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Bioassay
The document discusses bioassays and dose-response curves, detailing methods to measure the presence and potency of drugs using various biological and chemical assays. It outlines principles, types of assays, and the detailed procedures for conducting bioassays, emphasizing factors like sensitivity, specificity, and reproducibility. The content also includes information on the assembly of instruments used in bioassays, along with theoretical calculations for determining drug potency and effectiveness.
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Bioassay
- 1. Bioassay & DRC DrNaser Ashraf Tadvi Associate Professor
- 2. 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
- 3. Types of Assays •Biological Method (Bioassay) • Physico-chemical methods – Chromatography, spectrophotometry • Radioimmunoassay • Microbiological methods
- 4. 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
- 5. 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
- 6. 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
- 7. 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
- 8. 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
- 9. BIOASSAY Can beperformed IN VIVO -On intact animal IN VITRO -Isolated tissues -Specific cells -Organisms in culture
- 10. 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.
- 11. 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.
- 12. Assembly for recordingcontraction of isolated tissue • Lever: Simple lever is used. Stylus
- 13. Magnification and Adjustment Magnificationfor Frog rectus is 5-10 Fast contracting- low Slow contracting- high BA A B
- 14. 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)
- 15. Assembly of instrumentsfor bioassay Mariottes Bottle Sherringtons starling drum Simple writing lever Aeration tube Inner Organ bath Outer Organ bath Kymograph
- 16. 1 gm weightis added Writing lever Assembly of instruments for bioassay
- 17. 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.
- 18. Pithing and Dissectionof Frog
- 19. 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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- 21. 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)
- 22. 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.
- 23. 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
- 24. Types of Bioassay •A. Quantal (Direct end point) Assay • B. Graded Assay -Bracketing assay -Matching assay -Interpolation assay -Multiple point assay
- 25. 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.
- 26. Quantal response (Allor none assay)
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- 28. 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)
- 29. 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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- 31. Test 1/10 10 U 80U40 U20 U Test SS 64 U32 U16 U Dose response curve of test solution in graded doses
- 32. Latin square S1 S2t t S1 S2 S2 S1 t X
- 33. 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
- 34. • 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
- 35. • 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
- 36. 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
- 37. 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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- 39. LDR CURVES OF2 DRUGS WITH DIFFERENT POTENCY
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- 47. 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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Editor's Notes
- #5 Examples ach , histamine
- #7 Blood glucose lowering responses in rabbit
- #8 Constant tissue bath temperature, oxygenation, use of standard perfusion fluid, maintenance of time sequence Comparison of main pharmacological effect Unknown and standard drug should have same mode of action and pharmacological effects so that the drc runs parallel and potency ratios can be compared conveniently
- #9 Minimize errors due to biological variations by using animals of same species, sex, weight, and if isolated tissue is used should be sensitive and number of animals should be large enough for applying statistics
- #19 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.
- #37 Add cc/U taken in syringe
- #43 TI=LD50/ED50
- #44 CSF=LD1/ED99