IN VIVO STUDIES
Consistent performance of a controlled release formulation upon dosing is critical to a quality product. In vivo evaluation of any drug delivery system is quite essential because many factors like pH of different biological organs, enzyme systems and variable affinity of carrier system for the various biological fluids including the tissues are expected to influence its performance. These factors affect the in vivo biological distribution and the drug release profile from a novel carrier system. In vivo studies are important in evaluating the bioavailability of drug from the designed formulations.
Screening of the anti-inflammatory activity
The screening methods for the evaluation of anti inflamatory activity have been classified as follows :-
A. Nonimmunological methods
1. Evaluation of acute inflammation
Carrageenan induced paw edema model (Winter et al.,1962)
Histamine induced hind paw method.
Carrageenan granuloma pouch technique.
2. Evaluation of chronic inflamation
Formaldehyde induced arthritis.
B. Immunological methods
1. Adjuvant induced arthritis
Complete Freunds adjuvant induced arthritic model.
2. Collagen induced arthritic model.
3. Borrelia burgdorferi induced arthritic model.
U.V. erythema inflamatory model.
MATERIAL AND METHODS
Male albino rats
In vivo study was performed on the albino rats (av.wt.100±20 g).The animal studies were conducted with the permission of Institutional Animal Ethical Committee of Dr. Hari Singh Gour University, Sagar (M.P.). Animals were housed in plastic cages in the thermoneutral environment, and were supplied with feed pellet and water ad libitum.
Induction of arthritis
Carrageenan induced arthritis method was selected for present study (Winter et al., 1962). Carrageenan acts as phlogistic agent which causes the formation of edema due to stimulation of release of various proinflamatory agents like prostaglandins, histamine and serotonin, bradykinin. tachykinins, reactive oxygen and nitrogen species etc.
The rats were divided into four groups with each group comprising of three animals and labelled properly. After induction of arthritis plain drug suspended in PBS (pH 7.4), ETX-NPs and f-ETX-NPs in a dose equivalent to etoricoxib of 0.5 mg/kg body weight were administered through tail vein to albino rats. After 6 hr following administration of formulation, blood of animals was collected from retro orbital plexus of the eye, the rats were sacrificed and various organs such as liver, lung, kidney, spleen and arthritic knee joint were isolated. The organs were weighed, washed in PBS (pH 7.4) and stored at -20°C until further required.
Table 7.1 Data of biodistribution of etoricoxib in albino rats after i.v. administration of formulations
Distribution of % injected dose /whole organ or tissue at 6 hr
Each value is expressed as mean ±SD (n=3)
Figure 7.1(A) % concentration of etoricoxib in blood after 6 hr of i.v. injection
Figure 7.1(B) % concentration of etoricoxib in liver after 6 hr of i.v. injection
Figure 7.1(C) % concentration of etoricoxib in spleen after 6 hr of i.v. injection
Figure 7.1(D) % concentration of etoricoxib in kidney after 6 hr of i.v. injection
Figure 7.1(E) % concentration of etoricoxib in lung after 6 hr of i.v. injection
Figure 7.1(F) % concentration of etoricoxib in noninflamed joint after 6 hr of i.v. injection
Figure 7.1(G) % concentration of etoricoxib in inflamed joint after 6 hr of i.v. injection
Carrageenan-induced Paw edema in rats
This model is based on the principle of release of various inflamatory mediators by carrageenan. Edema formation due to carrageenan in rat paw is biphasic event. The initial phase is attributed to the release of histamine and serotonin. The second phase of edema is due to the release of prostaglandins, protease and lysosome.
Assay was performed by single subcutaneous injection of 1% (0.1 ml) carrageenan as phlogistic agent and inflamation was determined by measuring change in the volume of inflamed paw , using a well calibrated plethysmometer (UGO,BASILE 7140, Italy).
The carrageenan edema test was performed for drug loaded BSA nanoparticles as described by (Winter et al., 1962).
Albino rats selected for the present study were weighed, numbered and left paw was marked with ink at the level of tibiotarsic articulation, so that every time, the paw was dipped into the plethysmometer up to the fix mark to ensure the constant paw volume. Basal paw volume was measured plethysmographically by volume displacement method using Plethysmometer (UGO Basile 7140 Italy ) by immersing the paw till the level of tibiotarsic articulation The weight of each animal was taken, averaged and were found to be around 120 g .The day time was chosen for the study to avoid any significant changes in the circardian rhythyms. Animals were divided into four groups (n=3) including one controlled group starved overnight with water ad libitum prior to the day of experiment. Test formulation of drug loaded BSA NPs (0.2%w/v) and plain drug suspended in PBS (pH 7.4) in dose of 0.5 mg/kg body wt. was administered through iv route to albino rats of respective groups excluding control group. The control group was injected with normal saline (PBS, pH 7.4). After administration of test formulations of drug loaded NPs of BSA, the rats were challenged by a subcutaneous injection of 0.1 ml of 1% solution of carrageenan into the sub-plantar side of the right hind paw. The paw volume was measured every hour till 4th hr and subsequently readings were taken at 8, 12, 18, 24, 36, 48 and 72 hrs after challenge. The increase in paw volume was calculated as percentage compared with the basal volume. The difference of average values between treated animals and control group is calculated for each time interval and evaluated statistically. The percent Inhibition for each group was calculated using the formula as follows.
% inhibition of edema = ————————-? 100
Where Vcontrol =mean edema volume of rats in control group,
Vtreated mean edema volume of each rat in test group.
The results are reported in the Table 7.2. A graph was plotted between % inhibition of edema Vs time ( Figure 7.2).
Table 7.2 Screening Data for anti-inflamatory activity of plain drug solution, drug loaded nanoparticulate system and ligand conjugated drug loaded nanoparticulate system using carrageenan induced paw edema model.
% Inhibition of edema
Plain drug solution
Drug loaded nanoparticulate system
Ligand conjugated drug loaded nanoparticulate system
ND= Not detectable; each value is expressed as mean ±SD (n=3)
Figure 7.2 Plot of % inhibition of edema Vs time
Results were expressed as mean ±standard deviation (SD) and statistical analysis was performed with PSS 10.1 Chicago (USA).
The biodistribution parameters were calculated with the help of (pk analyst) scientific programme from Micromath Inc. (UK).
RESULTS AND DISCUSSION
In vivo studies are important in evaluating the therapeutic efficacy of designed dosage forms and also help in establishing the correlation between the results obtained from the in vitro experimentation to that of in vivo conclusions.
In order to understand the fate of drug loaded NPs in vivo, the biodistribution of drug in various major organs was investigated. The amount of drug in the body depends upon its release, distribution metabolism and excretion from body. The concentration of drug in inflamatory knee joint was found about 3 folds higher in case of drug loaded NPs and about 10 folds higher in case of folic acid conjugated system as compare to the free drug administration. These results evidenced the site specific targeting of drug in inflammatory region.
Concentration of drug in blood was found to be 53.17±2.11 % in case of plain drug solution, while in case of ETX-NPs and f-ETX-NPs it was found to be about 29.23±1.39 % and 24.39±1.10 % respectively of the whole injected dose which proved the sustained effect of formulations. The concentration of drug in liver was found to be 17.0±2.10%, 18.2±2.43% and 21.2±2.119% of the whole injected dose in case of plain drug, ETX-NPs and f-ETX-NPs respectively. Slight increase in the amount of drug (although very less) in various organs from formulations ETX-NPs and f-ETX-NPs suggested the RES uptake of nanoparticulate formulations in those organs. Concentration of drug in inflamed joint was raised up to 3 fold in case of plain ETX-NPs and 10 fold in case of f-ETX-NPs as compared to plain drug administration. This proved the targeting efficiency of nanoparticulate formulations both uncoupled as well as folate conjugated nanoparticles.
The % inhibition of edema was found to be significantly higher from f-ETX-NPs as compared to the ETX-NPs and plain drug .The folic acid (folate) attached to the surface of NPs might have carried the NPs to folate receptors over expressed on the activated macrophages that is responsible for the release of various inflamatory cytokines including prostaglandins (PGs).
The above data suggested that the development of folate-conjugated therapeutic agents in treatment of arthritis may further enhance its site specific drug delivery at inflamed joints and may also be used as sustained drug delivery system in rheumatoid arthritis.