5 13.2  2.1*,1 20.2  1.9zy1,2

19.7  2.9 14.5  1.5*1 9.82  2.5yô1,2

2.69 1.10 0.49

FEX, GD, TD, Papp, M to S and S to M represents pure fexofenadine hydrochloride, Gelucire 44/14 dispersions, TPGS dispersions, apparent permeability coefficient in 120 min, mucosal to serosal and serosal to mucosal directions, respectively. 1, 2 indicate comparison with FEX, GD (1:4 ratio) formulation, respectively. *, y, z, ô indicates significant difference at p50.05, p50.01, p50.001 and not significant, respectively. xNumbers represent the weight/weight ratios of FEX/Gelucire 44/14. jjNumbers represent the weight/weight ratios of FEX/vitamin E TPGS.

The T1/2 (elimination half-life) of FEX was slightly prolonged with dispersions compared to pure drug. The AUC values for the pure drug, GD and TD were 951  418, 2376.6  501 and 3341.3  534 (ng h mL1). The relative bioavailability values for the dispersions were 2.51  0.46 and 3.54  0.50. These values indicating significantly (p50.001) higher extent of absorption and relative bioavailability of FEX from the dispersions following oral administration as compared to pure drug and ranked in the order TD (1:4)4GD (1:4)4FEX. The enhanced bioavailability of FEX from dispersions was explained by several mechanisms as follows (a). As the solid state of the drug was changed from crystalline to amorphous state (confirmed from scanning electron microscopy studies) with faster dissolution rate, more drug will be available in solution form at the site of absorption. (b). As these excipients are water dispersible surfactants, used in the self-emulsifying lipidic formulations, they forms emulsion upon contact with aqueous media. (c). Reduced efflux of the drug from the intestine due to P-gp inhibitory activity of these excipients. All these mechanisms

Table 5. In-situ parameters of fexofenadine hydrochloride from dispersions across rat intestine (mean  SD; n ¼ 3). Formulation FEX GD (1:4)ô TD (1:4)x

Peff(rat)(cm/s)  106

Peff(human)(cm/s)  105

ka(min1)

ER

7.04  0.56 19.7  0.39 z1 37.7  0.68 z1,2

2.61  0.18 6.72  0.12z1 12.59  0.22z1,2

0.0157  0.001 0.0403  0.0007*1 0.0755  0.0013zy1,2

– 2.8 5.4

Peff(rat), Peff(human), Ka and ER represents effective permeability coefficient in 90 min and enhancement ratio, respectively. 1, 2 indicate comparison with FEX, GD (1:4) formulation, respectively. *, y, z indicate significant difference at p50.05, p50.01 and p50.001, respectively. ôNumbers represent the weight/weight ratios of FEX/Gelucire 44/14. xNumbers represent the weight/weight ratios of FEX/vitamin E TPGS.

Figure 9. Effective permeability coefficient (Peff) values of pure fexofenadine hydrochloride (FEX), dispersion of FEX and Gelucire 44/14 at 1:4 ratio (GD 1:4) and dispersion of FEX and vitamin E TPGS at 1:4 ratio (TD 1:4) using single pass intestinal perfusion. Inset shows representative plot of the concentration ratio of the outlet and inlet concentrations (Cout/Cin)0 versus time. Steady state is achieved when (Cout/Cin)0 of FEX remains constant. (mean  SD; n ¼ 3).

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Figure 10. Histological sections of rat intestine: without treatment (A); after single pass intestinal perfusion studies with pure fexofenadine hydrochloride (FEX) (B); dispersions of FEX and Gelucire 44/14 (GD 1:4) (C) and dispersions of FEX and vitamin E TPGS (TD 1:4) (D), showing intact layers of intestine (Hematoxylin/Eosin stain, 100X  magnification).

Figure 11. Pharmacokinetic profiles of fexofenadine hydrochloride (FEX) in serum following oral administration of pure FEX, dispersion of FEX and Gelucire 44/14 at 1:4 ratio (GD 1:4) and dispersion of FEX and vitamin E TPGS at 1:4 ratio (TD 1:4). Each value represents mean  SD; n ¼ 6.

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Table 6. Pharmacokinetic parameters of fexofenadine hydrochloride after an oral administration of dispersions (10 mg/kg) to rats (mean  SD, n ¼ 6). Pharmacokinetic parameters

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Cmax (ng/mL) Tmax (h) K (h1) T1/2 (h) AUC0–t (ng h mL1) AUC0–1 (ng h mL1) RA

Formulations FEX

GD (1:4)ô 1

250  32 510  65z 1.0  0.0 1.0  0.0 0.428  0.21 0.269  0.026 1.89  0.86 2.586  0.24 879.5  345.2 2207.4  407z1

TD (1:4)x 798  77z12 1.0  0.0 0.265  0.025 2.636  0.25 3111.1  441.12zy1,2

951  418

2376.6  501z1

3341.3  534zy1,2

1.0  0.0

2.51  0.46z1

3.54  0.50z1,2

AUC, area under the curve; K, elimination rate constant; RA, relative bioavailability. 1, 2 indicate FEX, GD (1:4) formulation, respectively. *, y, z indicates significant difference at p50.05, p50.01 and p50.001, respectively. ôNumbers represent the weight/weight ratios of FEX/Gelucire 44/14. xNumbers represent the weight/weight ratios of FEX/vitamin E TPGS.

either alone or in combination, improve the bioavailability of the drug.

Conclusion From the results of present study, it can be concluded that both the carriers GLC and TPGS have improved the oral bioavailability of the FEX with enhanced solubility, dissolution and permeability. Reprecipitation studies and phase solubility studies revealed solubility enhancement of the FEX. The dissolution rate of the dispersions prepared with GLC and TPGS showed improved dissolution rate compared to pure FEX, would provide rapid absorption. Solid state studies confirmed the transformation of drug from crystalline state to amorphous state. Ex vivo permeation studies showed decreased efflux of the drug from the dispersions as these carriers are reported to be P-gp inhibitory in nature. In situ permeation and in vivo pharmacokinetic studies showed improved absorption and oral bioavailability of the FEX from dispersions compared to pure drug. In conclusion, the dispersions prepared with TPGS (1:4 ratio) showed improved dissolution, permeation, absorption and oral bioavailability compared to dispersions prepared with GLC.

Acknowledgements The authors would like to thank Ami Life Sciences Pvt. Ltd, Gujarat, India for the generous gift sample of fexofenadine hydrochloride. The authors are also grateful to Gattefosse, France and Isochem, France for the donation of excipients. The financial assistance to Basanth Babu Eedara by All India Council of Technical Education (New Delhi, India) in the form of Junior Research Fellowship is duly acknowledged. The authors also thank Mr. T. Jayapal Reddy, Chairman, St. Peter’s Institute of Pharmaceutical Sciences, Hanamkonda for providing the necessary facilities.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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