Enhanced brain distribution and pharmacodynamics of rivastigmine by liposomes following intranasal administration

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International Journal of Pharmaceutics xxx (2013) xxx–xxx

Contents lists available at SciVerse ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical nanotechnology

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Enhanced brain distribution and pharmacodynamics of rivastigmine by liposomes following intranasal administration

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Zhen-Zhen Yang a,b , Yan-Qing Zhang c , Zhan-Zhang Wang a,b , Kai Wu a,b , Jin-Ning Lou d , Xian-Rong Qi a,b,∗ a

Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China c Department of Pharmaceutical Engineering, Tianjin University of Commerce, Tianjin 300134, China d Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing 100029, China b

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Article history: Received 28 February 2013 Received in revised form 11 April 2013 Accepted 2 May 2013 Available online xxx

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Keywords: Intranasal administration Liposomes Cell penetrating peptide (CPP) Biodistribution Pharmacodynamics Rivastigmine

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1. Introduction

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Alzheimer’s disease (AD) is a common progressive neurodegenerative disorder associated with cholinergic neurons degeneration. The blood–brain barrier (BBB) not only provides protection for the brain but also hinders the treatment and diagnosis of this neurological disease, because the drugs must cross BBB to reach the lesions. The present work was aimed at formulating rivastigmine liposomes (Lp) and cellpenetrating peptide (CPP) modified liposomes (CPP-Lp) to improve rivastigmine distribution in brain and proceed to enhance pharmacodynamics by intranasal (IN) administration and minimize side effects. The results revealed that Lp especially the CPP-Lp can enhance the permeability across the BBB by murine brain microvascular endothelial cells model in vitro. IN administration of rivastigmine solution and rivastigmine liposomes demonstrated the capacity to improve rivastigmine distribution and adequate retention in CNS regions especially in hippocampus and cortex, which were the regions most affected by AD, than that of IV administration. Importantly, the lagging but intense inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities were relative to the extended release, absorption and retention. In addition, there was very mild nasal toxicity of liposomal formulations. The data suggest that rivastigmine liposomes especially CPP-Lp improve the brain delivery and enhance pharmacodynamics which respect to BBB penetration and nasal olfactory pathway into brain after IN administration, and simultaneously decrease the hepatic first pass metabolism and gastrointestinal adverse effects. © 2013 Published by Elsevier B.V.

Alzheimer’s disease (AD), the most common neurodegenerative disease, is characterized by synaptic loss and degeneration of cholinergic neurons in the cortex and other areas of the brain, which are resulting deficits in cholinergic transmission and acetylcholine (ACh) level (Pakaski and Kalman, 2008). Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) can hydrolyze ACh in the brain (Mutlu and Degim, 2005). Cholinesterase inhibitors (ChEIs) catalyze the breakdown of AChE in synaptic cleft, thus enhancing ACh level to moderate AD. Rivastigmine is a reversible, noncompetitive and carbamate-type dual ChEIs of brain AChE and BuChE simultaneously (Spencer and Noble, 1998), which is widely

∗ Corresponding author at: Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China. Tel.: +86 10 82801584; fax: +86 10 82801584. E-mail addresses: [email protected], [email protected] (X.-R. Qi).

used as the symptomatic treatment of AD with mild-to-moderate dementia. Rivastigmine is presently on the market delivered orally in the form of tablets and capsules. Unfortunately, there are limitations of oral therapy of rivastigmine including hepatic first pass metabolism and clearance, gastrointestinal destruction of the drug by digestive enzymes and acidic pH conditions of the digestive tract, inferior and unpredictable uptake and bioavailability, and gastrointestinal adverse effects, in severe cases, irreparable esophageal tears (Tenovuo, 2005; Venkatesh et al., 2007). More seriously, the blood–brain barrier (BBB) provides protection for the brain but hinders the treatment and diagnosis of neurological diseases because the drugs must cross the BBB to reach the lesions. Systemic drug delivery by nasal route is currently receiving considerable attention because this route has shown a noninvasive and acceptable administration of various drugs, avoidance of the hepatic first pass metabolism and the preferential drug delivery to brain via olfactory pathway while bypassing the BBB (Illum, 2003). This alternate route from nasal mucosa to brain has been achieved faster and higher drug absorption and has been used feasibly with tacrine

0378-5173/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ijpharm.2013.05.009

Please cite this article in press as: Yang, Z.-Z., et al., Enhanced brain distribution and pharmacodynamics of rivastigmine by liposomes following intranasal administration. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.05.009

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(Jogani et al., 2008), nimodipine (Zhang et al., 2004), diazepam (Kaur and Kim, 2008) in the treatment of central nervous system (CNS) diseases and so on. In our previous study, the distribution of rivastigmine and its relation to pharmacodynamic effects, the inhibition of AChE and BuChE following intranasal (IN) and intravenous (IV) administration in rats were investigated (Yang et al., 2012). It has been shown that IN administration of rivastigmine had the capacity to improve distribution and pharmacological effect of rivastigmine in CNS regions compared to IV administration and the IN route can be an advantageous strategy for delivering rivastigmine into brain. Like other CNS disease, the treatment of AD is particularly challenging because the therapeutic molecules must be transported not only across the brain cell membrane but also across the BBB. Nanoparticles have promising applications for drug delivery as well as for the diagnosis and treatment of several pathologies, such as those related to the CNS. Cell-penetrating peptides (CPPs) are a collection of different families of short peptides believed to enter cells by penetrating cell membranes (Trabulo et al., 2010; Zhang et al., 2009). CPPs have been widely exploited for the intracellular delivery of various cargoes such as proteins, siRNA and nanocarrier systems including liposomes and nanoparticles. Although the explicit mechanism of internalization of CPPs is unclear, there appears to be two kinds of mechanisms, transduction pathway and endocytosis pathway (Ma et al., 2011; Torchilin, 2008). In order to catch on whether liposomes and CPP improve brain distribution of rivastigmine, minimize peroral side effects by IN routes, the rivastigmine liposomes (Lp) and CPP-modified liposomes (CPP-Lp) were prepared. Transport efficiency cross BBB of liposomal rivastigmine was comparatively evaluated with rivastigmine solution in murine brain microvascular endothelial cells (BMVECs) model. Distributions in CNS regions, plasma, and peripheral tissues as well as pharmacodynamic effects of these formulations were compared by determination of rivastigmine concentration, and AChE and BuChE activity in rats. Finally, toxicities of liposomal formulations were observed by mucosa lesion, ciliary movement and hemolysis evaluation.

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2. Materials and methods

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2.1. Materials and animals

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Rivastigmine was obtained from Beijing Intermediate Imp. & Exp. Co., Ltd. (Beijing, China). 1,2-Distearoyl-sn-glycero-3phosphoethanolamine-N-[amino (polyethylene glycol)2000 ] (DSPE-PEG-NHS) was purchased from NOF Corporation (Tokyo, Japan). Egg phosphatidylcholine (EPC) was got from Sigma–Aldrich (St. Louis, MO, USA). Cholesterol (Chol) was from Wako Pure Chemical Industries, Ltd. (Odaka, Japan). Sephadex G-50 was obtained from Pharmacia Biotech (Piscataway, NJ, USA). The CPP peptide (Gly-Leu-Pro-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg) was synthesized by GL Biochem Ltd. (Shanghai, China). AChE and BuChE Assay Kit were purchased from Nanjing Jiancheng Bioengineering Institution (Nanjing, China). Antipyrine was purchased from Beijing Huamaike Biotechnology Co., Ltd. (Beijing, China). All other chemicals used were of analytical or HPLC grade. Murine brain microvascular endothelial cells (BMVECs, China–Japan Friendship Hospital, Beijing, China) were maintained in the endothelial cell culture medium (DMEM, 20% fetal calf serum, 100 U/mL penicillin, 100 ␮g/mL streptomycin, 2 mmol/L l-glutamine, 100 ␮g/mL endothelial cell growth factor (ECGF) and 40 U/mL heparin). Male Sprague-Dawley (SD) rats weighing 200 ± 30 g were obtained from Peking University Health Science Center (license No. SCXK (Jing) 2006–0008) and were housed under standard

conditions with free access to food and water. The animals were fasted for at least 12 h prior to the experiment and were given water freely. Toads were obtained from Fangyuanyuan farm (Beijing, China). All of the animal experiments adhered to the principles of care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of Peking University. 2.2. Synthesis of DSPE-PEG-CPP CPPs were reacted with DSPE-PEG-NHS (1:1.25, mol/mol) in dimethyl formamide adding minute quantity of triethylamine under stirring for 24 h at ambient temperature. In the reaction, the terminal amino of peptide was covalently attached to the functional NHS of the lipid-PEG derivative. The reacted mixture were further put into a dialysis bag with cutoff molecular weight of 3500 Da and dialyzed against deionized water over 24 h in order to remove unreacted materials. The final solution was lyophilized to dry powder. The molecular weight (MW) of the resulting product was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). 2.3. Liposomes preparation Preparation of rivastigmine liposomes was carried out using ammonium sulfate gradient loading method. Firstly, two kinds of blank liposomes were prepared as following procedures: EPC, Chol (1:1, mol ratio) and EPC, Chol, DSPE-PEG-CPP (1:1:0.06, mol ratio) were dissolved with chloroform and methanol (4:1, v/v) in a pear-shaped flask and were subsequently evaporated to dry film using a rotary evaporator under vacuum. The lipid film was rehydrated and sonicated with 250 mM ammonium sulfate solution. The suspensions after hydration were successively extruded through polycarbonate membranes with the pore size of 400 nm, and 200 nm for 3 times, respectively. After extrusion, the suspensions were further dialyzed by the phosphate buffered saline (PBS, 50 mM KH2 PO4 , 15.2 mM NaOH, pH 6.5) over 6 h to obtain the blank liposomes. Secondly, rivastigmine was loaded into the blank liposomes. Namely, 5 mg of rivastigmine was added into 1 mL blank liposomes suspensions (50 mg lipid/mL) and incubated at 50 ◦ C with intermittently shaken for 20 min to obtain the rivastigmine liposomes (Lp) and CPP-modified rivastigmine liposomes (CPP-Lp), respectively. 2.4. Liposomes characterization and drug release in vitro 2.4.1. Determination of rivastigmine concentration in vitro by HPLC The rivastigmine concentration was determined by high performance liquid chromatography (HPLC) method with UV detector (Waters, USA). The mobile phase was consisted of acetonitrile: water (20 mM NaH2 PO4 ·2H2 O, 10 mM Na2 HPO4 ·12H2 O) (25: 75, v/v) at 1.0 mL/min of flow rate and 218 nm of wavelength using ODS column (Bonchrom, 250 mm × 4.6 mm). 2.4.2. Liposomes characterizations The size, zeta potential and polydispersity index (PDI) of two liposomes were determined by dynamic light scattering (DLS) using Malvern Zetasizer 3000HSA (Malvern Instruments Ltd., UK). The total content of rivastigmine (Wtotal ) in liposomes suspension was determined after destroyed the liposomes by adding methanol. The rivastigmine encapsulated in liposomes (Wencapsulate ) was determined after separated encapsulating rivastigmine from free one by gel filtration through a Sephadex G-50 column and eluted with pH 6.5 PBS. The encapsulation

Please cite this article in press as: Yang, Z.-Z., et al., Enhanced brain distribution and pharmacodynamics of rivastigmine by liposomes following intranasal administration. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.05.009

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efficiency of rivastigmine (EE%) was estimated with the formula: EE% = (Wencapsulate /Wtotal ) × 100%.

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2.4.3. Drug release in vitro In vitro release of two rivastigmine-loaded liposomes was performed by the dialysis against the release medium in pH 6.5 PBS. A volume of 1 mL liposomes suspensions in dialysis tubing was immersed in 100 mL of the release medium, and oscillated with a shaker at a rate of 100 times per minute at 37 ◦ C. 1 mL release medium was sampled at 5, 15, 30, 45, 60, 90, 120, 180, 240 and 360 min, respectively, and immediately replaced with equal volume of the medium after each sampling. Released rivastigmine was determined by HPLC method described above. The cumulative release percentage (Release%) was calculated.

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2.5. BBB model and transport across the BBB

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BMVECs were seeded at a density of 1 × 105 cells/cm2 on 2% gelatin coated 12-insert cell incubator (Corning, NY, USA, 0.4 ␮m pore size, 12 mm diameter). After about 8 days’ culture, the culture medium was changed every other day and the cells were checked under the microscope for complete confluence. The cell monolayer integrity was monitored by a transendothelial electrical resistance (TEER) instrument (Word Precision Instruments, Inc., Sarasota, FL, USA) to measure the TEER. Only cell monolayer with TEER exceeding 200  cm2 were selected for this experiment (Cecchelli et al., 1999). In addition, adding slightly less culture medium into the out slot of the incubator than that of the inner slot to observe the change between bilateral liquid surface of outside and inside slot. If the differential liquid level can keep over 4 h, we can also infer the cell monolayer have formed the tight junction (Xie et al., 2004). Then experimental samples, including rivastigmine solution, Lp, CPP-Lp, respectively, were added in the apical compartment of the BBB model. Transport ratio (%) was determined by using DMEM as a transport medium. A volume of 500 ␮L sample was taken from the outside compartment at 0, 0.25, 1, 2, 4, 8, 12 and 24 h, and replaced with 500 ␮L fresh DMEM immediately after each sampling. All collected samples were analyzed by HPLC as described above. The apparent permeability coefficient (Papp ) was calculated at the time point of 24 h. As the method mentioned by Van Bree et al. (van Bree et al., 1988), the Papp (cm/s), which represented the permeability of the drug across the BBB, was calculated on the basis of the formula Papp = (dQ/dt)/(C0 × A), where dQ/dt was the rate at which the drug appeared in the receiver compartment, C0 was the initial concentration of the drug in the donor compartment and A was the diffuse surface of transwell (1.12 cm2 ).

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2.6. Administration via nasal route and animal treatment

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Lp and CPP-Lp at a rivastigmine concentration of 5 mg/mL was used for IN administration. Rivastigmine dissolved in PBS (containing 2.5% poloxamer 188) at 10 mg/mL was used for IN administration. Rivastigmine dissolved in physiological saline at 0.4 mg/mL was used for IV administration. Poloxamer 188 used here was to accommodate viscosity similar to liposomes and enhance penetration of rivastigmine to abate the discrepancies between liposomes and solution in vivo, since it has good permeation enhancing effect during intranasal delivery (Na et al., 2010). The animals were anesthetized with an intraperitoneal dose of 20% (w/v) urethane (1 g/kg) and kept under anesthesia throughout the whole experiment. For IN administration, a modified procedure described by Hirai et al. (1981) was used. Rats were placed in a supine position. The trachea was cannulated with polyethylene (PE) tube to allow free breathing and another PE tube was placed in the esophagus to seal the nasal cavity to prevent drainage of the drug.

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Rivastigmine solution, Lp and CPP-Lp were given via a PE tube that attached to a microsyringe and inserted into each nostril of rats about 5 mm at a 2 mg/kg dose. For IV administration, 2 mg/kg doses were administered via tail vein. After administration the blood and tissue samples were collected at predetermined sampling points (15 min, 60 min and 240 min, respectively). The blood samples were taken by heparinized tubes from the orbit venous plexus of rats at each time point and then the samples were harvested by immediately centrifugation at 10,000 rpm for 10 min and stored at −20 ◦ C until analysis. After blood sampling, the rats were executed, then the liver, kidney and spleen were promptly removed and the removed brain was immediately dissected into the olfactory region, cortex, hippocampus, cerebrum and cerebellum. All tissues were quickly rinsed and blotted up to get rid of blood-taint and visible blood vessels as much as possible and weighed. The whole tissues were homogenized and stored at −20 ◦ C prior to analysis.

2.7. Determination of rivastigmine concentration in vivo by HPLC/MS Rivastigmine in various biological samples was assayed using the HPLC-mass spectrometry (HPLC/MS, Agilent 1100, USA) method. Extraction of rivastigmine from the plasma and tissue samples was carried out using the liquid-liquid extraction technique described in the previous study (Yang et al., 2012). The combined extracts were evaporated at 45 ◦ C under nitrogen stream and redissolved in 200 ␮L mobile phase, and 10 ␮L was for HPLC/MS analysis. Quantitative analysis was performed by selected ion recording over the respective protonated molecular ions [M−H+ ] of rivastigmine (m/z 251.4 ± 0.5), antipyrine (m/z 189.1 ± 0.5). The mobile phase consisted of methanol and water (80:20, v/v) at a flow rate of 0.3 mL/min using Agilent C18 column (250 mm × 4.6 mm, 5 ␮m) as the analytical column. The MS conditions were as follows: ionization ESI positive polarity, capillary voltage set to 4 kV, fragmentor voltage (cone) 40 V, source temperature 350 ◦ C.

2.8. Pharmacodynamic study Male SD rats weighing 200 ± 30 g were used. The animals were anesthetized with an intraperitoneal dose of 20% (w/v) urethane (1 g/kg) and kept under anesthesia throughout the whole experiment. Rats of the control group (n = 10) received water only. Drug preparation and administration of rats were the same as described above. After blood sampling, rats were euthanized, then the liver, kidney and spleen were immediately removed and the olfactory region, cortex, hippocampus, cerebrum and cerebellum were immediately excised on ice. All samples were stored at −80 ◦ C as soon as possible until analysis. Determination of AChE and BuChE activity was performed based on the colorimetric method originally described by Scali et al. (2002). The whole samples were homogenized in 9 volumes of ice-cold physiological saline. The homogenates were subjected to centrifugation (14,000 rpm for 25 min) and supernatant were collected appending for assay. AChE and BuChE activity in the homogenate supernatant were assayed according to the instructions of AChE and BuChE Assay Kit by a modification of the standard spectrophotometric method at 412 nm. Protein concentration of the homogenate supernatant was measured by the Coomassie Brilliant Blue protein-binding method using bovine serum albumin as a standard (Bradford, 1976). The inhibition of AChE and BuChE activity was converted into the percentage by comparison with the normal control rats.

Please cite this article in press as: Yang, Z.-Z., et al., Enhanced brain distribution and pharmacodynamics of rivastigmine by liposomes following intranasal administration. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.05.009

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2.9. Nasal toxicity evaluation 2.9.1. Morphology of nasal mucosal The rat was used to evaluate the effect to the morphology of nasal mucosal. In brief, 12 male SD rats were divided into four groups (n = 3). The rats in Group 1 were given intranasally through left nostril with physiological saline (negative control) and through right nostril with 1% SDS solution (positive control), respectively, twice daily for seven consecutive days. The rats in Groups 2–4 were given intranasally through right nostril with 5 mg/kg rivastigmine solution, Lp and CPP-Lp twice daily for seven consecutive days, respectively. The rats were sacrificed 24 h after the last administration, and the nasal septum with the epithelial cell membrane was carefully separated from the bone for optical microscopic and morphological examination. The septum was fixed with 10% formalin, sliced on a microtome and stained with hematoxylin–eosin, and the morphology was examined under an optical microscope (XSP-2C, Shanghai optical instrument factory, China).

2.9.2. Ciliary movement The duration of the ciliary movement were estimated through the in situ toad palate model modified according to the previous report (Puchelle et al., 1982). 20 toads weighing 30–40 g were divided into five groups in the toad experiment (n = 4). Groups 1–5 was given physiological saline, 1% SDS solution, 5 mg/mL rivastigmine solution, Lp and CPP-Lp, respectively. All animals received the following performance: the upper palate of toad was exposed and treated with 0.5 mL of test solutions for 0.5 h, and then rinsed with physiological saline. Finally, the palate was dissected into small

patches of the same size (1 cm × 1 cm), after which, the mucocilia was spread on the glass slide and examined under an optical microscope (Nikon Fx-35A, Japan), and the duration of the ciliary movement was recorded. 2.9.3. Haemolysis Haemolytic experiment was used to assess the effects of formulations to cell membrane. The whole blood was firstly harvested from sheep and the erythrocytes were obtained by washing and centrifuging for 10 min at 2500 r/min for several times until the supernatant was clear. Then, physiological saline was added to obtain a 2% erythrocyte suspension. Subsequently, 2.5 mL erythrocyte suspension was added to each tube, which contained different volumes (0.5, 0.3 and 0.1 mL) of rivastigmine solution, Lp and CPP-Lp, respectively. Then physiological saline was added in each tube to obtain a final volume of 5 mL. And the final rivastigmine concentration of three formulations in each tube was 5, 3 and 1 mg/mL, respectively. The positive and negative control groups were obtained by mixing 2.5 mL distilled water and physiological saline with 2.5 mL 2% erythrocyte suspension, respectively. All the mixtures were incubated for 1 h, 2 h and 3 h at 37 ◦ C and then centrifuged for 5 min at 2500 rpm, respectively. The absorbance of the supernatant was measured at 540 nm (UV1100 Spectrophotometer, Shanghai Mapada Instruments Co., Ltd., China). All experiments were conducted in triplicate. The hemolysis rate was calculated using the following formula: Hemolysis rate (%) = (ODt − ODnc )/(ODpc − ODnc ) × 100%, where ODt refers to the absorbance of test solutions, ODnc refers to the absorbance of negative group and ODpc refers to the absorbance of positive group.

Fig. 1. (A) Principle of the synthesis of DSPE-PEG-CPP. (B) MALDI-TOF-MS of DSPE-PEG-CPP mixture. The arrow pointed to the peak of the targeting compound. The molecular weight of DSPE-PEG-CPP was about 4662.2.

Please cite this article in press as: Yang, Z.-Z., et al., Enhanced brain distribution and pharmacodynamics of rivastigmine by liposomes following intranasal administration. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.05.009

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Data are presented as the mean ± SD in vitro and are presented as the mean ± SEM in vivo. One way ANOVA was used to determine significance among groups after which post hoc tests with the Bonferroni correction were used for comparison between individual groups. A value of p < 0.05 was considered statistically significant.

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3. Results and discussion

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Synthesis route of DSPE-PEG-CPP was shown in Fig. 1A. MALDITOF-MS showed the MW of the resulting product around 4662.2 was in accordance with the theoretical MW of DSPE-PEG-CPP (4572) (Fig. 1B).

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a medium for in vitro release, since the nasal cavity has the pH of 5.5–6.5 (Dahl and Mygind, 1998). All of the liposomes tested in this research displayed uniform sizes and shapes. The mean particle size, zeta potential, PDI and entrapment efficiency of Lp and CPP-Lp are shown in Table 1. The modification of CPP did not significantly change zeta potential of CPP-Lp may due to the shielding effect of PEGylation. The drug release (Fig. 2C) from all liposomes showed a similar pattern, but it was significantly prolonged in comparison to that of drug solution (data not shown). The extended release would have availability in prolonged pharmacological effect in vivo. The similar physicochemical characteristics and drug release of the two liposomes allowed us to specifically compare the effects of CPP modification on transport across the BBB, biodistribution and therapeutic abilities. In addition, our results revealed that rivastigmine liposomes could maintain their characteristics without significant changes in the size, PDI and surface potential after 4 weeks storage in 4 ◦ C.

3.2. Liposomes preparation, characterization and drug release in vitro

3.3. Transport across the BBB

Rivastigmine is a water-soluble and weakly alkaline drug with the MW of 400.43. The encapsulation of rivastigmine in liposomes with high encapsulation was difficult. As indicated as Schnyder and Huwyler (2005), the choice, the optimization, and the validation of a specific loading technique may be a complex problem depending on the physicochemical properties of a given drug. In the present study, rivastigmine tartrate was used and ammonium sulfate gradient method which is considered suitable for encapsulating this kind drug (Fritze et al., 2006; Haran et al., 1993) was chosen to prepare rivastigmine liposomes after screening. The modification of CPP to liposomes (Fig. 2A) expects to increase the penetration of liposomes into the rat brain. We select pH 6.5 PBS as a solvent of rivastigmine and its liposomes for IN administration and also as

BMVECs, the main cell which make up the BBB, can form tight junction hampering most molecules to transport across the BBB into the brain parenchyma (Xie et al., 2005). Drug transport across the BBB model (Fig. 3) showed that the transport of three rivastigmine formulations was in a time-dependent manner. And, there was significant difference between both Lp and CPP-Lp with rivastigmine solution at 24 h (P < 0.05 and P < 0.01, respectively). After 24 h, the Papp of rivastigmine solution, Lp and CPP-Lp for the BBB model was (3.36 ± 0.10) × 10−6 cm/s, (3.77 ± 0.29) × 10−6 cm/s and (3.96 ± 0.09) × 10−6 cm/s (P < 0.05 compared with rivastigmine solution group), respectively. The results demonstrated that CPP modified liposomes can improve drug transmembrane effect owing to transcytosis and penetrating cell membranes (Ma et al., 2011;

Fig. 2. (A) Schematic diagram of rivastigmine liposomes (Lp) and CPP modified rivastigmine liposomes (CPP-Lp). (B) Size distribution of Lp and CPP-Lp determined by dynamic light scattering. (C) Rivastigmine release from Lp and CPP-Lp. The results represent as mean ± SD (n = 3).

Please cite this article in press as: Yang, Z.-Z., et al., Enhanced brain distribution and pharmacodynamics of rivastigmine by liposomes following intranasal administration. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.05.009

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Table 1 Characteristics of rivastigmine liposomes (Lp) and CPP-modified rivastigmine liposomes (CPP-Lp). Liposomes

Diameter (nm)

Polydispersity index

Lp CPP-Lp

166.3 ± 17.4 178.9 ± 11.7

0.255 ± 0.041 0.333 ± 0.032

Zeta potential (mV) -10.5 ± 2.4 -8.6 ± 2.4

Entrapment efficiency (%) 33.4 ± 6.6 30.5 ± 8.0

The data are presented as the mean ± SD value for at least three different preparations.

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Saw et al., 2010). Du et al. (Du et al., 2011) showed that by attaching an arginine-rich CPP to antisense morpholino oligonucleotides (AMOs), the AMO targeting efficiency was greatly improved and demonstrated efficient uptake in the brain, and they considered that CPP improved cellular AMO uptake through endocytosis. Khafagy el et al. (2009) studied four types of CPP and found that l-penetratin markedly increased the permeability of insulin across the nasal membrane without causing marked damage to the integrity of cells in the nasal respiratory mucosa.

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3.4. Pharmacokinetics and biodistribution

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IN delivery holds great promise for delivery of drugs into the CNS system. There is high vascularity of the nasal mucosa available for rapid absorption of drugs to systemic circulation in the nasal cavity (Dahl and Mygind, 1998). Galantamine, also as a ChEIs, showed a good feasibility after IN delivery in vitro and in vivo (Leonard et al., 2005). Previously, we have shown that IN administration of rivastigmine had the capacity to improve CNS distribution and cholinesterase inhibition compared to IV administration (Yang et al., 2012). In this study, the nasal delivery of liposomal rivastigmine was developed to improve its brain delivery. The concentration profiles of rivastigmine in CNS regions, i.e., olfactory region, cortex, hippocampus, cerebrum, cerebellum, and peripheral tissues, i.e., plasma, liver, kidney and spleen after IN application of rivastigmine solution, Lp and CPP-Lp and IV application of rivastigmine solution at 15, 60 and 240 min after administration were determined and were shown in Fig. 4. Among all determined tissues, the amount of rivastigmine was the highest in kidney, while was the lowest in liver (Fig. 4A–C) and this may be due to that rivastigmine was renal excretion and extensively biotransformed in the liver to NAP 226-90 (Polinsky, 1998). Fig. 4D clearly demonstrates that both IN and IV administration of rivastigmine formulations resulted in extremely rapid distribution into the systemic circulation followed by a quick decline in the plasma concentrations. And the concentration of rivastigmine in plasma after IV administration was lower than that of IN administration. There was shown similar plasma-versus-time behavior between CPP-Lp and rivastigmine solution group after IN administration, indicating

that either CPP or poloxamer 188 (Kabanov et al., 2002) in formulation can enhance absorption of rivastigmine in plasma through IN route. Additionally, rivastigmine can promptly distribute into peripheral tissues reaching a certain concentration. In order to better evaluate the delivery of rivastigmine in brain, we divided the whole brain into olfactory region, cortex, hippocampus, cerebrum and cerebellum. The levels of rivastigmine after IN administration were higher in all CNS regions than that of IV administration (Fig. 4A–C). Furthermore, the average rivastigmine concentration of CPP-Lp in CNS tissues were higher than that of Lp following IN administration and significantly higher in hippocampus, cortex and olfactory region at 15 min and 60 min (Fig. 4E), indicating that CPP can promote absorption of rivastigmine to brain through the nasal cavity. Additionally, IN administration of rivastigmine solution showed high level of rivastigmine among CNS region may owing to the role of the poloxamer 188 which was considered as a enhancer to enhance the drug absorption through mucosa and even can improve transport of drugs to the brain (Kabanov et al., 2002). Compared to IV administration, the concentration of drug in CNS regions was just slightly decreased from 15 min to 60 min for IN administration, which suggested that there was adequate retention of the drug via IN route, while they all had reduced to a comparatively low level at 240 min. It is believed that there are two different pathways for drug uptake from the nasal cavity into the brain. One is that drugs can directly transport into brain via the olfactory pathway, and the other is that drugs reach the brain by crossing the BBB (Illum, 2000). Some studies have shown the olfactory pathway has great potential in the nose-to-brain delivery (Nonaka et al., 2008; Westin et al., 2006). If the concentrations of rivastigmine were higher in the brain after IN delivery than after an IV injection, a direct pathway into the brain, from the nasal olfactory area, must exist. In summary, the pharmacokinetics and biodistribution results demonstrated IN application of rivastigmine formulations significantly increased the distribution of rivastigmine into the plasma and CNS regions compared with IV administration of rivastigmine solution, which can deduce that the amount of drug in CNS regions may attribute to these two routes simultaneously. And rivastigmine concentration of CPP-Lp were significantly higher in hippocampus, cortex and olfactory region at 15 min and 60 min than that of Lp following IN administration, indicating that CPP can promote absorption of rivastigmine to brain through the nasal cavity. 3.5. Pharmacodynamic study

Fig. 3. The transport ratio (%) across the BBB model in vitro of CPP-modified rivastigmine liposomes (CPP-Lp), rivastigmine liposomes (Lp) and rivastigmine solution. The results represent as mean ± SD (n = 3). *P < 0.05, **P < 0.01 vs rivastigmine.

Rivastigmine has been reported to inhibit AChE and BuChE with equal potency by carbamylation of a serine in the catalytic triad (Bar-On et al., 2002; Jann, 2000). Both AChE and BuChE were associated with formation of amyloid plaque and neurofibrillary tangles, the two pathologic criteria for the diagnosis of AD (Ballard, 2002; Parnetti et al., 2011). Thus, measurement of activities of the two enzymes is used as the pharmacodynamic biomarker. All animals have not been observed any abnormal phenomenon during the experimental process. Fig. 5 illustrates the AChE and BuChE activity detected in CNS regions (i.e., olfactory region, cortex, hippocampus, cerebrum and cerebellum), plasma and peripheral tissues (i.e., liver, kidney and spleen) after IN application of rivastigmine solution, Lp and CPP-Lp and IV application of rivastigmine solution. Comparing

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Fig. 4. Biodistribution in plasma, olfactory region, cortex, hippocampus, cerebrum, cerebellum, liver, kidney and spleen of rats at a dose of 2 mg/kg of rivastigmine formulations following intranasal (IN) and intravenous (IV) administration at 15 min (A), 60 min (B) and 240 min (C) respectively. (D) Plasma concentration versus time profile of rats at a dose of 2 mg/kg of rivastigmine formulations following IN and IV administration at 15 min, 60 min and 240 min. (E) Rivastigmine concentration in olfactory region, cortex and hippocampus versus time profile of rats at a dose of 2 mg/kg of Lp and CPP-Lp following IN administration at 15 min, 60 min and 240 min. Data were presented the mean ± SEM (n = 3–5 at each time point). (a) P < 0.05 vs IV rivastigmine; (b) P < 0.05 vs IN rivastigmine; (c) P < 0.05 vs IN Lp; (d) P < 0.05 vs IN CPP-Lp.

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with the control rats, the activity of AChE and BuChE were all found different degrees of decrease after IN and IV administration. In CNS regions, the activity of two enzymes were rapidly decreased at 15 min following IV administration of rivastigmine

solution, especially the activity of AChE and BuChE in cerebrum and the activity of BuChE in cortex had a significant difference compared to IN groups (Fig. 5). However, the activity of two enzymes after IV administration were higher than that of IN groups at 60 min,

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particularly the activity of AChE in cortex and cerebrum and the activity of BuChE in cerebellum, which suggested that IN administration has lagging but intense inhibition of two enzymes than that of IV administration. It was well established that rivastigmine generally had greater activity in the hippocampus and cortex of brain which were the regions most affected by AD (Spencer and Noble, 1998). In present study, the rank trend of two enzymes’ activity in the cortex and hippocampus at 15 min and 60 min was following rivastigmine solution > Lp > CPP-Lp after IN administration of three rivastigmine formulations, in according with the drug distribution in the regions (Fig. 4), indicating that liposomes, as a drug delivery system, and the modification of CPP can be used to enhance the pharmacodynamic efficacy of rivastigmine, and this was also consistent with the results of transport across the BBB model in vitro (Fig. 3). After IN and IV administration, AChE and BuChE activities in plasma and peripheral tissues were also found visible decrease comparing with the control rats and generally revealed the Utype drawing in plasma, decreased at 15 and 60 min while slightly reversed at 240 min. The activity of AChE and BuChE until 240 min in plasma after IN administration still showed the significant decrease, but not for the IV administration at 240 min in plasma, possibly implying the IN administration had longer action on inhibiting the activity of AChE and BuChE than that of IV administration. And this also corresponded with the extended absorption of IN route shown in concentration in plasma–time curve (Fig. 4D). Drugs following IV injection can directly distribute into the system circulation and CNS and play a role, whereas there is an absorption phase for IN administration, which may contribute the lag inhibiting effect of two enzymes. The non-synchronization between the CNS distribution and the activity of two enzymes in vivo may be for the reason that AChE and BuChE both have different subtypes and these subtypes may play various roles in different tissues. It also revealed regional differences in the effects of AChE and BuChE inhibition for rivastigmine formulations possibly due to the preferential selectivity of the enzymes toward different tissues, although no completely firm conclusions can be drawn regarding the differences due to assay variability and individual variation in rats. And at 240 min after IN administration, the drug content of CPP-Lp group in CNS regions was lower than those of Lp group and the activity of AChE and BuChE have also increased in olfactory region, cortex, hippocampus, liver and kidney, which might due to the faster clearance rate of CPP-Lp in vivo. In early stages of AD, inhibition of AChE is more important, but as the disease progresses, BuChE inhibition contributes more to reducing the cholinergic deficit (Williams et al., 2003). On the basis of our data, we also observe the value of assessing the effects of appropriate pharmacological interventions on both AChE and BuChE activity in CNS regions, plasma and peripheral tissues, which would be beneficial in treating the cognitive decline of AD. Our results are corresponding with the opinion that rivastigmine commonly have greater activity in the cortex and hippocampus of brain and this property of rivastigmine may indicate its potential clinical implications, since the cortex and hippocampus are the main brain regions affected in AD.

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3.6. Nasal toxicity study

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In the study, three respects including the morphology of nasal mucosa in rats, the duration of ciliary movement in the palate

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Table 2 Duration of ciliary movement for various formulations on the toad palate model. Treatment

Duration of ciliary movement (min)

Normal saline Rivastigmine solution Lp CPP-Lp 1% SDS

672 626 598 580 173

± ± ± ± ±

28 44 48 36* 53***

Q2

Relative (%) 100.0 93.3 88.9 86.7 25.7

Data are presented as mean ± SD (n = 3). * P < 0.05 vs. normal saline group. ** P < 0.01 vs normal saline group. *** P < 0.001 vs normal saline group.

of toads and the sheep erythrocytes hemolytic experiment were used to evaluate the toxicity of rivastigmine solution and its liposomes. The nasal cavity is covered with pseudostratified columnar epithelium cells. Most of these cells are covered with a layer of cilia which move in a well-organized and coordinated way to propel the overlying mucus layer toward the pharynx and also contribute to propel potentially hazardous substances such as dust, microorganisms, allergens and carcinogens (Illum, 2003; Mistry et al., 2009). After intranasal administration of various rivastigmine formulations to rats twice daily for seven consecutive days, the optical photomicrographs of rat mucosa were shown in Fig. 6. The cilia were all orderly arranged on the surface of the mucosa after intranasal administration of physiological saline (Fig. 6A), whereas the epithelia of positive group exposed to 1% SDS exhibited severe disruption and complete loss of some parts of the epithelium (Fig. 6B). Compared to control groups, the pseudostratified ciliated columnar epithelium of three formulations were almost unbroken and the cilia were arranged orderly on the surface of the mucosa, which indicated that rivastigmine and its liposomes were relatively safe without causing marked damage to the nasal mucosa. The duration of ciliary movement lasted for 626 and 598 min for mucosa treated with rivastigmine and Lp respectively, which were not significantly different from that for physiological saline (672 min) (Table 2). In contrast, the mucociliary movement of toad palate ceased quickly after exposing to 1% SDS. The duration of ciliary movement lasted 580 min for mucosa treated with CPP-Lp, which was significantly different from the negative group. However, there was no difference between CPP-Lp and rivastigmine solution, demonstrating that CPP-Lp had mild effect toward the duration of ciliary movement. Generally, the concentration inducing hemolysis is smaller, the membrane damage is greater. In an attempt to reflect hemolysis effect more intuitively, the present study measured the percentage method to evaluate hemolysis. If the ratio is more than 5%, it can be regarded as hemolysis. The hemolysis ratio of three rivastigmine formulations at test concentrations (5 mg/mL) were all less than 5%, indicating they played tiny role to the destruction of cell membrane. Overall, results indicated that rivastigmine solution, Lp and CPP-Lp on morphology of nasal mucosa, movement of cilia and hemolytic effect toward cell membrane was not significantly different from that of the physiological saline, indicating that there was no prominently nasal toxicity of rivastigmine formulations. But it was worth mentioning that the effects of rivastigmine formulations was examined only according to the concentration given in vivo, thus the further study should be carried out.

Fig. 5. Inhibition of acetylcholinesterase (AChE) and butyrylcholinersterase (BuChE) activity in CNS tissues (olfactory region, cortex, hippocampus, cerebrum, cerebellum) and plasma and peripheral tissues (liver, kidney and spleen) of rats at a dose of 2 mg/kg of rivastigmine formulations following intranasal (IN) and intravenous (IV) administration at 15 min, 60 min and 240 min, respectively. Data were expressed with mean ± SEM. The rats used in this study were 10 for control groups, 5 for IN and IV administration except for IV administration at 15 min where the rats was 4. *P < 0.05, **P < 0.01, ***P < 0.001 vs control; (a) P < 0.05 vs IV rivastigmine; (b) P < 0.05 vs IN rivastigmine; (c) P < 0.05 vs IN Lp; (d) P < 0.05 vs IN CPP-Lp.

Please cite this article in press as: Yang, Z.-Z., et al., Enhanced brain distribution and pharmacodynamics of rivastigmine by liposomes following intranasal administration. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.05.009

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Fig. 6. Optical microscopic images of rat nasal mucosal membranes in vertical sections following IN administration of (A) normal saline, (B) 1% SDS, (C) rivastigmine solution, (D) rivastigmine liposomes (Lp) and (E) CPP modified rivastigmine liposomes (CPP-Lp) for seven days. The bar indicates 50 ␮m. Tissues were stained with hematoxylin–eosin followed by fixation in 4% paraformaldehyde solution (n = 3).

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Conflict of interest The authors declare no competing financial interest. Acknowledgments

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We would like to acknowledge National Basic Research Program (No. 2013CB932501, No. 2009CB930303), NSFC (No. 30970785, No. 81273454), Beijing NSF (No. 7132113), Doctoral Foundation of the Ministry of Education (No. 20100001110056) and Innovation Team of Ministry of Education (No. BMU20110263) for funding of these works.

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References

619 620 621 622 623

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Ballard, C.G., 2002. Advances in the treatment of Alzheimer’s disease: benefits of dual cholinesterase inhibition. Eur. Neurol. 47, 64–70. Bar-On, P., Millard, C.B., Harel, M., Dvir, H., Enz, A., Sussman, J.L., Silman, I., 2002. Kinetic and structural studies on the interaction of cholinesterases with the anti-Alzheimer drug rivastigmine. Biochemistry 41, 3555–3564. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Cecchelli, R., Dehouck, B., Descamps, L., Fenart, L., Buee-Scherrer, V.V., Duhem, C., Lundquist, S., Rentfel, M., Torpier, G., Dehouck, M.P., 1999. In vitro model for evaluating drug transport across the blood–brain barrier. Adv. Drug Deliv. Rev. 36, 165–178. Dahl, R., Mygind, N., 1998. Anatomy, physiology and function of the nasal cavities in health and disease. Adv. Drug Deliv. Rev. 29, 3–12. Du, L., Kayali, R., Bertoni, C., Fike, F., Hu, H., Iversen, P.L., Gatti, R.A., 2011. Arginine-rich cell-penetrating peptide dramatically enhances AMO-mediated ATM aberrant splicing correction and enables delivery to brain and cerebellum. Hum. Mol. Genet. 20, 3151–3160. Fritze, A., Hens, F., Kimpfler, A., Schubert, R., Peschka-Suss, R., 2006. Remote loading of doxorubicin into liposomes driven by a transmembrane phosphate gradient. Biochim. Biophys. Acta 1758, 1633–1640. Haran, G., Cohen, R., Bar, L.K., Barenholz, Y., 1993. Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases. Biochim. Biophys. Acta 1151, 201–215. Hirai, S., Yashika, S., Matsuzawa, T., Mima, H., 1981. Absorption of drugs from the nasal mucosa of rat. Int. J. Pharm. 7, 317–325. Illum, L., 2000. Transport of drugs from the nasal cavity to the central nervous system. Eur. J. Pharm. Sci. 11, 1–18. Illum, L., 2003. Nasal drug delivery—possibilities, problems and solutions. J. Control. Release 87, 187–198. Jann, M.W., 2000. Rivastigmine, a new-generation cholinesterase inhibitor for the treatment of Alzheimer’s disease. Pharmacotherapy 20, 1–12. Jogani, V.V., Shah, P.J., Mishra, P., Mishra, A.K., Misra, A.R., 2008. Intranasal mucoadhesive microemulsion of tacrine to improve brain targeting. Alzheimer Dis. Assoc. Disord. 22, 116–124.

Kabanov, A.V., Batrakova, E.V., Alakhov, V.Y., 2002. Pluronic block copolymers as novel polymer therapeutics for drug and gene delivery. J. Control. Release 82, 189–212. Kaur, P., Kim, K., 2008. Pharmacokinetics and brain uptake of diazepam after intravenous and intranasal administration in rats and rabbits. Int. J. Pharm. 364, 27–35. Khafagy el, S., Morishita, M., Isowa, K., Imai, J., Takayama, K., 2009. Effect of cellpenetrating peptides on the nasal absorption of insulin. J. Control. Release 133, 103–108. Leonard, A.K., Sileno, A.P., MacEvilly, C., Foerder, C.A., Quay, S.C., Costantino, H.R., 2005. Development of a novel high-concentration galantamine formulation suitable for intranasal delivery. J. Pharm. Sci. 94, 1736–1746. Ma, D.X., Shi, N.Q., Qi, X.R., 2011. Distinct transduction modes of arginine-rich cell-penetrating peptides for cargo delivery into tumor cells. Int. J. Pharm. 419, 200–208. Mistry, A., Stolnik, S., Illum, L., 2009. Nanoparticles for direct nose-to-brain delivery of drugs. Int. J. Pharm. 379, 146–157. Mutlu, N.B., Degim, Z., 2005. Bioavailability file: rivastigmine tartrate. J. Pharm. Sci. 30, 150–157. Na, L., Mao, S., Wang, J., Sun, W., 2010. Comparison of different absorption enhancers on the intranasal absorption of isosorbide dinitrate in rats. Int. J. Pharm. 397, 59–66. Nonaka, N., Farr, S.A., Kageyama, H., Shioda, S., Banks, W.A., 2008. Delivery of galanin-like peptide to the brain: targeting with intranasal delivery and cyclodextrins. J. Pharmacol. Exp. Ther. 325, 513–519. Pakaski, M., Kalman, J., 2008. Interactions between the amyloid and cholinergic mechanisms in Alzheimer’s disease. Neurochem. Int. 53, 103–111. Parnetti, L., Chiasserini, D., Andreasson, U., Ohlson, M., Huls, C., Zetterberg, H., Minthon, L., Wallin, A.K., Andreasen, N., Talesa, V.N., Blennow, K., 2011. Changes in CSF acetyl- and butyrylcholinesterase activity after long-term treatment with AChE inhibitors in Alzheimer’s disease. Acta Neurol. Scand. 124, 122–129. Polinsky, R.J., 1998. Clinical pharmacology of rivastigmine: a new-generation acetylcholinesterase inhibitor for the treatment of Alzheimer’s disease. Clin. Ther. 20, 634–647. Puchelle, E., Zahm, J.M., Sadoul, P., 1982. Mucociliary frequency of frog palate epithelium. Am. J. Physiol. 242, C31–C35. Saw, P.E., Ko, Y.T., Jon, S., 2010. Efficient liposomal nanocarrier-mediated oligodeoxynucleotide delivery involving dual use of a cell-penetrating peptide as a packaging and intracellular delivery agent. Macromol. Rapid Commun. 31, 1155–1162. Scali, C., Casamenti, F., Bellucci, A., Costagli, C., Schmidt, B., Pepeu, G., 2002. Effect of subchronic administration of metrifonate, rivastigmine and donepezil on brain acetylcholine in aged F344 rats. J. Neural Transm. 109, 1067–1080. Schnyder, A., Huwyler, J., 2005. Drug transport to brain with targeted liposomes. NeuroRx 2, 99–107. Spencer, C.M., Noble, S., 1998. Rivastigmine. A review of its use in Alzheimer’s disease. Drugs Aging 13, 391–411. Tenovuo, O., 2005. Central acetylcholinesterase inhibitors in the treatment of chronic traumatic brain injury—clinical experience in 111 patients. Prog. Neuropsychopharmacol. Biol. Psychiatry 29, 61–67. Torchilin, V.P., 2008. Tat peptide-mediated intracellular delivery of pharmaceutical nanocarriers. Adv. Drug Deliv. Rev. 60, 548–558.

Please cite this article in press as: Yang, Z.-Z., et al., Enhanced brain distribution and pharmacodynamics of rivastigmine by liposomes following intranasal administration. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.05.009

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G Model IJP 13331 1–11

ARTICLE IN PRESS Z.-Z. Yang et al. / International Journal of Pharmaceutics xxx (2013) xxx–xxx

715 716 717 718 719 720 721 722 723 724 725 726 727

Trabulo, S., Cardoso, A.L., Mano, M., MCP, D.L., 2010. Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems. Pharmaceuticals 3, 961–993. van Bree, J.B., de Boer, A.G., Danhof, M., Ginsel, L.A., Breimer, D.D., 1988. Characterization of an “in vitro” blood–brain barrier: effects of molecular size and lipophilicity on cerebrovascular endothelial transport rates of drugs. J. Pharmacol. Exp. Ther. 247, 1233–1239. Venkatesh, K., Bullock, R., Akbas, A., 2007. Strategies to improve tolerability of rivastigmine: a case series. Curr. Med. Res. Opin. 23, 93–95. Westin, U.E., Bostrom, E., Grasjo, J., Hammarlund-Udenaes, M., Bjork, E., 2006. Direct nose-to-brain transfer of morphine after nasal administration to rats. Pharm. Res. 23, 565–572. Williams, B.R., Nazarians, A., Gill, M.A., 2003. A review of rivastigmine: a reversible cholinesterase inhibitor. Clin. Ther. 25, 1634–1653.

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Xie, Y., Ye, L.Y., Zhang, X.B., Hou, X.P., Lou, J.N., 2004. Establishment of an in vitro> model of brain–blood barrier. Beijing Da Xue Xue Bao 36, 435–438. Xie, Y., Ye, L.Y., Zhang, X., Cui, W., Lou, J., Nagai, T., Hou, X., 2005. Transport of nerve growth factor encapsulated into liposomes across the blood–brain barrier: in vitro and in vivo studies. J. Control. Release 105, 106–119. Yang, Z.Z., Zhang, Y.Q., Wu, K., Wang, Z.Z., Qi, X.R., 2012. Tissue distribution and pharmacodynamics of rivastigmine after intranasal and intravenous administration in rats. Curr. Alzheimer Res. 9, 315–325. Zhang, Q., Jiang, X., Jiang, W., Lu, W., Su, L., Shi, Z., 2004. Preparation of nimodipineloaded microemulsion for intranasal delivery and evaluation on the targeting efficiency to the brain. Int. J. Pharm. 275, 85–96. Zhang, X., Jin, Y., Plummer, M.R., Pooyan, S., Gunaseelan, S., Sinko, P.J., 2009. Endocytosis and membrane potential are required for HeLa cell uptake of R.I.-CKTat9, a retro-inverso Tat cell penetrating peptide. Mol. Pharmacol. 6, 836–848.

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