Bioassay

Bioassay

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  Bioassay & DRC DrNaser Ashraf Tadvi Associate Professor
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  Assay • Analysis doneto determine the presence of a substance & amount of that substance (Known or unknown) in biological fluids is known as Assay. • Determines Activity and potency of drug
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  Types of Assays •Biological Method (Bioassay) • Physico-chemical methods – Chromatography, spectrophotometry • Radioimmunoassay • Microbiological methods
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  Bioassay • Measurement ofthe concentration or potency of a drug from magnitude of its main biological effect Main Pharmacological effect of drug Compared with Pharmacological effect of standard drug Potency ratios are compared quantitatively
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  Indications(Uses) of Bioassay •Measure pharmacological activity of new/ chemically undefined substance • Measure concentration of known substance in tissue extract or body fluid e.g Ach, histamine • Measure ED50 / LD50 of a drug • Biological standardization of natural drugs which cannot be obtained in chemically pure form e.g Vasopressin, oxytocin
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  Parameters recorded • Bloodpressure (Dogs and cats): Sympathomimetics • Ach: contraction of rabbit duodenum or frog rectus abdominis • Skeletal muscle relaxants: rabbit head drop method • Histamine: Guinea pig ileum • Serotonin on rat colon
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  Principles of Bioassay •Experimental conditions should be constant • Standard should be in pure form and known conc. Should be available • Unknown and standard drug should have same mode of action and pharmacological effects • Use a specified pharmacological technique like rectus muscle preparation for Ach • System should be insensitive to other drugs
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  Characteristics of agood bioassay • Sensitivity - ability to detect smallest concentration • Specificity – the response which is being measured should specific. • Reproducibility - same observations using different instrument and operator, over longer time periods. • Stability – sensitivity of preparation should be constant. • Availability – the particular tissue should be easily available
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  BIOASSAY Can beperformed IN VIVO -On intact animal IN VITRO -Isolated tissues -Specific cells -Organisms in culture
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  Advantages of isolatedtissue experiments over intact animal experiments Several preparations can be obtained from a single animal. Small amount of the test material is required. Cheap, less time consuming. Interference due to pharmacokinetic factors and compensatory reflexes is avoided.
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  Assembly for recordingcontraction of isolated tissue Organ Bath: • Rudolph Magnus was first to design organ bath. Outer organ bath: made up of perplex with electrical thermostat and stirrer for uniform temperature. Inner organ bath: made of glass , capacity of bath is 30ml.
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  Assembly for recordingcontraction of isolated tissue • Lever: Simple lever is used. Stylus
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  Magnification and Adjustment Magnificationfor Frog rectus is 5-10 Fast contracting- low Slow contracting- high BA A B
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  Role of Aeration/ Aeration tube 1). Provides oxygen/ air to mounted tissue. 2). Allows uniform distribution of drug in organ bath through bubbles. 3). Areation tube helps in mounting tissue in organ bath. Aeration – 30-40 bubbles / min (air)
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  Assembly of instrumentsfor bioassay Mariottes Bottle Sherringtons starling drum Simple writing lever Aeration tube Inner Organ bath Outer Organ bath Kymograph
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  1 gm weightis added Writing lever Assembly of instruments for bioassay
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  Dissection and mountingthe tissue Procedure:  A pithed frog is laid on its dorsal surface in a dissection tray.  The skin of the abdomen cut to expose the whole abdomen.  The rectus muscle is identified, and the border of the muscle dissected out from adjacent abdominal wall.  A thread is passed with the help of needle through the pubic attachment of muscle and tied ,muscle is cut.  The upper end of muscle i.e, xiphisternum part also tied and cut.  The lower end of the muscle is tied to aeration tube at ‘s’ bent.
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  Pithing and Dissectionof Frog
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  Dissection and mountingthe tissue  Before mounting the tissue make sure the blood vessels are removed.  Aeration tube with mounted tissue is placed in inner organ bath filled with PSS.  The upper end of muscle is tied to lever with thread.  A load of 1gm is attached to the other side of lever at same distance as that of tissue from the fulcrum. This helps the muscle to relax in between contraction.  Tissue is aerated with air i.e 30-40 bubbles /min. and allowed to relax for 30- 45 min before recording responses.
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  Mounting the tissue
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  Acetyl choline Dilutions Stock solution- 1 mg/ml Acetylcholine(1000μg/ml) The dilutions are made as follows- 1 ml stock soln. +9 ml distilled water= 1/10 dilution(100 μg/ml) 1 ml 1/10 dilution +9 ml distilled water= 1/100 dilution(10 μg/ml) 1 ml 1/100dilution +9 ml distilled water= 1/1000 dilution(1μg/ml)
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  Recording cycle  Baseline:30 seconds(s) (start drum & allow to run freely)  Contractions: 90 s (contact period of Ach with tissue)  Relaxation: 3 minutes (tissue washed 2-3 times after 90 sec contact period)  Total cycle is 5 mins  Soon after contraction of 90 secs the weight of 1gm is lowered down to allow muscle to relax slowly, until lever comes to base line.  The contractions are recorded with each dose of solution till ceiling effect is obtained i.e no further increase in height of contraction with subsequent doses.
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  Recording cycle  Afterthe ceiling effect is obtained with standard Ach solution, the procedure is repeated with the test solution(unknown strength of Ach) in different dilutions.  The further experiment is done by Multiple point assay i.e. 2+1 assay Sensitivity of Rectus muscle can be increased by adding Physostigmine(0.5μg/ml)/ Alcohol to perfusion fluid
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  Types of Bioassay •A. Quantal (Direct end point) Assay • B. Graded Assay -Bracketing assay -Matching assay -Interpolation assay -Multiple point assay
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  Quantal response (Allor none assay) • In this assay, dose of standard and unknown which provide predetermined ‘all or none’ response, then their potency ratios compared. Ex: Digitalis induced cardiac arrest in cats, insulin induced hypoglycemic convulsions in mice.
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  Quantal response (Allor none assay)
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  Graded response assay
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  Multiple Point Assay •Bracketing and Matching assay methods are not ideal because lack of accuracy, sensitivity • So , to minimize those errors multiple point assays preferred (3,4 or 6 point assays)
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  0.2 100 25.612.86.43.21.6 51.20.80.410 U 400 200 40 U 20 U 80 U 16 U 32 U 64 U S2S1 S1 S1 S1S2 S2 S2 t t t t 2+1 Bioassay of Ach on Isolated Frog Rectus Muscle S2S1 T
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  6 Dose response curve
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  Test 1/10 10 U 80U40 U20 U Test SS 64 U32 U16 U Dose response curve of test solution in graded doses
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  Latin square S1 S2t t S1 S2 S2 S1 t X
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  Calculations Response Height of contraction (mm) Meanheight (mm) Dose (ml) 1 2 3 S1 34 32 37 35.2 0.1 S2 67 65 80 72.7 0.2 T 42 42 43 40.7 0.32 Dose ratio(d) = = = 2 S1 S2 0.2 0.1
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  • From dataobtained log potency ratio (M) is calculated from the formula: M = × log d M = × log 2 M = × 0.301 = 0.047 Calculations T – S1 S2 – S1 41 - 35 73 - 35 6 38
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  • From M,the strength of the unknown can be calculated – Strength of = × antilog of M test solution = × 1.114 = 0.348125 mg/ml = 348 μg/ml The given concentration was 300 μg/ml , so the error is 16% Percentage error(%)= × 100 Calculations S1 t 0.1 0.32 ACT ACT - OCT
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  Calculation DRUG DOSE (μg)RESPONSE (mm) LOG DOSE 2 0 0.30103 4 0 0.60206 8 0 0.90309 16 4 1.20412 32 8 1.50515 64 24 1.80618 128 30 2.10721 256 40 2.40824 512 42 2.70927 1024 45 3.0103 2048 45 3.31133
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  Dose Response Curve -10 0 10 20 30 40 50 0500 1000 1500 2000 2500 RESPONSE (mm) DOSE (μg) RESPONSE(mm)
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  Calculation of ED50
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  LDR CURVES OF2 DRUGS WITH DIFFERENT POTENCY
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  EFFICACY & POTENCY
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  FREQUENCY DISTRIBUTION CURVE
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  Therapeutic Index ED50 LD50
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  Certain Safety Factor
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  Therapeutic window
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  Matching/ Bracketing Assay
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  Matching/ Bracketing Assay
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  Matching/Bracketing Assay Advantage : Faster  Can be completed when amount of test drug available is small  Does not involve complicated calculations Disadvantage:  Match is subjective  Exact match may not always be possible
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  Interpolation method
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  Interpolation method
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  Thank You