Pyrogenic substances

Distilled water is often used in the formulahon of oral and topical pharmaceutical preparations and a low bacterial count is desirable. It is also used after distillation with a specially designed still, often made of glass, for the manufacture of parenteral preparations and a post-distillation heat sterilization stage is commonly included in the process. Water for such preparahons is often stored at 80°C in order to prevent bacterial growth and the production of pyrogenic substances which accompany such growth. 

One pyrogen test is a qualitative biological test based on the fever response of rabbits. If a pyrogenic substance is injected into the vein of a rabbit, a temperature elevation will occur within 3 hours. Many imitative medical agents will also cause a fever. 

A preferred method for the detection of pyrogens is the limulus amebocyte lysate (LAL) test. A test sample is incubated with amebocyte lysate from the blood of the horseshoe crab, Limulus polyphemus. A pyrogenic substance will cause a gel to form. This is a result of the... 

Historically, the rabbit pyrogen test constituted the most widely used method. This entails parenteral administration of the product to a group of healthy rabbits, with subsequent monitoring of rabbit temperature using rectal probes. Increased rabbit temperature above a certain point suggests the presence of pyrogenic substances. The basic rabbit method, as outlined in the European Pharmacopoeia, entails initial administration of the product to three rabbits. The product is considered to have passed the test if the total (summed) increase of the temperature of all three animals is less than 1.15 °C. If the total increase recorded is greater than 2.65 °C then the product has failed. However, if the response observed falls between these two limits... 

This test is popular because it detects a wide spectrum of pyrogenic substances. However, it is also subject to a number of disadvantages, including ... 

Bovine colostrum as well as milk whey contain essentially bacteria and pyrogenic substances and may contain viruses. Humanized or specifically reconstructed antibodies from transgenic animals can contain traces of endogenous antibodies from the host animal that are very difficult to separate from the newly expressed molecules. 

The pyrogenic activity of LPS is much higher than that of most other pyrogenic substances. This is the reason why an in-vitro limit test for LPS (the Limulus Amoebocyte Lysate, or LAL test) generally suffices for quality control piuposes of parenteral medicines and raw materials, including water for injection. 

So not all pyrogens are endotoxins, see Sect. 19.3.4. Endotoxins are however the most common cause of toxic reactions that are attributed to contamination of pharmaceutical products with pyrogens their pyrogenic activity is much greater than that of most other pyrogenic substances. 

Materials intended for intravenous use should be free from undue amounts of pyrogenic substances and this condition may be established by making intravenous injection of prescribed quantities into rabbits and noting the effect on the rectal temperature compared with a control level established for each animal. 

The General Tests and Assays. This section of the USP gives methods for tests that are general in nature and apply to a number of the substances. Procedures are iacluded for such tests as heavy metals, melting point, chloride, sulfate, sterility, bacterial endotoxins, and pyrogens. Also iacluded are descriptions of various analytical techniques, such as spectrophotometry, chromatography, and nmr, and descriptions of tests to be used on glass or plastic containers, mbber closures, etc. 

Pyrogenic A fever-producing substance. The presence of these substances is determined by the Limulus Amebocyte Lysate (LAL) test and measured in EU/ml (endotoxin units per milliliter). 

The inherent mechanism that prevents the height of fever from reaching a potentially dangerous level. It is mediated by substances liberated both systemically and within the brain during fever that counter the formation or action of endogenous pyrogens, or inhibit the activity of neural circuits that modulate febrigenesis. 

Pyrogens are substances that, when they enter the blood stream, influence hypothalamic regulation of body temperature, usually resulting in fever. Medical control of pyrogen-induced fever proves very difficult, and in severe cases results in patient death. 

Pyrogens may be detected in parenteral preparations (or other substances) by a number of methods. Two such methods are widely employed in the pharmaceutical industry. 

Disadvantages The protein expressed is accumulated within the cell matrix (intracellular) the protein does not undergo posttranslational modifications (resulting in proteins that may be structurally different or less useful to humans) the presence of lipopolysaccharides (pyrogens—microbial substances that cause fever) is likely to contaminate the product and there is a need for more extensive chromatographic purification. 

While the simultaneous presence of benzofuran and benzothiophene is established in some fractions from coal pyrogenation and from the distillation of some petroleums, benzothiophene is entirely absent in heterocyclic compounds extracted from natural substances of vegetable origin. However, numerous benzofuran or hydrobenzofuran derivatives of biogenetic or metabolic origin have been isolated. 

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