Pyrogen
As pyrogenic (from ancient Greek πῦρ p „ r "fire" and γεννάειν gennáein "produce") one describes on the one hand "inflammable substances", on the other hand " substances produced in fire ".
In medicine are pyrogenic substances which at parenteral administration fever can produce.
General
A distinction is made between exogenous and endogenous pyrogens. To the exogenous pyrogens molecular compounds such as can lipopolysaccharide ( bacteria - endotoxins ) are counted, but also particles such as rubber abrasion of injection vials and microscopic plastic particles. For this reason, the test for endotoxins and the detection of particles saves a further check for pyrogens in parenteral solutions. A distinction is made between the following exogenous types of pyrogens:
- bacterial pyrogens
- Endotoxins of gram-negative bacteria (lipopolysaccharides (LPS) of the outer bacterial membrane with high heat resistance)
- Components of gram-positive bacteria (such as teichonic acids )
- Virus pyrogens
- are particularly formed by myxoviruses and have a low heat resistance
- Pyrogens as components of mushrooms
- Pyrogens of non-biological origin
- Metal compounds in elastomers, rubber abrasion
The group of endogenous pyrogens consists of fever-inducing substances that the body produces itself, such as the interleukins IL-1 and IL-6 or TNF-alpha . These set off a cascade of immune reactions, as a result of which prostaglandins are formed as endogenous pyrogens.
proof
Evidence is provided by the rabbit test , the Limulus test (also known as the LAL test for short) and, since 2013, increasingly by the monocyte activation test (MAT). For reasons of animal and species protection , the MAT is preferable.
Rabbit test
The rabbit test for testing for pyrogens was included in the British Pharmacopeia (BP) as early as 1912 and in the US Pharmacopeia (USP) in 1942.
The test measures the increase in body temperature in rabbits which is produced after intravenous injection of a sterile solution of the substance to be tested.
The rabbit test is carried out as follows according to Ph. Eur .: 1 to 3 days before the main test, a pyrogen-free isotonic saline solution (10 ml / kg body weight) is injected in the so-called preliminary test and the course of the body temperature is determined. If there are no deviations (less than 0.6 K ), three rabbits are injected with a test solution in the main test. The body temperatures are determined and compared 90 minutes before and 3 hours after the injection. The test substance corresponds to the test and is pyrogen-free if the sum of the individual values in three animals is not greater than 1.15 K. If the sum is greater than 2.65 K, the test substance is not pyrogen-free. If the total is in between, the test is repeated. In 2012, 110,000 rabbits were used for the rabbit test in Europe, but it is no longer required by national pharmacopoeia in the USA and Europe.
Limulus test (LAL test)
The LAL test is based on the coagulation or coagulation of the lysed amebocytes and lipopolysaccharides of the cell wall of gram-negative rod bacteria that are found in the blood of the horseshoe crab ( Limulus polyphemus ) . This test is about 100 times more sensitive than the rabbit test, but only responds to endotoxin (LPS) from gram-negative bacteria.
- LAL: L imulus- A möbocyten- L ysat
- Test systems available for sale already react to 100–1000 gram-negative bacteria per ml
- indicated internationally as EU = endotoxin units
- Applications: review of injectable drugs, analysis of milk , dairy and egg products , Dialysatwasser
To carry out the LAL test, the blood of around 400,000 caught horseshoe crabs is taken each year; these are then released back into the sea. An estimated 50,000 animals die annually for US production alone.
A test has also been available since 2003 that is based on a genetically engineered version of the relevant horseshoe crab protein and therefore does not use live animals. However, this test was only slowly adopted, which only changed in 2016 when the European Pharmacopoeia recognized this test.
Monocyte Activation Test (MAT)
The monocyte activation test is an in vitro test based on the human fever reaction with human blood cells (monocytes). It combines the advantages of the rabbit test and the LAL test without their specific disadvantages. The MAT not only detects endotoxic pyrogens, but also non-endotoxin pyrogens (not possible in the LAL), is just as easy to carry out as the LAL test and, for reasons of animal welfare, is definitely preferable to the rabbit test. An additional advantage of the MAT is that its results can be directly transferred to humans, in contrast to all other pyrogen tests (e.g. on rabbits or even on horseshoe crab amoebocytes, whether in the LAL or BET), since they are species-specific based on human monocytes. It was legitimized in the European Pharmacopoeia in 2010 as MAT monograph 2.6.30 and in 2012 by the FDA for the USA. The MAT was validated internationally in 2013 and has since gained acceptance and widespread use in the USA and Europe, as it is not only a legal and full-fledged alternative to the rabbit test, but its results are now reliably viewed worldwide. The test can be carried out using standardized and commercially available test kits.
Pyrogenic action
By their effect on the macrophages are mediators released, leading to an induction of COX-2 expression in the capillary endothelium of the strongly vaskularisierbaren vascular organ of lamina terminalis lead (OVLT), whereby the PGE 2 synthesis is stimulated. PGE 2 activates prostanoid EP3 receptors in the heat regulation center of the anterior hypothalamus and thus leads to an increased formation of cyclic AMP (cAMP). This causes an increase in the target value for the core body temperature, with the consequence of the temperature rise due to a reduced heat release (constriction of the skin vessels) and increased heat production (muscle tremors, chills ).
Web links
Individual evidence
- ↑ a b c d e f g h i j Thomas Hartung, Nina Haswia, Mardas Daneshian, Bodo Holtkamp, Gabriele Schmitz, Anke Hossfeld: A really human determination of endotoxins and non-endotoxin pyrogens. In: Pharm. Ind. Volume 75, No. 5, 2013, pp. 825-834.
- ↑ ZIM success example: bacteriophages instead of horseshoe crabs - test system for the specific detection of endotoxins (PDF; 885 kB).
- ↑ Ph. Eur. 5.0, 2005, 2.6.8 Testing for pyrogens.
- ^ Sarah Zhang: The Last Days of the Blue-Blood Harvest . In: The Atlantic . May 9, 2018 ( theatlantic.com [accessed May 14, 2018]).
- ↑ FDA: Guidance for Industry: Pyrogen and endotoxins testing: Questions and answers , June 2012 (PDF).
- ↑ Aktories: General and special pharmacology and toxicology, 9th edition