SCREENING METHODS IN PHARMQACOLOGY pyrogen testing TOPIC.pptx
- 1. PYROGEN TESTING 09/18/2024 By: Ch. Sandeep Reddy Assoc. Professor Department of Pharmacology
- 2. Dept of Pharmacology 09/18/2024 2 Pyrogens Pyrogens - fever inducing organic substances Responsible for many febrile reaction These are Endotoxin. Having nature Endogenous (inside body) Exogenous (outside body) Exogenous pyrogens – mainly lipopolysaccharides bacterial origin, but not necessary
- 3. Dept of Pharmacology 09/18/2024 3 Endotoxin characteristic thermostable water-soluble unaffected by the common bactericides non-volatile These are the reasons why pyrogens are
- 4. Dept of Pharmacology 09/18/2024 4 Test for pyrogens = Rabbit test the development of the test for pyrogens reach in 1920 a pyrogen test was introduced into the USP XII (1942) The test consists of measuring the rise in body
- 5. Dept of Pharmacology 09/18/2024 5 Why the Rabbit? Reproducible pyrogenic response Other species not predictable Similar threshold pyrogenic response to humans
- 6. Dept of Pharmacology 09/18/2024 6 Rabbit Pyrogen Test Rabbits must be healthy and mature New Zealand or Belgian Whites used mostly Either sex may be used Must be individually housed between 20 and 23°C Not varies more than ± 3º c. Free from disturbances likely to excite them.
- 7. Dept of Pharmacology 09/18/2024 7 equipment and material used in test (glassware, syringes, needles etc) Must be free from Pyrogens by heating at 250º c for not less then 30 minutes or any other method retaining boxes (comfortable for rabbits as possible) Thermometers or thermistor probe (standardized position in rectum, precision of ± 0.1°C) Rabbit Pyrogen Test
- 9. Dept of Pharmacology 09/18/2024 9 Rabbit pyrogen test Preliminary test (Sham Test) intravenous injection of sterile pyrogen-free saline solution Warm the pyrogen free solution up to 38.5ºc to exclude any animal showing an unusual response to the trauma (shock) of injection any animal showing a temperature variation greater than 0.6C is not used in the main
- 10. Dept of Pharmacology 09/18/2024 10 Rabbit pyrogen test - main test: group of 3 rabbits preparation and injection of the product: warming the product dissolving or dilution duration of injection: not more than 4 min the injected volume: not less than 0.5 ml per 1 kg and not more than 10 ml per kg of body mass determination of the initial and maximum temperature all rabbits should have initial Temperature: from 38.0 to 39.8C the differences in initial Temperature should not differ from one another by more than 1C
- 11. Dept of Pharmacology 09/18/2024 11 Interpretation of the results: the test is carried out on the first group of 3 rabbits; if necessary on further groups of 3 rabbits to a total of 4 groups, depending on the results obtained intervals of passing or failing of products are on the basis of summed temperature response
- 12. Dept of Pharmacology 09/18/2024 12 The result of pyrogen test: No.of Rabbits Test 3 rabbits Individual Tempt. rise (°c) 0.6 Tempt. Rise in group (°c) 1.4 Passes If above not passes 0.6 3.7 Passes 3+5 = 8 rabbits If above test not passes the sample is said to pyrogenic.
- 13. Dept of Pharmacology 09/18/2024 13 LAL Test Limulus amebocyte lysate test. to measure the concentration of endotoxins of gram-negative bacterial origin reagent: amoebocyte lysate from horseshoe crab, Limulus polyphemus
- 14. Dept of Pharmacology 09/18/2024 14 Limulus polyphemus = horseshoe crab
- 15. Dept of Pharmacology 09/18/2024 15 Principle The addition of solution containing endotoxin to a solution of lysate produce turbidity. The rate of reaction depends upon concentration of endotoxin , the pH and the temperature. The endotoxin reference standard is the freeze dried. The test is based on the primitive blood- clotting mechanism of the horseshoe crab
- 16. Dept of Pharmacology 09/18/2024 16 Commercially derived LAL reagents bleeding adult crabs into an anticlotting solution washing and centrifuging to collect the amebocyte lysing in 3% NaCl lysate is washed and lyophilized for storage activity varies on a seasonal basis and standardization is necessary.
- 17. Test performance (short) avoid endotoxin contamination Before the test: interfering factors should not be present equipment should be depyrogenated the sensitivity of the lysate should be known Test: equal volume of LAL reagent and test solution (usually 0.1 ml of each) are mixed in a depyrogenated test-tube incubation at 37°C, 1 hour remove the tube - invert in one smooth motion (180°) - read (observe) the result
- 18. Dept of Pharmacology 09/18/2024 18 Endotoxin concentration monitoring Following method are used to monitor the endotoxin concentration Method A: El-clot method: limit test Method B: semi-quantitative gel-clot method Method C: kinetic turbidimetric method Method D: kinetic chromogenic method Method E: end-point chromomeric method
- 19. Dept of Pharmacology 09/18/2024 19 different techniques: the gel-clot technique - gel formation the turbidimetric technique - the development of turbidity after cleavage of an endogenous substrate the chromogenic technique - the development of color after cleavage of a synthetic peptide- chromogen complex
- 20. Dept of Pharmacology 09/18/2024 20 REFERENCES U.S.PHARMACOPEIA Pharmaceutical Formulations by M.E.Aulton, H.C. Ansel.page no 195- 196.