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    Bioassay 112070804012 Bioassay 112070804012 Presentation Transcript

    • A seminar onBioassay of official drugsBy :PARTHM.pharma-1(Q.A)APMC Pharmacy College 1
    • Content1. Definition2. Principle of bioassay3. Importance of bioassay4. Types of bioassay5. Methods of bioassay6. Limitation of bioassay7. Bioassay of official drugs 2
    • Definition:Estimation of the conc. or potency of a substance by measuring its biological response in living systems. Observation of pharmacological effects on [1] living tissues, or cells [2] microorganisms [3] animals 3
    • Principle of bioassay:Compare the biological effect produced by the test substance with that of standard preparation and find out how much test substance is required to produce same biological effect as produced by the standard. 4
    • Importance of bioassay: Active principle of drug is unknown Active principle cannot be isolated, e.g posterior pituitary extract etc. Chemical method is either ◦ not available ◦ if available, too complex, ◦ insensitive to low doses e.g. Histamine can be bioassay in microgram conc. Unknown Chemical composition, e.g. long acting thyroid stimulator. Chemical composition of drug is different but has same pharmacological action e.g. cardiac glycosides isolated from diff sources, catecholamines etc. 5
    • To ascertain the potency of a drug.Stability studies are also conducted by bioassays.It also measure toxicity.Bacterial products like toxins, antitoxins, vaccines, are assayed by only bioassay. 6
    • Limitation of bioassay: 7
    • Bioassay of official drugs:1)HEPARIN SODIUM:(IP’96) by comparing the conc. necessary to prevent the clotting of sheep, goat or human plasma with the conc. of the std. preparation. Standard preparation: The freeze-dried sodium salt of the purified active principle from bovine intestinal mucous membranes. 8
    • Special reagent:Prepared plasma:collect the blood in to vessel containing 8%w/v sol. Of sodium citrate &blood(1:19) mix &centrifuge to pool out plasma To 1 ml of plasma add 0.2 ml of 1% w/v of calcium chloride sol. mix it The plasma is suitable if clot forms within 5 mins.Solution of standard preparation:The minimum quantity of std. preparation of heparin sodiumwhich, when added in 0.8 ml of saline solution, maintain fluidity in 1ml of prepared plasma for 1 hour after the addition of 0.2 ml of 1%w/v calcium chloride.On the day of assay prepare a solution of std. preparation such thatit contains in each 0.8ml of saline solution the above determined qty.of the std. preparation. 9
    • Test solution: Weight accurately about 25mg of the test sample dissolved in sufficient saline solution to give the conc. of 1mg/ml dilute to concentration corresponds to that of standardMethod: To very clean test tubes add graded amt. of std. preparation, the largest dose doesn’t exceed 0.8 ml. add sufficient saline solution to make volume 0.8ml & add 1.0 ml of prepared plasma to each test tube. add 0.2 ml of 1% of calcium chloride, note the time mix properly so that entire inner surface of test tubes is wet In the same manner set up a series of test preparation 10
    • completing the entire process within 20 minute after addition of prepared plasmaafter 1 hour the addition of calcium chloride solution, determined the extent of clotting in each test tubes, recognize three grades between zero and full clotting.Dilution of test preparation which contain same concentration as that of standard show same degree of clottingIf the degree of clotting in dilution of the std. preparation lies between that observed in 2 of the dilution of test preparation, the potency of later is estimated.If there is no correspondence between the degree of clotting by standard & test, new dilution prepared & assay is repeated.Calculate the estimated potency of the test preparation by combining the result of assay with standard statical methods. 11
    • 2)OXYTOIN:(IP’07) The potency of oxytocin is determined by comparing its activity withthat of the Standard Preparation of oxytocin under the conditions of a suitablemethod of assay.Standard Preparation:consisting of freeze-dried synthetic oxytocin peptide with human albumin andcitric acid (supplied in ampoules containing 12.5 Units).Method:By contraction of the rat uterus: Inject 100 mg of oestradiol benzoate intramuscularly into a female rat weighing 120 to 200 g 18 to 24 hours before the assay. Kill the rat and suspend one horn of the uterus in a bath containing a solution of the following composition. 12
    • Composition (% w/v)Sodium chloride 0.662Potassium chloride 0.045Calcium chloride 0.007Sodium bicarbonate 0.256Disodium hydrogen phosphate 0.029Sodium dihydrogen phosphate 0.003Magnesium chloride 0.010Dextrose 0.050 Maintain the bath at 32o C at which spontaneous contractions ofthe uterus are abolished and the preparation maintains its sensitivity. Oxygenate the solution with a mixture of 95% of oxygen and 5% of carbon dioxide record the contractions of the muscle using a suitable instrumentgiving a linear response Record the contractions produced by the addition of two doses ofthe Standard Preparation suitably diluted with the above solution. 13
    • The doses should be such as to produce clearly discriminatedcontractionsThe required doses normally lie between 10 and 50 micro Units per ml ofbath liquid. The doses should be added at regular intervals of 3 to 5 minutesdepending upon the rate of recovery of the muscle. Dilute test preparation so as to produce same response as that of standard The ratio between the two doses of the preparation being examined should be the same as that of the Standard Preparation and this ratioshould be kept constant throughout the assay. The two doses of Standard Preparation and the preparation beingexamined should be given according to a randomized block or a Latinsquare design and at least six responses to each should be recorded. calculate the result of the assay by standard statistical methods. 14
    • 3)STERPTOKINASE:(IP’96)Bioassay by comparing its ability to activate human plasminogen to formplasmin with that of the Standard Preparation. The plasmin generated isdetermined by measurement of the time taken to lyse a fibrin clot underthe conditions of a suitable method of assay.Standard Preparation:The Standard Preparation is consisting of freeze-dried streptokinase(supplied in ampoules containing 700 Units of streptokinase activity).Suggested Method:Use citro-phosphate buffer pH 7.2 containing 3% w/v of bovineserum albumin for the preparation of solutions and dilutions. Prepare the Standard Preparation to contain 1000 Units ofstreptokinase activity per ml and prepare a solution of the preparationbeing examined of the same concentration; keep the solutions in ice and use within 6 hours. 15
    • Prepare three serial dilutions of the Standard Preparation solongest clot-lysis time is less than 20 minutes. prepare three similar dilutions of the solution of the preparationbeing examined. Keep the solutions in ice and use within 1 hour take 24 tubes(8 mm), three for the dilutions of the Standard Preparation and three for the dilutions of the Standard Preparation being examined, allocating four tubes to each dilution Add 0.2ml of dilution, 0.2 ml of citro-phosphate buffer pH 7.2 containing 3% w/v of bovine serum albumin and 0.1 ml of a solution containing 20 Units of thrombin per ml,Place the tubes in a water-bath at 37o and allow to stand for 2 minutes to attain temperature equilibrium. 16
    • Add 0.5 ml of a 1% w/v solution of human euglobulins in each tubes at interval of 5-sec. measure the time in seconds that elapses between the addition of the euglobulin and the lysis of the clot. calculate the result of the assay by standard statistical methods.4)Vitamin D: (USP 23 NF 18) It is bioassayed by measuring the ability of vitamin D to stimulatecalcification of the rachitic metaphysis in rats. Assay uses young rats (not less than 55 days old) that have developedrickets on a rachitogenic diet. These rats are divided in to groups and fed the rachitogenic dietwith either USP cholecalciferol reference standard, unknown or no supplementation (control).One half the dose of vitamin D as cholecalciferol standard or unknowngiven to rats on day 1 to 3 of assay period. 17
    • At the end of fixed period (7to10 days) they are weighted& scarifies.Any rats whose wt. decrease has removed from further analysis.The leg bones of remaining rats are dissected out & assayed for amt.ofrecalcification of bones.The activity of vitamin D may be determined by amt. of recalcificationin relation with reference standard.5) Plague vaccine:The potency of plague vaccine is estimated by determining the dosenecessary to protect mice against a lethal dose of a virulent strain ofYersinia pestis.Test Animals:Use white mice, 6 to 7 weeks old, each weighing between 20 and 28 gand of a strain susceptible to plague infection. 18
    • •The animals should be healthy and free from intercurrent infectionwith organisms such as Salmonella.Suggested Method:Selection of suitable virulent strain:•A freeze-dried virulent culture of Y. pestis is revived by subculturing0.5 ml in 9.5 ml of nutrient broth in test-tube and incubating at 28ofor exactly 48 hours.•Such a culture should contain 300 to 600 million organisms per ml.•Make 10-fold dilutions in nutrient broth and test for virulence. 19
    • Standard challenge dose:Freshly reconstitute the freeze-dried culture and dilute with nutrientbroth to strength such that 0.2 ml contains 60 to 120 organisms.Measurement of protective power:Prepare a series of five graded doses of the preparation beingexamined arranged in such a manner that the 50% protective dose(ED50) lies about the middle of the selected series. 16 mice are used for each dose.Inject subcutaneously the selected dose in two equal parts with aninterval of 7 days between them.Inject subcutaneously the standard challenge dose in each group ofmice 7 day after second half of dose. 20
    • Observe the animals for 15 days and record the number of deaths ineach group. Carry out a post-mortem, look for signs of plague .If plague organisms are not seen, such deaths are excluded from thecalculation.After observation kill all the surviving animals and examine forsigns of plague.Calculate the median effective immunising dose, ED 50, by standardstatistical methods.The vaccine passes the test if it has an ED50 of 0.004 ml or less permouse. 21
    • 6) RABIES ANTISERUM:The potency of rabies antiserum is determined by comparingthe dose necessary to protect mice against a lethalintracerebral dose of rabies virus with the dose of theStandard Preparation of rabies antiserum necessary to give thesame protection.Standard Preparation:The standard preparation is a dried serum the potency ofwhich has been determined in relation to the InternationalStandard.Suggested Method:Test animals:Use healthy mice of either sex weighing between 10 and 14gm. 22
    • Test virus:Any suitable strain of rabies virus of known potency such as theCVS strain may be used.Determination of potency of the rabies antiserum:Prepare a series of 2-fold dilutions of the Standard Preparation and ofthe preparation being examined with water containing 2% v/v of heatinactivated normal horse serumAdd a quantity of a suspension of the test virus containing the testdose. keep the mixtures at 37o for 1 hour. Inject intracerebrally 0.03 ml of each mixture into 10 mice. Observe the mice for 14 days after the injection. 23
    • Mice dying before the fifth day after inoculation with the virus are eliminated from the test. All the mice dying between the fifth and fourteenth days after showing signs of rabies are considered to have died of rabies.Mice living up to the fourteenth day but showing signs of rabies are also counted as having died from rabies. Calculate the result of the test by standard statistical methods.  24
    • References :Indian pharmacopoeia 1996,VOL.I, page no. 361,550.Indian pharmacopoeia 1996, VOL.II, page no. 602,656.United state pharmacopoeia 23,National Formulary 18,asian edition.Elements of pharmacology ,15th edition by Dr. R.K. Goyal ,page no. 578-582. 25
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