G.RAJESWARI

M.PHARMACYPHARMACOLOGY.Y11MPH15111M.A.M College Of Pharmacy

Quantitative estimation can be done by..

BIOASSAY:

Estimation of concentration or potency of a substance by measurement of biological response it produces.i.e. Observation of pharmacological effects on [1] living tissues, or cells [2] microorganisms [3] animals

Also known as BioStandardization.

Bioassay generally employed on..Chemical assay is not available.Quantity of sample too small.Estimate concentration of active principle in tissue extract.e.g..insulin.Estimate pharmacological activity of unidentified substance,Measure drug toxicity,Diagnosis & research.Dose of drug required to produce therapeutic effect.

If active principle of drug is unknown,e.g. insulin. Chemical method not available. Chemical composition not known. Purification for chemical assay not possible. To ascertain the potency hence served as quantitative part of screening procedure. Investigate function of endogenous mediators. Measure drug toxicity & unwanted effects.

1) All bioassays(lab.studies , toxicity studies , clinical trials)must be comparative against a standard drug or preparation, Way of minimizing error.Standard Preparation:- Representative of substance serves as basis for comparative measurement of activity.

In India maintained & distributed by:

Central Drug Research Laboratory,Kolkata. Central Research Institute,Kasauli.

4)Method should estimate as far as possible the error due to biological variation.

2) Standard & new drug should be as far as possible identical to each other.

So,dose response curve will have same slope & parallel.

3) Method of comparison preferably(not essentially) test therapeutic property of drug.

[1] Quantal Assays [ Direct endpoint ]Elicits an All or None response in different animalsE.g.. Digitalis induced cardiac arrest in guinea pigs hypoglycaemic convulsions in mice. Digitalis induced head drop in rabbitsCalculation of LD50 in mice or rats[2] Graded Response Assays [mostly on tissues]Graded responses to varying dosesUnknown dose response measured on same tissue

Graded:Here proportionate increase in responses due to increase in dose or concentration.

E.g. contraction of smooth muscle for histamine assay.

It can be done by following methods..MATCHING BIOASSAY:-Firstly responses of test is taken & is matched with response of standard drugs.Done till a closed matching is observed.Corresponding concentration calculated.Employed for small sample size.

INTERPOLATION:- Concentration response curve ofstandard established. 2-3 responses of testis recorded. Selection such that they lie on LINEAR PORTION of CRC of standard drug. Precision & reliability is better.

MULTIPLE POINT BIOASSAYThree point bioassay:- [2+1 dose assay] two standard & one test responses are taken down.Fast & convenientProcedure [E.g. Ach bioassay] Log dose response [LDR] curve plotted with varying conc of std Ach solutions and given test solutionSelect 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 4 sets data [Latin square: Randomisation reduces error] as followss1s2tts1s2s2ts1s1s2tPlot mean of S1, S2 and T against dose. Calculate Log Potency ratio [ M ] = [ (T S1) / (S2-S1) ] X log d [d = dose ratio]

Four point bioassay:-two standards & two test responses are recorded.

Two responses of standard should lie on linear portion of CRC in ratio of 1:2.

Test response is by trial & error method.

Employing statistics responses are recorded.

Most common method used.

SIX POINT BIOASSAY:-Three concentration of standard & test are used.More time consuming method.BRACKETTING METHOD:- Used when test sample is too small.

Response of test is bracketed between two responses ( greater & smaller ) of standard substances.

Precision & reliability is poor.

MERITS:Biological products like toxin,anti toxin,sera can be conveniently assayed.

Measure minute(nano mole & Pico mole)quantities of active substances.

Can detect active substance without prior extraction or other treatment.

DEMERITS:Key problem is variability in response.Large number of animal to be used.Expertise in experimental design,execution of assay & analysis of data required.Leads to expensive & time consuming.Time related changes ins sensitivity of test organ.Tachyphylactic responses of substance being assayed.

Bioassay of vasopressin:Principle : Vassopressor activity is estimated by comparing the avtivity of test with that of std under the conditions of suitable methods of bioassay. Std preparation:It is the 1st International std preparation.Methodology:Freeze dried synthetic arginine vasopressin peptide acetate with human albumin & citric acid(supplied in ampoules containing 8.20 units Inject slowly in to tail vein of male albino rat, a solution of -adreno receptor blocking agent(10ml/kg BW of solution prepared by dissolving 5mg of phenoxybenzamine Hcl in 0.1ml 95% ethanol), 1M Hcl & dil to 5ml with saline.

After 18hrs, anaesthetise the rat, ater 45-60min tie the rat on its back to the operating table by its hind legs. Cannulate the trachea with short polyethylene tube of external diameter 2.5mm & dissect carotid artery , ready for cannulation.Then cannulate the femoral vein close to the inguinal ligament.Dissect femoral vein, when dissecting a deep branch found & tied to prevent bleeding. Tie a short polyethylene cannula into femoral vein by 2 ligatures & join with an thistle funnel containing saline at about 37 c. Fix wet absorbent cotton swab to thigh so as to cover incision & cannula.At this stage inject 200 units of heparin, dissolved in saline/100g of body wt through venous cannula. Then tie in a carotid cannula of external diameter about 1mm & connect by a column of saline solution containing heparin with a suitable pressure measuring device such as a mercury manometer of internal diameter about 2-3mm.

CNS & PNS including both vagus & associated with sympathetic nerves is left intact.No air is injected, inject all solutions through venous cannula by means of syringe & wash it with saline. Dilute std & test preparation with saline & choose 2 doses of std & test preparation should given in randomised block or latin square design & 4-5 responses to each are recorded.Measure all responses & calculate results by std statistical methods.Bioassay of hyaluronidase:Method: Hyaluronic acid is measured by its ability to form turbidity with an acid albumin solution. Turbidity is a function of hyaluronic acid concentration and can hence be related to enzyme activity. One unit is based on the change in absorbency (turbidity) at 540nm of an internal standard assayed concurrently with each lot. Internal standard replaces USP/NF reference no longer available.

Reagents:

0.1 M Sodium phosphate buffer, pH 5.3 with 0.15 M sodium chloride (HSE buffer) 0.5 M Sodium acetate buffer, pH 4.2 Albumin reagent: Prepare by dissolving 2.5 grams of bovine serum albumin, Fraction V in 250 ml of 0.5 M sodium acetate buffer, pH 4.2. Adjust pH to 3.0 with 2 N HCl and heat at 93C for 30 minutes. Cool and adjust final volume to 1000 ml with 0.5 ml sodium acetate buffer, pH 4.2. Standard: Prepare stock solutions of 1.0 and 0.5 mg/ml. Hyaluronic acid (HA): Dissolve 10 mg Worthington hyaluronic acid (Code: VHHA) in 25 ml 0.1 M sodium phosphate buffer: pH 5.3 with 0.15 M sodium chloride. Note: This solution can be prepared by allowing VHHA to dissolve overnight. Heating in a boiling water bath for 10 - 15 minutes is the preferred method if the material is not immediately soluble.

Enzyme:

Prepare stock solution of enzyme at one mg/ml in 0.1 M sodium phosphate buffer pH 5.3 with 0.15 M sodium chloride. Immediately prior to use dilute further in the same buffer. For crude grade material concentrations of 0.01 - 0.05 mg/ml are recommended. For purified grade concentration of 0.001 - 0.01 mg/ml are recommended.Procedure:

I. Standard Curve

Into a series of numbered tubes, pipette as follows:

Corresponding toTube #1ml HAmg HAml HSE buffer10.000.001.0020.100.040.9030.200.080.8040.250.100.7550.300.120.7060.400.160.60

Place all tubes in a boiling water bath for 5 minutes. Cool to room temperature. Add 9.0 ml of albumin reagent and allow to stand for 10 minutes. Read absorbance at 540 nm. Plot absorbance at 540 nm versus mg HA to form standard curve. Hyaluronic acid should be soluble under the defined conditions and should produce a standard curve with a slope of 1.5 or greater.II. Test Procedure

Pipette 0.5 ml of a 0.4 mg/ml hyaluronic acid solution into a series of test tubes. Incubate at 37C for 4 - 5 minutes to achieve temperature equilibrium. Incubate one blank tube with one ml of 0.1 M sodium phosphate buffer, pH 5.3 with 0.15 M sodium chloride. At timed intervals add 0.5 ml of appropriately diluted enzyme or standard to respective tubes. Incubate each tube exactly 10 minutes and cool in an ice bath to room temperature. Add 9.0 ml of albumin reagent to each tube and incubate at room temperature for 10 minutes. Read A540of each tube versus the blank.

70.500.200.5080.600.240.4090.700.280.30100.800.320.20

Calculation:Determine the amount of hyaluronic acid remaining after digestion from the standard curve. Calculate the amount of hyaluronic acid digested as follows:Calculate turbidity reducing units/mg of enzyme or standard as follows:Calculate units/mg enzyme as follows:

Bioassay of corticotrophin:Principle: Potency of corticotrophin is determined by comparing its adrenal ascorbic acid depletion property with that of the std preparation of corticotrophin under the conditions of suitable methods of assay.Std preparation & unit: Std preparation is a quantity of dry corticotrophin. The unit is contained in 0.88mg of the std preparation at present in use.Methods:Method-I: (when it is intended for admin by subcutaneous) Rats of either sex(100-200g) & kept under uniform conditions for atleast a week before test. On the day before the test, weigh & hypophysectomise the rats.

After the operation allow access to a 5% solution of anhydrous dextrose in saline solution in addition to rat diet & water & keep the animals in a quiet room at a constant temp b/w 24-27.Carry out the rest b/w 18 & 36 hrs after hypophysectomy.On the day of test reweigh the animals & assign them at random to 6 groups of 8-10 animals.Choose 3 doses of std & test, administer in random by sc injection , doses adjusted to the body wt of animals(1,0.5,&0.25 units/100g of body wt). Dissolve the praparations in 0.01N Hcl & make subsequent dilutions with water containing 15% w/v of gelatin & should be injected with in 4 hrs.3hrs after injection remove both adrenal glands from the anaesthetised rat & weigh. Kill the rat & examine for hypophysectomy.Homogenise the adrenal glands in freshly prepared 2.5% w/v solution of metaphosphoroc acid & adjust to 10ml with same solution.

Allow it to stand for 30min & centrifuge, add 7ml of 4,53% w/v solution of sodium acetate adjusted to pH 7 by the addition of 5N acetic acid,3ml of water,& 2ml of std 2,6-dichlorophenolindophenol solution. 30sec after mixing, measure the extinction of resulting solution at the maximum at about 520nm.Calculate the wt of ascorbic acid from a std curve prepared by treating suitable aliquots of a solution of ascorbic acid in a 2.5% w/v solution of metaphosphoric acid by the same process, & express in mg/100g of adrenal gland.Calculate the results of the assay by std statistical methods.Method-II:Follow the method I with the following modifications: Administer the std & test preparation by iv injection in saline solution with out adding gelatin & acidify by adding 0.25% v/v of 5N acetic acid. Doses of the order of 1,0.5,&0.25 milliunit/100g of body wt are usually suitable. Remove the adrenal glands b/w 55 & 65min after injection.(when it is intended for admin by intravenous)

Limit of error: The fiducial limits of error of the estimated potency (p=0.95) are not less than 64% & not more than 156% of stated potency.Bioassay of plague vaccine:Principle: The potency of palgue vaccine is estimated by determining the dose required to protect mice against a lethal dose of virulent strain of yersinia pertis.Test animals: Use white mice(6-7 weeks old),each weighing b/w 20-28g, susceptible to plague infection.Method:Prepare a series of 5 graded doses of preparation being examined, 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 2 equal parts with an interval of 7days b/w them. Inject subcutaneously the std dose in each group of mice. 7 days after 2nd half of dose.Observe the animals for 15days & record the number of deaths in each group.Carry out postmartum, observe it for signs of plague. If plague organisms are not seen, such deaths are excluded from the experiment.After observation kill all the surviving animals & examine for signs of plague.Calculate the median effective immunizing dose, ED50 by std statistical methods.Vaccine passes the test if it has an ED50 of 0.004ml or less/mouse. Bioassay of rabies antiserum:Principle: The potency of rabies antiserum is determined by comparing the dose required to protect mice against a lethal intracerebral dose of rabies virus with the dose of std preparation of rabies antiserum required to give the same protection.

Std preparation: Std preparation is a dried serum,the potency of which has been determined in relation to the International std.Method:Prepare a series of 2 fold dilutions of std&test preparation with water containing 2%v/v of heat inactivated normal horse serum. Add a quantity of suspension of test virus containing the test dose.Keep the mixture at 37 for 1hr.Inject intracerebrally 0.03ml of each mixture into 10 mice, observe the mice for 14days after injection.If mice die before 5th day after inoculation with the virus are eliminated from the test. All the mice die b/w 5th-14th day after showing signs of rabies are considered as they died by rabies. Mice live upto the 14th day but showing signs of rabies are also counted as they died from rabies.Calculate the results by std statistical methods.

REFERENCES:Elements of pharmacology,Goyal,R.k.Essentials of pharmacotherapeutics,Barar,F.S.K.Hand book of experimental pharmacology,kulkarni.s.k.Indian pharmacopoeia, 1996.Internet.