For the parenteral products, the most usual vehicle is water. Water is most abundantly available, cheap, safe, non-toxic and non-irritating. But anything that is to be administered parenterally has to be sterile and apyrogenic. Apyrogenesity means lack of pyrogens. Pyrogens are the metabolic products of bacteria. In making a vehicle suitable for parenteral preparation, we may sterilize it by autoclaving, which would kill the vegetative organisms as well as the spores. But the water once had organisms living in it and their metabolic products now stay in the water. These products are not destroyed by autoclaving. Since they cannot be destroyed at the usual autoclaving temperature, they must be prevented from getting into water. So the water being taken for parenteral preparation must be apyrogenic.
Pyrogens can cause a lot of damage, if they are injected into a human being. Hence every batch of injections is tested for the presence of pyrogens. The word "pyrogens" means, "fever producing".
What is the Definition of Pyrogen
Pyrogens are fever producing substances, which are metabolic products of microorganisms. Chemically, they are lipid substances associated with a carrier molecule, which is usually a polysaccharide. The carrier may also be a peptide. These carriers increase the solubility of the lipid. Pyrogens are produced by many microorganisms including bacteria, yeasts and moulds. Most potent pyrogens are the endotoxins produced from the cell walls of the Gram- negative bacteria. Pyrogens have a high molecular weight, often, more than 1,000,000. 1
What is the source of Pyrogen
Sources of Pyrogens: Solvents, drugs, additives apparatus used in manufacture, containers; all or any of these may be sources of pyrogens. The method of storage in between preparation and sterilization also may cause the development of pyrogens.
Hence every item must be apyrogenic and method of storage must not allow any bacterial growth.
What PYROGENS do : ?
If unintentionally, pyrogens are injected into a patient, they may bring about the following physiological changes:
? Erythema at the injection site
? Pain in the legs and trunk
? General discomfort
? High temperature
Pyrogens, if present in large volume parenterals, can be especially dangerous, as they would be present in large quantities and are given intravenously.
? Pyrogens in large volume parenterals may make the patients very ill and the effect of rise of temperature may be fatal.
Hence there is a need for testing parenteral solutions for their apyrogenesity and Pharmacopeias make it compulsory.
What is the history of Pyrogens :
In the years gone by, there was only one way in which pyrogens were tested for, i.e., by the Rabbits' test. But today much easier methods such as the limulus amoebocyte lysate test are available for testing for pyrogens. If we look at the history of pyrogens, we find that since the time of introduction of intravenous therapy, in 1656, by Sir Christopher Wren, the awareness of a fever producing agent was there. The documented research into the development of medicines contains many references to episodes of fever following intravenous infusions. But the scientists could not find the mechanism by which intravenous therapy was producing fever. Since fever frequently followed sepsis in wounds, physicians thought that the tissues in the wounds were decomposing, then putrefying by fermentation and in the process were causing the fever. Louis Pasteur (1822-1895), discovered that bacteria caused fermentation. Then scientists associated fever and pyrogens with fermentation and with bacteria. But they had no idea about the origin of pyrogens, whether they were produced by bacteria or whether they were a part of bacteria.
Research into the nature of pyrogens started showing good results in 1894, when Centanni extracted pyrogens from many types of bacteria. He extracted pyrogens from Esherichia coli too. Scientists started looking into the chemical and physiological nature of pyrogens. Centanni showed that pyrogens were not proteinous and that they were not susceptible to heat. It was the publications of Hort and Penfold, in the year 1912, that provided the best understanding of the nature of pyrogens. They started the Rabbit Assay and standardized it for fever. By correlating the fever effect in rabbits with the extent of pyrogens, and by characterizing the bacteria by staining as Gram positive and Gram negative Hort and Penfold could prove that the pyrogenic bacteria were predominantly Gram negative whereas the Gram positive were mostly apyrogenic. Then they could correlate between the bacterial count in the injected distilled water and the extent of fever caused and also prove that dead bacteria also caused fever. Then, Hort and Penfold, concluded that the real cause of injection fevers was a filterable substance which was stable to heat, and was produced by bacteria. Seibert's investigations in the years1923-1925, proved these findings conclusively. Seibert could also develop a process for the production of apyrogenic infusion fluids.
Hospital pharmacists were very happy to follow the method introduced by Siebert to produce large volume parenterals that were very safe for use in patients. Seibert's methods also launched the large commercial LVP solutions industry. Today's huge LVP production industry depends on the availability of large volumes of sterile and apyrogenic water which is produced by distillation.
A typical production procedure for manufacture of injectables goes like this:
Apyrogenic raw materials are dissolved in apyrogenic water for injection.
Mixing is done in depyrogenated tanks.
The prepared solution is dispensed into depyrogenated containers.
These containers are immediately autoclaved in order to prevent the growth of any microorganisms that might be present in the solution.
What is the Nature of Pyrogens :
A pyrogen is a fever producing substance. The most important pyrogen is the endotoxin produced by the Gram negative bacteria. Some pyrogens are there, which are not of microbial origin. They are in fact pharmacological agents, such as bleomycin and colchicines. But it is the microbial pyrogens that pose a problem to the manufacturers. While many organisms including live and dead bacteria, fungi, viruses, malarial parasites, and the bacterial products such as streptococcal exotoxins, staphylococcal enterotoxin, bacterial endotoxin lipopolysaccharide, and fungal products can act as pyrogens, it is the Gram negative bacteria produced endotoxin that is the most significant pyrogen for the manufacturers of injectables. This endotoxin is the most significant because its lipopolysaccharide component is very toxic in very small quantities. A quantity as small as 1ng/kg of the USP Reference endotoxin can cause fever.
Fever inducing mechanism:
Endotoxin pyrogen enters the bloodstream.
It binds to Lipopolysaccharide (LPS) binding protein (LPB).
LPB takes it to the reticuloendothelial system (RES).
The receptor cells in the RES are the circulating mononuclear cells.
This attachment to the receptor cells causes the production of proinflammatory cytokines.
These cytokines are interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-a (TNFa).
These factors produce inflammation and fever.
Nature of Bacterial Endotoxin: This endotoxin is present almost everywhere and is very difficult to remove. Its nature and omnipresence is as following:
This is a high molecular weight complex molecule.
This comes from the outer cell wall membrane of gram negative bacteria.
These GNB keep shedding the endotoxin into the environment in which live.
The endotoxin is shed by the bacteria as they live, as they multiply and also after they die and disintegrate.
So endotoxin may be present in a system in a form associated with live organisms or unassociated, in a free manner.
Endotoxin associated with microorganisms may be removed by microporous sterilizing filters.
But unassociated endotoxin passes through these filters also.
It cannot be destroyed by autoclaving as it is heat stable.
Endotoxin can be found in food materials, in ground waters, in soil and in substances.
It is found on laboratory glassware, research apparatus, and water baths.
It can be found in all places where moisture may be there.
Chemical Nature of endotoxin:
Bacterial endotoxins contain lipid, protein and carbohydrate.
The primary chemical structure is a polysaccharide structure which is covalently bound to a lipid, called lipid A.
Because of this structure, the bacterial endotoxin is called as lipopolysccharide or LPS.
The lipid A part is in the bacterial cell wall.
The polysaccharide projects into the environment.
The polysaccharide consists of two parts; a core oligosaccharide which is connected to Lipid A and a longer oligosaccharide O- specific chain which is attached to the core.
This second oligosaccharide O- specific chain is the variable part of the molecular structure.
The minimum variable component is the lipid A.
Lipid A consists of a disaccharide of glucosamine.
The glucosamine is highly substituted with ester linked and amide linked long chain fatty acids.
Each fatty acid has a backbone of 14 carbon atoms.
Lipid A is responsible for the harmful and useful activities of endotoxin.
1. The Theory and Practice of industrial Pharmacy, Leon Lachman and Herbert A. Lieberman, Special Indian Edition, 2009, CBS Publishers and distributors, page 641.
2. James Swarbrick, Encyclopedia of Pharmaceutical Technology, Volume 1,Third edition, , Informa Health care, Newyork: 2007: pages 3052- 3053
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