International Journal of Biological Macromolecules 67 (2014) 418–425
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Donepezil nanosuspension intended for nose to brain targeting: In vitro and in vivo safety evaluation Bhavna a , Shadab Md b , Mushir Ali b , Rashid Ali c , Aseem Bhatnagar c , Sanjula Baboota b , Javed Ali b,∗ a
Department of Pharmaceutics, Dehradun Institute of Technology (DIT), Dehradun 248009, Uttarakhand, India Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi 110062, India c Department of Nuclear Medicine, Institute of Nuclear Medicine and Allied Sciences, Brig. S K Mazumdar Marg, Delhi 110054, India b
a r t i c l e
i n f o
Article history: Received 7 January 2014 Received in revised form 5 March 2014 Accepted 11 March 2014 Available online 3 April 2014 Keywords: Cholinesterase inhibitor Intranasal delivery Ionic crosslinking Nanosuspension Alzheimer’s disease
a b s t r a c t The present study was to develop donepezil loaded nanosuspension for direct olfactory administration which reaches the brain and determining safety profile in Sprague–Dawley rats. Nanosuspension was prepared by ionic-crosslinking method. The developed nanosuspension was intranasally instilled into the nostrils of rats with the help of cannula (size 18/20) so that drug reached into the brain directly via nose to brain pathway. The nanosuspension had an average size of 150–200 nm with a polydispersity index of 0.341. The donepezil concentration was estimated in the brain homogenate using HPLC method. The Cmax showed concentration of donepezil in brain and plasma as 7.2 ± 0.86 and 82.8 ± 5.42 ng/ml, respectively, for drug suspension and concentration of donepezil in brain and plasma as 147.54 ± 25.08 and 183.451 ± 13.45 ng/ml, respectively, for nanosuspension at same dose of 0.5 mg/ml when administered intranasally (p < 0.05). The in vivo safety evaluation studies showed that no mortality, hematological changes, body weight variations and toxicity in animals was observed, when nanosuspension was administered in different doses as compared to control group (normal saline). Donepezil loaded chitosan nanosuspension is a potential new delivery system for treatment of Alzheimer’s disease, when transported via olfactory nasal pathway to the brain. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The global prevalence of Alzheimer’s disease (AD) has been dramatically increased and it is estimated that approximately 25 million people worldwide were attacked by AD [1]. Still the etiology of AD is not clear, free radicals have been recognized to be one of the important factors for progression of AD [2]. At present, most of the protective and therapeutic strategies against AD are still very limited and requires the more effective strategy. A reversible cholinesterase inhibitor exhibits high specificity for centrally active cholinesterase [3–5]. The cholinergic deficit is one of the major pathological features of Alzheimer’s disease [6]. This deficit has been associated with the loss of cognition and memory, the primary symptoms of this disorder [7]. Intranasal (IN) delivery is considered as a viable and attractive route of administration for various therapeutic agents [8]. Advantages of IN administration include a large surface area for delivery, rapid achievement of target drug levels,
∗ Corresponding author. Tel.: +91 9811312247; fax: +91 11 26059663. E-mail addresses: [email protected], [email protected] (J. Ali). http://dx.doi.org/10.1016/j.ijbiomac.2014.03.022 0141-8130/© 2014 Elsevier B.V. All rights reserved.
circumvent blood brain barrier (BBB) and avoidance of first pass metabolism; furthermore, this delivery route is non-invasive, maximizing patient comfort and compliance. In addition, IN dosing may facilitate transport of central nervous system (CNS) drugs into the brain [9–13]. Although this concept has not been universally established [14–16] and may depend on physicochemical properties of the drug [17,18]. The selected cholinesterase inhibitor donepezil is freely soluble in chloroform, soluble in water and in glacial acetic acid, slightly soluble in ethanol and in acetonitrile while it is practically insoluble in ethyl acetate and in n-hexane. Currently marketed acetyl-cholinesterase inhibitors are found entirely in oral dosage form. However, alternative routes, in particular IN administration, may provide benefits relative to oral dosing. Since, oral dose of cholinesterase inhibitors available currently in the market is as once a daily tablet or capsule (5 mg or 10 mg/day) [19]. Though this daily repeated oral administration is convenient for some of patients, it is very difficult for AD’s patient who suffers memory disorder to miss scheduled medication. In addition, these cholinesterase inhibitors also showed the gastrointestinal side effects such as diarrhoea, nausea, anorexia and muscle convulsions etc. As a result, it is very important to
Bhavna et al. / International Journal of Biological Macromolecules 67 (2014) 418–425
develop a long-term, non-gastrointestinal delivery system of these cholinesterase inhibitors for treatment of AD [20]. The purpose of this work was to prepare nanosuspension (NS) of donepezil and determining its particle size and surface morphology, and assessment of the safety of cholinesterase inhibitor nanosuspension by nasal route. 2. Materials and methods 2.1. Chemicals and reagents Donepezil a cholinesterase inhibitor ((RS)-1-benzyl-4-(5,6dimethoxy-1-indanon)-2-yl methylpiperidine) as a drug candidate was received as a gift sample from Ranbaxy Research Laboratory, Gurgaon, India. Chitosan, 400 kD MW and 85% deacetylated and polysorbate-80 was purchased from Sigma Aldrich (New Delhi, India). All other chemicals were of analytical grade or highest grade commercially available. 2.2. Animals Healthy, young, male Sprague–Dawley rats weighing 200–220 g were selected for the study and acclimatized to the laboratory conditions for at least five days prior to the test and randomly assigned to weight-matched experimental groups. Animals were obtained from Central Animal House Facility of Institute of Nuclear Medicine and Allied Sciences (INMAS), DRDO, Delhi, India. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) of INMAS protocol number (INM/TS/IEC/007/07). The animals were housed in polypropylene cages in groups of six in each cage and were kept in a room maintained at 25 ± 2 ◦ C with a 12 h light/dark cycle. They were provided standard laboratory animal feed (Golden Feed, Mehrauli, Delhi) and water ad libitum. 3. Experimental 3.1. Preparation of cholinesterase inhibitor nanosuspension Cholinesterase inhibitor nanoparticles were prepared by ionic crosslinking method [21,22]. Chitosan was dissolved in 1.0% (w/v) acetic acid, and donepezil was added to the chitosan solution (10 ml) in different amount of 5, 10 and 15 mg. The nanoparticles were prepared using tripolyphosphate (TPP) as a cross linker. The TPP solution was added drop wise by 1 ml syringe in drug containing chitosan solution and stirred at 500 rpm for an hour using magnetic stirrer (Remi, Mumbai, India). Then the solution was filtered with membrane filter (0.45 m) to remove the residual TPP”. The resultant nanoparticles were concentrated by ultracentrifugation at 15,000 rpm at 4 ◦ C for 40 min. The supernatant was used to determine the encapsulation efficiency and the pellets were dispersed in phosphate buffer saline. The nanosuspension was used for further study. 3.2. Characterization of cholinesterase inhibitor nanosuspension 3.2.1. Transmission electron microscopy The particle size of nanosuspension was determined using transmission electron microscopy (TEM). The nanosuspension (5–10 l) was placed on a paraffin sheet with a carbon coated grid placed over it and the sample left was for a min to allow the nanoparticles to adhere on the carbon substrate. Then the grid was placed on the drop of phosphotungstate for 10 s. The remaining solution was removed by absorbing the liquid with a piece of filter paper and sample was air-dried. The sample was examined by TEM (FEI Phillips Morgagni 268-D TEM Germany) and
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the photographic images were captured at 60–80 kV at different magnifications (1550× and 44,000×). The images were analyzed using Soft Imaging Viewer software. 3.2.2. Scanning electron microscopy The NS was examined for surface morphology by scanning electron microscopy (SEM). To the dried sample of nanosuspension gold sputter coating was carried out under reduced pressure in an inert argon gas atmosphere (Agar Sputter Coater P7340). After sputter coating the sample on the carbon coated grid was examined under scanning electron microscope (Leo 435 VP Cambridge, UK) operated at 15–25 kV and photographs were recorded. 3.2.3. Donepezil loaded nanoparticles encapsulation efficiency and loading capacity Donepezil-loaded nanoparticles were separated from aqueous suspension by centrifugation using cooling centrifuge (C24, Remi Centrifuge, Mumbai, India) at 20,000 g and 14 ◦ C for 60 min. The supernatant was collected and drug content (free drug) in supernatant was determined by the UV spectrophotometric method (UV-1601) Shimadzu, Kyoto, Japan) at 271 nm. The donepezil encapsulation efficiency (EE) and loading capacity (LC) of nanoparticles were calculated as follows, while all the measurements were performed in triplicate and averaged. %EE =
%LC =
(A − B) × 100 A
(A − B) × 100 C
where A is the total amount of donepezil, B is the free amount of donepezil andC is the weight of nanoparticles. 3.2.4. In vitro release studies The in vitro dissolution studies were carried out to compare the release of drug form the optimized nanoparticulate formulation to observe the release pattern. The nanoparticulate suspension and drug solution each having same quantity (5 mg) of donepezil was taken. The in vitro drug release study was performed using dialysis bag method [23]. Nanoparticles equivalent to 5 mg of drug was placed in a cellulose dialysis bag, (MWCO 12,000 g/mole; Sigma, St. Louis, USA) and to this a little amount of dissolution media was added, which was then sealed at both ends. The dialysis bag was dipped into the receptor compartment containing the dissolution medium, which was stirred continuously at 100 rpm maintained at 37 ◦ C. The receptor compartment was closed to prevent evaporation of the dissolution medium. Samples were withdrawn at regular time intervals and the same volume was replaced with fresh dissolution medium. The samples were measured by UV spectrophotometer at 271 nm. 3.2.5. Estimation of drug content in the brain by HPLC method Male Sprague–Dawley rats (n = 6) weighing between 200 ± 10 g were selected for the present study. The dose of donepezil solution and optimised nanosuspension of donepezil (0.1 ml) was administered at same dose of 0.5 mg/ml through the intranasal route with the help of 18/20 gauze cannula fitted in the 1 ml syringe and the rats were kept in upright position at an angle of 90◦ , so that maximum drug concentration can reach to the brain [24]. After 20 min of intranasal dosing the animals were sacrificed under ether anaesthesia and the whole brain was excised, isolated and weighed. The brain tissue was then homogenized in 1% acetic acid using tissue homogenizer (T-25 Ultra-Turrax, Bangalore, India), and particulate matter removed by centrifugation and filtration. The clarified supernatant was analyzed for drug content in brain via HPLC method [25]. Chromatographic separation was achieved
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with a Lichrocart® C18 reversed-phase column (250 mm × 4.6 mm, particle size 5 m). The mobile phase consisted of methanol, 0.02 M buffer phosphate (pH 7.4) and triethylamine in a ratio of (50:50:0.5). The mobile phase was set at a flow rate of 1 ml/min. The run time of the sample was kept 15 min. The temperature was kept at 20 ◦ C ± 5 ◦ C. The ultra-violet detection of donepezil was performed at 280 nm. Pharmacokinetic parameters were evaluated using PK solution 2.0 software (non-compartmental modeling) and statistical evaluation was carried out using the GraphPad Prism software package (Version 4.03). Pharmacokinetic parameters such as Cmax , tmax , Ke , AUC0→24 , AUC0→∞ , AUMC0→24 and MRT were calculated for both the drug suspension and nanosuspension group. 3.2.6. In vivo safety evaluation of donepezil nanosuspension Twenty-four male Sprague–Dawley rats were taken and divided into four groups, containing six animals in each. The groups were designated as Control, I–III. donepezil loaded nanosuspension was administered intranasally to the animals of groups (I–III) at a drug concentration of 0.5, 1.0 and 1.5 mg/ml, respectively. The intranasal administration of normal saline was administered as a control group. The dose volume for each group was 0.1 ml (100 l) daily up to 7 weeks. On 49th day animals were sacrificed; blood and vital organs (lungs, liver, kidney heart, spleen, and brain) were collected. The animal safety studies of donepezil nanosuspension evaluated on the basis of neurobehavioral, body weight, hematological and histopathological findings in animals. 3.2.6.1. Gross pathology. Animals were observed for mucous membrane (nasal secretion), eye irritation, tear secretion, excessive blinking, salivation, cyanosis, lethargy, pyloerection (ruffled fur), paralysis, skin irritation, edema, erythema, nose switching, as well as directed and non-directed movements within the cage prior to, during and immediately following each exposure throughout the experiment as a sign and symptoms of toxicity. The animals were observed for any mortality during the experiment. 3.2.6.2. Food, water intake and body weight variation analysis. Food and water intake of experimental animals was determined on a periodic basis throughout the study. From these determinations, mean individual daily food and water consumption, and its efficiency were calculated. Body weights of all animals were observed on weekly basis individually and the percentage weight gain of the treated animals were compared with that of the control group to observe clinical signs of toxicity, just prior to exposure, throughout the study. 3.2.6.3. Hematological analysis. Blood samples were collected directly from the cardiac puncture in a tube containing EDTA (anticoagulant) for analysis of hematological parameters. Values
of red blood cells (RBC), white blood cells (WBC), hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean cell hemoglobin (MCH) and platelets counts were determined and compared with control using an auto analyzer (Roche Integra, 400 Plus, Diagnostic Systems, IN, USA). 3.2.6.4. Organ/body weight ratio analysis. The organ/body weight ratio for different vital organs of treated groups was compared with the control, and was observed for any changes. 3.2.6.5. Histopathological analysis. The nasal mucosa and brain tissues were collected and fixed in 10% buffered formalin. Sections (5 m) were cut of nasal mucosa and from the middle lobes of brain of each group more or less from similar positions. The paraffin embedded nasal mucosa and brain tissue sections were then deparaffinised using xylene and ethanol. The deparaffinised sections were stained with hematoxylin and eosin. The histopathological images were analysed for any microscopic changes under florescence microscope (Olympus BX 60, NY, USA) at 40× magnification. 4. Statistical analysis The statistical analysis of the samples was undertaken using a Student’s t-test, and p-value 0.05) in body weights of treated group as compared to control (Fig. 5). No treatment related changes in behavioral parameters were found when compared with control group. 5.4.3. Hematological analysis The values of RBC, WBC, Hb, HCT, MCV, MCH and platelets were found to be within normal range in treated animals and there were no significant changes (p > 0.05) as compared to control animals (Fig. 6). 5.4.4. Organ/body weight ratio analysis There were found no significant differences (p > 0.05) in the organ/body weight ratio of vital organs with the donepezil loaded
5.4.5. Histopathological analysis It was observed from the histopathological images that there is no microscopic change in the nasal mucosa and brain tissues of treated animals when compared with the control animals. The obtained images of histopathological studies are shown in Fig. 8. 6. Discussion The blood brain barrier (BBB) denies many therapeutic agents access to brain and diseases of the CNS; even more than 98% of drugs of smaller molecular size do not cross the BBB [30]. The BBB is formed by the endothelial cells of the cerebral capillaries and comprises the major exchange interface between the blood and the brain [31]. There are many methods potentially to overcome the obstacle of BBB, with the purpose of improving drug delivery into the brain including osmotic opening of the tight junctions, nose to brain pathway is a conduit for transport of agents into the CNS, and the direct surgical administration of drugs into the brain which is an area of on-going research. It has been suggested that there is free communication between the nasal sub mucosal interstitial space and the olfactory perineuronal space, which appears to be continuous with a subarachnoid extension that surrounds the olfactory nerve as it penetrates the cribriform plate [32]. One of the most promising approaches appears to be the employment of nanotechnology by using liposomes and nanoparticles [33]. Nanoparticles have been proved to deliver a great variety of drugs across the BBB. The non-invasive administration by intranasal, intravenous or infusion of drug-loaded nanoparticles enables the brain delivery of agents, including macromolecules, low molecular drugs, and few biological entities, that cannot independently permeate the BBB in therapeutically effective concentrations. Binding to the particles also may lead to a reduction in side effects of relatively toxic drugs such as doxorubicin. In addition, due to a significantly more effective brain delivery by biodegradable polymeric nanoparticles, the drug dose might be decreased, together leading to a significant improvement of the patients’ quality of life [34]. The mechanism for the brain uptake of nanoparticles
Fig. 7. Variation in body weight gain of different groups of experimental animals. Control: normal saline; Group I: donepezil nanosuspension (0.5 mg/ml); Group II: donepezil nanosuspension (1 mg/ml); Group III: donepezil nanosuspension (1.5 mg/ml).
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Fig. 8. Histopathology images of sections of: (A) nasal mucosa, (B) brain comparing normal group with the treated groups of animals.
now appears to be receptor-mediated endocytosis by the brain capillary endothelial cells followed by transcytosis [35]. One important major requirement for nanoparticulate brain delivery systems is that they are rapidly biodegradable, i.e. over a time frame of a few days. Non-degradable particles such as fullerenes, toxic systems as quantum dots, or potentially risky pointed delivery systems such as carbon nanotubes, which may have hazardous effects similar to asbestos, therefore, are not useful [34]. The route of existence for nose to brain can be entirely explained when the effect of toxicity parameters will be taken into consideration while administering the formulations through intranasal route. In this study we examined the safety evaluation of donepezil nanosuspension administered to the brain via intranasal administration. The animal toxicity studies of donepezil loaded chitosan nanosuspension were evaluated on the basis of body weight, hematological and histopathological changes in rat model. The whole body weight of treated animals from control and groups of donepezil nanosuspension 0.5 mg/ml (I), 1 mg/ml (II) and 1.5 mg/ml (III) were observed on weekly basis and the percentage weight gain of the treated animals were compared with that of the control group. No significant difference was observed (p > 0.05). Hematological evaluation was performed on the blood samples of group I–III animals and were compared with the control group. The hematological values were found to be in the normal ranges. Organ/body weight ratio showed no significant difference (p > 0.05) in the weight of organs of all groups after 7 weeks. Hence no effect was observed on organ/body weight ratio with the donepezil nanoformulation, when administered intranasally. It was observed from the histopathological images that there was no microscopic change in the brain tissues, of treated animals when compared with the control animals. Thus, the nanosuspension of donepezil concentrations of 0.5, 1 and 1.5% showed no changes in body weight, hematological parameters and histopathology. Hence, intranasal route of administration was an achievement to deliver nanoparticulate suspension and thus can be used for treating neurodegenerative disorders of brain. This research effort has three components. Firstly we developed a novel formulation of cholinesterase inhibitor in the form of a nanosuspension, which has the potential utility for treatment of neurodegenerative disorders, by reducing the dose and avoiding the first pass effect thus reducing the side effects which have been reported for the marketed formulations. Secondly we used
this formulation to address the question of the relevance of the olfactory transport route to brain for cholinesterase inhibitor uptake following intranasal administration. Thirdly, the in vivo safety evaluation studies of nanosuspension in animal models to show whether any toxicity occurred when the nanosuspension of cholinesterase inhibitor was administered intranasally. The nasal cavity has about a total volume of 15–20 ml with a total surface area of 150 cm2 ; therefore, volume that can be delivered into the nasal cavity is restricted to 25–200 l [36,37]. The total volume of developed cholinesterase inhibitor nanosuspension administered to the nasal cavity of the animals was 0.1 ml (100 l), which showed a safe delivery system since; no toxicity in nasal mucosa and brain was observed, with the treated animals. Thus nanosuspension is a good approach to deliver drugs to brain in neurodegenerative disorder via nasal route of administration. The data showed that the nanosuspension of concentration (0.5, 1 and 1.5 mg/ml) of donepezil loaded chitosan nano particulate suspension is safe which has been proved by morphological, hematological and histopathological parameters. Hence, developing a delivery system via intranasal route to brain can be an achievement for treating neurodegenerative disorders of brain. 7. Conclusion In the present study, we described the development of donepezil loaded nanosuspension using the ionic cross linking method. This formulation was able to show higher drug concentration in brain and no mortality, hematological changes, body weight variations and histopathological changes in animals, when formulation was administered in different doses as compared to normal saline administered intranasally. Thus it is concluded that donepezil loaded nanosuspension is capable of providing direct nose-to-brain delivery, thereby enhancing drug concentration in the brain. It is anticipated that the present manuscript will assist researchers in implementing the utility of in vivo safety studies which must be performed for different drugs, vaccines and challenge studies for drug safety in the nasal mucosa while administration through intranasal route. Conflicts of interest statement The authors declare that there are no conflicts of interest.
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Acknowledgments The authors are thankful for financial support provided by Indian Council of Medical Sciences (ICMR), to carry out the research work under Project No. 45/26/2007-PHA/BMS, New Delhi, India. The authors are also thankful to Department of Nuclear Medicine, Institute of Nuclear Medicine and Allied Sciences (INMAS), DRDO, (Proposal No. INM/TS/IEC/007/07). Delhi for providing experimental animals for in vivo studies. References [1] [2] [3] [4] [5]
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Donepezil nanosuspension intended for nose to brain targeting: In vitro and in vivo safety evaluation Bhavna a , Shadab Md b , Mushir Ali b , Rashid Ali c , Aseem Bhatnagar c , Sanjula Baboota b , Javed Ali b,∗ a
Department of Pharmaceutics, Dehradun Institute of Technology (DIT), Dehradun 248009, Uttarakhand, India Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi 110062, India c Department of Nuclear Medicine, Institute of Nuclear Medicine and Allied Sciences, Brig. S K Mazumdar Marg, Delhi 110054, India b
a r t i c l e
i n f o
Article history: Received 7 January 2014 Received in revised form 5 March 2014 Accepted 11 March 2014 Available online 3 April 2014 Keywords: Cholinesterase inhibitor Intranasal delivery Ionic crosslinking Nanosuspension Alzheimer’s disease
a b s t r a c t The present study was to develop donepezil loaded nanosuspension for direct olfactory administration which reaches the brain and determining safety profile in Sprague–Dawley rats. Nanosuspension was prepared by ionic-crosslinking method. The developed nanosuspension was intranasally instilled into the nostrils of rats with the help of cannula (size 18/20) so that drug reached into the brain directly via nose to brain pathway. The nanosuspension had an average size of 150–200 nm with a polydispersity index of 0.341. The donepezil concentration was estimated in the brain homogenate using HPLC method. The Cmax showed concentration of donepezil in brain and plasma as 7.2 ± 0.86 and 82.8 ± 5.42 ng/ml, respectively, for drug suspension and concentration of donepezil in brain and plasma as 147.54 ± 25.08 and 183.451 ± 13.45 ng/ml, respectively, for nanosuspension at same dose of 0.5 mg/ml when administered intranasally (p < 0.05). The in vivo safety evaluation studies showed that no mortality, hematological changes, body weight variations and toxicity in animals was observed, when nanosuspension was administered in different doses as compared to control group (normal saline). Donepezil loaded chitosan nanosuspension is a potential new delivery system for treatment of Alzheimer’s disease, when transported via olfactory nasal pathway to the brain. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The global prevalence of Alzheimer’s disease (AD) has been dramatically increased and it is estimated that approximately 25 million people worldwide were attacked by AD [1]. Still the etiology of AD is not clear, free radicals have been recognized to be one of the important factors for progression of AD [2]. At present, most of the protective and therapeutic strategies against AD are still very limited and requires the more effective strategy. A reversible cholinesterase inhibitor exhibits high specificity for centrally active cholinesterase [3–5]. The cholinergic deficit is one of the major pathological features of Alzheimer’s disease [6]. This deficit has been associated with the loss of cognition and memory, the primary symptoms of this disorder [7]. Intranasal (IN) delivery is considered as a viable and attractive route of administration for various therapeutic agents [8]. Advantages of IN administration include a large surface area for delivery, rapid achievement of target drug levels,
∗ Corresponding author. Tel.: +91 9811312247; fax: +91 11 26059663. E-mail addresses: [email protected], [email protected] (J. Ali). http://dx.doi.org/10.1016/j.ijbiomac.2014.03.022 0141-8130/© 2014 Elsevier B.V. All rights reserved.
circumvent blood brain barrier (BBB) and avoidance of first pass metabolism; furthermore, this delivery route is non-invasive, maximizing patient comfort and compliance. In addition, IN dosing may facilitate transport of central nervous system (CNS) drugs into the brain [9–13]. Although this concept has not been universally established [14–16] and may depend on physicochemical properties of the drug [17,18]. The selected cholinesterase inhibitor donepezil is freely soluble in chloroform, soluble in water and in glacial acetic acid, slightly soluble in ethanol and in acetonitrile while it is practically insoluble in ethyl acetate and in n-hexane. Currently marketed acetyl-cholinesterase inhibitors are found entirely in oral dosage form. However, alternative routes, in particular IN administration, may provide benefits relative to oral dosing. Since, oral dose of cholinesterase inhibitors available currently in the market is as once a daily tablet or capsule (5 mg or 10 mg/day) [19]. Though this daily repeated oral administration is convenient for some of patients, it is very difficult for AD’s patient who suffers memory disorder to miss scheduled medication. In addition, these cholinesterase inhibitors also showed the gastrointestinal side effects such as diarrhoea, nausea, anorexia and muscle convulsions etc. As a result, it is very important to
Bhavna et al. / International Journal of Biological Macromolecules 67 (2014) 418–425
develop a long-term, non-gastrointestinal delivery system of these cholinesterase inhibitors for treatment of AD [20]. The purpose of this work was to prepare nanosuspension (NS) of donepezil and determining its particle size and surface morphology, and assessment of the safety of cholinesterase inhibitor nanosuspension by nasal route. 2. Materials and methods 2.1. Chemicals and reagents Donepezil a cholinesterase inhibitor ((RS)-1-benzyl-4-(5,6dimethoxy-1-indanon)-2-yl methylpiperidine) as a drug candidate was received as a gift sample from Ranbaxy Research Laboratory, Gurgaon, India. Chitosan, 400 kD MW and 85% deacetylated and polysorbate-80 was purchased from Sigma Aldrich (New Delhi, India). All other chemicals were of analytical grade or highest grade commercially available. 2.2. Animals Healthy, young, male Sprague–Dawley rats weighing 200–220 g were selected for the study and acclimatized to the laboratory conditions for at least five days prior to the test and randomly assigned to weight-matched experimental groups. Animals were obtained from Central Animal House Facility of Institute of Nuclear Medicine and Allied Sciences (INMAS), DRDO, Delhi, India. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) of INMAS protocol number (INM/TS/IEC/007/07). The animals were housed in polypropylene cages in groups of six in each cage and were kept in a room maintained at 25 ± 2 ◦ C with a 12 h light/dark cycle. They were provided standard laboratory animal feed (Golden Feed, Mehrauli, Delhi) and water ad libitum. 3. Experimental 3.1. Preparation of cholinesterase inhibitor nanosuspension Cholinesterase inhibitor nanoparticles were prepared by ionic crosslinking method [21,22]. Chitosan was dissolved in 1.0% (w/v) acetic acid, and donepezil was added to the chitosan solution (10 ml) in different amount of 5, 10 and 15 mg. The nanoparticles were prepared using tripolyphosphate (TPP) as a cross linker. The TPP solution was added drop wise by 1 ml syringe in drug containing chitosan solution and stirred at 500 rpm for an hour using magnetic stirrer (Remi, Mumbai, India). Then the solution was filtered with membrane filter (0.45 m) to remove the residual TPP”. The resultant nanoparticles were concentrated by ultracentrifugation at 15,000 rpm at 4 ◦ C for 40 min. The supernatant was used to determine the encapsulation efficiency and the pellets were dispersed in phosphate buffer saline. The nanosuspension was used for further study. 3.2. Characterization of cholinesterase inhibitor nanosuspension 3.2.1. Transmission electron microscopy The particle size of nanosuspension was determined using transmission electron microscopy (TEM). The nanosuspension (5–10 l) was placed on a paraffin sheet with a carbon coated grid placed over it and the sample left was for a min to allow the nanoparticles to adhere on the carbon substrate. Then the grid was placed on the drop of phosphotungstate for 10 s. The remaining solution was removed by absorbing the liquid with a piece of filter paper and sample was air-dried. The sample was examined by TEM (FEI Phillips Morgagni 268-D TEM Germany) and
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the photographic images were captured at 60–80 kV at different magnifications (1550× and 44,000×). The images were analyzed using Soft Imaging Viewer software. 3.2.2. Scanning electron microscopy The NS was examined for surface morphology by scanning electron microscopy (SEM). To the dried sample of nanosuspension gold sputter coating was carried out under reduced pressure in an inert argon gas atmosphere (Agar Sputter Coater P7340). After sputter coating the sample on the carbon coated grid was examined under scanning electron microscope (Leo 435 VP Cambridge, UK) operated at 15–25 kV and photographs were recorded. 3.2.3. Donepezil loaded nanoparticles encapsulation efficiency and loading capacity Donepezil-loaded nanoparticles were separated from aqueous suspension by centrifugation using cooling centrifuge (C24, Remi Centrifuge, Mumbai, India) at 20,000 g and 14 ◦ C for 60 min. The supernatant was collected and drug content (free drug) in supernatant was determined by the UV spectrophotometric method (UV-1601) Shimadzu, Kyoto, Japan) at 271 nm. The donepezil encapsulation efficiency (EE) and loading capacity (LC) of nanoparticles were calculated as follows, while all the measurements were performed in triplicate and averaged. %EE =
%LC =
(A − B) × 100 A
(A − B) × 100 C
where A is the total amount of donepezil, B is the free amount of donepezil andC is the weight of nanoparticles. 3.2.4. In vitro release studies The in vitro dissolution studies were carried out to compare the release of drug form the optimized nanoparticulate formulation to observe the release pattern. The nanoparticulate suspension and drug solution each having same quantity (5 mg) of donepezil was taken. The in vitro drug release study was performed using dialysis bag method [23]. Nanoparticles equivalent to 5 mg of drug was placed in a cellulose dialysis bag, (MWCO 12,000 g/mole; Sigma, St. Louis, USA) and to this a little amount of dissolution media was added, which was then sealed at both ends. The dialysis bag was dipped into the receptor compartment containing the dissolution medium, which was stirred continuously at 100 rpm maintained at 37 ◦ C. The receptor compartment was closed to prevent evaporation of the dissolution medium. Samples were withdrawn at regular time intervals and the same volume was replaced with fresh dissolution medium. The samples were measured by UV spectrophotometer at 271 nm. 3.2.5. Estimation of drug content in the brain by HPLC method Male Sprague–Dawley rats (n = 6) weighing between 200 ± 10 g were selected for the present study. The dose of donepezil solution and optimised nanosuspension of donepezil (0.1 ml) was administered at same dose of 0.5 mg/ml through the intranasal route with the help of 18/20 gauze cannula fitted in the 1 ml syringe and the rats were kept in upright position at an angle of 90◦ , so that maximum drug concentration can reach to the brain [24]. After 20 min of intranasal dosing the animals were sacrificed under ether anaesthesia and the whole brain was excised, isolated and weighed. The brain tissue was then homogenized in 1% acetic acid using tissue homogenizer (T-25 Ultra-Turrax, Bangalore, India), and particulate matter removed by centrifugation and filtration. The clarified supernatant was analyzed for drug content in brain via HPLC method [25]. Chromatographic separation was achieved
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with a Lichrocart® C18 reversed-phase column (250 mm × 4.6 mm, particle size 5 m). The mobile phase consisted of methanol, 0.02 M buffer phosphate (pH 7.4) and triethylamine in a ratio of (50:50:0.5). The mobile phase was set at a flow rate of 1 ml/min. The run time of the sample was kept 15 min. The temperature was kept at 20 ◦ C ± 5 ◦ C. The ultra-violet detection of donepezil was performed at 280 nm. Pharmacokinetic parameters were evaluated using PK solution 2.0 software (non-compartmental modeling) and statistical evaluation was carried out using the GraphPad Prism software package (Version 4.03). Pharmacokinetic parameters such as Cmax , tmax , Ke , AUC0→24 , AUC0→∞ , AUMC0→24 and MRT were calculated for both the drug suspension and nanosuspension group. 3.2.6. In vivo safety evaluation of donepezil nanosuspension Twenty-four male Sprague–Dawley rats were taken and divided into four groups, containing six animals in each. The groups were designated as Control, I–III. donepezil loaded nanosuspension was administered intranasally to the animals of groups (I–III) at a drug concentration of 0.5, 1.0 and 1.5 mg/ml, respectively. The intranasal administration of normal saline was administered as a control group. The dose volume for each group was 0.1 ml (100 l) daily up to 7 weeks. On 49th day animals were sacrificed; blood and vital organs (lungs, liver, kidney heart, spleen, and brain) were collected. The animal safety studies of donepezil nanosuspension evaluated on the basis of neurobehavioral, body weight, hematological and histopathological findings in animals. 3.2.6.1. Gross pathology. Animals were observed for mucous membrane (nasal secretion), eye irritation, tear secretion, excessive blinking, salivation, cyanosis, lethargy, pyloerection (ruffled fur), paralysis, skin irritation, edema, erythema, nose switching, as well as directed and non-directed movements within the cage prior to, during and immediately following each exposure throughout the experiment as a sign and symptoms of toxicity. The animals were observed for any mortality during the experiment. 3.2.6.2. Food, water intake and body weight variation analysis. Food and water intake of experimental animals was determined on a periodic basis throughout the study. From these determinations, mean individual daily food and water consumption, and its efficiency were calculated. Body weights of all animals were observed on weekly basis individually and the percentage weight gain of the treated animals were compared with that of the control group to observe clinical signs of toxicity, just prior to exposure, throughout the study. 3.2.6.3. Hematological analysis. Blood samples were collected directly from the cardiac puncture in a tube containing EDTA (anticoagulant) for analysis of hematological parameters. Values
of red blood cells (RBC), white blood cells (WBC), hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean cell hemoglobin (MCH) and platelets counts were determined and compared with control using an auto analyzer (Roche Integra, 400 Plus, Diagnostic Systems, IN, USA). 3.2.6.4. Organ/body weight ratio analysis. The organ/body weight ratio for different vital organs of treated groups was compared with the control, and was observed for any changes. 3.2.6.5. Histopathological analysis. The nasal mucosa and brain tissues were collected and fixed in 10% buffered formalin. Sections (5 m) were cut of nasal mucosa and from the middle lobes of brain of each group more or less from similar positions. The paraffin embedded nasal mucosa and brain tissue sections were then deparaffinised using xylene and ethanol. The deparaffinised sections were stained with hematoxylin and eosin. The histopathological images were analysed for any microscopic changes under florescence microscope (Olympus BX 60, NY, USA) at 40× magnification. 4. Statistical analysis The statistical analysis of the samples was undertaken using a Student’s t-test, and p-value 0.05) in body weights of treated group as compared to control (Fig. 5). No treatment related changes in behavioral parameters were found when compared with control group. 5.4.3. Hematological analysis The values of RBC, WBC, Hb, HCT, MCV, MCH and platelets were found to be within normal range in treated animals and there were no significant changes (p > 0.05) as compared to control animals (Fig. 6). 5.4.4. Organ/body weight ratio analysis There were found no significant differences (p > 0.05) in the organ/body weight ratio of vital organs with the donepezil loaded
5.4.5. Histopathological analysis It was observed from the histopathological images that there is no microscopic change in the nasal mucosa and brain tissues of treated animals when compared with the control animals. The obtained images of histopathological studies are shown in Fig. 8. 6. Discussion The blood brain barrier (BBB) denies many therapeutic agents access to brain and diseases of the CNS; even more than 98% of drugs of smaller molecular size do not cross the BBB [30]. The BBB is formed by the endothelial cells of the cerebral capillaries and comprises the major exchange interface between the blood and the brain [31]. There are many methods potentially to overcome the obstacle of BBB, with the purpose of improving drug delivery into the brain including osmotic opening of the tight junctions, nose to brain pathway is a conduit for transport of agents into the CNS, and the direct surgical administration of drugs into the brain which is an area of on-going research. It has been suggested that there is free communication between the nasal sub mucosal interstitial space and the olfactory perineuronal space, which appears to be continuous with a subarachnoid extension that surrounds the olfactory nerve as it penetrates the cribriform plate [32]. One of the most promising approaches appears to be the employment of nanotechnology by using liposomes and nanoparticles [33]. Nanoparticles have been proved to deliver a great variety of drugs across the BBB. The non-invasive administration by intranasal, intravenous or infusion of drug-loaded nanoparticles enables the brain delivery of agents, including macromolecules, low molecular drugs, and few biological entities, that cannot independently permeate the BBB in therapeutically effective concentrations. Binding to the particles also may lead to a reduction in side effects of relatively toxic drugs such as doxorubicin. In addition, due to a significantly more effective brain delivery by biodegradable polymeric nanoparticles, the drug dose might be decreased, together leading to a significant improvement of the patients’ quality of life [34]. The mechanism for the brain uptake of nanoparticles
Fig. 7. Variation in body weight gain of different groups of experimental animals. Control: normal saline; Group I: donepezil nanosuspension (0.5 mg/ml); Group II: donepezil nanosuspension (1 mg/ml); Group III: donepezil nanosuspension (1.5 mg/ml).
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Fig. 8. Histopathology images of sections of: (A) nasal mucosa, (B) brain comparing normal group with the treated groups of animals.
now appears to be receptor-mediated endocytosis by the brain capillary endothelial cells followed by transcytosis [35]. One important major requirement for nanoparticulate brain delivery systems is that they are rapidly biodegradable, i.e. over a time frame of a few days. Non-degradable particles such as fullerenes, toxic systems as quantum dots, or potentially risky pointed delivery systems such as carbon nanotubes, which may have hazardous effects similar to asbestos, therefore, are not useful [34]. The route of existence for nose to brain can be entirely explained when the effect of toxicity parameters will be taken into consideration while administering the formulations through intranasal route. In this study we examined the safety evaluation of donepezil nanosuspension administered to the brain via intranasal administration. The animal toxicity studies of donepezil loaded chitosan nanosuspension were evaluated on the basis of body weight, hematological and histopathological changes in rat model. The whole body weight of treated animals from control and groups of donepezil nanosuspension 0.5 mg/ml (I), 1 mg/ml (II) and 1.5 mg/ml (III) were observed on weekly basis and the percentage weight gain of the treated animals were compared with that of the control group. No significant difference was observed (p > 0.05). Hematological evaluation was performed on the blood samples of group I–III animals and were compared with the control group. The hematological values were found to be in the normal ranges. Organ/body weight ratio showed no significant difference (p > 0.05) in the weight of organs of all groups after 7 weeks. Hence no effect was observed on organ/body weight ratio with the donepezil nanoformulation, when administered intranasally. It was observed from the histopathological images that there was no microscopic change in the brain tissues, of treated animals when compared with the control animals. Thus, the nanosuspension of donepezil concentrations of 0.5, 1 and 1.5% showed no changes in body weight, hematological parameters and histopathology. Hence, intranasal route of administration was an achievement to deliver nanoparticulate suspension and thus can be used for treating neurodegenerative disorders of brain. This research effort has three components. Firstly we developed a novel formulation of cholinesterase inhibitor in the form of a nanosuspension, which has the potential utility for treatment of neurodegenerative disorders, by reducing the dose and avoiding the first pass effect thus reducing the side effects which have been reported for the marketed formulations. Secondly we used
this formulation to address the question of the relevance of the olfactory transport route to brain for cholinesterase inhibitor uptake following intranasal administration. Thirdly, the in vivo safety evaluation studies of nanosuspension in animal models to show whether any toxicity occurred when the nanosuspension of cholinesterase inhibitor was administered intranasally. The nasal cavity has about a total volume of 15–20 ml with a total surface area of 150 cm2 ; therefore, volume that can be delivered into the nasal cavity is restricted to 25–200 l [36,37]. The total volume of developed cholinesterase inhibitor nanosuspension administered to the nasal cavity of the animals was 0.1 ml (100 l), which showed a safe delivery system since; no toxicity in nasal mucosa and brain was observed, with the treated animals. Thus nanosuspension is a good approach to deliver drugs to brain in neurodegenerative disorder via nasal route of administration. The data showed that the nanosuspension of concentration (0.5, 1 and 1.5 mg/ml) of donepezil loaded chitosan nano particulate suspension is safe which has been proved by morphological, hematological and histopathological parameters. Hence, developing a delivery system via intranasal route to brain can be an achievement for treating neurodegenerative disorders of brain. 7. Conclusion In the present study, we described the development of donepezil loaded nanosuspension using the ionic cross linking method. This formulation was able to show higher drug concentration in brain and no mortality, hematological changes, body weight variations and histopathological changes in animals, when formulation was administered in different doses as compared to normal saline administered intranasally. Thus it is concluded that donepezil loaded nanosuspension is capable of providing direct nose-to-brain delivery, thereby enhancing drug concentration in the brain. It is anticipated that the present manuscript will assist researchers in implementing the utility of in vivo safety studies which must be performed for different drugs, vaccines and challenge studies for drug safety in the nasal mucosa while administration through intranasal route. Conflicts of interest statement The authors declare that there are no conflicts of interest.
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Acknowledgments The authors are thankful for financial support provided by Indian Council of Medical Sciences (ICMR), to carry out the research work under Project No. 45/26/2007-PHA/BMS, New Delhi, India. The authors are also thankful to Department of Nuclear Medicine, Institute of Nuclear Medicine and Allied Sciences (INMAS), DRDO, (Proposal No. INM/TS/IEC/007/07). Delhi for providing experimental animals for in vivo studies. References [1] [2] [3] [4] [5]
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