pharmacoscintigraphic evaluation of potential of lipid nanocarriers for nose-to-brain delivery of...

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International Journal of Pharmaceutics xxx (2014) xxx–xxx

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Pharmacoscintigraphic evaluation of potential of lipid nanocarriers fornose-to-brain delivery of antidepressant drug

M.Intakhab Alam a,b, Sanjula Baboota a,*, Alka Ahuja a, Mushir Ali a, Javed Ali a,Jasjeet K. Sahni a, Aseem Bhatnagar c

aDepartment of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi, IndiabDepartment of Pharmaceutics, College of Pharmacy, Jazan University, Jazan, Saudi ArabiacDepartment of Nuclear Medicine, Institute of Nuclear Medicine and Allied Sciences (INMAS), Brig SK Mazumdar Marg, Delhi, India

A R T I C L E I N F O

Article history:Received 2 January 2014Received in revised form 30 April 2014Accepted 3 May 2014Available online xxx

Keywords:PharmacoscintigraphyDTEDTPBiodistributionRadio-labelling efficiency

A B S T R A C T

Efficacy of antidepressants relies upon their continued presence at the site of action (brain) over aprolonged period of time. The BBB restricts the access of antidepressants to the brain on oral as well asintravenous administration. Direct delivery (by-passing the BBB) of antidepressant drugs can increasethe CSF concentration with concomitant reduction in dose and side effects. Intranasal administration ofnanostructured lipid carriers (NLC) containing antidepressant drug circumvent the BBB and maintain theprolonged release at the site of action. The aim of the present study was to evaluate the enhancement inbrain uptake of NLC containing duloxetine (DLX) after intranasal administration. Duloxetine loaded NLC(DLX-NLC) was evaluated pharmacoscintigraphically for drug targeting potential (DTP), drug targetingefficiency (DTE) and biodistribution studies in different organs including brain. The radio-labellingefficiency of DLX and DLX-NLC was found to be 98.41 �0.96 and 98.87 � 0.82 after 30 min, respectively.The biodistribution studies exhibited higher percentage of radioactivity/g for DLX-NLC formulations inbrain as compared with the DLX. The higher DTP (86.80%) and DTE (757.74%) suggested that DLX-NLCformulation has a better brain targeting efficiency than DLX solution (DTP = 65.12%; DTE = 287.34%) whenadministered intranasally. Moreover, the intranasal administration exhibited about 8-times higherconcentration of DLX in brain when compared with the intravenous administration of DLX solution. Theintranasal NLC containing DLX can be employed as an effective method for the treatment of depression.

ã 2014 Published by Elsevier B.V.

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

journal homepage: www.elsev ier .com/locate / i jpharm

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1. Introduction

Pharmacoscintigraphic technique has been widely used tostudy the in vivo behaviour of drug and drug delivery systems usingemitted radiations from the radioactive materials. This technologyhas proven to be of great value in the assessment of a wide range ofpharmaceutical formulations and new drug delivery systems. Theradiometric detection of drugs labelled with a suitable radiotraceris the best technique for the detection and concentration of thedrugs given through nasal route. Pharmacoscintigraphy studyincludes gamma-counts in different organs and gamma-imaging ofintact animal after administering the calculated dose of drug.Gamma-counts of gamma-radiation emitted by the depositedradiolabelled DLX and NLC in different organs were done by thegamma-counter. Moreover, the gamma-imaging was done by the

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40* Corresponding author. Tel.: +91 11 26059688/5634; fax: +91 11 26059663.E-mail address: [email protected] (S. Baboota).

http://dx.doi.org/10.1016/j.ijpharm.2014.05.0040378-5173/ã 2014 Published by Elsevier B.V.

Please cite this article in press as: Alam, M.I., et al., Pharmacoscintigradelivery of antidepressant drug, Int J Pharmaceut (2014), http://dx.doi.o

gamma-camera which gives images to provide the functional mapof physiological processes. For scintigraphic studies the formula-tions are usually labelled with the gamma-ray emitting radionu-clide 99mTc (technetium), which has ideal radiation energy(140 keV) for use with a gamma-camera (Newman and Wilding,1998). The short half-life of 99mTc (6 h), coupled with a very cleanand safe radiation emission profile which contains few beta-particles results in very low radiation doses, so that satisfactoryscintigraphic data can be obtained using only a fraction of theradiation dose required for diagnostic X-ray procedures (Newmanand Wilding, 1998).

Neurotransmitters (e.g., serotonin) are chemical messengerswithin the brain that facilitate communication among nerve cells.Inadequate supplies lead to the symptoms that are known asdepression. It is a serious medical condition and is associated withdecrease in functioning and well-being, high levels of disability,and high work absenteeism and health care costs. According toWHO estimates, depression will become the second-largest causeof the global health burden by 2020. Yet, depression remains one of

phic evaluation of potential of lipid nanocarriers for nose-to-brainrg/10.1016/j.ijpharm.2014.05.004

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e most under diagnosed conditions. Over 60 percent of suicidese attributed to major depressive disorder. It is a common mentalness with lifetime occurrence rates close to 20% which isritable as well (Glahn et al., 2012). Duloxetine (DLX), an SNRI ise first in the class of anti-depressants that ensures rapid andstained efficacy in the treatment of both emotional and physicalmptoms of depression. DLX promises treatment of physicalmptoms that accompany major depressive disorder (MDD) such

aches, pains, and gastrointestinal disturbance as well. On oralministration DLX undergoes hepatic first pass metabolism ands a systemic bioavailability of 50% (Lantz et al., 2003). Moreovere drug is acid labile at gastric pH. Oral administration of the drugso causes side effects including nausea, dry mouth, headache,zziness, orthostatic hypotension fatigue. Efficacy of antidepres-nts relies upon their continued presence at the site of actionrain) over a prolonged period of time (Kilts, 2003). On oral as well intravenous administration the BBB restricts the access oftidepressant drugs to the brain. Brain targeting can increase theF concentration of the drug with concomitant reduction in dosed side effects (Misra et al., 2003).Intranasal administration offers a non-invasive alternativeute to the central nervous system (CNS) for drug delivery,fectively bypassing the BBB (Graff and Pollack, 2005). The nasalute is one of the most permeable and highly vascularized site forug administration ensuring rapid absorption and onset oferapeutic action. The neural connections between the nasalucosa and the brain provide a unique pathway for noninvasivelivery of therapeutic agents to the CNS (Thorne and Frey, 2001;um, 2000). The olfactory and trigeminal nerve components in thesal epithelium provide pathways to deliver therapeutic agents toe olfactory bulb and brainstem, respectively, where dispersion toher CNS areas may be possible via pulsatile flow within therivascular spaces of cerebral blood vessels (Thorne et al., 2004;orne et al., 2008).NLCs are particles produced from the blend of solid and liquid

il) lipids. It possesses many “imperfections” increasing drugading capacity and minimizing or avoiding drug expulsion duringorage (Muchow et al., 2008). Being lipophilic in nature NLC hasen expected for the transport of therapeutic substances to theain. They are composed of physiological and biodegradable lipidshibiting low toxicity that means an excellent tolerability. Theid nanoparticles are able to enhance the chemical stability ofmpounds sensitive to light, oxidation and hydrolysis (Pardeike

al., 2009).Thus, the present study was designed to deliver DLX, antidepressant drug to the brain through nose to brain route ofug delivery using NLC as drug delivery system and theantification and biodistribution studies were performed byarmacoscintigraphic method.

Materials and method

. Drugs and reagents

DLX was provided by Dr. Reddy’s Laboratories (Hyderabad,dia). Glyceryl monostearate (solid lipid) (Loba chemie Pvt. Ltd.,umbai, India), pluronic F-68 (surfactant) and capryol PGMCquid lipid) (Sigma Chemical Company, MO, US), bile saltodium taurocholate) (co-surfactant) (Thomas Baker, chemicals,d., Mumbai, India), and mannitol (cryoprotectant) (S.D. fine-em Ltd., Mumbai, India) were used as received from suppliers.dium pertechnetate, separated from molybdenum-99 (99Mo) bylvent extraction method, was provided by Regional Centre fordiopharmaceutical Division (Northern Region), Board of Radia-n and Isotope Technology (New Delhi, India). Stannous chloridehydrate (SnCl2 2H2O) was purchased from Sigma–Aldrich

Please cite this article in press as: Alam, M.I., et al., Pharmacoscintigrdelivery of antidepressant drug, Int J Pharmaceut (2014), http://dx.doi

(St. Louis, MO, USA). Instant thin layer chromatography (ITLC)silicic acid (ITLC-SA) strips were purchased from Gelman SciencesInc. (Ann Arbor, MI, USA). All other chemicals and solvents wereof analytical reagent grade and were used without furtherpurification.

2.2. Animals

Swiss albino Wistar rats of either sex (200–250 g) were used forperforming the gamma count in different organs. All animals weregiven free access to water and food and kept under standardlaboratory conditions, temperature at 25 � 2 �C with a natural light–dark cycle and relative humidity of 55 � 5%. The animals werehoused in polypropylene cages, six per cage with free access tostandard laboratory diet (Lipton feed, Mumbai, India; providing3630 kcal/g energy and containing 22.10% crude protein, 4.10%crude oil, 4.05% crude fiber,10.05% ash, 0.75% sand silica) and waterad libitum. Ethical clearance for performing biodistribution studieswas taken from the Institutional Animal Ethics Committee, JamiaHamdard, New Delhi and the study was performed at INMAS(Delhi, India).

New Zealand rabbits weighing between 2.00 and 2.50 kg(female) were employed for performing the gamma-imagingstudies. Animals were procured from the animal house ofINMAS (Delhi, India). The guidelines of Committee for thePurpose of Control and Supervision of Experiments on Animals(CPCSEA) (Ministry of Culture, Govt. of India) were followedthroughout the study and maximum care was taken to makecertain that animals were treated in the most human andethically acceptable method.

3. Experiments

3.1. Preparation of DLX-NLC and lyophilization

DLX-NLC was prepared by dissolving DLX (2 g/l) in a mixture ofmelted solid lipid (glyceryl monostearate) and liquid lipid (capryolPGMC). The lipid concentration and the ratio of liquid lipid to totallipid was optimized to 2 (% w/w of aqueous phase) and 0.94:1,respectively. The lipid mixture was homogenized (Heidolph, Diax900, Schwabach, Germany) at 10,000 rpm for 20 min with hotaqueous solution (80 �C) of surfactants {pluronic F-68 = 1.5% w/w;and bile salt (sodium taurocholate) = 0.5% w/w} followed byultrasonication (10 min) and lyophilization (at –70 �C) usingmannitol (3% w/w) as cryoprotectant (Alam et al., 2011).

3.2. Procedure for radio-labelling

Radio-labelling was performed using sodium pertechnetate.Radioactivity was eluted out from Mo–Tc generator in saline.Ethanol was chosen as a solvent to extract out the radioactivity. Asuitable method of radio-labelling was chosen by which DLX waslabelled with 99mTc. The method of radiolablleing was standard-ized by gamma-imaging technique so as to visualize thedistribution of radiolabelled drug in animal models. The selectionof 99mTc was based on a number of properties including its shorthalf-life of 6.02 h, cost effective, easily eluted from the generator,soluble in solvents like acetonitrile and methyl ethyl ketone (MEK)and the dried form of activity is easily leached out from the glassbeaker with the help of acetonitrile.

3.3. Radio-labelling of DLX and DLX-NLC

radio-labelling was done using 99mTc by a direct labellingmethod. DLX (5 mg) or DLX-NLC (equivalent to 5 mg of DLX) wasaccurately weighed in a vial and 1 ml of distilled water was added.

aphic evaluation of potential of lipid nanocarriers for nose-to-brain.org/10.1016/j.ijpharm.2014.05.004


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Stannous chloride was dissolved in ethanol to make a solution of1 mg/ml and 100 ml of this solution was added to the vialcontaining DLX or DLX-NLC. To the resultant mixture 99mTcpertechnetate (200 mci) was added carefully with continuousmixing and incubated at 25 �C for 30 min. Quality control of radio-labelling was done using ITLC-SA strips using acetone as mobilephase to determine radio-labelling efficiency (%). The strip wasspotted with labelled complex (2–3 ml) above 1 cm from thebottom. The labelled complexes along with the reduced/hydro-lyzed 99mTc stay behind at the bottom of the strip; the freepertechnetate migrates to the top. The developed strip wasevaluated for radioactivity by a well-type gamma ray spectrometer(type GRS23C, Electronics Corporation of India Ltd., Hyderabad,India). The excess of stannous chloride was studied in terms ofradio-colloids which was determined in acetic acid:pyridine:water(5:3.5:1). The excess of stannous chloride utilized for reduction of99mTc may produce the radio-colloids which is undesirable. Thefree pertechnetate as well as the labelled complex migrated withthe solvent front to the top of the strip, leaving behind the radio-colloids at the bottom. The radioactivity in the strip wasdetermined with the solvent front using acetone as well as usingthe mixture of acetic acid:pyridine:water and compared todetermine the net amount of labelled complexes. Moreover theethanol was used in sufficient quantity just to dissolve stannouschloride.

3.4. Optimization of radio-labelling parameters

The various radio-labelling parameters required to optimize theradio-labelling of DLX or DLX-NLC included incubation time, pHand temperature and reducing agent (SnCl2), to achieve the desiredreaction condition for radio-labelling. The radiochemical puritywas determined on ITLC using acetone as mobile phase.

3.5. Effect of stannous chloride (SnCl2) strength

The solution of concentration 1 mg/ml was made by dissolvingan accurately weighed sample of SnCl2 in ethanol. The effect ofstannous chloride (SnCl2) strength on radio-labelling of DLX andDLX-NLC was observed at different concentrations varied from 50to 500 ml of 1 mg/ml solution.

3.6. Effect of pH

The effect of pH was determined for pH range of 3.0, 5.5, 7.4 and10.0 and the pH was optimized for determining the radiochemicalpurity of the DLX or DLX-NLC complex.

3.7. Effect of temperature

The optimum temperature for radio-labelling was obtained bydetermining the radio-labelling efficiency at different temper-atures of 20, 25, 30 and 35 �C.

3.8. Effect of incubation time

The radiolabelled complex of DLX or DLX-NLC with theoptimized strength of SnCl2 solution, pH and temperature wereincubated at room temperature for different time intervals of 10,20, 30, 40, 50, and 60 min.

3.9. Radiochemical stability of the radiolabelled complexes

The radiochemical stability of the radiolabelled complexes ofDLX and DLX-NLC performed in saline as well as serum. The in vitrostability study was performed by mixing 200 ml of radiolabelled


DLX or DLX-NLC with 1 ml of saline or serum. Small aliquots werewithdrawn at different time intervals up to 24 h and radiochemicalstability of DLX or DLX-NLC was evaluated by ITLC using acetone asmobile phase. The developed strips were cut into 7:3 ratios andradioactivity in each part was measured using gamma rayspectrometer to calculate the stability of the product.

3.10. Biodistribution studies

The biodistribution studies were carried out using Wistar rats ofeither sex (200–250 g). Just before the experiment, the rats wereweighed and restrained in rat restrainers. All the rats were markedwith picric acid solution for identification and randomly dividedinto three groups consisting of three rats at each time point (i.e.,nine rats in each group). Group 1 was given DLX-loaded NLC (DLX-NLC) intranasally, group 2 received DLX solution intranasally andgroup 3 received DLX solution intravenously. Doses of 0.54 mg/daywere administered in case of intranasal and intravenous DLXsolution whereas a dose equivalent to 0.54 mg/day of DLX wasgiven in case of DLX-NLC. DLX-NLC was administered intranasallyto rats, compared to intranasal and intravenous administration ofDLX solution.

Radiolabelled formulation (200 mCi/100 ml) was administeredin each nostril intravenously. Formulations containing DLXsolution and DLX-NLC suspension (DLX-NLC was suspended inwater containing chitosan �0.6% w/w) was instilled into thenostrils with the help of micropipette (100 ml) fitted with micro tipat the delivery site (olfactory region of the nose). The rats were heldfrom the back, in upright position during nasal administration.These were anaesthetized with diethyl ether and sacrificed atdifferent time intervals (6, 12, 24 h) and the blood was collected bycardiac puncture. The ethics committee approved only three timepoints for the study. Moreover, the brain and other organs (heart,liver, lungs, spleen, intestine and kidney) were collected, washedtwice using normal saline, made free from adhering tissue/fluid,dried and weighed. Radioactivity present in each tissue/organ wasmeasured using shielded well-type gamma scintillation counter.Radiopharmaceutical uptake per gram in each tissue/organ wascalculated as a fraction of administered dose using the followingequation (Saha, 1993).

%Radioactivity=g of tissue

¼ countsinsampleweightofsample � totalcountsinjected

� 100

3.11. Pharmacokinetic studies

Blood samples were withdrawn via cardiac puncture at 0 (pre-dose), 6, 12, 24 h in microcentrifuge tubes in which 8 mg of EDTAwas added as an anticoagulant. The collected blood was mixedproperly with the anticoagulant and centrifuged at 4000 rpm for20 min. The plasma was separated and analysed by measuring theradioactivity using the gamma scintillation counter. Variouspharmacokinetic parameters including Cmax, MRT, AUC0–24 andAUMC0–24 were calculated using Kinetica software1 (ThermoFisher Scientific, Berman, Germany). The pharmacokinetic dataamong different formulations were compared for statisticalsignificance by one way analysis of variance (ANOVA) followedby Tukey–Kramer multiple comparison tests using GraphPad Instatsoftware (GraphPad Software Inc., CA, USA). The brain targetingefficiency (DTE%) and nose to brain direct transport percentage(DTP%) were also calculated (Kumar et al., 2008).

DTEð%Þ ¼ ðAUCbrain=AUCbloodÞi:n:ðAUCbrain=AUCbloodÞi:v:

� 100



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Fig. 1. (a) TEM and, (b) SEM images of lyophilized DLX-NLC. These micrographs revealed the nanoparticulate (80.17–127.73 nm) and spherical nature of DLX-NLC.

Table 1Effect of pH, temperature, incubation time and strength of SnCl2 solution on radio-labelling efficiency of DLX and DLX-NLC.

Parameters Value REa of DLX � SD(n = 3)

REa of DLX-NLC � SD(n = 3)

pH of medium 3.0 94.32 � 2.31 88.97 � 3.226.0 93.89 � 1.42 95.63 � 2.517.4 97.77 � 2.23 98.93 � 1.36

10 89.63 � 1.21 94.57 � 3.34Temperature(�C)

20 88.71 � 3.12 91.64 � 4.11

25 98.73 � 2.24 98.95 � 1.6230 96.96 � 3.61 98.32 � 2.2335 94.87 � 2.23 95.64 � 3.14

Incubation time (min) 10 71.33 � 2.37 86.45 � 2.5420 90.12 � 5.28 93.37 � 3.7330 97.88 � 1.17 98.31 � 1.2140 97.37 � 1.85 98.82 � 1.3250 96.97 � 2.19 97.64 � 2.7360 96.13 � 2.11 95.76 � 1.84

Strength of reducing agent(mg/ml)

50 83.97 � 5.41 93.69 � 1.21

100 98.41 � 0.96 96.53 � 1.62200 97.78 � 1.31 98.87 � 0.82400 95.32 � 3.72 97.81 � 0.45500 89.57 � 2.34 96.45 � 0.97

a RE = radio-labelling efficiency.


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Pð%Þ ¼ Bi:n: � Bx

Bi:n:� 100

here Bx = (Bi.v./Pi.v.) � Pi.n.Bx is the brain AUC fraction contributed by systemic circulationrough the BBB following intranasal administration.Bi.v. is the AUC0–24 (brain) following intravenous administration.Pi.v. is the AUC0–24 (blood) following intravenous administration

Bi.n. is the AUC0–24 (brain) following intranasal administration.Pi.n. is the AUC0–24 (blood) following intranasal administration.AUC is the area under the curve.

2. Gamma-imaging studies

The healthy New Zealand rabbits (n = 3) weighing between 2.00d 2.50 kg (female) were selected for the study. The rabbits wereaesthetized using ketamine hydrochloride intramuscular injec-n (1 ml of 50 mg/ml) and held from the back in slanted positionring intranasal administration. The radiolabelled formulationsLX-NLC and DLX solution) was administered via nasal route andaced on the imaging platform. The localization all through thedy was visualized using Single Photon Emission Computerizedmography (SPECT) gamma camera, provided by GE healthcarestem (Hawkeye Millennium VG, GE Medical Systems, Milwau-e, WI, USA) and the images were recorded using the eNTEGRAftware.

3. Statistical analysis

All the data were expressed as the mean standard deviationSD). The data was analyzed using Kruskal–Wallis test (non-rametric test). A Dunn’s multiple comparison test were used tompare different formulations and a p-value of less than 0.10

< 0.10) was considered to be significant.

Results and discussions

The prepared DLX-NLCs were evaluated for different param-ers including particle size, particle size distribution, polydis-rsity index, entrapment efficiency, drug loading, surfaceorphology, in vitro release, stability studies, pharmacodynamicudies and estimation in brain and blood as described in oureceding report (Alam et al., 2011; Alam et al., 2012). Theansmission electron microscopy (TEM) and scanning electronicroscopy (SEM) images are shown in Fig. 1. The TEM micro-aphs revealed the spherical shape and size below 130 nm (80.17–7.73 nm) in diameter of lyophilized DLX-NLC. The SEM


micrographs also suggested the nanoparticulate and sphericalnature of DLX-NLC.

4.1. Optimization of radio-labelling parameters

The radio-labelling of DLX and DLX-NLC was performed bydirect labelling method. The radio-labelling parameters includingstrength of SnCl2 solution (reducing agent), pH, temperature andincubation time were optimized based on the radio-labellingefficiency (% RE). The maximum RE was found to be 98.41% and98.87% after 30 min and 40 min at 100 mg/ml and 200 mg/mlconcentration of SnCl2 solution as reducing agent for DLX and DLX-NLC, respectively (Table 1). The possible existence of unwantedradiochemical impurity in the form of free pertechnetate andcolloids was found up to 0.59 � 0.14% and 0.88 � 0.31% for DLX-NLCand 0.41 �0.21% and 0.69 � 0.11% for DLX, respectively. The poorlabelling efficiency was observed at lower concentrations ofstannous chloride because of the partial reduction of pentavalentpertechnetate from its heptavalent oxidation state; however, thehigher amounts bring about the greater creation of undesirableradio-colloids. It is an essential requirement to change techne-tium’s oxidation state for preparing 99mTc-labelled compounds.



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Saline-DLX Serum-DLX Saline-DNLC Serum-DNLC

Fig. 2. Stability of radiolabelled DLX and DLX-NLC in saline and serum. DLX andDLX-NLC were found to be stable by exhibiting more than 90% of radio-labellingefficiency in saline as well as serum for a time period of 24 h.


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This was accomplished by using a reducing agent in the radio-labelling procedure. In early radio-labelling studies, reducingagents such as ascorbic acid and ferrous iron were used thatresulted to incomplete reduction, requiring removal of unreactedpertechnetate. However, stannous chloride is considered to becompetent of generating high yields of 99mTc-labelled compounds,abolishing the need to remove free pertechnetate (Eckelman andRichards, 1970).

Similarly the observed results of studies of effects of pH,temperature and incubation time on the radio-labelling efficiencyof DLX and DLX-NLC are shown in Table 1. When incubated for30 min, at pH 7.4 and temperature 25 �C the radio-labelling of DLXwas found to be stable and optimized. The optimized conditions forradio-labelling of DLX-NLC were found to be 40 min, 25 �C and 7.4for incubation time, temperature and pH, respectively. The radio-labelling efficiency at different values of the selected conditionswas found to be more than 80 percent.

The extent of nasal absorption depends on pH at the absorptionsite that may also affect the integrity of nasal mucosa. It has beenreported that when pH ranges from 3 to 10 minimal quantities ofproteins and enzymes are released from cells, demonstrating nocellular damages i.e., if pH values are below 3 or above 10 damageshave been observed intracellularly and at membrane level (Pujaraet al., 1995). The human nasal mucosal pH is approximately 5.5–6.5(England et al.,1999) and the nasal pH of rat is 7.39 (Hiraiet al.,1981).

The chemistry of DLX and DLX-NLC radio-labelling with 99mTcmay be explained on the basis of binding at electron donatingfunctional groups (e.g., hydroxyl and amine functional groups oflipids and DLX, respectively). The powerful reducing agentstannous ion (Sn++) reduces 99mTc to the more reactive oxidationstate (+5) from nonreactive species (+7) to promote binding. The99mTc-labelling may occur by binding at electron donatingfunctional groups because 99mTc favours ligands that are

Table 2Biodistribution of intranasally administered DLX-NLC formulation, intranasal DLX solu

Organs Percent injected dose per gram of tissue (% ID/g)

Intranasal DLX-NLC Intranasal DLX

6 h � SD(n = 3)

12 h � SD (n = 3) 24 h � SD (n = 3) 6 h � SD(n = 3)

Blood 6.45 � 1.73 3.50 � 0.89 3.01 � 1.41 1.39 � 0.81

Brain 10.75 � 1.34 6.28 � 1.11 3.82 � 0.78 0.82 � 0.62

Heart 4.69 � 1.56 2.48 � 0.88 2.13 � 0.94 1.21 � 0.19

Lungs 5.43 � 1.80 3.19 � 1.78 3.21 � 1.31 1.37 � 0.53

Liver 2.89 � 1.17 2.20 � 1.69 3.16 � 1.27 0.64 � 0.75

Kidney 13.87 � 2.27 11.54 � 2.11 5.40 � 1.18 1.21 � 0.98

Spleen 12.96 � 2.38 8.00 � 1.86 5.28 � 1.23 1.64 � 0.39

Intestine 15.40 � 3.14 6.87 � 1.43 3.67 � 1.33 1.44 � 0.71


competent to recompense for the high positive charge of thecentral atom (Soane et al., 1999).

4.2. Radiochemical stability of the radiolabelled complexes

The DLX and DLX-NLC were found to be stable in saline as wellas serum by exhibiting the radio-labelling efficiency of more than90% for a time period of 24 h (Fig. 2). The radio-labelling of DLX wasfound to be 94.47 � 4.28% and 97.70 � 1.01% in saline and serum,respectively when determined after 30 min. Similarly for DLX-NLCit was found to be 98.45 � 2.01 and 99.76 � 1.21 in saline and serum, respectively after 30 min. The radiolabelled complexes werefound to be stable and there was no significant breaking of thecomplexes. In most radiopharmaceuticals, a 90% limit of ‘bound’99mTc chelate is suggested (Saha, 1996). It is significant todetermine the stability in serum to make sure the intactness ofthe labelled complexes in the presence of proteins and severalother substances there in serum. It also supports the stability invivo upon administration into the body and their use indetermining the biodistribution models. Thus it was concludedthat these radiolabelled complexes could be administered viaintranasal route for gammascintigraphic studies.

4.3. Biodistribution studies of DLX and DLX-NLC formulation

The biodistribution studies were performed to investigate theamount of drug reaching in various vital organs including brain,liver, kidney, intestine, heart, spleen, intestine and blood. Thepercent injected dose per gram of tissue (% ID/g) was determined atdifferent time intervals. The biodistribution of radiolabelledintranasal DLX-NLC, compared with a positive control of radio-labelled intranasal DLX solution and intravenous DLX solution areshown in Table 2.

The biodistribution data of DLX-NLC and DLX solution viaintranasal route resulted in a higher % ID/g in the brain for the NLCformulation as compared with the DLX solution (p < 0.10).Biodistribution studies revealed more localization in kidney,spleen and liver for DLX-NLC. The higher level of intranasalDLX-NLC in different organs may be explained on the basis of itsnanoparticulate and lipophilic nature and avoidance from thedegrading environment in the nasal cavity (Cf. intranasal DLXwhich is devoid of these characteristics). In addition, differentorgans may be approached by different mechanisms by themolecules administered in the body. The estimation of higheramount in case of intranasal DLX-NLC in brain could be due to thedirect pathways of transport (neuronal and non-neuronal) to thebrain (Cf. intravenous DLX where BBB restricts the transport to thebrain). The accumulation in the liver and the spleen is generallyascribed to uptake by the reticuloendothelial system (RES) likemacrophage cells (Dobrovolskaia et al., 2008), whereas the

tion and intravenous DLX solution.

Intravenous DLX

12 h � SD(n = 3)

24 h � SD(n = 3)

6 h � SD(n = 3)

12 h � SD(n = 3)

24 h � SD(n = 3)

1.06 � 0.81 0.79 � 0.48 0.646 � 0.05 0.598 � 0.07 0.449 � 0.080.66 � 0.92 0.49 � 0.59 0.132 � 0.02 0.124 � 0.06 0.105 � 0.091.25 � 0.32 1.51 � 0.72 0.398 � 0.04 0.216 � 0.04 0.181 � 0.061.44 � 0.27 0.83 � 0.63 0.361 � 0.07 0.268 � 0.07 0.093 � 0.011.18 � 0.78 0.91 � 0.81 0.178 � 0.09 0.186 � 0.08 0.224 � 0.011.67 � 0.61 1.30 � 0.64 0.100 � 0.06 0.185 � 0.05 0.112 � 0.041.56 � 0.82 1.43 � 0.94 0.177 � 0.04 0.259 � 0.04 0.116 � 0.032.02 � 0.54 1.37 � 0.52 0.363 � 0.07 0.246 � 0.03 0.161 � 0.05



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IJP 14061 1–8

esence of particles in the lungs may be the results from theirglomeration caused by the adsorption of plasma proteins (Alexis

al., 2008). Moreover, DLX undergoes extensive metabolism tomerous metabolites involving oxidation of the naphthyl ringllowed by conjugation and further oxidation. Metabolically mostbout 70%) of the DLX dose appears in the urine as metabolites ofX; about 20% is excreted in the faeces (Lantz et al., 2003). Veryw concentrations of the 99mTc-labelled complexes of DLX-NLCd DLX were recovered from liver and showed constancy withe suggesting the intactness and in vivo stability of the

mplexes. The in-vivo stability can be predicted based on the vitro stability results (Sun et al., 2002). DLX-NLC exhibitedcellent in vitro stability hence expected in vivo also. The higher invo stability can be indicated by rapid clearance of intact complexr low concentration of complex) from the liver or RES organsver, spleen, kidneys etc.). However the higher uptake of DLX-NLC

RES organs can be explained based on the fact that the GMSntaining nanoparticles exhibited higher uptake by the RESgans as reported by (Pandey et al., 2005), (Sankar et al., 2012) andoni et al., 2014).

4. Effect of route of administration on nose-to-brain delivery of DLX

The DLX solution was administered by both intranasal as well astravenous route of administration. The brain concentration ofX was compared after 6 h of administration. The intranasalministration exhibited more than 6-times higher concentration

DLX in brain (0.82% ID/g) when compared with the intravenousministration (0.134% ID/g) of DLX solution (p < 0.10). The higherncentration of DLX in brain may be explained on the basis ofrect pathway of transport (neuronal and non-neuronal) fromse to brain. BBB restrict the transport of drug molecules to theain upon intravenous administration. Intranasal administrationfers a non-invasive alternative route to the brain for druglivery effectively bypassing the BBB (Graff and Pollack, 2005).is method allows drugs that do not cross the BBB to be delivered

the CNS and eliminates the need for systemic delivery.

5. Effect of NLC on biodistribution of DLX

The organ distribution of DLX administered as intranasal DLX-C was compared with intranasal DLX solution. It revealednificant differences (p < 0.10) in the biodistribution of DLX. TheX-NLC exhibited higher distribution not only in brain but also inher organs as compared to pure DLX solution. The higherstribution of DLX-NLC than DLX solution after intranasalministration may be explained because of nanoparticulated lipophilic nature of NLC and enhancement in permeation/sorption by the surfactant and co-surfactant added during theeparation of NLC.

ble 3an pharmacokinetic parameters (�S.D., n = 3), drug targeting efficiency (DTE%) andranasal DLX-NLC suspension, intranasal DLX solution and intravenous DLX solution

ethod Organ/tissue

Cmax

(% ID/g)AUC0 ! 24

(h % ID/g)AUC0 ! 1(h % ID/g)

ntranasal DLX-NLC Blood 6.45 � 1.34 68.91 � 9.16 147.93 � 11.17

Brain 10.75 � 1.73a 111.69 � 7.27a 180.90 � 8.31a

ntranasal DLX Blood 1.39 � 0.82 18.45 � 2.16 44.43 � 9.23

Brain 0.82 � 0.51b 11.34 � 1.63b 28.79 � 2.11b

ntravenous DLX Blood 0.646 � 0.07 10.01 � 3.16 31.66 � 9.53

Brain 0.132 � 0.05 2.14 � 0.86 10.29 � 0.77

p < 0.10 vs intranasal/intravenous DLX solution.p < 0.10 vs intravenous DLX solution.p < 0.10 vs DLX solution.


4.6. Effect of NLC on nose-to-brain delivery of DLX

DLX was administered as DLX-NLC and DLX solution throughthe same route of intranasal administration. The concentration ofDLX was determined in the brain after 6 h of administration andcompared. DLX encapsulated in NLC exhibited higher concentra-tion in brain (10.75% ID/g) as compared to pure DLX solution (0.82%ID/g) (p < 0.10). NLC significantly affected the permeation/absorp-tion of DLX through nose-to-brain route of drug administration.The superiority of NLC for nose to brain delivery over the solutionmay be attributed to its lipidic nature (facilitating permeation/absorption), nanoparticulate nature (different mechanisms areinvolved) and avoidance of DLX from degrading environment innasal cavity (e.g., lytic enzymes and pH present in nasal secretions).

4.7. Pharmacokinetic studies

The different pharmacokinetic parameters of intranasal DLX-NLC suspension, intranasal DLX solution and intravenous DLXsolution were calculated by determining the concentration (% ID/g)of DLX in blood and brain. The results of different pharmacokineticparameters are given in Table 3.

The pharmacokinetic parameters for DLX-NLC were comparedwith that of drug solution administered by intravenous route. Itwas observed that the Cmax of DLX in blood (6.45% ID/g) and brain(10.75% ID/g) was higher in case of intranasal DLX-NLC formulationas compared with that of the DLX solution administered byintravenous and intranasal routes (p < 0.10). Moreover, theintranasal administration of DLX solution exhibited higheraccumulation of DLX in brain (p < 0.10) than the DLX solutionadministered intravenously. Thus the intranasal administrationprovided higher concentration in brain than administered throughintravenous route. Moreover, the lipid nanocarriers exhibitedhigher concentration than the DLX solution per se.

Intranasal administration provides a non-invasive method forbypassing the BBB and delivering therapeutic drugs along theolfactory and trigeminal nerves directly to the brain (Alam et al.,2011). Furthermore, DLX-NLC contributed higher uptake in thebrain as compared to pure DLX solution because of nanoparticulatenature and lipophilicity of NLC. Nano-delivery systems have greatpotential to facilitate the movement of drugs across barriers (e.g.,BBB). Numerous mechanisms are reported by which nanoparticlesattain maximum drug concentration in brain including increasedretention of drugs in brain-blood capillaries combined with anadsorption to capillary walls as higher concentration gradientincrease transport, increasing the fluidization of BBB membrane,opening of tight junctions among endothelial cells (an endocytoticevents occur due to upfolding of the cell membrane), inhibiting theP-glycoprotein efflux system (e.g., by poloxamer containingnanoparticles) (Alam et al., 2010).

Q6 direct nose to brain transport (DTP%) following administration of.

AUMC0–24

(% ID.h2/ml)Kel (1/h) Drug targeting

efficiency(DTE %)

Direct nose-to-braintransport(DTP %)

1043.64 � 33.51 0.038 � 0.0018 757.74c 86.80c

1615.32 � 22.42a 0.055 � 0.0012a

278.28 � 11.63 0.030 � 0.0018 287.34 65.12171.36 � 18.26b 0.028 � 0.002b

152.49 � 23.13 0.020 � 0.007 – –

33.26 � 8.26 0.012 � 0.005



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Fig. 3. Gamma scintigraphic images after intranasal administration (6 h) of (a) DLX-NLC suspension, (b) DLX solution. These images are showing the localization of DLX indifferent organs including brain of rabbit. DLX-NLC exhibited better localization than DLX.


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IJP 14061 1–8

However, intravenous delivery gave little accumulation of thedrug in the brain because of the BBB. The availability of DLX insmall amount in brain after intravenous administration of DLXsolution may be attributed to its physicochemical properties.Some small molecules with appropriate lipophilicity, molecularweight (mw) and charge gain access through diffusion from bloodinto the CNS (Gabathuler, 2010). However, the majority of smallmolecules do not cross the BBB. The relative impermeability ofthe BBB results from tight junctions among capillary endothelialcells which are formed by cell adhesion molecules (Alam et al.,2010). Small lipophilic molecules can diffuse passively across theBBB into the brain but will be exposed to efflux pumps (P-glycoprotein, some multidrug resistance proteins, breast cancerresistance protein and others) expressed on the luminal side ofthe BBB and exposed to degrading enzymes (ecto- and endo-enzymes) localized in the cytoplasm of endothelial cells beforebrain penetration (Alam et al., 2010; Gabathuler, 2010). Inaddition, the brain availability of drug through intravenous routeis largely affected by the half-life of the drug in the plasma, rapidmetabolism, and level of non-specific binding to plasma proteinsand the permeability of the compound across the BBB and intoperipheral tissues (Patel et al., 2003).

Significantly higher AUC and Cmax for intranasal DLX-NLCcompared to intranasal as well as intravenous DLX solution wereobtained. The drug targeting efficiency (DTE%) and brain drugdirect transport percentage (DTP%) were also calculated for nasallyadministered formulations (Table 3). These were calculated usingtissue/organ distribution data following intranasal and intrave-nous administration. The DTP% and DTE% represent the percentageof drug directly transported to the brain via the olfactory pathway.The DLX-NLC showed the higher DTE (%) and DTP (%) values thanDLX solution. The higher DTE (%) and DTP (%) suggest that DLX-NLChas better brain targeting efficiency mainly because of substantialdirect nose-to-brain transport. These findings conclude that DLX-NLC increased nose-to-brain uptake of the DLX.

4.8. Gamma imaging studies

The study was performed to evaluate the localization of DLXafter intranasal administration of DLX-NLC suspension and DLX


solution. The images showing localization of DLX in differentorgans including brain of rabbit are shown in Fig. 3.

The uptake of DLX in brain and other organs was visualizedfollowing intranasal administration of radiolabelled formulations(99mTc-DLX-NLC suspension and 99mTc-DLX solution). The in-creased radiation intensity was shown from the radiolabeledformulation in the brain region for DLX-NLC suspension ascompared with that for DLX solution. The scintigraphy imageswere consistent with the results discussed in previous sections(biodistribution and pharmacokinetic studies) and high uptake ofDLX into the brain was observed.

5. Conclusion

The intranasal DLX-NLC formulation was successfully deliveredinto the brain of rat and appreciable amount of DLX was estimated.It exhibited its potential to be transported through nose-to-brainroute of drug administration. Furthermore it exhibited its potentialto be distributed throughout the body indicating its permeabilitythrough the biological barriers. The delivery system was found tobe useful to avoid probable systemic side effects due to DLX.Consequently, the present study visibly established that intranasalNLC is a suitable method for the effective delivery of DLX for thetreatment of behavioural disorders including depression. Accord-ingly, it may also be used as an effective method for the treatmentof other CNS disorders.

Contributors

M. Intakhab Alam: designed the study and wrote the protocol;Aseem Bhatnagar: carried out the pharmacocsintigraphic studies;Alka ahuja and Mushir Ali: managed the literature searches andanalyses; Javed Ali and Jasjeet K Sahni: undertook the statisticalanalysis; Sanjula Baboota: wrote the first draft of the manuscript.All authors contributed to and have approved the final manuscript.

Conflict of interest

The authors declare no conflict of interest. The authors alone areresponsible for the content and writing of the paper.



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le of the funding source

Council of Scientific and Industrial Research (CSIR) and Jamiamdard, New Delhi, (India) funded for carrying out thesearch.

knowledgements

Authors are thankful to Dr. Reddy’s Laboratories, Hyderabad,dia) for providing gift sample of the drug and CSIR, New Delhi,r providing a Senior Research Fellowship to M. Intakhab Alam.thors are also thankful to Institute of Nuclear Medicine andlied Sciences (INMAS) for providing facilities for pharmacoscinti-aphic studies.

ference

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