Pyrogen Tests in Pharmaceuticals
Introduction to Pyrogen
tests
By: Shant Dermesrobian
Definition
In
theory
a
pyrogen
is any substance that,
when injected into a mammal, elicits a
rise
in body temperature
,
and substances produced by some
Gram-
positive bacteria, mycobacteria, fungi and
also viruses conform to this definition.
•
The most common pyrogens, however,
and those of major significance to the
pharmaceutical industry, are produced
by
Gram-negative
bacteria and are
termed
endotoxins
; they are
lipopolysaccharides (
LPS
) found in the
cell envelope.
Characteristic effects
•
It is dose dependent.
They cause:
Increase in circulating levels of inflammatory
cytokines followed by fever, blood coagulation,
hypotension, neutrophilia, increases levels of
cortisol.( low & moderate doses).
High pyrogenic doses
shock characterized by
cardiovascular dysfunction, failure and death.
Pharmacopoeial tests
That for pyrogens uses
rabbits
to assess
pharmacological activity and therefore
the prescence of pyrogens of
all
kinds.
The test for bacterial endotoxins uses
freshly
lysed amoebocytes
(blood cells) of
the horseshoe crab and is therefore
termed the
L
imulus
A
moebocyte
L
ysate
test (
LAL
).
The rabbits test
According to BP:
Three rabbits
Samples are injected into the margineal
ear (10ml/kg).
Monitored for 3 hours, at 30 minutes
intervals.
Criteria for pass or fail, as the number of
rabbits increase to 12 rabbits.
LAL test
Principle
:
Coagulation
The endotoxin-induced coagulation of its blood
is based on an enzyme-mediated interaction of
LAL with endotoxins.
LAL (limulus amebocyte lysate) test reagent
comes/ extracted from the american horseshoe
crab
Limulus polyphemus.
Test
Positive
prescence of endotoxins.
LAL vs. Rabbits
LAL is estimated to be 1000-fold sensitive.
But, LAL detects only endotoxins of G-ve
bacteria and
not all pyrogens.
Major limitation of the rabbits test is the
endotoxin tolerance because of repeated
use of animals.
There is low reactivity to the endotoxin
produced by certain species,
Legionella .
Pyrogen tests are crucial in pharmaceuticals to detect substances that can cause fever and adverse effects. Learn about pyrogens, endotoxins, pharmacopoeial tests using rabbits and LAL, and their significance in ensuring pharmaceutical safety.
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Presentation Transcript
Introduction to Pyrogen tests By: Shant Dermesrobian
Definition In theory a pyrogen is any substance that, when injected into a mammal, elicits a rise in body temperature, and substances produced by some Gram- positive bacteria, mycobacteria, fungi and also viruses conform to this definition.
The most common pyrogens, however, and those of major significance to the pharmaceutical industry, are produced by Gram-negative bacteria and are termed endotoxins; they are lipopolysaccharides (LPS) found in the cell envelope.
Characteristic effects It is dose dependent. They cause: Increase in circulating levels of inflammatory cytokines followed by fever, blood coagulation, hypotension, neutrophilia, increases levels of cortisol.( low & moderate doses). High pyrogenic doses shock characterized by cardiovascular dysfunction, failure and death.
Pharmacopoeial tests That for pyrogens uses rabbits to assess pharmacological activity and therefore the prescence of pyrogens of all kinds. The test for bacterial endotoxins uses freshly lysed amoebocytes (blood cells) of the horseshoe crab and is therefore termed the Limulus Amoebocyte Lysate test (LAL ).
The rabbits test According to BP: Three rabbits Samples are injected into the margineal ear (10ml/kg). Monitored for 3 hours, at 30 minutes intervals. Criteria for pass or fail, as the number of rabbits increase to 12 rabbits.
LAL test Principle: Coagulation The endotoxin-induced coagulation of its blood is based on an enzyme-mediated interaction of LAL with endotoxins. LAL (limulus amebocyte lysate) test reagent comes/ extracted from the american horseshoe crab Limulus polyphemus. Test Positive prescence of endotoxins.
LAL vs. Rabbits LAL is estimated to be 1000-fold sensitive. But, LAL detects only endotoxins of G-ve bacteria and not all pyrogens. Major limitation of the rabbits test is the endotoxin tolerance because of repeated use of animals. There is low reactivity to the endotoxin produced by certain species, Legionella .
