SCREENING METHODS IN PHARMQACOLOGY pyrogen testing TOPIC.pptx

SCREENING METHOD OF PYROGEN TESTING

1. PYROGEN TESTING 4/29/2021 Dept of Pharmacology 1 By: Ch. Sandeep Reddy Assoc. Professor Department of Pharmacology

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 4/29/2021 Dept of Pharmacology 2

3. Endotoxin characteristic  thermostable  water-soluble  unaffected by the common bactericides  non-volatile  These are the reasons why pyrogens are difficult to destroy once produced in a product 4/29/2021 Dept of Pharmacology 3

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 temperature in healthy rabbits by the intravenous injection of a sterile solution of the substance under the test. 4/29/2021 Dept of Pharmacology 4

5. Why the Rabbit?  Reproducible pyrogenic response  Other species not predictable  Similar threshold pyrogenic response to humans  Rabbit chosen for economic purposes 4/29/2021 Dept of Pharmacology 5

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. 4/29/2021 Dept of Pharmacology 6

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) 4/29/2021 Dept of Pharmacology 7 Rabbit Pyrogen Test

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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 0.6  C is not used in the main test 4/29/2021 Dept of Pharmacology 9

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 39.8  C the differences in initial Temperature should not differ from one another by more than 1  C 4/29/2021 Dept of Pharmacology 10

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 4/29/2021 Dept of Pharmacology 11

12. The result of pyrogen test: No.of Rabbits T e st 3 rabbits Individual Tempt. rise (°c) 0.6 Tempt. Rise in group (°c) 1.4 P as s e s I f a b o v e n ot p a s s e s 0. 6 3. 7 P as s e s 3+5 = 8 rabbits If above test not passes the sample is said to pyrogenic. 4/29/2021 Dept of Pharmacology 12

13. LAL Test  Limulu s ameboc y t e l y s ate tes t .  t o m ea s ure th e conc e n t ration of endotoxin s of gram-negative bacterial origin  reag e nt: a moeboc y te l y sate fr om horseshoe crab, Limulus polyphemus 4/29/2021 Dept of Pharmacology 13

14. Limulus polyphemus = horseshoe crab 4/29/2021 Dept of Pharmacology 14

15. P r i nc i p le 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 4/29/2021 Dept of Pharmacology 15

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. 4/29/2021 Dept of Pharmacology 16

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 4/29/2021 Dept of Pharmacology 17

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 4/29/2021 Dept of Pharmacology 18

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 4/29/2021 Dept of Pharmacology 19

20. R E FE R EN C ES  U.S.PHARMACOPEIA  Pharmaceutical Formulations by M.E.Aulton, H.C. Ansel.page no 195-196. 4/29/2021 Dept of Pharmacology 20

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