Pyrogens and Parenteral Pharmaceuticals*
by Peter Grandics
Endotoxin (ET) produced by Gram-negative bacteria is an increasing concern in biotechnology. Endotoxin is highly toxic to mammalian cells and is one of the most potent modulators of the immune system.
Endotoxin, also called lipopolysaccharide (LPS), is composed of hydrophobic fatty acid and hydrophilic carbohydrate domains (1). The primary hydrophobic domain, also called Lipid A, carries many of the biological activities associated with LPS. Lipid A contains fatty acid chains attached to a phosphorylated disaccharide. The lipid composition of Lipid A exhibits strain-specific variations. The core is made of carbohydrates, some of which are phosphorylated and ethanolaminylated. The repeat units are trisaccharides, the number of which also varies in a strain-specific manner.
The chemical nature of LPS makes pyrogen removal problematic (2). LPS is unusually thermostable and fairly insensitive to pH changes. High concentrations of acids or bases are necessary to destroy LPS within a reasonably short time. The size of LPS varies depending on the environment. Molecular weights ranging from 2 kDa to several million Da have been reported. Also, naturally occurring LPS has a Stokes radius lower than purified endotoxin (3) typically used to qualify filters. This adds to the uncertainty of developing effective LPS removal methods. The heterogeneity of LPS is substantial, and its implications are not fully appreciated in the area of endotoxin removal.
The mechanism of endotoxin action has been studied extensively both in vitro and in vivo. LPS is released both by live and dead bacteria and is bound by the LPS-binding protein (LPB). Both LPB (4-6) and BPI (bactericidal/permeability increasing protein) (7,8) play an important role in the host response to endotoxin. The LPB-LPS complex binds to the CD 14 receptor on the cell surface leading to activation of the cell (9). The main target cells are the circulating mononuclear cells which produce pro-inflammatory cytokines, such as IL-1 and TNF-alpha (10,11). The pro-inflammatory cytokines are involved in acute and chronic inflammation, and modulate the host response to bacterial infection (12).
Even in the absence of fever, IL-1 and TNF-alpha induces IL-6 and acute phase proteins, such as C-reactive protein. This has recently been suggested as a marker for the increased mortality and morbidity for end-stage-renal-disease (ESRD) patients (13). Therefore, even in the absence of fever, increased cytokine production due to LPS contamination can reflect an inflammatory reaction with potentially detrimental consequences.
Besides LPS, Gram-negative bacteria release peptides, such as exotoxin A (from Pseudomonas), peptidoglycans, muramyl peptides and other still-unidentified substances. These bacterial products act similarly to LPS in terms of inducing the secretion of cytokines. The molecular size of these products vary greatly. The peptidoglycans and LPS are large molecules, while LPS subunits and muramyl peptides may be as small as 2000 Da. In the supernatant of Pseudomonas, a major group of water-borne bacteria, a very low molecular weight (<1000 Da) pyrogen was identified recently (14,15). In actual product samples, a mixture of pyrogens are always present, suggesting that no single modality of pyrogen removal may be adequate.
This heterogeneity of pyrogenic bacterial products raises the question of effective detection of pyrogens in parenteral pharmaceuticals. Both the United States Pharmacopoeia and the European Pharmacopoeia specify the rabbit pyrogen test and the Limulus amoebocyte lysate (LAL) test (16,17). However, the LAL test detects only LPS, and gives false negatives with certain products. The rabbit test, technically, is an acute toxicity test with an arbitrarily selected end-point. There are reports that some parenteral products (human serum albumin) have caused pyrogenic reactions in patients even after passing the rabbit test and the LAL test (18). Therefore, it is possible that even the rabbit test cannot detect all pyrogenic bacterial products.
An alternative pyrogen test is the monocyte activation/cytokine assay (19,20). In this assay, monocytes isolated from human peripheral blood are incubated with test samples and their supernatant is assayed for pyrogenic cytokines, such as IL-1 or TNF-alpha (21). A variation of this test is the whole blood/IL-6 assay (22). A monocytic cell-based assay has also been developed (18).
The disadvantage of the monocyte activation/cytokine assay is that the sensitivity of freshly isolated monocytes can vary depending on the immunological responsiveness of the donors (20). To make this assay suitable for quality control purposes, a well-defined monocytic cell line is needed which retains its ability to synthesize and secrete cytokines. This could lead to the development of a quantitative pyrogen test with a broader range of applicability. The MONO-MAC-6 (23) and the THP-1 (24) cell lines have been characterized for their suitability in a cell-based pyrogen assay, and were found to have the ability to detect bacterial pyrogens (18).
The MONO-MAC-6 cell line has been successfully used to detect pyrogenicity in human serum albumin lots which passed both the LAL and rabbit pyrogenicity tests but gave adverse (pyrogenic) reactions in humans (18). This suggests that a monocytic cell-based assay is more effective in detecting pyrogens than the standard methods, and could successfully complement existing methods.
The physiological effects of pyrogens in humans are diverse and dose-dependent. First, pyrogens elevate the circulating levels of inflammatory cytokines, e.g., IL-1, IL-6, TNF-alpha, and IL-8 (10) followed by the clinically relevant events of fever, hypotension, lymphopenia, neutrophilia, elevated levels of plasma cortisol and acute phase proteins, e.g., C-reactive protein (13,25). Low doses of pyrogens induce inflammatory reactions without any clinically significant symptoms. Moderate doses of pyrogens induce fever and significant changes in plasma composition (26,27). High dose of pyrogen administration can lead to septic shock characterized by cardiovascular dysfunction including myocardium depression and dilatation, vasodilation, vasoconstriction, endothelium dysfunction and organ dysfunction (kidney, liver, lung, brain) followed by multiple organ failure and death (28,29).
Endothelial cells play a major role in the regulation of hemostasis by maintaining an antithrombotic barrier. Endotoxin-mediated endothelial cell injury is implicated in the pathogenesis of septic shock (28). Endothelial cells change in response to pyrogenic stimulus and develop prothrombic properties. This involves the production of tissue factor (30, 31), the downregulation of thrombomodulin and the inhibition of factor C activation (31), leucocyte adhesion, and increased platelet adherence to endothelial cells (32). The denudation of endothelium alters vessel permeability and flow, and exposes subadjacent tissue to inflammatory cells and mediators.
The molecular mechanism of endothelial damage by LPS has been recently explained (33). The damage process involves at least three different parallel mechanisms. LPS-activated PBMCs cause apoptosis of endothelial cells (33,34) through membrane-bound TNF-alpha (m-TNF). The cell-free supernatant of activated PBMCs also trigger programmed cell death in the absence of TNF-alpha. In addition, activated PBMCs have the ability to induce endothelial cell-cycle arrest in G0/1 (33). This is due to the production of TGF-beta (35) which inhibits endothelial cell proliferation and repair. The data strongly suggest that LPS causes irreversible damage to the endothelium.
Introducing LPS intravenously into healthy humans suppressed the cytokine response by PBMCs when re-challenged in vitro with LPS, IL-1-beta or TSST-1 (36). This confirmed that the reduced cytokine synthesis was not due to endotoxin tolerance but a true immune suppression reaction. The cytokine synthesis in CD 14+ monocytes did not return to the normal control levels even up to 24h, the duration of the experiment.
This observation is quite interesting in light of the low dose of LPS used (3 ng/kg or 30 EU/kg body weight ). Current endotoxin standard for parenteral pharmaceutical lot release is 350 EU/dose. Characterization of Pseudomonas culture filtrates have demonstrated that only about 40-50% of the cytokine producing activity is caused by LPS-like material (37,38). Since Pseudomonas is a major water-borne microorganism, the total LPS-equivalent pyrogenicity is more accurately expressed as 700-875 EU/dose. This is in the same order of magnitude as the amount of endotoxin administered by Granowitz et al. (36), i.e., 1950 EU for a 65 kg average weight male.
Therefore, the possibility exists that injectable pharmaceuticals meeting current pyrogenicity standards are immunosuppressive. Current testing methods (16,17) are insufficient to answer this question. To accurately determine sample pyrogenicity, both LPS and non-LPS pyrogenicity should be quantified. We also have to consider that inflammatory reactions occur in the absence of fever. This makes it even more important to monitor these reactions at the cellular level.
A patient population most exposed to this risk are those on replacement therapies, e.g., hemophiliacs, or diabetics. Another patient group at risk include people in severe trauma, e.g., septic shock. Studying the cytokine-producing capability of CD14+ monocytes of hemophiliacs or diabetics could shed light on potential inflammatory reactions induced by the therapy. In septic shock patients, the infusion of the plasma extender serum albumin, although meeting current pyrogenicity standards, could potentially diminish the already depleted resources of their immune system. This could lead to a prolonged recovery period and, in some cases, contribute to the high rate of mortality among this patient group.
A discovery program based on the studies of Granowitz et al. (36) and Lindner et al. (33) would, at the cellular level, clarify the responses of patients to the administration of pyrogens in parenteral pharmaceuticals. Such discovery program could follow a general outline described below.
First, the cytokine-inducing ability of the pharmaceutical in question on isolated PMBC and/or a monocytic cell line could be studied in vitro. The secondary cytokine response of product-treated monocytes to endotoxin, IL-1 beta and TSST-1 would reveal any potential immunosuppressive effect of the product. Activated monocytes, when co-cultured with endothelial cells, would indicate if apoptosis-inducing effects are present. Experiments using pyrogen-depleted product as well as uninduced monocytes would provide controls. Technologies are available to deplete pyrogens from these products. This in vitro portion of the research could be carried out with monocytes from healthy donors and/or a monocytic cell line.
The second phase research could be performed in a clinical trial with patients treated with standard and pyrogen-depleted products. Monocytes would be isolated before and after administration of the product. The ability of these monocytes to secrete inflammatory cytokines before and after an in vitro LPS challenge would reveal if any immunosuppressive effect is noticeable solely due to the administration of the product. Co-culturing of endothelial cells with the activated monocytes would indicate if any apoptosis of endothelial cells has occured.
In conclusion, experimental data suggest that the pyrogenicity limits for parenteral pharmaceuticals may be higher than desired for maximal patient benefit. A discovery study with patients at risk of pyrogen administration should be conducted to obtain additional data. Conclusive evidence of the immunosuppressive effects of currently acceptable pyrogen levels in parenteral pharmaceuticals would require the introduction of new testing procedures and a re-evaluation of pyrogenicity standards.
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*A revised version of this paper will be published in the April issue of Pharmaceutical Technology.
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