PREPARATION AND EVALUATION OF NANOPARTICLES CONTAINING

AN ANTI-ALZHEIMER’S DRUG

SYNOPSIS FOR

M. PHARM DISSERTATION

SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA

BY

AJAY KUMAR K.R

I M. PHARM

Department of Pharmaceutics

Dayananda Sagar College of PharmacY

2011

ANNEXURE-II

PROFAMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. / Name of the candidate and address (in block letters) /

AJAY KUMAR K.R

I M. PHARM
DEPARTMENT OF PHARMACEUTICS
DAYANANDA SAGAR COLLEGE OF PHARMACY
KUMARASWAMY LAYOUT
BANGALORE-560078
PERMANENT ADDRESS
AJAY KUMAR K.R
s/o ramesha k
#732, krushika, 11TH CROSS
CHAMUNDESHWARI NAGAR
MANDYA – 571 401
KARNATAKA.
2. / Name of the institute / Dayananda Sagar College of Pharmacy, Shavige Malleswara Hills,
Kumaraswamy Layout,
Bangalore-560078,
Karnataka.
3. / Course of study and subject / Master of Pharmacy in Pharmaceutics
4. / Date of admission to course / 29 July 2011
5. / Title of the project:
PREPARATION AND EVALUATION OF NANOPARTICLES CONTAINING AN ANTI-ALZHEIMER’S DRUG
6. / Brief resume of the intended work
6.1 Need of the study
Alzheimer’s disease1 represents the most common form of dementia worldwide, affecting more than 35 million people. It is a slowly progressive disease of the brain that is characterized by impairment of memory and eventually by disturbances in reasoning, planning, language, and perception. It is believed that Alzheimer's disease results from an increase in the production/accumulation of a specific protein called beta-amyloid protein in the brain that leads to nerve cell death. The main risk factor for Alzheimer's disease is increased age. As the population ages, the frequency of Alzheimer's disease continues to increase.
Drug targeting especially targeting of drugs by nanoparticles2 have been getting much attention by the researchers for treating various central nervous system disorders including Alzheimer’s disease. Drugs are incorporated into nanoparticles which have been fabricated in conventional ways. Drug delivery into the brain using nanoparticles has several advantages which includes: (1) reducing the dose of a therapeutic drug which, when given peripherally, maintains the biological potency in the nervous system, (2) allowing drugs that normally do not cross the blood-brain barrier to penetrate into the nervous system, and (3) reducing the peripheral side effects by increasing the relative amount of the drug reaching the brain.
Hence, in the present study, considering the effectiveness of nanoparticles for treating Alzheimer’s disease, an attempt will be to deliver an Anti-Alzheimer’s drug using nanoparticles.
6.2 Review of literature
Luppi et al3. prepared albumin nanoparticles using cyclodextrins for nasal delivery of the anti-Alzheimer drug tacrine. Bovine serum albumin nanoparticles were prepared using a coacervation method, followed by thermal cross-linking, starting from protein solution at alkaline pH. After preparation, nanoparticles were loaded by soaking from solutions of tacrine hydrochloride and lyophilised. Thermal analysis supported by Fourier Transform infrared spectroscopy were performed in order to confirm protein crosslinking in nanosphere structure and possible drug/carrier interaction occurred after the loading process. Moreover, size, polydispersity, zeta potential and morphology of the nanoparticles were investigated. Results indicated that all the nanoparticles presented a mean size and a polydispersity lower than 300 nm and 0.33 nm, respectively, were spherical shaped and negatively charged even after drug loading.
Wilson et al4. investigated the possibility of targeting tacrine into the brain using polymeric nanoparticles. The drug coated poly(n-butylcyanoacrylate) nanoparticles were prepared by emulsion polymerization and further coated with 1% polysorbate 80. The animal studies revealed that, in the brain a significant increase in tacrine concentration was observed in the case of poly(n-butylcyanoacrylate) nanoparticles coated with 1% polysorbate 80 compared to the uncoated nanoparticles and the free drug. The study concluded that the brain concentration of intravenously injected tacrine can be enhanced by binding to poly(n-butylcyanoacrylate) nanoparticles, coated with 1% the nonionic surfactant polysorbate 80.
Joshi et al5. prepared rivastigmine loaded nanoparticles using poly(lactic–co-glycolic acid) (PLGA) and polybutylcyanoacrylate (PBCA) as carriers. The pharmacodynamic performances of the nanoparticles were evaluated for brain targeting and memory improvement in scopolamine-induced amnesic mice using Morris Water Maze Test. PLGA nanoparticles were prepared by nanoprecipitation technique, while PBCA nanoparticles were prepared by emulsion polymerization technique. Effect of key formulation variables on particle size and percentage drug entrapment of nanoparticles was studied by using factorial design. Differential scanning colorimetry (DSC) thermograms indicated that rivastigmine was dispersed as amorphous state in both PLGA and PBCA nanoparticles. Transmission electron microscopy (TEM) studies indicated that the nanoparticles were spherical. Pharmacodynamic study demonstrated faster regain of memory loss in amnesic mice with both PLGA and PBCA nanoparticles when compared to rivastigmine solution.
Cui et al6. made an attempt to conjugate the Cu (I) chelator D-penicillamine covalently to nanoparticles via a disulfide bond or a thioether bond. Nanoparticle-chelator conjugates were stable between pH 6–8 in aqueous suspension if stored at 480C, and did not aggregate when challenged with salts and serum. Release of D-penicillamine from the nanoparicles was achieved using reducing agents such as dithiothreitol (as a model for glutathione). These studies indicated that nanoparticles have potential to deliver D-penicillamine to the brain for the prevention of b-amyloid accumulation, as well as to reduce metal ion accumulation in other CNS diseases.
Zhang et al7. prepared donepezil microparticles and evaluated its advantage as a sustained release delivery system with subcutaneous injection once a month. Donepezil microparticle was prepared using poly (D,L-lactide-co-glycolide) (PLGA) by an oil-water emulsion solvent evaporation technique. Donepezil microparticles showed the loading ratio 13.272.1% (w/w) and yield 54.870.8% with mean particle size about 75 mm. In vitro release of donepezil microparticles showed that donepezil completely released within 28 days in water, but the cumulative release percentages up to day 30 were 98.4% and 49.1% for phosphate buffer saline (PBS, pH 5.8) and PBS (pH 7.4), respectively. The in vivo experiment demonstrated that donepezil microparticles (90 mg/kg) produced a sustained release process in rats, and reached steady-state concentration at day 8 and maintained until day 27 with steady-state levels of donepezil between 130.3778 and 12179.8 ng/ml. donepezil microparticles (90 mg/kg) by subcutaneous infusion in rats produced the same pharmacological role as free donepezil (3 mg/kg day) by oral application route. These results implicated that donepezil microparticles as a sustained release delivery.
Park et al8. made an attempt to deliver the inorganic–organic hybrid as a drug delivery system by intercalating donepezil molecules into smectite clays (laponite XLG, saponite, and montmorillonite). According to the powder x-ray diffraction (XRD) patterns, Thermo gravimetric (TG) profiles, and Fourier transform infrared spectra (FTIR), it was confirmed that donepezil molecules were well stabilized in the interlayer space of clay via mono or double layer stacking. The adsorption amount and molecular structure of donepezil appeared to depend on the cation exchange capacity of the clay, which in turn, tailored the drug release patterns. Especially in the presence of a bulky cationic polymer (Eudragit® E-100) in the release media, the release rate was found to be improved due to its effective replacement with intercalated donepezil molecules. Therefore, to formulate a complete drug delivery system, the hybrids were coated with Eudragit® E-100 using a spray dryer, which also showed great enhancement in the release rate during a short period of time (180 min).
Mittal et al9. made an attempt to develop Tween 80 (T-80) coated polylactide-co-glycolide (PLGA) nanoparticles that can deliver estradiol to the brain upon oral administration. Estradiol containing nanoparticle were made by a single emulsion technique and T-80 coating was achieved by incubating the re-constituted nanoparticles at different concentrations of T-80. The nanoparticles were then evaluated in an ovariectomized (OVX) rat model of Alzheimer’s disease that mimics the postmenopausal conditions. The nanoparticles bound T-80 were found to proportionally increase from 9.72 ± 1.07 mg to 63.84 ± 3.59 mg with an increase in the initial concentration T-80 from 1% to 5% and were stable in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Orally administered T-80 coated nanoparticles resulted in significantly higher brain estradiol levels after 24 hours as compared to uncoated one at a dose of 0.2 mg/rat. Also, the nanoparticle treated group was successful in preventing the expression of amyloid beta-42 (Aβ42) immunoreactivity in the hippocampus region of brain. Together, the results indicate the potential of nanoparticles for oral delivery of estradiol to brain.
Wilson et al10. investigated chitosan nanoparticles as a delivery system for the delivery of tacrine. Tacrine-loaded chitosan nanoparticles were prepared by spontaneous emulsification. The particle size and zeta potential was determined by scanning probe microscopy and zetasizer, respectively. The prepared particles showed good drug-loading capacity. The in vitro release studies showed that after the initial burst, all the drug-loaded batches provided a continuous and slow release of the drug. Coating of nanoparticles with Polysorbate 80 slightly reduced the drug release from the nanoparticles. Release kinetics studies showed that the release of drug from nanoparticles was diffusion-controlled, and the mechanism of drug release was Fickian. The biodistribution of these particles after intravenous injection in rats showed that of nanoparticles coated with 1% polysorbate 80 altered the biodistribution pattern of nanoparticles.
Dong et al11. studied whether long-term administration of donepezil would slow amyloid plaque deposition in a mouse model of Alzheimer’s disease. Quantitative light and electron microscopy were used to investigate the effects of long-term administration (from 3 to 9months of age for 6months of treatment) of donepezil (1, 2, 4mg/kg, in drinking water) on tissue amyloid-β (Aβ) protein, plaque deposition, synaptic protein and synapse density in the hippocampus of Tg2576 mice. Administration of the 4mg/kg dose of donepezil, as compared to vehicle and lower doses of donepezil, significantly reduced brain tissue soluble Aβ1–40 and Aβ1–42, Aβ plaque number, and burden at the study end point in Tg2576 mice. The dose of 4mg/kg of donepezil also significantly increased synaptic density in the molecular layer of the dentate gyrus in Tg2576 mice. However, a significant change of the synaptophysin-positive bouton in the hippocampus was not observed. These results suggest that a higher dose of donepezil may have a impact on tissue level of Aβ protein and plaque deposition and may prevent synapse loss in the Tg2576 mouse model of Alzheimer’s disease.
6.3 Objective of the study
The objective of the study is to develop polymeric nanoparticles containing an anti-Alzheimer’s drug, which is expected to,
Ø  Improve site specificity.
Ø  Maintain the therapeutic drug concentration at the site of action for a prolonged period of time.
Ø  Improve the drug’s efficiency.
Ø  Reduce the dose related side effects.
6.4 Plan of work
The work will be executed as follows :
Ø  Selection of suitable drug and polymer for the preparation of nanoparticles.
Ø  Preformulation studies.
Ø  Optimizing the procedure for the preparation of nanoparticles.
Ø  Formulation of different batches of nanoparticles of anti-Alzheimer’s drug.
Ø  Evaluation of prepared nanoparticles, include :
·  Process yield
·  Particle size analysis
·  Percentage of drug loading
·  In vitro drug release studies
·  Release kinetics
Ø  Animal studies – optional
7. / Materials and methods
7.1 Source of data
Official Pharmacopoeia, Standard books, Pharmaceutical databases, internet, etc.
7.2 Drug
Anyone of the following drugs will be selected based on its suitability
1. Donepezil
2. Tacrine
3. Rivastigmine
4. Galantamine
5. Memantine
7.3 Polymer
Anyone of the following polymers will be selected based on its suitability for making nanoparticles
1. Chitosan
2. Poly(n-butyl cyanoacrylate)
3. Albumin
4. Gelatin
5. PLGA
7.4 Method of preparation
Anyone of the following methods will be used. The selection of suitable method will be based on the nature of the drug and polymer used.
1. Ionic gelation
2. Spontaneous emulsification
3. Emulsion polymerization
4. Emulsion cross-linking
7.5 Animal studies
The drug loaded nanoparticles will be evaluated for their effectiveness in improving the memory by the scopolamine-induced amnesic mice using Morris Water Maze Test. The effectiveness of the prepared nanoparticles to deliver the drug into the brain will be determined on rats.
7.6 Method of collection of the data (including sampling procedure, if any)
The pharmacological details of the drug will be collected from various standard books, journals and other sources like research literature databases such as Medline, Science Direct, etc.
Experimental data will be collected from the evaluation of designed formulation and then subjecting the formulation to different studies such as preformulation, process yield, particle size, percentage of drug loading, release profile, stability studies, etc.
The outline of such methods that would be adopted includes
1.  Selection of drug and polymer for the development of nanoparticles.
2.  Pre-formulation studies..
3.  Selection of suitable drug polymer ratio for the study.
4.  Development of nanoparticles based on studies in step 2 and 3.
5.  Evaluation of the prepared nanoparticles.
7.7. Does it require any investigation or interventions to be conducted or patients or other humans or animals? If so please describe briefly:
Yes – Swiss Albino Rats and Albino mice.
Animals are required to find out the effectiveness of the prepared formulations.
7.8. Has ethical clearance been obtained from your institute in case of 7.7
Yes
8. / List of references
1.  Rang HP, Dale MM, Ritter JM, Flower RJ. Rang and Dale’s Pharmacology. 6th ed. Philadelphia: Churchill livingstone; 2007:pp 514-17.
2.  Margaret F, Bennewitz, Mark Saltzman W. Nanotechnology for the delivery of drugs to the brain for epilepsy. J Nutr 2009;6:323–36.
3.  Luppi B, Bigucci F, Corace G, Delucca A, Cerchiara T, Sorrenti M, et al. Albumin nanoparticles carrying cyclodextrins for nasal delivery of the anti-Alzheimer drug tacrine. Eur J Pharm 2011;44:559–65.
4.  Wilson B, Samanta MK, Santhi K, SampathKumar KP, Paramakrishnan N, Suresh B. Targeted delivery of tacrine into the brain with polysorbate 80-coated poly(n butylcyanoacrylate) nanoparticles. Eur J Pharm Biopharm 2008;70:75–84.
5.  Joshi SA, Chavhan SS, Krutika K, Sawant. Rivastigmine loaded PLGA and PBCA nanoparticles: Preparation, optimization, characterization, in vitro and pharmacodynamic studies. Eur J Pharm Biopharm 2010;76:189–99.
6.  Cui Z, Lockman PR, Atwood CS, Hsu CH, Gupte A, David D, et al. Novel D-penicillamine carrying nanoparticles for metal chelation therapy in Alzheimer’s and other CNS diseases. Eur J Pharm Biopharm 2005;59:263–72.