About Authors:
Bajaj L.*, Chopra D.1
*PCTE Institute of Pharmacy, Jhandey, Ludhiana-142021, Punjab, India.
1Department of Pharmaceutical Sciences and Drug Reasearch,
Punjabi University. Patiala.
*lotika.bajaj@gmail.com / lotika@pcte.edu.in
Abstract
Alzheimer’s Disease is a neuropathological disorder that causes dementia by progressively degenerating the neurons that are responsible for learning and memory processes. Rivastigmine has demonstrated favorable efficacy and safety in patients with dementia. Nanostructure mediated drug delivery enhances drug bioavailability, improves the timed release of drug molecules, and enables precision drug targeting. Because of its cationic charges, biocompatibility, and low toxicity, chitosan has been used as a vehicle system for genes, protein and drugs. The present study has been undertaken to investigate the targeted delivery of rivastigmine loaded chitosan nanoparticles in streptozotocin induced dementia in mice. Rivastigmine loaded chitosan nanoparticles were prepared by ionic gelation method. The nanoparticles were evaluated for size, shape, zeta potential, microscopy, transmission electron microscopy. Drug-polymer compatibility was determined using differential scanning calorimetry. The amount of drug entrapped within the nanoparticles was determined spectrofluorometrically and in vitro drug release studies were done by spectrofluorometer. Morris Water Maze Test was used to evaluate in vivo activity of rivastigmine nanoparticles in mice. The mean hydrodynamic diameter of chitosan nanoparticles prepared was found to be 258 nm with positive zeta potential value of about 35.1 mV. The entrapment efficiency for various batches of rivastigmine nanoparticles showed a range of 42-84% w/w. Prolonged drug release was observed in case of chitosan nanoparticles. The best release pattern was seen in case of batch A with 1:1 drug : polymer ratio. Rivastigmineloaded Chitosan nanoparticles were found to be effective in STZ induced dementia in mice.
REFERENCE ID: PHARMATUTOR-ART-1742
Introduction
Among several causes of dementia, Alzheimer’s Disease is the most common. It involves progressive degeneration of neurons that are responsible for learning and memory processes (Standridgeet al., 2004; Iwata et al., 2005 and Goldberg, 2007). According to WHO it is estimated that there are currently about 18 million people worldwide suffering from Alzheimer’s Disease. This figure is estimated to nearly double by 2025 to 34 million. Symptoms include gradual development offorgetfulness, progressing to disturbances in language, disability to calculate, difficulty to judge location of objects in space and problem in moving (Giordano et al., 2007 and Shah et al.,2008).
Neuropathologically, the disease ischaracterized by a progressive loss of neurons and synapses with the presence of largenumbers of extracellular amyloid plaques and intracellular neurofibrillary tangles(Jacobsen et al., 2005). The earliest pathological event that occurs in the process of AD is the deposition of the amyloid-β peptide in insoluble forms within the brain. Other pathological features include extracellular senile plaques (mainly composed of amyloid-b peptide),intracellular neurofibrillary tangles, synaptic loss, and brain atrophy (Holtzman, 2001; Ling et al., 2003 and Fisher, 2008).
Loss and severe functional impairment of cholinergic axons is associated with reduction of the transmitter Acetylcholine (ACh), which is an essential neurotransmitter in the central and the peripheral nervous system. In neurodegenerative disease, cholinergic nuclei in the basal forebrain with diffuse projections into virtually all cortical areas appear to be most important. Degeneration and loss of trophic support for the cholinergic neurons of the basal forebrain and their projections is an early and pivotal event in AD, it has interaction with amyloid deposition and plaque formation (Herholz, 2008).
Two classes of drugs are approved for the treatment of Alzheimer’sdisease (AD). The first were the cholinesterase inhibitors (ChEI) (Pakasaki et al., 2008). The first drug of this class was tacrine in 1994, followed by donepezil, rivastigmine, and galantamine. All thesecholinesterase inhibitors are approved for the treatment of mild to moderate AD. Tacrine has shown severe hepatic side effects. Donepezil has a long half-life and is effective as a once daily drug administration with very less side effects (Herholz, 2008). It is therefore a poor candidate for sustained drug delivery dosage form. Galantamine also exhibits very long half-life of 7 hours (Lilienfeld, 2007). Rivastigmine has demonstrated favorable efficacy and safety in patients with dementia of the Alzheimer type and is widely approved for the treatment of mild to moderate AD (Tekin et al., 2006). It also inhibits Butyrylcholinestrase. In patients with dementia related to Alzheimer’s disease, rivastigmine has symptomatic effects that enable patients to do their work individually. Administered orally, it has short half-life of 1.5 hours due to hepatic first pass metabolism (Williams et al., 2003 and Arumugam et al., 2008).
Nanostructure mediated drug delivery enhances drug bioavailability, improves the timed release of drug molecules, and enables precision drug targeting (Hughes, 2005; Blasi et al., 2007 and Modi et al., 2009). They havedesired temporal and spatial deposition; drug release pattern, and decrease excessive and unfavorable biodistribution. This leads to delayed drug clearance and retards drug metabolism, resulting in their prolonged action (Craparo et al., 2008).
Chitosan is biocompatible, bioactive, and biodegradable polymer and can be easily engineered. It is widely used in preparing micro and nanoparticles. Because of its cationic charges, biocompatibility, and low toxicity, chitosan has been used as a vehicle system for genes, protein (including antibodies), and drugs (Prashanth et al., 2007; Malhotra et al., 2009; Singh et al., 2009 and Wilson et al., 2010).
In the present study chitosan nanoparticles of rivastigmine were prepared. They were evaluated for particles size, particle shape, zeta potential, entrapment efficiency and in vitro drug release (Craparo et al., 2008). Streptozotocin was used to induce dementia in mice. These animals were further used to determine in vivo efficacy of Rivastigmine nanoparticles in treatment of dementia associated with Alzheimer’s disease.
Materials and Methods
Materials
Rivastigmine was obtained as a gift sample from Sun Pharma Ltd. (Gujarat, India). Chitosan (MW=250 KDa; Degree of deacetylation=80%) was a gift from India Sea Foods (Cochin, India). Sodium Tripolyphosphate was purchased from Central Drug House Ltd., New Delhi. All other chemicals were of analytical grade and were used without further purification.
Preparation of chitosan Nanoparticles
Chitosan nanoparticles were prepared using Ionotropic gelation of chitosan with TPP anions (Soppimath et al., 2001; Agnihotri et al., 2004 and Chenguang et al., 2007). Ionotropic gelation happens when the positively charged amino groups in chitosan interact with the negatively charged TPP (Urrusuno et al., 1999). Chitosan was dissolved in purified water 0.1%, 0.15%, 0.2% and 0.25% w/v. Sodium triployphosphate (TPP) was also dissolved in purified water at various concentrations to obtain final ratios chitosan/TPP of 8/1, 7/1, 6/1, 5/1, and 4/1 w/w. The nanoparticles were formed spontaneously upon the incorporation of variable amount of the TPP solution into 3 ml of the chitosan solution, under magnetic stirring at room temperature (Chenguang et al.,2007).
For the association of rivastigmine to chitosan nanoparticles, rivastigmine was dissolved in purified water and then incorporated into TPP solution. The amount of rivastigmine was added such that various batches were obtained having drug/polymer ratio of 1/1, 1/2, 1/3, 1/5 and 1/7 w/w. Nanoparticles were centrifuged at 5000 x g for one hour. Supernatants were discarded and nanoparticles were resuspended in purified water for lyophilization and for in vitro characterization in phosphate buffer (pH 7.4) (Urrusuno et al., 1999). Further evaluation studies were carried out with lyophilized nanoparticles.
Physicochemical Characterization of nanoparticles
Particle Size and Polydispersity index
Measurements of particle size and polydispersity index were performed by Photon Correlation Spectroscopy (PSC) (Janes et al., 2001). Also known as Dynamic Light Scattering using a Zetasizer® 3000 (Malvern Instruments, NIPER, Mohali). All the samples were diluted with ultra-purified water. The size measurement was performed at 25°C at a 90° scattering angle, and it was recorded for 180 s for each measurement. For each sample, the mean diameter was triplicate. The mean hydrodynamic diameter was generated by cumulative analysis.
Surface Charge determination
Nanoparticles were characterized with Zeta potential (ζ) using a Zeta Sizer 4 (Malvern Instruments ltd., Malvern UK)(Craparo et al., 2008). The zeta potential measurements were performed by using an aqueous dip cell in an automatic mode. Samples were diluted in ultra-purified water and placed in the capillary measurement cell, with the cell position adjusted.
Morphology
The morphological examination of nanoparticles was performed by transmission electron microscopy (TEM) (Tecnai 20 G2 S TWIN at IIT Roorkee) set at 200 kV (Li et al., 2010). Nanoparticle suspension was placed on copper electron microscopy grids (Formvar filmed), air-dried and then viewed.
Structure
A good physicochemical understanding of the formulation is an absolute necessity for rational formulation design and properly interpreting in vivo results. From structural point of view, the arrangement of components and orientation of molecules within the nanoparticle can determine its behavior and stability and for this purpose scanning electron microscopy (SEM) (Tecnai 20 G2 S TWIN at Punjab University, Chandigarh; set at 200 kV) (Abdelwahed et al., 2006) was employed.
Differential scanning calorimetry
DSC studies were performed to understand the behavior of the cross-linked chitosan on application of thermal energy. Polysaccharides usually have a strong affinity for water and in solid state these macromolecules may have disordered structures that can be easily hydrated. The hydration properties of polysaccharides depend on primary and supramolecular structures. The endotherm related to evaporation of water is expected to reflect the molecular changes brought in after cross-linking. The cross-linking of chitosan with TPP at different pH modifies the crystalline nature of chitosan. Another thermal event observed was an exotherm due to the decomposition of the polymer. Owing to the differences in the chemical characteristics, changes in the exothermic peak of chitosan and cross-linked chitosan were also observed. The presence of free unsubstituted amine groups showed higher ΔH values.
Differential scanning calorimetry (DSC) was performed on a DSC-7 (Perkin-Elmer Corp., USA). A heating rate of 10°C/min was employed in the temperature range of 0-350°C. An empty aluminum pan was used as reference standard (Bhumkar et al., 2006).
Evaluation of Entrapment Efficiency
It 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 Spectrofluorometer (SL 174, Elico India) (Boonsongrit et al., 2005). Association efficiency was then calculated as follows:
EE or AE% = Total amount of drug added – Non-bound drug X 100
Total amount of drug
In vitro drug release studies
The Drug release profiles from nanoparticles were measured in vitro (Craparo et al., 2008). Forty milligrams of lyophilized nanoparticles were dispersed in 100 ml dissolution media consisting of phosphate buffer pH 7.4 previously equilibrated at 37oC in a shaker incubator at 100 rpm. At definite time interval, 2 ml of the dispersion was withdrawn and replaced with equivalent volume of dissolution medium to maintain the sink conditions. The amount of drug released from the nanoparticles was then analyzed spectrofluorometrically.
In vivostudies
Acute toxicity studies:Estimation of LD50 of Rivastigmine
For estimating LD50 overnight fasted male swiss albino mice (20-30gm) were used. Total of seven groups were employed in the study and each group comprised of 10 animals. Seven groups were administered different concentrations of rivastigmine intraperitoneally (1, 2, 3, 4, 5, 6 and 7 mgkg-1). Animals were observed for 4 h for death due to acute toxicity. The correction factor was applied to 0 and 100 percent mortality group. The percent mortality values were converted to probit values by reading the corresponding probit units from the probit table. Graph was plotted between probit values Vs log dose. LD50 was then calculated from the plot (Akhila et al., 2007).
Estimation of safety of rivastigmine loaded chitosan nanoparticles
Male swiss albino mice (20-30 gm) were used. Five groups were employed in the study and each group comprised of 10 animals. Each group was injected different concentrations of rivastigmine nanoparticles intraperitoneally (3, 4, 5, 6 and 7 mgkg-1) and animals were observed for 4 h for mortality.
Animals
Male swiss albino mice (25-30 g; procured from PAU, Ludhiana, India) were employed in the present study and were housed in animal house with free access to water and standard laboratory pellet chow diet (Kisan Feeds Ltd., Mumbai, India). The mice were exposed to 12 h light and 12 h dark cycle. The experiments were conducted between 9.00 to 18.00 h in a semi sound-proof laboratory. The animals were acclimatized to the laboratory condition five days prior to behavioral study. The protocol of the study was duly approved by Institutional Animal Ethics Committee (IAEC) and care of the animals was taken as per Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA).
Drugs and reagents
All the drug solutions and other reagents were freshly prepared before use. Rivastigmine tartrate and nanoparticles were suspended in purified water and streptozotocin (STZ) was dissolved in artificial cerebrospinal fluid (CSF).
Intracerebroventricular (ICV) administration of streptozotocin (STZ)
Male Swiss albino mice weighing 25–30 g were anesthetized with anesthetic ether. A polyethylene tube was placed around(except 3 mm of tip region) the hypodermic needle of 0.4 mm external diameter and it was attached to a 10 µl Hamilton microlitre syringe (Top Syringe, Mumbai, India) which was inserted perpendicularly through the skull (not more than 3 mm) into the brain of mouse. The injection site was 1 mm to right or left midpoint on the line drawn through to the anterior base of the ears. Injections were performed into right or left ventricle randomly. The STZ (3 mgkg-1) was administered ICVbilaterally in two divided doses, on the first and the third day. The concentration was adjusted so as to deliver 10 µl at the site. The control group was administered ICV injection of artificial cerebrospinal fluid (CSF) (Sharma, 2010).
Morris water-maze test
Morris water-maze test (MWM) was employed to assess learning and memory of the animals. MWM is a swimming based model where the animal learns to escape on to a hidden platform (Ashe et al., 2010). It consists of large circular pool (150 cm in diameter, 45 cm in height, filled to a depth of 30 cm with water at 28 ±1° C). The water was made opaque with white coloured non-toxic dye. The tank was divided into four equal quadrants with help of two threads, fixed at right angle to each other on the rim of the pool. A submerged platform (10 cm²), painted in white was placed inside the target quadrants of this pool, 1 cm below surface of water. The position of platform was kept unaltered throughout the training session. Each animal was subjected to four consecutive training trials on each day with inter-trial gap of 5 min. The mouse was gently placed in the water between quadrants, facing the wall of pool with drop location changing for each trial, and allowed 120 seconds to locate submerged platform. Then, it was allowed to stay on the platform for 20 s. If it failed to find the platform within 120 s, it was guided gently onto platform and allowed to remain there for 20 s. Day 4 escape latency time (ELT) to locate the hidden platform in water maze was noted as index of acquisition or learning. Animal was subjected to training trials for four consecutive days, the starting position was changed with each exposure as mentioned below and target quadrant (Q4) remained constant throughout the training period.
On fifth day, platform was removed and each mouse was allowed to explore the pool for 120 s. Mean time spent in all four quadrants was noted. The mean time spent by the animal in target quadrant searching for the hidden platform was noted as index of retrieval. The experimenter always stood at the same position. Care was taken that relative location of water maze with respect to other objects in the laboratory serving, as prominent visual clues were not disturbed during the total duration of study. All the trials were completed between 09:00 to 18:00 h (Saluja V. et al, 2011).
Experimental Protocol
Nine groups of mice, each group comprising of six mice were employed in the present study.
Group I (Control group)
Mice were administered normal saline (10 ml kg-1 i.p.), 30 min before acquisition trials conducted from day 1 to day 4 and 30 min before retrieval trail conducted on day 5.
Group II (ICV CSF treated control group)
Mice were injected artificial CSF (25 mg ml-1, 10 µl, ICV) in two dosage schedules that is on first and on third dayfollowed by exposure to MWM test after 14 days.
Group III (ICV STZ treated group)
Mice were injected STZ (3 mg kg-1, 10 µl, ICV) in two dosage schedules that is on first and on third dayfollowed by exposure to MWM test after 14 days.
Group IV (Rivastigmine per se group)
Mice were administered rivastigminne (1.08 mg kg-1 i.p.) daily for 7 days and then subjected to MWM test, the administration of rivastigmine was also continued during acquisition trail. On day 5, the animals were administered vehicle only, before retrieval trial.
Group V (Blank nanoparticles per se group)
Mice were administered blank nanoparticles (1.08 mg kg-1 i.p.) daily for 7 days and rest of the procedure was same as described for group IV.
Group VI (Rivastigmine nanoparticles per se group)
Mice were administered rivastigmine nanoparticles (1.08 mg kg-1 i.p.) daily for 7 days and rest of the procedure was same as group IV.
Group VII (Rivastigmine + ICV STZ treated group)
ICV STZ mice were administered rivastigmine (1.08 mg kg-1i.p.) starting after seven days from the second dose of STZ, daily for 7 days and again next for four consecutive days (day 1 to day 4, 30 min before) during acquisition trials. On day 5, the mice were administered vehicle only, before retrieval trial.
Group VIII (Rivastigmine nanoparticles + ICV STZ treated group)
ICV STZ mice were administered rivastigmine nanoparticles (1.08 mg kg-1i.p.) starting after seven days from the second dose of STZ, daily for 7 days and rest of the procedure was same as described for group VII.
Group IX (Blank nanoparticles + ICV STZ treated group)
ICV STZ mice were administered blank nanoparticles (equivalent to rivastigmine nanoparticles) starting after seven days from the second dose of STZ, daily for 7 days and rest of the procedure was same as described for group VII.
Statistical analysis
All results were expressed as mean ± standard error of mean (SEM). Data were analyzed using one-way ANOVA followed by post hoc Tukey’s multiple range test using Sigma Stat Statistical Software, version 2.0. P < 0.05 was considered to be statistically significant.
Results and Discussion
Particle Size and Polydispersity index
Particle size analysis was performed by photon correlation spectroscopy. The PCS yields the mean diameter (z-average) of the bulk population of the particles and additionally a polydispersity index as measure for the width of the distribution (Janes et al., 2001). PCS is also known as Dynamic Light Scattering which is done using a Zetasizer® 3000 (Malvern Instruments, NIPER, Mohali). The polydispersity index (PI) ranges from 0 (monodisperse) to 0.500 (very broad distribution). The PCS gives a volume distribution weighing especially large particles. PCS is therefore highly sensitive to detect a few larger particles resulting from particle aggregation during storage of the suspensions. The ionic gelation of chitosan with sodium tripolyphosphate during ultasonication led to the formation of particles in the nanoscale size. Use of ultrasonication for increasing duration decreased the mean diameter and polydispersity of the nanoparticles but did not affect the zeta potential. The average particle size and polydispersity index of the obtained nanoparticles were 258 nm and 0.261 respectively (Figure 1).
Figure 1: Mean diameter and polydispersity index of Rivastigmine loaded chitosan nanoparticles
Surface Charge determination
Nanoparticles were characterized with Zeta potential (ζ) using a Zeta Sizer 4 (Malvern Instruments ltd., Malvern UK)(Craparo et al., 2008). Measurements were obtained at an angle of 90?. Scattering intensity data were analyzed by a digital correlator and ?tted by the method of inverse Laplace transformation. Measurements were made in triplicate. The distribution of zeta potential of rivastigmine loaded chitosan nanoparticles is presented in Figure 2. Zeta potential of nanoparticles was +35.1 mV. Zeta potential is used to predict stability of colloidal dispersions. This result demonstrated that the chitosan nanoparticles obtained by homogenization followed by ultrasonication is a physically stable system.
Figure 2: Zeta potential value of Rivastigmine loaded chitosan nanoparticles
Morphology
The morphological examination of nanoparticles was performed by transmission electron microscopy (TEM) (Tecnai 20 G2 S TWIN at IIT Roorkee) set at 200 kV (Li et al., 2009). One drop of nanoparticulate dispersion was placed on the grid, dried for 3 to 5 minutes, and drained on the filter paper. The grid was further dried by keeping it in the petri plate; then it was loaded in the transmission electron microscope, and areas were scanned for observation of nanoparticles. The picture was taken under the electron microscope and is shown in Figure 3. TEM was conducted to investigate the morphology of chitosan nanoparticles. The particle size of rivastigmine chitosan nanoparticles from TEM images accords with that from PCS. The images showed that rivastigmine chitosan nanoparticles exhibited spherical shape with hairy surface.
Figure 3: TEM of Rivastigmine loaded chitosan nanoparticles
Structure
From structural point of view, the arrangement of components and orientation of molecules within the nanoparticle can determine its behavior and stability and for this purpose scanning electron microscopy (SEM) (Tecnai 20 G2 S TWIN at Punjab University, Chandigarh; set at 200 kV) (Abdelwahed et al., 2006) was employed.Scanning electron micrographs revealed that lyophilized rivastigmine loaded chitosan nanoparticles were spherical in shape with most of the smaller particles exhibiting smooth surface (Figure 4).
Figure 4: SEM of Rivastigmine loaded chitosan nanoparticles
Differential scanning calorimetry
DSC is a tool to investigate the melting and recrystallization behavior of a given material. Differential scanning calorimetry (DSC) was performed on a DSC-7 (Perkin-Elmer Corp., USA). A heating rate of 10°C/min was employed in the temperature range of 0-350°C. An empty aluminum pan was used as reference standard (Bhumkar et al., 2006).
The DSC curves of chitosan, pure rivastigmine, rivastigmine loaded chitosan nanoparticles and physical mixture of rivastigmine and chitosan are shown (Figure 5). Rivastigmine shows a sharp endothermic peak at 122ºC which corresponds to its melting point (Figure 5 B). In the case of rivastigmine loaded chitosan nanoparticles (Figure 5 C), endothermic peak coincides at around 124ºC indicates molecular level dispersion of drug in nanoparticle matrix. The physical mixture of drug and nanoparticles (Figure 5 D) shows separate characteristic endothermic peaks for drug and nanoparticles.
Figure 5: DSC thermograms of (A) Chitosan (B) Rivastigmine (C) Rivastigmine loaded chitosan nanoparticles (D) Physical mixture of rivastigmine and chitosan nanoparticles.
Evaluation of Entrapment Efficiency
It 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 Spectrofluorometer (SL 174, Elico India) (Boonsongrit et al., 2006). Association efficiency was then calculated as follows:
EE or AE% = Total amount of drug added – Non-bound drug X 100
Total amount of drug
In the present study five different drug : polymer ratios (Batch A, 1:1; Batch B, 1:2; Batch C, 1:3; Batch D, 1:5: Batch E, 1:7) were employed to determine the drug release characteristics of polymeric nanoparticles. Entrapment efficiency of batch A, B, C, D and E was found to be 59.63%, 78.65%, 83.74%, 45.19% and 42.2% respectively.This shows that with increase in proportion of chitosan, Association efficiency increases to a certain extent. But, upon increase in chitosan concentration above drug : polymer ratio 1 : 3, the association efficiency significantly decreases (Figure 6).
|
BATCH (Drug to Polymer ratio) |
Association Efficiency |
|
A (1:1) |
59.63±1.9 |
|
B (1:2) |
78.65±2.2 |
|
C (1:3) |
83.74±3.1 |
|
D (1:5) |
45.19±2.8 |
|
E (1:7) |
42.2±2.5 |
Table 1:Entrapment Efficiency of different batches of Rivastigmine chitosan nanoparticles. Values represent Mean ± S.D (n=3).
Figure 6: Association efficiency of Rivastigmine-chitosan nanoparticles.
Values represent Mean ± S.D (n=3).
In vitro drug release studies
The drug release profiles from best batches of nanoparticles were measured in vitro (Craparo et al., 2008). Forty milligrams of lyophilized nanoparticles were dispersed in 100 ml dissolution media consisting of phosphate buffer pH 7.4 previously equilibrated at 37oC in a shaker incubator at 100 rpm. At definite time interval, 2 ml of the dispersion was withdrawn and replaced with equivalent volume of dissolution medium to maintain sink conditions. The amount of drug released from the nanoparticles was then analyzed spectrofluorometrically (Table 2). It was observed that the drug release rate decreases significantly with an increase in chitosan/drug ratio (Figure 7).
Table 2: In VitroDrug Release of Rivastigmine from chitosan nanoparticles. Values represent Mean ± S.D. (n=3)
|
S.No. |
Time (h) |
% Cumulative Drug Release |
||||
|
Batch A |
Batch B |
Batch C |
Batch D |
Batch E |
||
|
1 |
1 |
9.46±1.1 |
10.14±2.1 |
3.45±1.9 |
9.92±1.7 |
10.51±2.2 |
|
2 |
2 |
18.26±2.0 |
16.82±1.4 |
5.46±1.5 |
18.48±1.9 |
21.90±1.1 |
|
3 |
3 |
39.14±1.9 |
24.19±1.5 |
6.58±2.3 |
36.61±2.2 |
34.16±1.2 |
|
4 |
4 |
51.19±1.8 |
32.46±1.9 |
9.69±2.0 |
49.11±2.1 |
48.21±1.9 |
|
5 |
5 |
62.48±1.6 |
41.91±2.0 |
12.41±1.8 |
61.82±1.3 |
59.91±1.0 |
|
6 |
6 |
70.21±2.2 |
50.48±1.2 |
17.68±1.6 |
70.01±2.1 |
71.24±1.6 |
|
7 |
7 |
76.61±2.1 |
59.11±2.2 |
21.25±2.2 |
78.49±1.7 |
82.44±1.5 |
|
8 |
8 |
81.22±1.7 |
68.64±1.0 |
28.91±1.4 |
85.16±1.4 |
89.61±1.8 |
|
9 |
10 |
88.16±1.4 |
77.91±1.8 |
36.18±1.3 |
89.10±1.9 |
96.21±2.0 |
|
10 |
12 |
90.31±1.1 |
81.12±2.1 |
42.14±2.2 |
99.14±1.0 |
99.56±1.1 |
|
11 |
24 |
92.60±1.3 |
85.61±1.9 |
48.28±1.6 |
|
|
Figure 7: In vitro drug release profile of Rivastigmine from various batches of chitosan nanoparticles. Values represent Mean ± S.D. (n=3).
In vivo studies
Acute Toxicity Studies:Estimation of LD50 (dose lethal to 50% of the animal group)of rivastigmine
In toxicology, the median lethal dose, LD50 of substance is the dose required to kill half the members of a tested population after specified test duration. LD50 figures are frequently used as an indicator of substance's acute toxicity. The LD50 is usually expressed as the mass of substance administered per unit mass of test subject, such as grams of substance per kilogram of body mass. Stating it this way allows the relative toxicity of different substances to be compared, and normalizes for the variation in the size of the animals exposed. When the response of drug is quantal or all-or-none the ED50 value (the dose effective in producing certain expected response in 50% of the animal group) becomes the LD50. Ratio between LD50 and ED50 represents the therapeutic index. Greater the therapeutic index, safer is the drug.
For estimating LD50 of rivastigmine seven groups of male swiss mice were employed and each group was administered different concentrations (1, 2, 3, 4, 5, 6 and 7 mgkg-1) of rivastigmine solution. The first group which was administered 1 mgkg-1 of rivastigmine was the most tolerated dose (0% mortality). The last group with 7 mgkg-1 rivastigmine is the least tolerated dose (100% mortality). The following table shows the results of LD50 study done with rivastigmine in mice using Miller and Tainter method (Akhila et al., 2007). Graph was plotted between log dose and probit values and LD50 of rivastigmine calculated was 5 mgkg-1, i.p.
Figure 8: Graph between log dose and probit value for determination of LD50 value of rivastigmine.
Table 3: Estimation of LD50 of rivastigmine
|
Group |
Dose i.p mg/kg |
Log dose |
Dead/Total |
% Dead |
Corrected % |
Probit |
|
1 |
1 |
0 |
0/10 |
0 |
2.5 |
3.04 |
|
2 |
2 |
0.3010 |
1/10 |
10 |
10 |
3.72 |
|
3 |
3 |
0.4771 |
2/10 |
20 |
20 |
4.16 |
|
4 |
4 |
0.6020 |
3/10 |
30 |
30 |
4.48 |
|
5 |
5 |
0.6089 |
5/10 |
50 |
50 |
5.00 |
|
6 |
6 |
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