Folate-conjugated Therapeutic Agents for Arthritis

Folate- meld Therapeutic Agents for ArthritisIN VIVO STUDIESConsistent performance of a controlled release ordertion upon dosing is critical to a quality product. In vivo military rank of any medicine deliver-coloredy system is quite essential because umpteen factors like pH of different biological harmoniums, enzyme systems and variable affinity of carrier wave system for the conglomerate biological fluids including the tissues argon expected to influence its performance. These factors put on the in vivo biological distribution and the dose release profile from a novel carrier system. In vivo studies are important in evaluating the bioavailability of medicine from the designed formulations.Screening of the anti-inflammatory activityThe screening modes for the evaluation of anti inflamatory activity have been classified as follows -A. Nonimmunological methods1. Evaluation of acute fervidnessCarrageenan bring forth hired man dropsy model (Winter et al.,1962)Histamine i nduced back(prenominal) paw method.Carrageenan granuloma pouch technique.2. Evaluation of chronic inflamationFormaldehyde induced arthritis.B.Immunological methods1. Adjuvant induced arthritisComplete Freunds adjuvant induced rheumatoid model.2. Collagen induced arthritic model.3. Borrelia burgdorferi induced arthritic model.C.MiscellaneousU.V. erythema inflamatory model.MATERIAL AND METHODS mannish albino smokesIn vivo study was performed on the albino rats (av.wt.10020 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 prevail pellet and water ad libitum.Induction of arthritisCarrageenan induced arthritis method was selected for present study (Winter et al., 1962). Carrageenan acts as phlogistic agent which causes the fundamental law of oedema due to stimulation of release of various pro inflamatory agents like prostaglandins, histamine and serotonin, bradykinin. tachykinins, reactive oxygen and nitrogen species etc.BIODISTRIBUTION STUDYThe rats were divided into four classifys with separately group comprising of three animals and labelled properly. subsequently induction of arthritis plain medicate hang in phosphate buffer solution (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 retroactive orbital plexus of the eye, the rats were sacrificed and various organs such as liver, lung, kidney, irascibility and arthritic knee joint were isolated. The organs were weighed, washed in PBS (pH 7.4) and stored at -20C until advertise required.Table 7.1 Data of biodistribution of etoricoxib in albino rats later on i.v. administration of formulationsOrgansSystemDistribution of % injected dose /whole organ or tissue at 6 hrBlood out-and-out(a) dose53.172.11ETX-NPs29.231.39f-ETX-NPs24.391.10LiverPlain drug17.02.10ETX-NPs18.212.43f-ETX-NPs21.252.11SpleenPlain drug10.691.13ETX-NPs12.431.47f-ETX-NPs11.451.25KidneyPlain drug9.080.49ETX-NPs6.600.65f-ETX-NPs6.200.68LungPlain drug2.760.28ETX-NPs4.810.53f-ETX-NPs6.600.71Non- worsen jointPlain drug0.106.02ETX-NPs0.110.02f-ETX-NPs0.104.017Inflamed jointPlain drug0.110.020ETX-NPs0.350.058f-ETX-NPs1.420.049Each value is show as mean SD (n=3) meet 7.1(A) % absorption of etoricoxib in blood later on 6 hr of i.v. injection ensure 7.1(B) % tightness of etoricoxib in liver after 6 hr of i.v. injection framing 7.1(C) % soaking up of etoricoxib in spleen after 6 hr of i.v. injectionFigure 7.1(D) % concentration of etoricoxib in kidney after 6 hr of i.v. injectionFigure 7.1(E) % concentration of etoricoxib in lung after 6 hr of i.v. injectionFigure 7.1(F) % concentration of etoricoxib in noninflamed joint after 6 hr of i.v. injec tionFigure 7.1(G) % concentration of etoricoxib in inflamed joint after 6 hr of i.v. injectionPHARMACODYNAMIC STUDYCarrageenan-induced Paw edema in ratsThis model is rear on the principle of release of various inflamatory mediators by carrageenan. Edema formation due to carrageenan in rat paw is biphasic event. The initial material body is attributed to the release of histamine and serotonin. The second phase of edema is due to the release of prostaglandins, peptidase 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 quite a little of inflamed paw , development a well calibrated plethysmometer (UGO,BASILE 7140, Italy).The carrageenan edema test was performed for drug besotted BSA nanoparticles as exposit 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 victimization 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 base to be around 120 g .The daytime 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 way of life 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 dr ug 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 office of the right hind paw. The paw volume was measured every min 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 metric as percentage differentiated with the basal volume. The difference of average values amid treated animals and control group is calculated for each time time interval and evaluated statistically. The percent Inhibition for each group was calculated using the formula as follows.Vcontrol -Vtreated% inhibition of edema = - 100Vcontrol 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 lo aded nanoparticulate system and ligand conjugated drug loaded nanoparticulate system using carrageenan induced paw edema model.Time (hr)% Inhibition of edemaPlain drug solutionDrug loaded nanoparticulate systemLigand conjugated drug loaded nanoparticulate system1/215.141.96.710.817.541.1124.212.810.321.718.913.3237.423.812.541.823.015.2441.534.219.372.138.414.2832.273.526.573.247.503.91221.592.246.913.962.356.92412.011.828.123.455.018.3367.370.7919.972.648.727.648ND12.101.924.593.360ND7.530.8718.152.872NDND7.780.89ND= non detectable each value is uttered as mean SD (n=3)Figure 7.2 Plot of % inhibition of edema Vs timeStatistical analysisResults were denotative as mean standard deviation (SD) and statistical analysis was performed with PSS 10.1 lettuce (USA).The biodistribution parameters were calculated with the help of (pk analyst) scientific programme from Micromath Inc. (UK).RESULTS AND DISCUSSIONIn vivo studies are important in evaluating the therapeutic efficacy of designed dosage forms and alike 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 high(prenominal) in gaucherie of drug loaded NPs and about 10 folds higher in case of folic venereal disease 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 53.172.11 % in case of plain drug solution, while in case of ETX-NPs and f-ETX-NPs it was found to be about 29.231.39 % and 24.391.10 % respectively of the whole injected dose which prove the sustained effect of formulations. The concentration of drug in liver was found to be 17.02.10%, 18.22.43% and 21.22.119% of the whole injected dose in case of plain drug, ETX-NPs and f-ETX-NPs respectively. little(a) 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 i ncluding prostaglandins (PGs).CONCLUSIONThe supra 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.