A METHOD OF DEVELOPING MEMANTINE HCL LOADED CHITOSAN-SODIUM TRIPOLYPHOSPHATE (STPP) NANOPARTICLES

Abstract

To develop Memantine HCl loaded chitosan - sodium tripolyphosphate (STPP) nanoparticles using Ionic gelation method and evaluates their physicochemical properties and in-vitro release studies for possible targeted delivery to the brain. To fabricate polymeric nanoparticles for better controlled and targeting action of drug, which also overcome the problems associated with conventional formulations like multidose therapy, poor patient compliance and high cost. Memantine HCl loaded chitosan nanoparticles (F1 to F6) were prepared by Ionotropic gelation method. The formulated nanoparticles were evaluated for external morphological characters, determination of particle size analysis, zeta potential, drug content, entrapment efficiency and in-vitro release studies. The particle size varied from 148 to 317 nm and zeta potential was in negative and its value found to be - 46.4 mV. The drug content for the Memantine HCl loaded chitosan nanoparticles varied from 69.5 ± 7.2% to 87.9 ± 1.2%. The entrapment efficiencies were found to be minimum and maximum of 55.50 ± 2.4% and 86.30 ± 3.6%. The percentage yields of all formulations were in the range of 48.24 ± 1.24 to 86.13 ± 1.37%. In-vitro release of drug follows zero order and showed sustained release behaviour for a period of 24 hr. The optimized formulation contains 3:1 ratio of chitosan & STTP and demonstrated successful sustained release. Memantine HCl loaded chitosan nanoparticle is a potential new delivery system for treatment of Alzheimer’s disease.

Specification

Description:FIELD OF THE INVENTION
[0001] The present invention is related to develop Memantine HCl loaded chitosan-sodium tripolyphosphate (STPP) nanoparticles more particularly using Ionic gelation method.

BACKGROUND
[0002] The preparation of new simple Chitosan nanoparticle delivery system is very important and could have many applications particularly in pharmaceutical field to be used as a drug delivery system. The production of robust Chitosan nanoparticles has been developed and characterized by chemical crosslinking using sulfate anions. Approach: Chitosan polymer is considered one of the best polymers used in the field of Nanomedicine due to its safety, biocompatibility, biodegradability and environment friendly. Therefore, the development of a new method for the production of Chitosan nanoparticles should be of great importance for the pharmaceutical industry applications. The method was found to produce Chitosan sulfate capable carrying drug molecules which could explain the importance of such technique. Results: The size of Chitosan sulfate nanoparticles was determined using different LMW Chitosan HCl and sodium sulfate were confirmed by Laser diffraction, DSC and FTIR spectroscopy and it was tested for its dissolution rate. Conclusion/Recommendations: LMW Chitosan sulfate nanoparticles were relatively stable in aqueous medium and exert a slow rate of dissolution when placed in acidic medium. The properties of the Chitosan sulfate nanoparticles are considered suitable carriers in Nanomedicine and drug delivery technology.

[0003] Polymeric nanoparticles have been extensively studied as particulate carriers in the pharmaceutical and medical fields, because they show promise as drug delivery systems as a result of their controlled- and sustained-release properties, subcellular size, and biocompatibility with tissue and cells. Several methods to prepare nanoparticles have been developed during the last two decades, classified according to whether the particle formation involves a polymerization reaction or arises from a macromolecule or preformed polymer. In this review the most important preparation methods are described, especially those that make use of natural polymers. Advantages and disadvantages will be presented so as to facilitate selection of an appropriate nanoencapsulation method according to a particular application.

[0004] Polymeric nanoparticles have been extensively studied as particulate carriers in the pharmaceutical and medical fields, because they show promise as drug delivery systems as a result of their controlled- and sustained-release properties, subcellular size, and biocompatibility with tissue and cells. Several methods to prepare nanoparticles have been developed during the last two decades, classified according to whether the particle formation involves a polymerization reaction or arises from a macromolecule or preformed polymer. In this review the most important preparation methods are described, especially those that make use of natural polymers. Advantages and disadvantages will be presented so as to facilitate selection of an appropriate nanoencapsulation method according to a particular application.

SUMMARY
[0005] The various trials with change in the concentration ratio of chitosan and STPP had proven that, the best suitable concentration is 3:1. The particle size & zeta potential of optimized formulation (F3) was found to be 148 nm & - 46.4, which indicates that the formulation was having good stability. The nano formulations were designed for sustained release of the drug for a period of 24 h and this may reduce the frequency of dosing, thereby minimizing the occurrence of side effects. From this, it was concluded that F3 formulation was considered to be the best formulation and serves as a potential formulation for the treatment of Alzheimer's disease. But, more animal studies and extensive clinical studies are needed to examine and justify the efficacy of the prepared drug delivery system.

DESCRIPTION OF THE FIGURES:
[0006] Figure1: illustrates IR Spectra of memantine HCl.

[0007] Figure 2: illustrates λ max of Memantine HCl.

[0008] Figure 3: illustrates IR Spectra of Memantine HCl.

[0009] Figure 4: illustrates IR Spectra of Chitosan.

[0010] Figure 5: illustrates IR Spectra of STPP.

[0011] Figure 6: illustrates IR Spectra of Memantine HCl + Chitosan.

[0012] Figure 7: illustrates IR Spectra of Memantine HCl + Chitosan + STPP.

[0013] Figure 8: illustrates DSC thermo grams of (A) pure Memantine HCl (B) Chitosan and (C) Memantine HCl + Chitosan.

[0014] Table 1: illustrates standard calibration curve of memantine HCl.

[0015] Figure 9: illustrates the Standard Calibration Curve of Memantine HCl.

[0016] Figure 10: illustrates scanning electron micrographs of Nanoparticles formulations F1 - F6.

[0017] Figure 11: illustrates Size analysis of Nanoparticles formulations F1- F6.

[0018] Table 2: illustrates Size Analysis.

[0019] Figure 12: illustrates Zeta potential of nanoparticles formulation F3.

[0020] Table No 3: illustrates drug Content.

[0021] Table 4: illustrates drug entrapment efficiency.

[0022] Table - 5: illustrates percentage yield (Mean  S.D. of three determinations).

[0023] Table-6: illustrates percentage cumulative drug release.

[0024] Fig-13: illustrates In vitro drug release studies.

[0025] Table-7: illustrates kinetic modelling of drug release for F3 formulation.

[0026] Table-8: illustrates accelerated stability studies of optimized formulations F3.

DETAILED DESCRIPTION OF THE INVENTION:
[0027] Memantine Hydrochloride was purchased from A. S. Joshi & Company, Mumbai. Chitosan obtained from Rolex chemical Industries, Sodium Tripolyphosphate was obtained from Sisco research laboratory Pvt. Ltd, Mumbai and all other ingredients used were of analytical grade.

[0028] Identification of Pure Drug: FTIR spectroscopy was used for identification of pure drug. Determination of λ max: Preparation of Stock Solution: Accurately weighed 10 mg of Memantine Hydrochloride was transferred in a 100 ml volumetric flask. To the flask, phosphate buffer was added in small proportion so as to dissolve Memantine Hydrochloride. The volume was made up to 100 ml with phosphate buffer pH 6.8 to get a concentration of 100 μg/ml.

[0029] Determination of λ max: 20 μg/ml solution of Memantine Hydrochloride was prepared and transferred into a 10 ml volumetric flask to complete the volume to 7 ml followed by addition of 1.4 ml eosin reagent and 1.2 ml of 0.2 M acetate buffer pH 3.6. The resulting solution was scanned in UV - Vis spectrophotometer from 600 - 400 nm to determine the λ max.

[0030] Compatibility studies: A successful formulation of a stable and effective dosage form depends on selection of excipients that are added to promote the consistent release and bioavailability of the drug and protect it from degradation. If the excipients are new and not been used in formulation containing the active substance, the compatibility studies are mandatory.

[0031] Drug - polymer compatibility studies by FTIR: This was confirmed by infrared light absorption scanning spectroscopy (IR) studies. Infra-red spectra of pure drug and mixture of formulations were recorded by dispersion of drug and mixture of formulations in suitable solvent (KBr) using Fourier Transform Infrared Spectrophotometer.

[0032] Drug-polymer compatibility studies by DSC: The differential scanning calorimetry analysis was performed for the compatibility studies between the drug and the polymer. Each sample was sealed in Aluminium disc and purged with air at a flow rate of 40 ml / min and maintain the temperature at250C - 2000C. The DSC spectrum of the pure Memantine hydrochloride was compared with mixture of the Memantine hydrochloride and the chitosan.

[0033] Calibration of standard curve: Accurately weighed 100 mg of Memantine Hydrochloride was dissolved in 100 ml of pH 6.8 phosphate buffer solution. 10 ml of this solution was further diluted up to 100 ml with 6.8 pH phosphate buffer to give a solution of Concentration 100 µg/ml. This resultant solution is used as working stock solution and further dilutions were prepared from the same solution. Aliquots of 0.1 mg/ml Memantine Hydrochloride standard working solution were transferred into a set of 10 ml volumetric flasks to produce solutions within the concentration range of 10, 20, 40, 60, 80 and 100 µg/ml. The volumes were finally completed with buffer and the coloured solutions were measured for absorbance at 557 nm. A calibration curve of absorbance against concentration was plotted and the drug follows the Beer's & Lambert's law in the concentration range of 10 - 100 µg/ml. The regression equation and correlation coefficient was determined.

[0034] PREPARATION OF NP'S BY IONICALLY CROSSLINKED METHOD: Memantine hydrochloride (100 mg) was dissolved in aqueous solution containing STPP (1% w/v). This aqueous solution was sonicated and then added dropwise to the 1% v/v acetic acid containing chitosan (1-6%). This mixture of solution was stirred under gentle magnetic stirring. After 3 hours of crosslinking, nano¬particles were isolated by centrifugation at 5,000 RPM and 5°C for 30 minutes, and subsequently washed several times with water. The par¬ticles were lyophilized and stored in dry conditions at 25°C. The nanoparticles were prepared with different concentration ratio of chitosan and STPP.

[0035] CHARACTERISATION OF PREPARED NP's4: The obtained formulations of Memantine hydrochloride loaded chitosan nanoparticles are characterized for following parameters. Particle Size Analysis & Surface Morphology: Particle Size Analysis: Measurement of particle size was performed by Photon Correlation Spectroscopy (PCS) known as Dynamic Light Scattering using a Zetasizer® 3000 (Malvern Instruments, NIPER, Mohali). All samples were diluted with ultra purified water & measured at 25° and 90° scattering angle, recorded for 180 s. The mean diameter for each sample was generated by cumulative analysis in triplicate.

[0036] Morphological Studies: The morphological examination of nanoparticles was performed using scanning electron microscopy (SEM) (Tecnai 20 G2 S TWIN at Punjab University, Chandigarh; set at 200 kV). At structural point of view, the arrangement of components and orientation of molecules within the nanoparticle can determine its behavior and stability. For this purpose, scanning electron microscopy (SEM) was employed.

[0037] Surface Charge determination: Nanoparticles were characterized with Zeta potential (ζ) using a Zeta Sizer (Suralabs., Hyderabad). The measurements were performed using an aqueous dip cell in an automatic mode by placing diluted samples (with ultra-purified water) in the capillary measurement cell and cell position was adjusted.

[0038] Entrapment Efficiency: The Entrapment Efficiency (EE %) is also known as Association Efficiency. The drug loaded nanoparticles were centrifuged at a high speed of 3500 - 4000 rpm for 30 min and the supernatant was assayed for non-bound drug concentration by using spectrophotometer. Entrapment efficiency was then calculated as follows:
EE %=(Total amount of drug added - Non-bound drug )/(Total amount of drug added)× 100



[0039] Drug content: Drug content was determined by centrifugation method. The nanoparticles suspension was centrifuged at 15,000 rpm for 40 min at 25°C to separate the free drug in the supernatant. Concentration of Memantine Hydrochloride in the supernatant was determined by UV - Vis spectrophotometrically at 557 nm after suitable dilution.

[0040] Percentage yield: It is determined by the equation

Percentage yield=(Weight of dried nanoparticles recovered )/(Sum of initial dry weight of starting material )×100

[0041] In vitro release studies: In vitro drug release studies were conducted by means of incubator orbital shaker. 50 mg of each accurately weighed formulation was transferred into 250 ml conical flask containing 100 ml pH 6.8 phosphate buffer. They were kept in an orbital shaker at 50 rpm maintained at 37°C. Aliquots of 5 ml buffer were withdrawn at predefined time intervals and the medium was replaced with same volume of buffer. The withdrawn samples are centrifuged at 4000 rpm for 15 min. The supernatant was collected. This study was carried out for 24 h, and the concentration of drug release was estimated by determining the absorbance at 557 nm using UV spectrophotometer6.

[0042] Kinetic modelling: In order to understand the kinetics and mechanism of drug release from optimized formulation F3, the result of in vitro drug release study of nanoparticles were fitted with various kinetic equations like zero order (cumulative % release vs. time), first order (log % drug remaining vs. time), Higuchi's model (cumulative % drug release vs. square root of time). R2 and k values were calculated for the linear curve obtained by regression analysis of the above plots7.

[0043] Stability study: The stability study was carried using the batch F3. The stability of drug loaded nanoparticles was evaluated in terms of its drug content, entrapment efficiency and in vitro drug release. The stability of nanoparticles was evaluated in PBS (pH 6.8). Nanoparticles formulation was incubated at 37 ± 1°C for a period of 90 d. After specified time intervals, the suspension was centrifuged at 4,000 rpm for 1 h, supernatant was removed and the amount of drug was detected by UV - Vis spectrophotometrically method at 557 nm8.

[0044] Identification of Pure Drug: FT - IR spectroscopy was used to determine the functional group present in the pure drug sample. The FTIR spectrum of pure Memantine HCl has shown the characteristic peaks at 2978.73, 2941.58, 2859.39, 2896.81, 2838.91, 1511.78, 1455.27, 1355.83, 436.30 and 448.78 cm−1. The absorption bands between 2800 and 3200 cm-1was indicates presence of -NH stretching of amine or amide groups. The wave numbers observed at 1511 and 1455 may be assigned to the C = O and C - N bonds respectively and the sharp peak occurred at 1511 indicates presence of C = O group attached to -NH. IR spectra of Memantine HCl is as follows:

[0045] Determination of λmax: The Memantine HCl with ESN in acidic medium forming an orange-red ion-pair complex was scanned in UV - Vis spectrophotometer from 800 - 400nm to determine the λmax. The λmax was found to be at 557 nm, so the calibration curve of Memantine HCl was developed at this wavelength.

[0046] Drug - Excipient Compatibility Studies: Fourier Transform Infra Red Spectroscopy (FTIR):The interaction studies were carried out to ascertain any kind of interaction of drug with the excipients used in the preparation of polymeric nanoparticles. Physical mixture of memantine and each selected excipients were prepared in the 1:1 w/w ratio gently blending with spatula at room temperature. The blends were considered homogeneous mixture when the mixture is used for IR analysis.

[0047] The FTIR spectra of Memantine HCl were recorded on a FTIR multiscope spectrophotometer (Brooker) equipped with spectrum 11.0.0.0449 software using KBr pellet method. The spectrum for each sample was recorded over than 400 - 4000 cm-1. The FTIR spectra of the pure drug and formulations were shown in Figures 3 - 7.
Inference: The FTIR spectrum of formulations had shown characteristic absorption bands which were comparable with absorption bands of individual sample. The results illustrated that, there were no chemical instabilities in drug - excipient combinations.

[0048] Differential Scanning Calorimetry (DSC): Drug excipient interactions play a vital role with respect to release of drug from the formulation amongst others. DSC has been used here to study the physical and chemical interaction between the drug and excipients used. The DSC thermo grams obtained are displayed in Figure 8. It shows that the decomposition temperature of drug was 341oC and formulation was 342oC. It indicates there is no chemical interaction between Memantine HCl and the polymer Chitosan used.

[0049] Inference: The DSC thermogram revealed that the formulation showed superimposition of drug, however mild shift was observed. The DSC results revealed that there was no interaction between the drug and additives used in the formulation. Standard Graph of Memantine Hcl: The standard curve of Memantine HCl was done by using pH 6.8 PBS as the medium and the maximum absorbance was found at 557 nm. The standard graph was constructed by making the concentrations of 20 µg/ml, 40 µg/ml, 60 µg/ml, 80 µg/ml and 100 µg/ml solutions. The absorbance of solutions was examined under UV - visible spectrophotometer at an absorption maximum of 557 nm. The standard graph was constructed by taking the absorbance on Y - axis and concentrations on X - axis. The standard calibration curve of Memantine HCl in pH 6.8 PBS was shown in Fig 9. Drug concentration and absorbance followed linear relationship the curve obeyed Beer - Lambert's law and the correlation coefficient value (R2) is 0.997.

[0050] PREPARATION OF NANOPARTICLES: Nanoparticles of Memantine HCl were prepared by Ionotropic gelation technique using Chitosan and STPP polymers in varying concentration ratio like 1:1 to 6:1. The chitosan nanoparticles were prepared based on the ionic interaction of a positively charged chitosan solution and negatively charged STPP solution. The charge density of both chitosan and STPP solution has a great effect on the ionic interaction. Total six batches were formulated (F1, F2, F3, F4, F5 & F6) and all the formulations were investigated for various parameters like Scanning electron microscopy (SEM), particle size, Zeta potential, Drug content, Entrapment efficiency, Percentage yield, in vitro drug release and drug release kinetics.

[0051] Scanning Electron Microscopy (SEM): SEM analysis of the F1 showed that the nanoparticles has irregular structure and the view of F2 - F6 showed that the nanoparticles are hollow spherical structure with a large central cavity in which Memantine HCl was loaded. The outer surface of the nanoparticles was smooth and shell of the nanoparticles also showed some porous structure. SEM analysis of formulations F1 - F6 is shown in Figure 10.

[0052] Size Analysis: Six formulations have been developed by varying the concentration of chitosan and the effect of particle size has been determined. The concentration ratio of chitosan and STPP 3:1 the particle size was found to be 148 nm. The formulations of varying the concentration of chitosan to STPP such as 1:1, 2:1, 4:1, 5:1, 6:1 was developed and particle size was found to be 190 nm, 195 nm 220 nm, 284 nm and 317 nm. The results indicate that the particle size increases with increased concentration ratio of chitosan and STPP. Variations in particle size to increase the concentration are due to agglomeration of the particles. The formulation F3 showed minimum particle size of 148 nm compare to other formulations.

[0053] Surface Charge (Zeta Potential): The electrostatic repulsion between particles with the same electric charge prevents the aggregation of the particles. The zeta potential values of formulation F3 was in negative and this demonstrated that the anionic surface of drug delivery system would provide improved targeting ability as compared to the cationic carrier. The zeta potential of the formulation F3 with a concentration of chitosan 3:1 was found to be - 46.4, which implies that it is having good stability (Figure 12).

[0054] Drug Content: The drug content was evaluated for all the formulations and it was observed that the nanoparticles obtained from F1 formulation showed maximum drug content (87.9 ± 1.2%.) and F6 showed minimum drug content (69.5 ± 7.2%). The drug content was decreased with increase in chitosan concentration. This may be due to loss of drug during manufacturing stage or increase in entrapment efficiency, so that drug is not available for estimation. This result indicated that there was no drug loss by manufacturing process or by excipients used in the formulation.

[0055] Entrapment Efficiency: Prepared nano formulations were estimated for entrapment efficiency and the results are shown in the Table 4. Drug entrapment efficiency varied from 55.50 ± 2.4 to 86.30 ± 3.6%. This result indicated that drug entrapment efficiency increased with increasing concentration of polymer up to 0.3% (F3). After that, there was no significant increase in entrapment efficiency. This may be due to unavailability of drug for entrapment. This can be attributed to fact that higher extent of polymer resulted in formation of a more rigid network structure which prevent the leaching out of drug during preparation of nanoparticles. The optimum efficiency was based on the drug content and polymer usage. From drug content and entrapment efficiency results chitosan nanoparticles F3 were considered as optimum trials.

[0056] Percentage Yield: The percentage yield of nanoparticles prepared by ionotropic gelation method was recorded in Table 5 and it was determined by collected the nanoparticles and weighed. The measured weight was divided by the initial dry weight of starting materials, which were used for the preparation of the nanoparticles. The percentage yields of nanoparticles of all formulations were in the range of 48.24 ± 1.24 to 86.13 ± 1.37 %. It was found that when concentration of chitosan increased, the percentage yields also increased. The formulation F6 showed maximum percentage yield of 86.13 ± 1.37 % compare to other formulations.

[0057] In vitro release studies: From the in-vitro release studies of Memantine HCl loaded chitosan nanoparticles (F1 - F6), it was observed that release profiles in intestinal medium (pH 6.8 phosphate buffer) were found to have very good controlled efficacy. The drug release depends upon concentration of the polymer matrix and increase in polymer concentration produced much more time for release of drug for all formulations. High polymer concentration (≥0.4% chitosan - F4 to F6) showed slow drug release for more than 24 h. Low polymer concentration (≤0.2% chitosan - F1 to F2) showed quick drug release within short period. Hence the formulations (F1 to F2, F4 to F6) were considered to be not satisfactory for controlled delivery of Memantine HCl either by quick release or over retarding. Memantine HCl nanoparticles prepared with 0.3% chitosan (F3) showed controlled and sustained drug release for a period of 24 h. The percentage cumulative drug release of F3 at the end of 24 h was found to be 95.85± 0.54%. In vitro drug release of Memantine HCl loaded chitosan nanoparticles is shown in Figure 13.

[0058] Kinetic Modelling of Drug Release: Dissolution data of the optimized formulation F3 was subjected to regression analysis and were fitted to kinetic models (Table 7). The R2 value of zero order and first order was found as 0.9623 & 0.7106 respectively. This result suggests that the drug released by zero order kinetics. Further to ascertain the exact mechanism of drug release the dissolution data of the optimized formulation was subjected to Peppas and Higuchi's diffusion equation. The R2 value of Higuchi's and peppas diffusion equation was obtained as 0.9623 and 0.801 respectively. This result suggests that the drug released followed diffusion mechanism.

[0059] Accelerated Stability Studies: The optimized formulations F3 was wrapped and sealed in an aluminum foil. Results of stability studies are shown in Table-8. The % drug content, entrapment efficiency and In vitro release study of most satisfactory formulation was determined and result showed that there was no significant changes occurs during storage after 90 days.
, Claims:CLAIMS:
I/WE CLAIM:
1.A method of developing chitosan based polymeric nanoparticles of an anti-alzheimer's drug
memantine hydrochloride, comprising:
a 100 mg of memantine hydrochloride was dissolved in 100 ml of ph 6.8 phosphate buffer solution;
a 10 ml of this solution was further diluted up to 100 ml with 6.8 ph phosphate buffer to give a
solution of concentration 100 μg/ml;
whereby this resultant solution is used as working stock solution and further dilutions were
prepared from the same solution;
wherein aliquots of 0.1 mg/ml memantine hydrochloride standard working solution were
transferred into a set of 10 ml volumetric flasks to produce solutions within the concentration range
of 10, 20, 40, 60, 80 and 100 μg/ml;
wherein the volumes were finally completed with buffer and the coloured solutions were measured
for absorbance at 557 nm;
memantine hydrochloride (100 mg) was dissolved in aqueous solution containing stpp (1% w/v);
whereby this aqueous solution was sonicated and then added dropwise to the 1% v/v acetic acid
containing chitosan (1-6%);
wherein this mixture of solution was stirred under gentle magnetic stirring;
wherein after 3 hours of crosslinking, nanoparticles were isolated by centrifugation at 5,000 rpm
and 5°c for 30 minutes and washed several times with water;
the particles were lyophilized and stored in dry conditions at 25°c; and
the nanoparticles were prepared with different concentration ratio of chitosan and stpp3

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