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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
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Pharmacoscintigraphic evaluation of potential of lipid nanocarriers for nose-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 a
Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi, India Department of Pharmaceutics, College of Pharmacy, Jazan University, Jazan, Saudi Arabia c Department of Nuclear Medicine, Institute of Nuclear Medicine and Allied Sciences (INMAS), Brig SK Mazumdar Marg, Delhi, India b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 January 2014 Received in revised form 30 April 2014 Accepted 3 May 2014 Available online xxx
Efficacy of antidepressants relies upon their continued presence at the site of action (brain) over a prolonged period of time. The BBB restricts the access of antidepressants to the brain on oral as well as intravenous administration. Direct delivery (by-passing the BBB) of antidepressant drugs can increase the CSF concentration with concomitant reduction in dose and side effects. Intranasal administration of nanostructured lipid carriers (NLC) containing antidepressant drug circumvent the BBB and maintain the prolonged release at the site of action. The aim of the present study was to evaluate the enhancement in brain uptake of NLC containing duloxetine (DLX) after intranasal administration. Duloxetine loaded NLC (DLX-NLC) was evaluated pharmacoscintigraphically for drug targeting potential (DTP), drug targeting efficiency (DTE) and biodistribution studies in different organs including brain. The radio-labelling efficiency 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 in brain as compared with the DLX. The higher DTP (86.80%) and DTE (757.74%) suggested that DLX-NLC formulation has a better brain targeting efficiency than DLX solution (DTP = 65.12%; DTE = 287.34%) when administered intranasally. Moreover, the intranasal administration exhibited about 8-times higher concentration of DLX in brain when compared with the intravenous administration of DLX solution. The intranasal NLC containing DLX can be employed as an effective method for the treatment of depression. ã 2014 Published by Elsevier B.V.
Keywords: Pharmacoscintigraphy DTE DTP Biodistribution Radio-labelling efficiency
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1. Introduction Pharmacoscintigraphic technique has been widely used to study the in vivo behaviour of drug and drug delivery systems using emitted radiations from the radioactive materials. This technology has proven to be of great value in the assessment of a wide range of pharmaceutical formulations and new drug delivery systems. The radiometric detection of drugs labelled with a suitable radiotracer is the best technique for the detection and concentration of the drugs given through nasal route. Pharmacoscintigraphy study includes gamma-counts in different organs and gamma-imaging of intact animal after administering the calculated dose of drug. Gamma-counts of gamma-radiation emitted by the deposited radiolabelled DLX and NLC in different organs were done by the gamma-counter. Moreover, the gamma-imaging was done by the
* Corresponding author. Tel.: +91 11 26059688/5634; fax: +91 11 26059663. E-mail address: [email protected] (S. Baboota).
gamma-camera which gives images to provide the functional map of physiological processes. For scintigraphic studies the formulations are usually labelled with the gamma-ray emitting radionuclide 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 clean and safe radiation emission profile which contains few betaparticles results in very low radiation doses, so that satisfactory scintigraphic data can be obtained using only a fraction of the radiation dose required for diagnostic X-ray procedures (Newman and Wilding, 1998). Neurotransmitters (e.g., serotonin) are chemical messengers within the brain that facilitate communication among nerve cells. Inadequate supplies lead to the symptoms that are known as depression. It is a serious medical condition and is associated with decrease in functioning and well-being, high levels of disability, and high work absenteeism and health care costs. According to WHO estimates, depression will become the second-largest cause of the global health burden by 2020. Yet, depression remains one of
http://dx.doi.org/10.1016/j.ijpharm.2014.05.004 0378-5173/ ã 2014 Published by Elsevier B.V.
Please cite this article in press as: Alam, M.I., et al., Pharmacoscintigraphic evaluation of potential of lipid nanocarriers for nose-to-brain delivery of antidepressant drug, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.05.004
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the most under diagnosed conditions. Over 60 percent of suicides are attributed to major depressive disorder. It is a common mental illness with lifetime occurrence rates close to 20% which is heritable as well (Glahn et al., 2012). Duloxetine (DLX), an SNRI is the first in the class of anti-depressants that ensures rapid and sustained efficacy in the treatment of both emotional and physical symptoms of depression. DLX promises treatment of physical symptoms that accompany major depressive disorder (MDD) such as aches, pains, and gastrointestinal disturbance as well. On oral administration DLX undergoes hepatic first pass metabolism and has a systemic bioavailability of 50% (Lantz et al., 2003). Moreover the drug is acid labile at gastric pH. Oral administration of the drug also causes side effects including nausea, dry mouth, headache, dizziness, orthostatic hypotension fatigue. Efficacy of antidepressants relies upon their continued presence at the site of action (brain) over a prolonged period of time (Kilts, 2003). On oral as well as intravenous administration the BBB restricts the access of antidepressant drugs to the brain. Brain targeting can increase the CSF concentration of the drug with concomitant reduction in dose and side effects (Misra et al., 2003). Intranasal administration offers a non-invasive alternative route to the central nervous system (CNS) for drug delivery, effectively bypassing the BBB (Graff and Pollack, 2005). The nasal route is one of the most permeable and highly vascularized site for drug administration ensuring rapid absorption and onset of therapeutic action. The neural connections between the nasal mucosa and the brain provide a unique pathway for noninvasive delivery of therapeutic agents to the CNS (Thorne and Frey, 2001; Illum, 2000). The olfactory and trigeminal nerve components in the nasal epithelium provide pathways to deliver therapeutic agents to the olfactory bulb and brainstem, respectively, where dispersion to other CNS areas may be possible via pulsatile flow within the perivascular spaces of cerebral blood vessels (Thorne et al., 2004; Thorne et al., 2008). NLCs are particles produced from the blend of solid and liquid (oil) lipids. It possesses many “imperfections” increasing drug loading capacity and minimizing or avoiding drug expulsion during storage (Muchow et al., 2008). Being lipophilic in nature NLC has been expected for the transport of therapeutic substances to the brain. They are composed of physiological and biodegradable lipids exhibiting low toxicity that means an excellent tolerability. The lipid nanoparticles are able to enhance the chemical stability of compounds sensitive to light, oxidation and hydrolysis (Pardeike et al., 2009). Thus, the present study was designed to deliver DLX, an antidepressant drug to the brain through nose to brain route of drug delivery using NLC as drug delivery system and the quantification and biodistribution studies were performed by pharmacoscintigraphic method.
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2. Materials and method
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2.1. Drugs and reagents
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DLX was provided by Dr. Reddy’s Laboratories (Hyderabad, India). Glyceryl monostearate (solid lipid) (Loba chemie Pvt. Ltd., Mumbai, India), pluronic F-68 (surfactant) and capryol PGMC (liquid lipid) (Sigma Chemical Company, MO, US), bile salt (sodium taurocholate) (co-surfactant) (Thomas Baker, chemicals, Ltd., Mumbai, India), and mannitol (cryoprotectant) (S.D. finechem Ltd., Mumbai, India) were used as received from suppliers. Sodium pertechnetate, separated from molybdenum-99 (99Mo) by solvent extraction method, was provided by Regional Centre for Radiopharmaceutical Division (Northern Region), Board of Radiation and Isotope Technology (New Delhi, India). Stannous chloride dihydrate (SnCl2 2H2O) was purchased from Sigma–Aldrich
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(St. Louis, MO, USA). Instant thin layer chromatography (ITLC) silicic acid (ITLC-SA) strips were purchased from Gelman Sciences Inc. (Ann Arbor, MI, USA). All other chemicals and solvents were of analytical reagent grade and were used without further purification.
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2.2. Animals
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Swiss albino Wistar rats of either sex (200–250 g) were used for performing the gamma count in different organs. All animals were given free access to water and food and kept under standard laboratory conditions, temperature at 25 2 C with a natural light –dark cycle and relative humidity of 55 5%. The animals were housed in polypropylene cages, six per cage with free access to standard laboratory diet (Lipton feed, Mumbai, India; providing 3630 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 water ad libitum. Ethical clearance for performing biodistribution studies was taken from the Institutional Animal Ethics Committee, Jamia Hamdard, 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-imaging studies. Animals were procured from the animal house of INMAS (Delhi, India). The guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) (Ministry of Culture, Govt. of India) were followed throughout the study and maximum care was taken to make certain that animals were treated in the most human and ethically acceptable method.
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3. Experiments
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3.1. Preparation of DLX-NLC and lyophilization
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DLX-NLC was prepared by dissolving DLX (2 g/l) in a mixture of melted solid lipid (glyceryl monostearate) and liquid lipid (capryol PGMC). The lipid concentration and the ratio of liquid lipid to total lipid was optimized to 2 (% w/w of aqueous phase) and 0.94:1, respectively. The lipid mixture was homogenized (Heidolph, Diax 900, Schwabach, Germany) at 10,000 rpm for 20 min with hot aqueous solution (80 C) of surfactants {pluronic F-68 = 1.5% w/w; and bile salt (sodium taurocholate) = 0.5% w/w} followed by ultrasonication (10 min) and lyophilization (at –70 C) using mannitol (3% w/w) as cryoprotectant (Alam et al., 2011).
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3.2. Procedure for radio-labelling
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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. A suitable method of radio-labelling was chosen by which DLX was labelled with 99mTc. The method of radiolablleing was standardized by gamma-imaging technique so as to visualize the distribution of radiolabelled drug in animal models. The selection of 99mTc was based on a number of properties including its short half-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 glass beaker with the help of acetonitrile.
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3.3. Radio-labelling of DLX and DLX-NLC
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radio-labelling was done using 99mTc by a direct labelling method. DLX (5 mg) or DLX-NLC (equivalent to 5 mg of DLX) was accurately weighed in a vial and 1 ml of distilled water was added.
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Please cite this article in press as: Alam, M.I., et al., Pharmacoscintigraphic evaluation of potential of lipid nanocarriers for nose-to-brain delivery of antidepressant drug, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.05.004
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Stannous chloride was dissolved in ethanol to make a solution of 1 mg/ml and 100 ml of this solution was added to the vial containing DLX or DLX-NLC. To the resultant mixture 99mTc pertechnetate (200 mci) was added carefully with continuous mixing and incubated at 25 C for 30 min. Quality control of radiolabelling was done using ITLC-SA strips using acetone as mobile phase to determine radio-labelling efficiency (%). The strip was spotted with labelled complex (2–3 ml) above 1 cm from the bottom. The labelled complexes along with the reduced/hydrolyzed 99mTc stay behind at the bottom of the strip; the free pertechnetate migrates to the top. The developed strip was evaluated 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 of radio-colloids which was determined in acetic acid:pyridine:water (5:3.5:1). The excess of stannous chloride utilized for reduction of 99m Tc may produce the radio-colloids which is undesirable. The free pertechnetate as well as the labelled complex migrated with the solvent front to the top of the strip, leaving behind the radiocolloids at the bottom. The radioactivity in the strip was determined with the solvent front using acetone as well as using the mixture of acetic acid:pyridine:water and compared to determine the net amount of labelled complexes. Moreover the ethanol was used in sufficient quantity just to dissolve stannous chloride. 3.4. Optimization of radio-labelling parameters The various radio-labelling parameters required to optimize the radio-labelling of DLX or DLX-NLC included incubation time, pH and temperature and reducing agent (SnCl2), to achieve the desired reaction condition for radio-labelling. The radiochemical purity was 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 dissolving an accurately weighed sample of SnCl2 in ethanol. The effect of stannous chloride (SnCl2) strength on radio-labelling of DLX and DLX-NLC was observed at different concentrations varied from 50 to 500 ml of 1 mg/ml solution.
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3.7. Effect of temperature
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The optimum temperature for radio-labelling was obtained by determining the radio-labelling efficiency at different temperatures of 20, 25, 30 and 35 C.
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3.8. Effect of incubation time
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The radiolabelled complex of DLX or DLX-NLC with the optimized strength of SnCl2 solution, pH and temperature were incubated at room temperature for different time intervals of 10, 20, 30, 40, 50, and 60 min.
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3.9. Radiochemical stability of the radiolabelled complexes
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The radiochemical stability of the radiolabelled complexes of DLX and DLX-NLC performed in saline as well as serum. The in vitro stability study was performed by mixing 200 ml of radiolabelled
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DLX or DLX-NLC with 1 ml of saline or serum. Small aliquots were withdrawn at different time intervals up to 24 h and radiochemical stability of DLX or DLX-NLC was evaluated by ITLC using acetone as mobile phase. The developed strips were cut into 7:3 ratios and radioactivity in each part was measured using gamma ray spectrometer to calculate the stability of the product.
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3.10. Biodistribution studies
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The biodistribution studies were carried out using Wistar rats of either sex (200–250 g). Just before the experiment, the rats were weighed and restrained in rat restrainers. All the rats were marked with picric acid solution for identification and randomly divided into three groups consisting of three rats at each time point (i.e., nine rats in each group). Group 1 was given DLX-loaded NLC (DLXNLC) intranasally, group 2 received DLX solution intranasally and group 3 received DLX solution intravenously. Doses of 0.54 mg/day were administered in case of intranasal and intravenous DLX solution whereas a dose equivalent to 0.54 mg/day of DLX was given in case of DLX-NLC. DLX-NLC was administered intranasally to rats, compared to intranasal and intravenous administration of DLX solution. Radiolabelled formulation (200 mCi/100 ml) was administered in each nostril intravenously. Formulations containing DLX Q5 solution and DLX-NLC suspension (DLX-NLC was suspended in water containing chitosan 0.6% w/w) was instilled into the nostrils with the help of micropipette (100 ml) fitted with micro tip at the delivery site (olfactory region of the nose). The rats were held from the back, in upright position during nasal administration. These were anaesthetized with diethyl ether and sacrificed at different time intervals (6, 12, 24 h) and the blood was collected by cardiac puncture. The ethics committee approved only three time points for the study. Moreover, the brain and other organs (heart, liver, lungs, spleen, intestine and kidney) were collected, washed twice using normal saline, made free from adhering tissue/fluid, dried and weighed. Radioactivity present in each tissue/organ was measured using shielded well-type gamma scintillation counter. Radiopharmaceutical uptake per gram in each tissue/organ was calculated as a fraction of administered dose using the following equation (Saha, 1993).
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3.6. Effect of pH The effect of pH was determined for pH range of 3.0, 5.5, 7.4 and 10.0 and the pH was optimized for determining the radiochemical purity of the DLX or DLX-NLC complex.
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countsinsample 100 ¼ weightofsample totalcountsinjected 3.11. Pharmacokinetic studies
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Blood samples were withdrawn via cardiac puncture at 0 (predose), 6, 12, 24 h in microcentrifuge tubes in which 8 mg of EDTA was added as an anticoagulant. The collected blood was mixed properly with the anticoagulant and centrifuged at 4000 rpm for 20 min. The plasma was separated and analysed by measuring the radioactivity using the gamma scintillation counter. Various pharmacokinetic parameters including Cmax, MRT, AUC0–24 and AUMC0–24 were calculated using Kinetica software1 (Thermo Fisher Scientific, Berman, Germany). The pharmacokinetic data among different formulations were compared for statistical significance by one way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparison tests using GraphPad Instat software (GraphPad Software Inc., CA, USA). The brain targeting efficiency (DTE%) and nose to brain direct transport percentage (DTP%) were also calculated (Kumar et al., 2008).
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DTEð%Þ ¼
ðAUCbrain =AUCblood Þi:n: 100 ðAUCbrain =AUCblood Þi:v:
Please cite this article in press as: Alam, M.I., et al., Pharmacoscintigraphic evaluation of potential of lipid nanocarriers for nose-to-brain delivery of antidepressant drug, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.05.004
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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.
DTPð%Þ ¼ 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292
Bi:n: Bx 100 Bi:n:
where Bx = (Bi.v./Pi.v.) Pi.n. Bx is the brain AUC fraction contributed by systemic circulation through 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. 3.12. Gamma-imaging studies The healthy New Zealand rabbits (n = 3) weighing between 2.00 and 2.50 kg (female) were selected for the study. The rabbits were anaesthetized using ketamine hydrochloride intramuscular injection (1 ml of 50 mg/ml) and held from the back in slanted position during intranasal administration. The radiolabelled formulations (DLX-NLC and DLX solution) was administered via nasal route and placed on the imaging platform. The localization all through the body was visualized using Single Photon Emission Computerized Tomography (SPECT) gamma camera, provided by GE healthcare system (Hawkeye Millennium VG, GE Medical Systems, Milwaukee, WI, USA) and the images were recorded using the eNTEGRA software.
micrographs also suggested the nanoparticulate and spherical nature of DLX-NLC.
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4.1. Optimization of radio-labelling parameters
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The radio-labelling of DLX and DLX-NLC was performed by direct labelling method. The radio-labelling parameters including strength of SnCl2 solution (reducing agent), pH, temperature and incubation time were optimized based on the radio-labelling efficiency (% RE). The maximum RE was found to be 98.41% and 98.87% after 30 min and 40 min at 100 mg/ml and 200 mg/ml concentration of SnCl2 solution as reducing agent for DLX and DLXNLC, respectively (Table 1). The possible existence of unwanted radiochemical impurity in the form of free pertechnetate and colloids was found up to 0.59 0.14% and 0.88 0.31% for DLX-NLC and 0.41 0.21% and 0.69 0.11% for DLX, respectively. The poor labelling efficiency was observed at lower concentrations of stannous chloride because of the partial reduction of pentavalent pertechnetate from its heptavalent oxidation state; however, the higher amounts bring about the greater creation of undesirable radio-colloids. It is an essential requirement to change technetium’s oxidation state for preparing 99mTc-labelled compounds.
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Table 1 Effect of pH, temperature, incubation time and strength of SnCl2 solution on radiolabelling efficiency of DLX and DLX-NLC. Parameters
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3.13. Statistical analysis
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All the data were expressed as the mean standard deviation (SD). The data was analyzed using Kruskal–Wallis test (nonparametric test). A Dunn’s multiple comparison test were used to compare different formulations and a p-value of less than 0.10 (p < 0.10) was considered to be significant.
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pH of medium
4. Results and discussions The prepared DLX-NLCs were evaluated for different parameters including particle size, particle size distribution, polydispersity index, entrapment efficiency, drug loading, surface morphology, in vitro release, stability studies, pharmacodynamic studies and estimation in brain and blood as described in our preceding report (Alam et al., 2011; Alam et al., 2012). The transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images are shown in Fig. 1. The TEM micrographs revealed the spherical shape and size below 130 nm (80.17– 127.73 nm) in diameter of lyophilized DLX-NLC. The SEM
Temperature ( C)
Incubation time (min)
Strength of reducing agent (mg/ml)
a
Value REa of DLX SD (n = 3) 3.0 94.32 2.31 6.0 93.89 1.42 7.4 97.77 2.23 10 89.63 1.21 88.71 3.12 20
REa of DLX-NLC SD (n = 3) 88.97 95.63 98.93 94.57 91.64
3.22 2.51 1.36 3.34 4.11
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98.73 96.96 94.87 71.33 90.12 97.88 97.37 96.97 96.13 83.97
2.24 3.61 2.23 2.37 5.28 1.17 1.85 2.19 2.11 5.41
98.95 98.32 95.64 86.45 93.37 98.31 98.82 97.64 95.76 93.69
1.62 2.23 3.14 2.54 3.73 1.21 1.32 2.73 1.84 1.21
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98.41 97.78 95.32 89.57
0.96 1.31 3.72 2.34
96.53 98.87 97.81 96.45
1.62 0.82 0.45 0.97
RE = radio-labelling efficiency.
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Radiolabeling efficiency (%)
Saline-DLX
Serum-DLX
Saline-DNLC
Serum-DNLC
105.00
100.00 95.00 90.00 85.00 80.00 0
5
10
15 Time (h)
20
25
30
Fig. 2. Stability of radiolabelled DLX and DLX-NLC in saline and serum. DLX and DLX-NLC were found to be stable by exhibiting more than 90% of radio-labelling efficiency 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 radiolabelling procedure. In early radio-labelling studies, reducing agents such as ascorbic acid and ferrous iron were used that resulted to incomplete reduction, requiring removal of unreacted pertechnetate. However, stannous chloride is considered to be competent of generating high yields of 99mTc-labelled compounds, abolishing the need to remove free pertechnetate (Eckelman and Richards, 1970). Similarly the observed results of studies of effects of pH, temperature and incubation time on the radio-labelling efficiency of DLX and DLX-NLC are shown in Table 1. When incubated for 30 min, at pH 7.4 and temperature 25 C the radio-labelling of DLX was found to be stable and optimized. The optimized conditions for radio-labelling of DLX-NLC were found to be 40 min, 25 C and 7.4 for incubation time, temperature and pH, respectively. The radiolabelling efficiency at different values of the selected conditions was found to be more than 80 percent. The extent of nasal absorption depends on pH at the absorption site that may also affect the integrity of nasal mucosa. It has been reported that when pH ranges from 3 to 10 minimal quantities of proteins and enzymes are released from cells, demonstrating no cellular damages i.e., if pH values are below 3 or above 10 damages have been observed intracellularly and at membrane level (Pujara et 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 (Hirai et al.,1981). The chemistry of DLX and DLX-NLC radio-labelling with 99mTc may be explained on the basis of binding at electron donating functional groups (e.g., hydroxyl and amine functional groups of lipids and DLX, respectively). The powerful reducing agent stannous ion (Sn++) reduces 99mTc to the more reactive oxidation state (+5) from nonreactive species (+7) to promote binding. The 99m Tc-labelling may occur by binding at electron donating functional groups because 99mTc favours ligands that are
5
competent to recompense for the high positive charge of the central atom (Soane et al., 1999).
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4.2. Radiochemical stability of the radiolabelled complexes
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The DLX and DLX-NLC were found to be stable in saline as well as serum by exhibiting the radio-labelling efficiency of more than 90% for a time period of 24 h (Fig. 2). The radio-labelling of DLX was found to be 94.47 4.28% and 97.70 1.01% in saline and serum, respectively when determined after 30 min. Similarly for DLX-NLC it was found to be 98.45 2.01 and 99.76 1.21 in saline and serum , respectively after 30 min. The radiolabelled complexes were found to be stable and there was no significant breaking of the complexes. In most radiopharmaceuticals, a 90% limit of ‘bound’ 99m Tc chelate is suggested (Saha, 1996). It is significant to determine the stability in serum to make sure the intactness of the labelled complexes in the presence of proteins and several other substances there in serum. It also supports the stability in vivo upon administration into the body and their use in determining the biodistribution models. Thus it was concluded that these radiolabelled complexes could be administered via intranasal route for gammascintigraphic studies.
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4.3. Biodistribution studies of DLX and DLX-NLC formulation
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The biodistribution studies were performed to investigate the amount of drug reaching in various vital organs including brain, liver, kidney, intestine, heart, spleen, intestine and blood. The percent injected dose per gram of tissue (% ID/g) was determined at different time intervals. The biodistribution of radiolabelled intranasal DLX-NLC, compared with a positive control of radiolabelled intranasal DLX solution and intravenous DLX solution are shown in Table 2. The biodistribution data of DLX-NLC and DLX solution via intranasal route resulted in a higher % ID/g in the brain for the NLC formulation 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 intranasal DLX-NLC in different organs may be explained on the basis of its nanoparticulate and lipophilic nature and avoidance from the degrading environment in the nasal cavity (Cf. intranasal DLX which is devoid of these characteristics). In addition, different organs may be approached by different mechanisms by the molecules administered in the body. The estimation of higher amount in case of intranasal DLX-NLC in brain could be due to the direct pathways of transport (neuronal and non-neuronal) to the brain (Cf. intravenous DLX where BBB restricts the transport to the brain). The accumulation in the liver and the spleen is generally ascribed to uptake by the reticuloendothelial system (RES) like macrophage cells (Dobrovolskaia et al., 2008), whereas the
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Table 2 Biodistribution of intranasally administered DLX-NLC formulation, intranasal DLX solution and intravenous DLX solution. Organs
Percent injected dose per gram of tissue (% ID/g) Intranasal DLX-NLC 6 h SD (n = 3)
Blood Brain Heart Lungs Liver Kidney Spleen Intestine
6.45 10.75 4.69 5.43 2.89 13.87 12.96 15.40
Intranasal DLX
12 h SD (n = 3)
1.73 1.34 1.56 1.80 1.17 2.27 2.38 3.14
3.50 6.28 2.48 3.19 2.20 11.54 8.00 6.87
0.89 1.11 0.88 1.78 1.69 2.11 1.86 1.43
24 h SD (n = 3)
3.01 3.82 2.13 3.21 3.16 5.40 5.28 3.67
1.41 0.78 0.94 1.31 1.27 1.18 1.23 1.33
6 h SD (n = 3) 1.39 0.82 1.21 1.37 0.64 1.21 1.64 1.44
0.81 0.62 0.19 0.53 0.75 0.98 0.39 0.71
Intravenous DLX 12 h SD (n = 3) 1.06 0.66 1.25 1.44 1.18 1.67 1.56 2.02
0.81 0.92 0.32 0.27 0.78 0.61 0.82 0.54
24 h SD (n = 3) 0.79 0.49 1.51 0.83 0.91 1.30 1.43 1.37
0.48 0.59 0.72 0.63 0.81 0.64 0.94 0.52
6 h SD (n = 3) 0.646 0.132 0.398 0.361 0.178 0.100 0.177 0.363
12 h SD (n = 3) 0.05 0.02 0.04 0.07 0.09 0.06 0.04 0.07
0.598 0.124 0.216 0.268 0.186 0.185 0.259 0.246
0.07 0.06 0.04 0.07 0.08 0.05 0.04 0.03
24 h SD (n = 3) 0.449 0.105 0.181 0.093 0.224 0.112 0.116 0.161
0.08 0.09 0.06 0.01 0.01 0.04 0.03 0.05
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presence of particles in the lungs may be the results from their agglomeration caused by the adsorption of plasma proteins (Alexis et al., 2008). Moreover, DLX undergoes extensive metabolism to numerous metabolites involving oxidation of the naphthyl ring followed by conjugation and further oxidation. Metabolically most (about 70%) of the DLX dose appears in the urine as metabolites of DLX; about 20% is excreted in the faeces (Lantz et al., 2003). Very low concentrations of the 99mTc-labelled complexes of DLX-NLC and DLX were recovered from liver and showed constancy with time suggesting the intactness and in vivo stability of the complexes. The in-vivo stability can be predicted based on the in vitro stability results (Sun et al., 2002). DLX-NLC exhibited excellent in vitro stability hence expected in vivo also. The higher in vivo stability can be indicated by rapid clearance of intact complex (or low concentration of complex) from the liver or RES organs (liver, spleen, kidneys etc.). However the higher uptake of DLX-NLC by RES organs can be explained based on the fact that the GMS containing nanoparticles exhibited higher uptake by the RES organs as reported by (Pandey et al., 2005), (Sankar et al., 2012) and (Soni et al., 2014). 4.4. Effect of route of administration on nose-to-brain delivery of DLX
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The DLX solution was administered by both intranasal as well as intravenous route of administration. The brain concentration of DLX was compared after 6 h of administration. The intranasal administration exhibited more than 6-times higher concentration of DLX in brain (0.82% ID/g) when compared with the intravenous administration (0.134% ID/g) of DLX solution (p < 0.10). The higher concentration of DLX in brain may be explained on the basis of direct pathway of transport (neuronal and non-neuronal) from nose to brain. BBB restrict the transport of drug molecules to the brain upon intravenous administration. Intranasal administration offers a non-invasive alternative route to the brain for drug delivery effectively bypassing the BBB (Graff and Pollack, 2005). This method allows drugs that do not cross the BBB to be delivered to the CNS and eliminates the need for systemic delivery.
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4.5. Effect of NLC on biodistribution of DLX
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The organ distribution of DLX administered as intranasal DLXNLC was compared with intranasal DLX solution. It revealed significant differences (p < 0.10) in the biodistribution of DLX. The DLX-NLC exhibited higher distribution not only in brain but also in other organs as compared to pure DLX solution. The higher distribution of DLX-NLC than DLX solution after intranasal administration may be explained because of nanoparticulate and lipophilic nature of NLC and enhancement in permeation/ absorption by the surfactant and co-surfactant added during the preparation of NLC.
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4.6. Effect of NLC on nose-to-brain delivery of DLX
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DLX was administered as DLX-NLC and DLX solution through the same route of intranasal administration. The concentration of DLX was determined in the brain after 6 h of administration and compared. DLX encapsulated in NLC exhibited higher concentration in brain (10.75% ID/g) as compared to pure DLX solution (0.82% ID/g) (p < 0.10). NLC significantly affected the permeation/absorption of DLX through nose-to-brain route of drug administration. The superiority of NLC for nose to brain delivery over the solution may be attributed to its lipidic nature (facilitating permeation/ absorption), nanoparticulate nature (different mechanisms are involved) and avoidance of DLX from degrading environment in nasal cavity (e.g., lytic enzymes and pH present in nasal secretions).
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4.7. Pharmacokinetic studies
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The different pharmacokinetic parameters of intranasal DLXNLC suspension, intranasal DLX solution and intravenous DLX solution were calculated by determining the concentration (% ID/g) of DLX in blood and brain. The results of different pharmacokinetic parameters are given in Table 3. The pharmacokinetic parameters for DLX-NLC were compared with that of drug solution administered by intravenous route. It was 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 formulation as compared with that of the DLX solution administered by intravenous and intranasal routes (p < 0.10). Moreover, the intranasal administration of DLX solution exhibited higher accumulation of DLX in brain (p < 0.10) than the DLX solution administered intravenously. Thus the intranasal administration provided higher concentration in brain than administered through intravenous route. Moreover, the lipid nanocarriers exhibited higher concentration than the DLX solution per se. Intranasal administration provides a non-invasive method for bypassing the BBB and delivering therapeutic drugs along the olfactory and trigeminal nerves directly to the brain (Alam et al., 2011). Furthermore, DLX-NLC contributed higher uptake in the brain as compared to pure DLX solution because of nanoparticulate nature and lipophilicity of NLC. Nano-delivery systems have great potential to facilitate the movement of drugs across barriers (e.g., BBB). Numerous mechanisms are reported by which nanoparticles attain maximum drug concentration in brain including increased retention of drugs in brain-blood capillaries combined with an adsorption to capillary walls as higher concentration gradient increase transport, increasing the fluidization of BBB membrane, opening of tight junctions among endothelial cells (an endocytotic events occur due to upfolding of the cell membrane), inhibiting the P-glycoprotein efflux system (e.g., by poloxamer containing nanoparticles) (Alam et al., 2010).
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Table 3 Mean pharmacokinetic parameters (S.D., n = 3), drug targeting efficiency (DTE%) and direct nose to brain transport (DTP%) following administration of intranasal DLX-NLC suspension, intranasal DLX solution and intravenous DLX solution. Method
Organ/ tissue
Cmax (% ID/g)
AUC0 ! 24 (h % ID/g)
AUC0 ! 1 (h % ID/g)
AUMC0–24 (% ID.h2/ml)
Kel (1/h)
Drug targeting efficiency (DTE %)
Direct nose-to-brain transport (DTP %)
Intranasal DLX-NLC
Blood Brain Blood Brain Blood Brain
6.45 1.34 10.75 1.73a 1.39 0.82 0.82 0.51b 0.646 0.07 0.132 0.05
68.91 9.16 111.69 7.27a 18.45 2.16 11.34 1.63b 10.01 3.16 2.14 0.86
147.93 11.17 180.90 8.31a 44.43 9.23 28.79 2.11b 31.66 9.53 10.29 0.77
1043.64 33.51 1615.32 22.42a 278.28 11.63 171.36 18.26b 152.49 23.13 33.26 8.26
0.038 0.0018 0.055 0.0012a 0.030 0.0018 0.028 0.002b 0.020 0.007 0.012 0.005
757.74c
86.80c
287.34
65.12
–
–
Intranasal DLX Intravenous DLX
a b c
p < 0.10 vs intranasal/intravenous DLX solution. p < 0.10 vs intravenous DLX solution. p < 0.10 vs DLX solution.
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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 in different organs including brain of rabbit. DLX-NLC exhibited better localization than DLX. 502
solution. The images showing localization of DLX in different organs including brain of rabbit are shown in Fig. 3. The uptake of DLX in brain and other organs was visualized following intranasal administration of radiolabelled formulations (99mTc-DLX-NLC suspension and 99mTc-DLX solution). The increased radiation intensity was shown from the radiolabeled formulation in the brain region for DLX-NLC suspension as compared with that for DLX solution. The scintigraphy images were consistent with the results discussed in previous sections (biodistribution and pharmacokinetic studies) and high uptake of DLX into the brain was observed.
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5. Conclusion
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The intranasal DLX-NLC formulation was successfully delivered into the brain of rat and appreciable amount of DLX was estimated. It exhibited its potential to be transported through nose-to-brain route of drug administration. Furthermore it exhibited its potential to be distributed throughout the body indicating its permeability through the biological barriers. The delivery system was found to be useful to avoid probable systemic side effects due to DLX. Consequently, the present study visibly established that intranasal NLC is a suitable method for the effective delivery of DLX for the treatment of behavioural disorders including depression. Accordingly, it may also be used as an effective method for the treatment of other CNS disorders.
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Contributors
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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 and analyses; Javed Ali and Jasjeet K Sahni: undertook the statistical analysis; Sanjula Baboota: wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript.
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However, intravenous delivery gave little accumulation of the drug in the brain because of the BBB. The availability of DLX in small amount in brain after intravenous administration of DLX solution may be attributed to its physicochemical properties. Some small molecules with appropriate lipophilicity, molecular weight (mw) and charge gain access through diffusion from blood into the CNS (Gabathuler, 2010). However, the majority of small molecules do not cross the BBB. The relative impermeability of the BBB results from tight junctions among capillary endothelial cells which are formed by cell adhesion molecules (Alam et al., 2010). Small lipophilic molecules can diffuse passively across the BBB into the brain but will be exposed to efflux pumps (Pglycoprotein, some multidrug resistance proteins, breast cancer resistance protein and others) expressed on the luminal side of the BBB and exposed to degrading enzymes (ecto- and endoenzymes) localized in the cytoplasm of endothelial cells before brain penetration (Alam et al., 2010; Gabathuler, 2010). In addition, the brain availability of drug through intravenous route is largely affected by the half-life of the drug in the plasma, rapid metabolism, and level of non-specific binding to plasma proteins and the permeability of the compound across the BBB and into peripheral tissues (Patel et al., 2003). Significantly higher AUC and Cmax for intranasal DLX-NLC compared to intranasal as well as intravenous DLX solution were obtained. The drug targeting efficiency (DTE%) and brain drug direct transport percentage (DTP%) were also calculated for nasally administered formulations (Table 3). These were calculated using tissue/organ distribution data following intranasal and intravenous administration. The DTP% and DTE% represent the percentage of drug directly transported to the brain via the olfactory pathway. The DLX-NLC showed the higher DTE (%) and DTP (%) values than DLX solution. The higher DTE (%) and DTP (%) suggest that DLX-NLC has better brain targeting efficiency mainly because of substantial direct nose-to-brain transport. These findings conclude that DLXNLC increased nose-to-brain uptake of the DLX.
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4.8. Gamma imaging studies
Conflict of interest
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538
The study was performed to evaluate the localization of DLX after intranasal administration of DLX-NLC suspension and DLX
The authors declare no conflict of interest. The authors alone are responsible for the content and writing of the paper.
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Role of the funding source
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Council of Scientific and Industrial Research (CSIR) and Jamia Hamdard, New Delhi, (India) funded for carrying out the research.
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Acknowledgements
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Authors are thankful to Dr. Reddy’s Laboratories, Hyderabad, (India) for providing gift sample of the drug and CSIR, New Delhi, for providing a Senior Research Fellowship to M. Intakhab Alam. Authors are also thankful to Institute of Nuclear Medicine and Allied Sciences (INMAS) for providing facilities for pharmacoscintigraphic studies.
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Reference
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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
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Pharmacoscintigraphic evaluation of potential of lipid nanocarriers for nose-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 a
Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi, India Department of Pharmaceutics, College of Pharmacy, Jazan University, Jazan, Saudi Arabia c Department of Nuclear Medicine, Institute of Nuclear Medicine and Allied Sciences (INMAS), Brig SK Mazumdar Marg, Delhi, India b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 January 2014 Received in revised form 30 April 2014 Accepted 3 May 2014 Available online xxx
Efficacy of antidepressants relies upon their continued presence at the site of action (brain) over a prolonged period of time. The BBB restricts the access of antidepressants to the brain on oral as well as intravenous administration. Direct delivery (by-passing the BBB) of antidepressant drugs can increase the CSF concentration with concomitant reduction in dose and side effects. Intranasal administration of nanostructured lipid carriers (NLC) containing antidepressant drug circumvent the BBB and maintain the prolonged release at the site of action. The aim of the present study was to evaluate the enhancement in brain uptake of NLC containing duloxetine (DLX) after intranasal administration. Duloxetine loaded NLC (DLX-NLC) was evaluated pharmacoscintigraphically for drug targeting potential (DTP), drug targeting efficiency (DTE) and biodistribution studies in different organs including brain. The radio-labelling efficiency 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 in brain as compared with the DLX. The higher DTP (86.80%) and DTE (757.74%) suggested that DLX-NLC formulation has a better brain targeting efficiency than DLX solution (DTP = 65.12%; DTE = 287.34%) when administered intranasally. Moreover, the intranasal administration exhibited about 8-times higher concentration of DLX in brain when compared with the intravenous administration of DLX solution. The intranasal NLC containing DLX can be employed as an effective method for the treatment of depression. ã 2014 Published by Elsevier B.V.
Keywords: Pharmacoscintigraphy DTE DTP Biodistribution Radio-labelling efficiency
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1. Introduction Pharmacoscintigraphic technique has been widely used to study the in vivo behaviour of drug and drug delivery systems using emitted radiations from the radioactive materials. This technology has proven to be of great value in the assessment of a wide range of pharmaceutical formulations and new drug delivery systems. The radiometric detection of drugs labelled with a suitable radiotracer is the best technique for the detection and concentration of the drugs given through nasal route. Pharmacoscintigraphy study includes gamma-counts in different organs and gamma-imaging of intact animal after administering the calculated dose of drug. Gamma-counts of gamma-radiation emitted by the deposited radiolabelled DLX and NLC in different organs were done by the gamma-counter. Moreover, the gamma-imaging was done by the
* Corresponding author. Tel.: +91 11 26059688/5634; fax: +91 11 26059663. E-mail address: [email protected] (S. Baboota).
gamma-camera which gives images to provide the functional map of physiological processes. For scintigraphic studies the formulations are usually labelled with the gamma-ray emitting radionuclide 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 clean and safe radiation emission profile which contains few betaparticles results in very low radiation doses, so that satisfactory scintigraphic data can be obtained using only a fraction of the radiation dose required for diagnostic X-ray procedures (Newman and Wilding, 1998). Neurotransmitters (e.g., serotonin) are chemical messengers within the brain that facilitate communication among nerve cells. Inadequate supplies lead to the symptoms that are known as depression. It is a serious medical condition and is associated with decrease in functioning and well-being, high levels of disability, and high work absenteeism and health care costs. According to WHO estimates, depression will become the second-largest cause of the global health burden by 2020. Yet, depression remains one of
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Please cite this article in press as: Alam, M.I., et al., Pharmacoscintigraphic evaluation of potential of lipid nanocarriers for nose-to-brain delivery of antidepressant drug, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.05.004
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the most under diagnosed conditions. Over 60 percent of suicides are attributed to major depressive disorder. It is a common mental illness with lifetime occurrence rates close to 20% which is heritable as well (Glahn et al., 2012). Duloxetine (DLX), an SNRI is the first in the class of anti-depressants that ensures rapid and sustained efficacy in the treatment of both emotional and physical symptoms of depression. DLX promises treatment of physical symptoms that accompany major depressive disorder (MDD) such as aches, pains, and gastrointestinal disturbance as well. On oral administration DLX undergoes hepatic first pass metabolism and has a systemic bioavailability of 50% (Lantz et al., 2003). Moreover the drug is acid labile at gastric pH. Oral administration of the drug also causes side effects including nausea, dry mouth, headache, dizziness, orthostatic hypotension fatigue. Efficacy of antidepressants relies upon their continued presence at the site of action (brain) over a prolonged period of time (Kilts, 2003). On oral as well as intravenous administration the BBB restricts the access of antidepressant drugs to the brain. Brain targeting can increase the CSF concentration of the drug with concomitant reduction in dose and side effects (Misra et al., 2003). Intranasal administration offers a non-invasive alternative route to the central nervous system (CNS) for drug delivery, effectively bypassing the BBB (Graff and Pollack, 2005). The nasal route is one of the most permeable and highly vascularized site for drug administration ensuring rapid absorption and onset of therapeutic action. The neural connections between the nasal mucosa and the brain provide a unique pathway for noninvasive delivery of therapeutic agents to the CNS (Thorne and Frey, 2001; Illum, 2000). The olfactory and trigeminal nerve components in the nasal epithelium provide pathways to deliver therapeutic agents to the olfactory bulb and brainstem, respectively, where dispersion to other CNS areas may be possible via pulsatile flow within the perivascular spaces of cerebral blood vessels (Thorne et al., 2004; Thorne et al., 2008). NLCs are particles produced from the blend of solid and liquid (oil) lipids. It possesses many “imperfections” increasing drug loading capacity and minimizing or avoiding drug expulsion during storage (Muchow et al., 2008). Being lipophilic in nature NLC has been expected for the transport of therapeutic substances to the brain. They are composed of physiological and biodegradable lipids exhibiting low toxicity that means an excellent tolerability. The lipid nanoparticles are able to enhance the chemical stability of compounds sensitive to light, oxidation and hydrolysis (Pardeike et al., 2009). Thus, the present study was designed to deliver DLX, an antidepressant drug to the brain through nose to brain route of drug delivery using NLC as drug delivery system and the quantification and biodistribution studies were performed by pharmacoscintigraphic method.
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2. Materials and method
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2.1. Drugs and reagents
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DLX was provided by Dr. Reddy’s Laboratories (Hyderabad, India). Glyceryl monostearate (solid lipid) (Loba chemie Pvt. Ltd., Mumbai, India), pluronic F-68 (surfactant) and capryol PGMC (liquid lipid) (Sigma Chemical Company, MO, US), bile salt (sodium taurocholate) (co-surfactant) (Thomas Baker, chemicals, Ltd., Mumbai, India), and mannitol (cryoprotectant) (S.D. finechem Ltd., Mumbai, India) were used as received from suppliers. Sodium pertechnetate, separated from molybdenum-99 (99Mo) by solvent extraction method, was provided by Regional Centre for Radiopharmaceutical Division (Northern Region), Board of Radiation and Isotope Technology (New Delhi, India). Stannous chloride dihydrate (SnCl2 2H2O) was purchased from Sigma–Aldrich
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(St. Louis, MO, USA). Instant thin layer chromatography (ITLC) silicic acid (ITLC-SA) strips were purchased from Gelman Sciences Inc. (Ann Arbor, MI, USA). All other chemicals and solvents were of analytical reagent grade and were used without further purification.
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2.2. Animals
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Swiss albino Wistar rats of either sex (200–250 g) were used for performing the gamma count in different organs. All animals were given free access to water and food and kept under standard laboratory conditions, temperature at 25 2 C with a natural light –dark cycle and relative humidity of 55 5%. The animals were housed in polypropylene cages, six per cage with free access to standard laboratory diet (Lipton feed, Mumbai, India; providing 3630 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 water ad libitum. Ethical clearance for performing biodistribution studies was taken from the Institutional Animal Ethics Committee, Jamia Hamdard, 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-imaging studies. Animals were procured from the animal house of INMAS (Delhi, India). The guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) (Ministry of Culture, Govt. of India) were followed throughout the study and maximum care was taken to make certain that animals were treated in the most human and ethically acceptable method.
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3. Experiments
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3.1. Preparation of DLX-NLC and lyophilization
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DLX-NLC was prepared by dissolving DLX (2 g/l) in a mixture of melted solid lipid (glyceryl monostearate) and liquid lipid (capryol PGMC). The lipid concentration and the ratio of liquid lipid to total lipid was optimized to 2 (% w/w of aqueous phase) and 0.94:1, respectively. The lipid mixture was homogenized (Heidolph, Diax 900, Schwabach, Germany) at 10,000 rpm for 20 min with hot aqueous solution (80 C) of surfactants {pluronic F-68 = 1.5% w/w; and bile salt (sodium taurocholate) = 0.5% w/w} followed by ultrasonication (10 min) and lyophilization (at –70 C) using mannitol (3% w/w) as cryoprotectant (Alam et al., 2011).
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3.2. Procedure for radio-labelling
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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. A suitable method of radio-labelling was chosen by which DLX was labelled with 99mTc. The method of radiolablleing was standardized by gamma-imaging technique so as to visualize the distribution of radiolabelled drug in animal models. The selection of 99mTc was based on a number of properties including its short half-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 glass beaker with the help of acetonitrile.
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3.3. Radio-labelling of DLX and DLX-NLC
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radio-labelling was done using 99mTc by a direct labelling method. DLX (5 mg) or DLX-NLC (equivalent to 5 mg of DLX) was accurately weighed in a vial and 1 ml of distilled water was added.
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Stannous chloride was dissolved in ethanol to make a solution of 1 mg/ml and 100 ml of this solution was added to the vial containing DLX or DLX-NLC. To the resultant mixture 99mTc pertechnetate (200 mci) was added carefully with continuous mixing and incubated at 25 C for 30 min. Quality control of radiolabelling was done using ITLC-SA strips using acetone as mobile phase to determine radio-labelling efficiency (%). The strip was spotted with labelled complex (2–3 ml) above 1 cm from the bottom. The labelled complexes along with the reduced/hydrolyzed 99mTc stay behind at the bottom of the strip; the free pertechnetate migrates to the top. The developed strip was evaluated 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 of radio-colloids which was determined in acetic acid:pyridine:water (5:3.5:1). The excess of stannous chloride utilized for reduction of 99m Tc may produce the radio-colloids which is undesirable. The free pertechnetate as well as the labelled complex migrated with the solvent front to the top of the strip, leaving behind the radiocolloids at the bottom. The radioactivity in the strip was determined with the solvent front using acetone as well as using the mixture of acetic acid:pyridine:water and compared to determine the net amount of labelled complexes. Moreover the ethanol was used in sufficient quantity just to dissolve stannous chloride. 3.4. Optimization of radio-labelling parameters The various radio-labelling parameters required to optimize the radio-labelling of DLX or DLX-NLC included incubation time, pH and temperature and reducing agent (SnCl2), to achieve the desired reaction condition for radio-labelling. The radiochemical purity was 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 dissolving an accurately weighed sample of SnCl2 in ethanol. The effect of stannous chloride (SnCl2) strength on radio-labelling of DLX and DLX-NLC was observed at different concentrations varied from 50 to 500 ml of 1 mg/ml solution.
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3.7. Effect of temperature
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The optimum temperature for radio-labelling was obtained by determining the radio-labelling efficiency at different temperatures of 20, 25, 30 and 35 C.
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3.8. Effect of incubation time
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The radiolabelled complex of DLX or DLX-NLC with the optimized strength of SnCl2 solution, pH and temperature were incubated at room temperature for different time intervals of 10, 20, 30, 40, 50, and 60 min.
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3.9. Radiochemical stability of the radiolabelled complexes
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The radiochemical stability of the radiolabelled complexes of DLX and DLX-NLC performed in saline as well as serum. The in vitro stability study was performed by mixing 200 ml of radiolabelled
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DLX or DLX-NLC with 1 ml of saline or serum. Small aliquots were withdrawn at different time intervals up to 24 h and radiochemical stability of DLX or DLX-NLC was evaluated by ITLC using acetone as mobile phase. The developed strips were cut into 7:3 ratios and radioactivity in each part was measured using gamma ray spectrometer to calculate the stability of the product.
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3.10. Biodistribution studies
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The biodistribution studies were carried out using Wistar rats of either sex (200–250 g). Just before the experiment, the rats were weighed and restrained in rat restrainers. All the rats were marked with picric acid solution for identification and randomly divided into three groups consisting of three rats at each time point (i.e., nine rats in each group). Group 1 was given DLX-loaded NLC (DLXNLC) intranasally, group 2 received DLX solution intranasally and group 3 received DLX solution intravenously. Doses of 0.54 mg/day were administered in case of intranasal and intravenous DLX solution whereas a dose equivalent to 0.54 mg/day of DLX was given in case of DLX-NLC. DLX-NLC was administered intranasally to rats, compared to intranasal and intravenous administration of DLX solution. Radiolabelled formulation (200 mCi/100 ml) was administered in each nostril intravenously. Formulations containing DLX Q5 solution and DLX-NLC suspension (DLX-NLC was suspended in water containing chitosan 0.6% w/w) was instilled into the nostrils with the help of micropipette (100 ml) fitted with micro tip at the delivery site (olfactory region of the nose). The rats were held from the back, in upright position during nasal administration. These were anaesthetized with diethyl ether and sacrificed at different time intervals (6, 12, 24 h) and the blood was collected by cardiac puncture. The ethics committee approved only three time points for the study. Moreover, the brain and other organs (heart, liver, lungs, spleen, intestine and kidney) were collected, washed twice using normal saline, made free from adhering tissue/fluid, dried and weighed. Radioactivity present in each tissue/organ was measured using shielded well-type gamma scintillation counter. Radiopharmaceutical uptake per gram in each tissue/organ was calculated as a fraction of administered dose using the following equation (Saha, 1993).
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3.6. Effect of pH The effect of pH was determined for pH range of 3.0, 5.5, 7.4 and 10.0 and the pH was optimized for determining the radiochemical purity of the DLX or DLX-NLC complex.
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3
countsinsample 100 ¼ weightofsample totalcountsinjected 3.11. Pharmacokinetic studies
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Blood samples were withdrawn via cardiac puncture at 0 (predose), 6, 12, 24 h in microcentrifuge tubes in which 8 mg of EDTA was added as an anticoagulant. The collected blood was mixed properly with the anticoagulant and centrifuged at 4000 rpm for 20 min. The plasma was separated and analysed by measuring the radioactivity using the gamma scintillation counter. Various pharmacokinetic parameters including Cmax, MRT, AUC0–24 and AUMC0–24 were calculated using Kinetica software1 (Thermo Fisher Scientific, Berman, Germany). The pharmacokinetic data among different formulations were compared for statistical significance by one way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparison tests using GraphPad Instat software (GraphPad Software Inc., CA, USA). The brain targeting efficiency (DTE%) and nose to brain direct transport percentage (DTP%) were also calculated (Kumar et al., 2008).
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DTEð%Þ ¼
ðAUCbrain =AUCblood Þi:n: 100 ðAUCbrain =AUCblood Þi:v:
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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.
DTPð%Þ ¼ 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292
Bi:n: Bx 100 Bi:n:
where Bx = (Bi.v./Pi.v.) Pi.n. Bx is the brain AUC fraction contributed by systemic circulation through 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. 3.12. Gamma-imaging studies The healthy New Zealand rabbits (n = 3) weighing between 2.00 and 2.50 kg (female) were selected for the study. The rabbits were anaesthetized using ketamine hydrochloride intramuscular injection (1 ml of 50 mg/ml) and held from the back in slanted position during intranasal administration. The radiolabelled formulations (DLX-NLC and DLX solution) was administered via nasal route and placed on the imaging platform. The localization all through the body was visualized using Single Photon Emission Computerized Tomography (SPECT) gamma camera, provided by GE healthcare system (Hawkeye Millennium VG, GE Medical Systems, Milwaukee, WI, USA) and the images were recorded using the eNTEGRA software.
micrographs also suggested the nanoparticulate and spherical nature of DLX-NLC.
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4.1. Optimization of radio-labelling parameters
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The radio-labelling of DLX and DLX-NLC was performed by direct labelling method. The radio-labelling parameters including strength of SnCl2 solution (reducing agent), pH, temperature and incubation time were optimized based on the radio-labelling efficiency (% RE). The maximum RE was found to be 98.41% and 98.87% after 30 min and 40 min at 100 mg/ml and 200 mg/ml concentration of SnCl2 solution as reducing agent for DLX and DLXNLC, respectively (Table 1). The possible existence of unwanted radiochemical impurity in the form of free pertechnetate and colloids was found up to 0.59 0.14% and 0.88 0.31% for DLX-NLC and 0.41 0.21% and 0.69 0.11% for DLX, respectively. The poor labelling efficiency was observed at lower concentrations of stannous chloride because of the partial reduction of pentavalent pertechnetate from its heptavalent oxidation state; however, the higher amounts bring about the greater creation of undesirable radio-colloids. It is an essential requirement to change technetium’s oxidation state for preparing 99mTc-labelled compounds.
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Table 1 Effect of pH, temperature, incubation time and strength of SnCl2 solution on radiolabelling efficiency of DLX and DLX-NLC. Parameters
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3.13. Statistical analysis
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All the data were expressed as the mean standard deviation (SD). The data was analyzed using Kruskal–Wallis test (nonparametric test). A Dunn’s multiple comparison test were used to compare different formulations and a p-value of less than 0.10 (p < 0.10) was considered to be significant.
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pH of medium
4. Results and discussions The prepared DLX-NLCs were evaluated for different parameters including particle size, particle size distribution, polydispersity index, entrapment efficiency, drug loading, surface morphology, in vitro release, stability studies, pharmacodynamic studies and estimation in brain and blood as described in our preceding report (Alam et al., 2011; Alam et al., 2012). The transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images are shown in Fig. 1. The TEM micrographs revealed the spherical shape and size below 130 nm (80.17– 127.73 nm) in diameter of lyophilized DLX-NLC. The SEM
Temperature ( C)
Incubation time (min)
Strength of reducing agent (mg/ml)
a
Value REa of DLX SD (n = 3) 3.0 94.32 2.31 6.0 93.89 1.42 7.4 97.77 2.23 10 89.63 1.21 88.71 3.12 20
REa of DLX-NLC SD (n = 3) 88.97 95.63 98.93 94.57 91.64
3.22 2.51 1.36 3.34 4.11
25 30 35 10 20 30 40 50 60 50
98.73 96.96 94.87 71.33 90.12 97.88 97.37 96.97 96.13 83.97
2.24 3.61 2.23 2.37 5.28 1.17 1.85 2.19 2.11 5.41
98.95 98.32 95.64 86.45 93.37 98.31 98.82 97.64 95.76 93.69
1.62 2.23 3.14 2.54 3.73 1.21 1.32 2.73 1.84 1.21
100 200 400 500
98.41 97.78 95.32 89.57
0.96 1.31 3.72 2.34
96.53 98.87 97.81 96.45
1.62 0.82 0.45 0.97
RE = radio-labelling efficiency.
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Radiolabeling efficiency (%)
Saline-DLX
Serum-DLX
Saline-DNLC
Serum-DNLC
105.00
100.00 95.00 90.00 85.00 80.00 0
5
10
15 Time (h)
20
25
30
Fig. 2. Stability of radiolabelled DLX and DLX-NLC in saline and serum. DLX and DLX-NLC were found to be stable by exhibiting more than 90% of radio-labelling efficiency 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 radiolabelling procedure. In early radio-labelling studies, reducing agents such as ascorbic acid and ferrous iron were used that resulted to incomplete reduction, requiring removal of unreacted pertechnetate. However, stannous chloride is considered to be competent of generating high yields of 99mTc-labelled compounds, abolishing the need to remove free pertechnetate (Eckelman and Richards, 1970). Similarly the observed results of studies of effects of pH, temperature and incubation time on the radio-labelling efficiency of DLX and DLX-NLC are shown in Table 1. When incubated for 30 min, at pH 7.4 and temperature 25 C the radio-labelling of DLX was found to be stable and optimized. The optimized conditions for radio-labelling of DLX-NLC were found to be 40 min, 25 C and 7.4 for incubation time, temperature and pH, respectively. The radiolabelling efficiency at different values of the selected conditions was found to be more than 80 percent. The extent of nasal absorption depends on pH at the absorption site that may also affect the integrity of nasal mucosa. It has been reported that when pH ranges from 3 to 10 minimal quantities of proteins and enzymes are released from cells, demonstrating no cellular damages i.e., if pH values are below 3 or above 10 damages have been observed intracellularly and at membrane level (Pujara et 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 (Hirai et al.,1981). The chemistry of DLX and DLX-NLC radio-labelling with 99mTc may be explained on the basis of binding at electron donating functional groups (e.g., hydroxyl and amine functional groups of lipids and DLX, respectively). The powerful reducing agent stannous ion (Sn++) reduces 99mTc to the more reactive oxidation state (+5) from nonreactive species (+7) to promote binding. The 99m Tc-labelling may occur by binding at electron donating functional groups because 99mTc favours ligands that are
5
competent to recompense for the high positive charge of the central atom (Soane et al., 1999).
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4.2. Radiochemical stability of the radiolabelled complexes
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The DLX and DLX-NLC were found to be stable in saline as well as serum by exhibiting the radio-labelling efficiency of more than 90% for a time period of 24 h (Fig. 2). The radio-labelling of DLX was found to be 94.47 4.28% and 97.70 1.01% in saline and serum, respectively when determined after 30 min. Similarly for DLX-NLC it was found to be 98.45 2.01 and 99.76 1.21 in saline and serum , respectively after 30 min. The radiolabelled complexes were found to be stable and there was no significant breaking of the complexes. In most radiopharmaceuticals, a 90% limit of ‘bound’ 99m Tc chelate is suggested (Saha, 1996). It is significant to determine the stability in serum to make sure the intactness of the labelled complexes in the presence of proteins and several other substances there in serum. It also supports the stability in vivo upon administration into the body and their use in determining the biodistribution models. Thus it was concluded that these radiolabelled complexes could be administered via intranasal route for gammascintigraphic studies.
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4.3. Biodistribution studies of DLX and DLX-NLC formulation
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The biodistribution studies were performed to investigate the amount of drug reaching in various vital organs including brain, liver, kidney, intestine, heart, spleen, intestine and blood. The percent injected dose per gram of tissue (% ID/g) was determined at different time intervals. The biodistribution of radiolabelled intranasal DLX-NLC, compared with a positive control of radiolabelled intranasal DLX solution and intravenous DLX solution are shown in Table 2. The biodistribution data of DLX-NLC and DLX solution via intranasal route resulted in a higher % ID/g in the brain for the NLC formulation 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 intranasal DLX-NLC in different organs may be explained on the basis of its nanoparticulate and lipophilic nature and avoidance from the degrading environment in the nasal cavity (Cf. intranasal DLX which is devoid of these characteristics). In addition, different organs may be approached by different mechanisms by the molecules administered in the body. The estimation of higher amount in case of intranasal DLX-NLC in brain could be due to the direct pathways of transport (neuronal and non-neuronal) to the brain (Cf. intravenous DLX where BBB restricts the transport to the brain). The accumulation in the liver and the spleen is generally ascribed to uptake by the reticuloendothelial system (RES) like macrophage cells (Dobrovolskaia et al., 2008), whereas the
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Table 2 Biodistribution of intranasally administered DLX-NLC formulation, intranasal DLX solution and intravenous DLX solution. Organs
Percent injected dose per gram of tissue (% ID/g) Intranasal DLX-NLC 6 h SD (n = 3)
Blood Brain Heart Lungs Liver Kidney Spleen Intestine
6.45 10.75 4.69 5.43 2.89 13.87 12.96 15.40
Intranasal DLX
12 h SD (n = 3)
1.73 1.34 1.56 1.80 1.17 2.27 2.38 3.14
3.50 6.28 2.48 3.19 2.20 11.54 8.00 6.87
0.89 1.11 0.88 1.78 1.69 2.11 1.86 1.43
24 h SD (n = 3)
3.01 3.82 2.13 3.21 3.16 5.40 5.28 3.67
1.41 0.78 0.94 1.31 1.27 1.18 1.23 1.33
6 h SD (n = 3) 1.39 0.82 1.21 1.37 0.64 1.21 1.64 1.44
0.81 0.62 0.19 0.53 0.75 0.98 0.39 0.71
Intravenous DLX 12 h SD (n = 3) 1.06 0.66 1.25 1.44 1.18 1.67 1.56 2.02
0.81 0.92 0.32 0.27 0.78 0.61 0.82 0.54
24 h SD (n = 3) 0.79 0.49 1.51 0.83 0.91 1.30 1.43 1.37
0.48 0.59 0.72 0.63 0.81 0.64 0.94 0.52
6 h SD (n = 3) 0.646 0.132 0.398 0.361 0.178 0.100 0.177 0.363
12 h SD (n = 3) 0.05 0.02 0.04 0.07 0.09 0.06 0.04 0.07
0.598 0.124 0.216 0.268 0.186 0.185 0.259 0.246
0.07 0.06 0.04 0.07 0.08 0.05 0.04 0.03
24 h SD (n = 3) 0.449 0.105 0.181 0.093 0.224 0.112 0.116 0.161
0.08 0.09 0.06 0.01 0.01 0.04 0.03 0.05
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presence of particles in the lungs may be the results from their agglomeration caused by the adsorption of plasma proteins (Alexis et al., 2008). Moreover, DLX undergoes extensive metabolism to numerous metabolites involving oxidation of the naphthyl ring followed by conjugation and further oxidation. Metabolically most (about 70%) of the DLX dose appears in the urine as metabolites of DLX; about 20% is excreted in the faeces (Lantz et al., 2003). Very low concentrations of the 99mTc-labelled complexes of DLX-NLC and DLX were recovered from liver and showed constancy with time suggesting the intactness and in vivo stability of the complexes. The in-vivo stability can be predicted based on the in vitro stability results (Sun et al., 2002). DLX-NLC exhibited excellent in vitro stability hence expected in vivo also. The higher in vivo stability can be indicated by rapid clearance of intact complex (or low concentration of complex) from the liver or RES organs (liver, spleen, kidneys etc.). However the higher uptake of DLX-NLC by RES organs can be explained based on the fact that the GMS containing nanoparticles exhibited higher uptake by the RES organs as reported by (Pandey et al., 2005), (Sankar et al., 2012) and (Soni et al., 2014). 4.4. Effect of route of administration on nose-to-brain delivery of DLX
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The DLX solution was administered by both intranasal as well as intravenous route of administration. The brain concentration of DLX was compared after 6 h of administration. The intranasal administration exhibited more than 6-times higher concentration of DLX in brain (0.82% ID/g) when compared with the intravenous administration (0.134% ID/g) of DLX solution (p < 0.10). The higher concentration of DLX in brain may be explained on the basis of direct pathway of transport (neuronal and non-neuronal) from nose to brain. BBB restrict the transport of drug molecules to the brain upon intravenous administration. Intranasal administration offers a non-invasive alternative route to the brain for drug delivery effectively bypassing the BBB (Graff and Pollack, 2005). This method allows drugs that do not cross the BBB to be delivered to the CNS and eliminates the need for systemic delivery.
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4.5. Effect of NLC on biodistribution of DLX
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The organ distribution of DLX administered as intranasal DLXNLC was compared with intranasal DLX solution. It revealed significant differences (p < 0.10) in the biodistribution of DLX. The DLX-NLC exhibited higher distribution not only in brain but also in other organs as compared to pure DLX solution. The higher distribution of DLX-NLC than DLX solution after intranasal administration may be explained because of nanoparticulate and lipophilic nature of NLC and enhancement in permeation/ absorption by the surfactant and co-surfactant added during the preparation of NLC.
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4.6. Effect of NLC on nose-to-brain delivery of DLX
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DLX was administered as DLX-NLC and DLX solution through the same route of intranasal administration. The concentration of DLX was determined in the brain after 6 h of administration and compared. DLX encapsulated in NLC exhibited higher concentration in brain (10.75% ID/g) as compared to pure DLX solution (0.82% ID/g) (p < 0.10). NLC significantly affected the permeation/absorption of DLX through nose-to-brain route of drug administration. The superiority of NLC for nose to brain delivery over the solution may be attributed to its lipidic nature (facilitating permeation/ absorption), nanoparticulate nature (different mechanisms are involved) and avoidance of DLX from degrading environment in nasal cavity (e.g., lytic enzymes and pH present in nasal secretions).
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4.7. Pharmacokinetic studies
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The different pharmacokinetic parameters of intranasal DLXNLC suspension, intranasal DLX solution and intravenous DLX solution were calculated by determining the concentration (% ID/g) of DLX in blood and brain. The results of different pharmacokinetic parameters are given in Table 3. The pharmacokinetic parameters for DLX-NLC were compared with that of drug solution administered by intravenous route. It was 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 formulation as compared with that of the DLX solution administered by intravenous and intranasal routes (p < 0.10). Moreover, the intranasal administration of DLX solution exhibited higher accumulation of DLX in brain (p < 0.10) than the DLX solution administered intravenously. Thus the intranasal administration provided higher concentration in brain than administered through intravenous route. Moreover, the lipid nanocarriers exhibited higher concentration than the DLX solution per se. Intranasal administration provides a non-invasive method for bypassing the BBB and delivering therapeutic drugs along the olfactory and trigeminal nerves directly to the brain (Alam et al., 2011). Furthermore, DLX-NLC contributed higher uptake in the brain as compared to pure DLX solution because of nanoparticulate nature and lipophilicity of NLC. Nano-delivery systems have great potential to facilitate the movement of drugs across barriers (e.g., BBB). Numerous mechanisms are reported by which nanoparticles attain maximum drug concentration in brain including increased retention of drugs in brain-blood capillaries combined with an adsorption to capillary walls as higher concentration gradient increase transport, increasing the fluidization of BBB membrane, opening of tight junctions among endothelial cells (an endocytotic events occur due to upfolding of the cell membrane), inhibiting the P-glycoprotein efflux system (e.g., by poloxamer containing nanoparticles) (Alam et al., 2010).
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Table 3 Mean pharmacokinetic parameters (S.D., n = 3), drug targeting efficiency (DTE%) and direct nose to brain transport (DTP%) following administration of intranasal DLX-NLC suspension, intranasal DLX solution and intravenous DLX solution. Method
Organ/ tissue
Cmax (% ID/g)
AUC0 ! 24 (h % ID/g)
AUC0 ! 1 (h % ID/g)
AUMC0–24 (% ID.h2/ml)
Kel (1/h)
Drug targeting efficiency (DTE %)
Direct nose-to-brain transport (DTP %)
Intranasal DLX-NLC
Blood Brain Blood Brain Blood Brain
6.45 1.34 10.75 1.73a 1.39 0.82 0.82 0.51b 0.646 0.07 0.132 0.05
68.91 9.16 111.69 7.27a 18.45 2.16 11.34 1.63b 10.01 3.16 2.14 0.86
147.93 11.17 180.90 8.31a 44.43 9.23 28.79 2.11b 31.66 9.53 10.29 0.77
1043.64 33.51 1615.32 22.42a 278.28 11.63 171.36 18.26b 152.49 23.13 33.26 8.26
0.038 0.0018 0.055 0.0012a 0.030 0.0018 0.028 0.002b 0.020 0.007 0.012 0.005
757.74c
86.80c
287.34
65.12
–
–
Intranasal DLX Intravenous DLX
a b c
p < 0.10 vs intranasal/intravenous DLX solution. p < 0.10 vs intravenous DLX solution. p < 0.10 vs DLX solution.
Please cite this article in press as: Alam, M.I., et al., Pharmacoscintigraphic evaluation of potential of lipid nanocarriers for nose-to-brain delivery of antidepressant drug, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.05.004
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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 in different organs including brain of rabbit. DLX-NLC exhibited better localization than DLX. 502
solution. The images showing localization of DLX in different organs including brain of rabbit are shown in Fig. 3. The uptake of DLX in brain and other organs was visualized following intranasal administration of radiolabelled formulations (99mTc-DLX-NLC suspension and 99mTc-DLX solution). The increased radiation intensity was shown from the radiolabeled formulation in the brain region for DLX-NLC suspension as compared with that for DLX solution. The scintigraphy images were consistent with the results discussed in previous sections (biodistribution and pharmacokinetic studies) and high uptake of DLX into the brain was observed.
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The intranasal DLX-NLC formulation was successfully delivered into the brain of rat and appreciable amount of DLX was estimated. It exhibited its potential to be transported through nose-to-brain route of drug administration. Furthermore it exhibited its potential to be distributed throughout the body indicating its permeability through the biological barriers. The delivery system was found to be useful to avoid probable systemic side effects due to DLX. Consequently, the present study visibly established that intranasal NLC is a suitable method for the effective delivery of DLX for the treatment of behavioural disorders including depression. Accordingly, it may also be used as an effective method for the treatment of other CNS disorders.
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Contributors
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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 and analyses; Javed Ali and Jasjeet K Sahni: undertook the statistical analysis; Sanjula Baboota: wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript.
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However, intravenous delivery gave little accumulation of the drug in the brain because of the BBB. The availability of DLX in small amount in brain after intravenous administration of DLX solution may be attributed to its physicochemical properties. Some small molecules with appropriate lipophilicity, molecular weight (mw) and charge gain access through diffusion from blood into the CNS (Gabathuler, 2010). However, the majority of small molecules do not cross the BBB. The relative impermeability of the BBB results from tight junctions among capillary endothelial cells which are formed by cell adhesion molecules (Alam et al., 2010). Small lipophilic molecules can diffuse passively across the BBB into the brain but will be exposed to efflux pumps (Pglycoprotein, some multidrug resistance proteins, breast cancer resistance protein and others) expressed on the luminal side of the BBB and exposed to degrading enzymes (ecto- and endoenzymes) localized in the cytoplasm of endothelial cells before brain penetration (Alam et al., 2010; Gabathuler, 2010). In addition, the brain availability of drug through intravenous route is largely affected by the half-life of the drug in the plasma, rapid metabolism, and level of non-specific binding to plasma proteins and the permeability of the compound across the BBB and into peripheral tissues (Patel et al., 2003). Significantly higher AUC and Cmax for intranasal DLX-NLC compared to intranasal as well as intravenous DLX solution were obtained. The drug targeting efficiency (DTE%) and brain drug direct transport percentage (DTP%) were also calculated for nasally administered formulations (Table 3). These were calculated using tissue/organ distribution data following intranasal and intravenous administration. The DTP% and DTE% represent the percentage of drug directly transported to the brain via the olfactory pathway. The DLX-NLC showed the higher DTE (%) and DTP (%) values than DLX solution. The higher DTE (%) and DTP (%) suggest that DLX-NLC has better brain targeting efficiency mainly because of substantial direct nose-to-brain transport. These findings conclude that DLXNLC increased nose-to-brain uptake of the DLX.
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4.8. Gamma imaging studies
Conflict of interest
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The study was performed to evaluate the localization of DLX after intranasal administration of DLX-NLC suspension and DLX
The authors declare no conflict of interest. The authors alone are responsible for the content and writing of the paper.
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Please cite this article in press as: Alam, M.I., et al., Pharmacoscintigraphic evaluation of potential of lipid nanocarriers for nose-to-brain delivery of antidepressant drug, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.05.004
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Role of the funding source
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Council of Scientific and Industrial Research (CSIR) and Jamia Hamdard, New Delhi, (India) funded for carrying out the research.
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Acknowledgements
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Authors are thankful to Dr. Reddy’s Laboratories, Hyderabad, (India) for providing gift sample of the drug and CSIR, New Delhi, for providing a Senior Research Fellowship to M. Intakhab Alam. Authors are also thankful to Institute of Nuclear Medicine and Allied Sciences (INMAS) for providing facilities for pharmacoscintigraphic studies.
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Reference
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Please cite this article in press as: Alam, M.I., et al., Pharmacoscintigraphic evaluation of potential of lipid nanocarriers for nose-to-brain delivery of antidepressant drug, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.05.004
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