bioassay

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Slide 1:

12 July 2011 1 “BIOASSAY OF OFFICIAL DRUGS” SJTPC, RAJKOT. Prepared by: MONA PANKHANIYA M. Pharm. Sem-I Q.A. Department. Guided by: MR. VASANT KHASIA Assistant Professor Q.A. Department.

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12 July 2011 2 Content Definition Principle of bioassay Importance of bioassay Factors affecting bioassay Types of bioassay Methods of bioassay Limitation of bioassay Bioassay of official drugs References

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12 July 2011 3 1. Definition Estimation of the conc. or potency of a substance by measuring its biological response in living systems with respect to std. sub. Observation of pharmacological effects on [1] living tissues, or cells (part of an animal) [2] microorganisms [3] animals

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12 July 2011 4 Bioassays are always comparative with the reference standard which may be obtained from 1.Expert committee on biological standardization of W.H.O.- Geneva. 2.International lab for biostandars-Copenhagen. 3.National institute for medical research-U.K. 4.CDRI-Lucknow. 5.CDL-Calcutta.

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12 July 2011 5 Bioassay can be Micro bioassay, radioimmunoassay. Other synonyms are biometrics or biological standardization. Biological standardization can not be considered as synonym of bioassay because in Bioassay one simply estimates the potency in a preparation. However in biological standardization, one has to increase or decrease the potency (concentration) of the sample/batch to bring to the standard level.

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12 July 2011 6 2. Principles 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. Std. and new drug should be identical to each other i.e. DRC have same slope value and DRC should be linear. Method of comparison should be proper. Precaution should be taken to minimize the errors due to biological variations. Stastical analysis should done to minimize the errors The degree of pharmacological response produced should be reproducible under identical conditions [Eg. Adrenaline shows same rise in BP in the same species under identical conditions: wt, age, sex, strain etc]

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12 July 2011 7 3. Importance of bioassay Active principle of drug is unknown Active principle cannot be isolated, e.g. digitalis have complex mixture of varying structure and activity. Chemical method is either -not available -if available, too complex -insensitive to low doses Unknown Chemical composition, e.g. long acting thyroid stimulator. Chemical composition of drug variable but has same pharmacological action e.g. cardiac glycosides isolated from diff sources, catecholamine etc. Subs. for those chemical assay is not adequate even though chemical structure is known. E.g. insulin, oxytocin. Physical property of rotation is established and hence the biological activity is to be determined. E.g. l-adrenaline is 60 time more active than d-adrenaline. Estimation of dose of drug, required to produce a therapeutic or toxic response. ED50 or LD50 .

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12 July 2011 8 4. Factors affecting bioassay 1.Biological variation -Animals -Environment -Temperature -Diet -Season -Experience 2.Planning of experiment - Solvent or vehicle for drug sub. -Route of administration -Time of administration -No. of animals -Dose -Design -Selection of method -Selection of animals

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12 July 2011 9 5. Types of Bioassays [1] Quantal Response Assays [ Direct endpoint ] Elicits an ‘All or None’ response in different animals E.g.. Digitalis induced cardiac arrest in guinea pigs Digitalis induced head drop in rabbits Calculation of LD50 in mice or rats [2] Graded Response Assays [mostly on tissues] Graded responses to varying doses Unknown dose response measured on same tissue Both assays depend ultimately on our plotting or making assumptions concerning the form of DRC.

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12 July 2011 10 6. Methods of bioassay End point determination Graphical method (Interpolation) Matching method Multipoint method e.g. 3 point, 4 point, 6 point etc.

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12 July 2011 11 A. End point determination: The threshold dose producing a positive effect is measured on each animal and the comparison between the average results of two groups of animal (one receiving standard and other the test) is done. Concentration Threshold dose of Std. of Unknown = ------------------------------------- × Conc. of Std. Threshold dose of Test

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12 July 2011 7/12/2011 12 B. Graphical method: Based on dose-response relationship 5-6 response of the graded doses of std. are taken. Two equivalent response of test are taken. The ht. of contraction in mm . is measured and plotted against log dose .

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12 July 2011 13 Advantage of graphical method Simple method. Chances of error is less if the sensitivity of preparation is not changed. Disadvantage of graphical method In obtaining the responses to different doses of the std. and test , the order in which they are added to the tissue preparation, the sensitivity of preparation is changed.

C. Matching method::

12 July 2011 14 C. Matching method: In this method constant dose of test is bracketed by varying doses of standard till the exact match is obtained between test &standard. Initially, two responses of the standard are taken. The doses are adjusted such that one is giving response of approximately 20% and other 70% of the maximum. The response of unknown, which lies in between two responses of standard dose, is taken. The panel is repeated by increasing or decreasing the doses of std. till three equal responses are obtained. The dose of test is kept constant. In the end responses of the double dose of the std. & test are taken. They should give equal response

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12 July 2011 15 Used when test sample is too small. Conc. of the test sample can be determined as follows: Dose of Std. Conc. of Test = ---------------------- x Conc. of Std. Dose of Test Bioassay of histamine by matching method

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12 July 2011 16 Advantages of matching method : It does not depend on the assumption of the DRC. Disadvantages of matching method : Time consuming. Inaccurate & margin of error difficult to estimate It gives no parallelism of the DRC of std. and unknown, hence of the qualitative differences. since the effect are matched at only one dose level.

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12 July 2011 17 D. Multipoint method: 1. Three point assay [2+1 dose assay] # Fast & convenient # Procedure [ Eg Ach bioassay] - Log dose response [LDR] curve plotted with varying conc of std Ach solutions and given test solution - Select two std doses s1& s2 [ in 1:2 dose ratio] from linear part of LDR [Let the corresponding response be S1, S2] - Choose a test dose t with a response T between S1 & S2 - Record data [Latin square: Randomisation reduces error] as follows - Plot mean of S1, S2 and T against dose. - The strength of the test solution = s1 t s2 s2 s1 t t s2 s1 s1 s2 t s2 t s1 t s1 s2

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12 July 2011 18 2. Four point method [2+2 dose assay] # Procedure [Eg Ach bioassay] -Log dose response [LDR] curve plotted with varying conc of std Ach solutions and given test solution -Select two std doses s1& s2 from linear part of LDR [ Let the corresponding response be S1, S2] - Choose two test doses t1 & t2 with response T1 &T2 between S1 & S2 -Record 4 data sets [Latin square: Randomisation reduces error] s1 s2 t1 t2 s2 t1 t2 s1 t1 t2 s1 s2 t2 s1 s2 t1 - Plot mean of S1, S2 and T1, T2 against dose. - The strength of the test solution =

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12 July 2011 19 7. Limitation of bioassay Expensive Time consuming Require extreme skills Less accurate Less reproducible Always comparative More laborious Effect measured in the test animal is often not that which the drug is intended to produce in treating patients Recent studies have shown that when several active principles are present in a crude drug, those producing the maximum therapeutic effect are not necessarily ones chiefly responsible for the action measured in the assay.

8. Bioassay of official drugs:

12 July 2011 20 8. 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.

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12 July 2011 21 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 sodium which, when added in 0.8 ml of saline solution, maintain fluidity in 1 ml 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 that it contains in each 0.8ml of saline solution the above determined qty. of the std. preparation.

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12 July 2011 22 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 standard Method: 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

Slide 23:

12 July 2011 23 completing the entire process within 20 minute after addition of prepared plasma after 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 clotting If 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 Stastical methods.

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12 July 2011 24 2) OXYTOCIN :(IP’07) The potency of oxytocin is determined by comparing its activity with that of the Standard Preparation of oxytocin under the conditions of a suitable method of assay. 24 24 Standard Preparation: consisting of freeze-dried synthetic oxytocin peptide with human albumin and citric 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.

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12 July 2011 25 Composition (% w/v) Sodium chloride 0.662 Potassium chloride 0.045 Calcium chloride 0.007 Sodium bicarbonate 0.256 Disodium hydrogen phosphate 0.029 Sodium dihydrogen phosphate 0.003 Magnesium chloride 0.010 Dextrose 0.050 Maintain the bath at 32 o C at which spontaneous contractions of the 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 instrument giving a linear response Record the contractions produced by the addition of two doses of the Standard Preparation suitably diluted with the above solution.

Slide 26:

12 July 2011 26 The doses should be such as to produce clearly discriminated contractions The required doses normally lie between 10 and 50 micro Units per ml of bath liquid. The doses should be added at regular intervals of 3 to 5 minutes depending 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 ratio should be kept constant throughout the assay. The two doses of Standard Preparation and the preparation being examined should be given according to a randomized block or a Latin square design and at least six responses to each should be recorded. calculate the result of the assay by standard statistical methods.

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12 July 2011 27 3) STERPTOKINASE :(IP’96) Bioassay by comparing its ability to activate human plasminogen to form plasmin with that of the Standard Preparation. The plasmin generated is determined by measurement of the time taken to lyse a fibrin clot under the 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 bovine serum albumin for the preparation of solutions and dilutions. Prepare the Standard Preparation to contain 1000 Units of streptokinase activity per ml and prepare a solution of the preparation being examined of the same concentration; keep the solutions in ice and use within 6 hours.

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12 July 2011 28 Prepare three serial dilutions of the Standard Preparation so longest clot-lysis time is less than 20 minutes. prepare three similar dilutions of the solution of the preparation being 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 37 o and allow to stand for 2 minutes to attain temperature equilibrium.

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12 July 2011 29 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. Vitamin D: (USP 23 NF 18) It is bioassayed by measuring the ability of vitamin D to stimulate calcification of the rachitic metaphysis in rats. Assay uses young rats (not less than 55 days old) that have developed rickets on a rachitogenic diet. These rats are divided in to groups and fed the rachitogenic diet with either USP cholecalciferol reference standard, unknown or no supplementation (control). One half the dose of vitamin D as cholecalciferol standard or unknown given to rats on day 1 to 3 of assay period.

Slide 30:

12 July 2011 30 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.of recalcification of bones. The activity of vitamin D may be determined by amt. of recalcification in relation with reference standard. 5) Plague vaccine: The potency of plague vaccine is estimated by determining the dose necessary to protect mice against a lethal dose of a virulent strain of Yersinia pestis. Test Animals: Use white mice, 6 to 7 weeks old, each weighing between 20 and 28 g and of a strain susceptible to plague infection.

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12 July 2011 31 The animals should be healthy and free from intercurrent infection with organisms such as Salmonella. Suggested Method: Selection of suitable virulent strain: A freeze-dried virulent culture of Y. pestis is revived by subculturing 0.5 ml in 9.5 ml of nutrient broth in test-tube and incubating at 28 o for 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.

Slide 32:

12 July 2011 32 Standard challenge dose: Freshly reconstitute the freeze-dried culture and dilute with nutrient broth 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 being examined arranged in such a manner that the 50% protective dose (ED 50 ) 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 an interval of 7 days between them. Inject subcutaneously the standard challenge dose in each group of mice 7 day after second half of dose.

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12 July 2011 33 Observe the animals for 15 days and record the number of deaths in each group. Carry out a post-mortem, look for signs of plague . If plague organisms are not seen, such deaths are excluded from the calculation. After observation kill all the surviving animals and examine for signs of plague. Calculate the median effective immunizing dose, ED50, by standard statistical methods. The vaccine passes the test if it has an ED50 of 0.004 ml or less per mouse.

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12 July 2011 34 6) RABIES ANTISERUM: The potency of rabies antiserum is determined by comparing the dose necessary to protect mice against a lethal intracerebral dose of rabies virus with the dose of the Standard Preparation of rabies antiserum necessary to give the same protection. Standard Preparation: The standard preparation is a dried serum the potency of which has been determined in relation to the International Standard. Suggested Method: Test animals : Use healthy mice of either sex weighing between 10 and 14 gm.

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12 July 2011 35 Test virus : Any suitable strain of rabies virus of known potency such as the CVS strain may be used. Determination of potency of the rabies antiserum : Prepare a series of 2-fold dilutions of the Standard Preparation and of the preparation being examined with water containing 2% v/v of heat inactivated normal horse serum Add a quantity of a suspension of the test virus containing the test dose. keep the mixtures at 37 o for 1 hour. Inject intracerebrally 0.03 ml of each mixture into 10 mice. Observe the mice for 14 days after the injection.

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12 July 2011 36 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. 7) INSULIN: Std. preparation and unit: -Pure, dry and crystalline. One unit contain 0.04082 mg. -Accurately weighed 20 units of insulin is dissolve in normal saline. Acidify with Hcl to pH 2.5.Add 1.4% to 1.8% glycerin. Final volume should contain 20 units per ml. store in cool place and use within six months.

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12 July 2011 Test preparation: - Same as std. solution. Methods of bioassay: 1) Rabbit method 2) Mouse method 1. Rabbit method Selection of rabbits: -Should be healthy, weighing about 1800-3000 gms. -Should be maintained on uniform diet but are fasted for 18 hrs. before assay. -water is withdrawn during the experiment. Standard and sample dilutions: - Freshly prepared by diluting with normal NaCl sol. So as to contain 1 and 2 units/ml.

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12 July 2011 Doses: - The dose which can produce suitable fall in blood sugar level is calculated for the std. Principle: - The potency of a test sample is estimated by comparing the hypoglycemic effect of the sample with that of the std. preparation of insulin. Procedure: 12 rabbits divided into 4 groups, each containing 3 rabbits. Two of std. dilution and two of test dilution. A sample of blood is taken from the marginal ear vein of each rabbit. Presence of reducing sugar is estimated per 100 ml of blood, which is ‘Initial Blood Sugar Level’ From each rabbit a sample of blood is withdrawn upto 5 hrs. at the interval of 1 hr. again blood sugar is estimated which is known as ‘Final Blood Sugar Level’ Same animals are used after one week but grouping is reversed, that is animals which received the less dose of the std. are given the higher dose of the test sample and vice versa. This test is known as ‘ Twin Cross Over Test’ Mean percentage decrease in blood sugar is calculated.

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12 July 2011 2. Mouse method: Mice show characteristic convulsions after s.c. inj. Of insulin at elevated temp. The % convulsions produced by the test and std. preparations are compared. Selection of mice: - Should be healthy, weighing about 18-22 gms. -Should be maintained on uniform diet but are fasted for 18 hrs. before assay. Standard and sample dilutions: Dilutions are prepared with sterile saline solution. So as to contain 0.064 - units/ml(s1) and 0.096 units/ml(s2). Similarly test sample solutions are also prepared.

Slide 40:

12 July 2011 40 Procedure: Mice are divided into 4 groups, each containing 25 mice. Insulin is injected s.c. as two group of std. and two group of test solution. % convulsions produced by the test sample are compared with those of the std. sample.

9. References ::

12 July 2011 41 9. 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 ,15 th edition by Dr. R.K. Goyal ,page no. 578-582.

Slide 42:

12 July 2011 42 THANK YOU