The desire to make available an in vitro pyrogen test as an alternative method to the rabbit pyrogen test is prompted by ethical considerations of animal protection as well as to matters of patient safety. It is known long that the raise in temperature in the rabbit represents an unreliable parameter due to stress-induced hyperthermia, hypothermia (29), sensitivity differences of rabbit strains (27), age, sex and even between individuals of the same breeds who have been found to be high, low, and intermediate responders (174). Therefore, the LAL was introduced in pyrogen testing in the 70s and is now widely used. The major limitations of this test is, as its alternative name bacterial endotoxin test (BET) implies, being limited to the pyrogens derived from Gram-negative bacteria and missing known pyrogens as lipoteichoic acid and peptidoglycans as well as exotoxins and other non-endotoxin pyrogens. Pyrogenic reactions in healthy volunteers have been reported during a clinical study when a preparation of human growth hormone was injected that had tested negative in the LAL and in the rabbit (133). Adverse reactions after the application of vaccine against early summer meningoencephalitis have been observed, although the LAL had tested negative (59). An assay capable of detecting all relevant pyrogens should mirror closely the physiological events which take place in the mammalian organism during a pyrogenic reaction.
Upon contact with so-called pathogen-associated molecular patterns (PAMPS) (127), e.g. bacterial, fungal or viral components, the blood monocytes are activated. The Cluster of Differentiation (CD) 14 receptor, a glycoprotein whose significance was recognized in 1990, exists in a membrane-bound (mCD14, a 53 kDa protein) and a soluble form. The former is embedded in the plasma membrane, the latter, sCD14, is capable of inhibiting LPS activity (175), but additionally LPS/sCD14 complexes can induce biological responses in certain CD14-negative cells such as endothelium (176). LPS binding takes place via the Lipid A component and is of high affinity (177).
SUMMARIZING DISCUSSION
The innate immune system of mammals uses the family of toll-like receptors (TLR) to engage microorganisms by recognizing PAMPS (178). For the recognition of LPS, the TLR4 receptor is crucial (179). The role of TLR4 was determined in TLR4 deficient mice which proved to be unresponsive to LPS (180). Upon binding, the TLR4 receptor dimerizes and the intracellular signal cascade is initiated.
In contrast, the pyrogen LTA, but also peptidoglycans and lipoproteins, bind to the TLR2 (181, 182) and cause fever along the same lines as LPS. The receptor engagement activates intracellular signaling cascades which lead to the formation of proinflammatory cytokines such as Interleukin-1β (IL-1β), IL-6 and others by the blood monocytes. The IPT measures the endpoint IL-1β.
The IL-1β exists in the mononuclear cells as a preformed molecule, the pro-IL-1β. Upon stimulation, a protease is activated, the IL-1 Converting Enzyme (ICE), whose catalytic function is essential for the generation of mature, extracellular IL-1β (183). It is an interesting phenomenon that in studies with human subjects, no IL-1β could be found in the blood stream after LPS injection or in naturally occurring sepsis, whereas IL-6 was always detectable (184-186). Messengers this potent are probably quickly bound to their corresponding receptors, e.g. to the soluble Interleukin-1 receptors (187), or internalized, e.g. by binding to/crossing the blood-brain barrier (188, 189), making them disappear fast from the blood serum. The individual roles of the proinflammatory cytokines in the generation of fever are still controversially discussed, but several lines of evidence suggest that IL-1β is indeed the most important and potent inducer of fever. Several studies in humans (190-193) have demonstrated the pyrogenic potency of this cytokine and studies in rabbits have shown clearly that the doses of IL-1β required are 100fold lower than those of IL-6 and around 10fold lower than those of TNF-α (46, 47).
Furthermore, the cytoplasmic domains of TLRs and the IL-1β receptor share the same signaling areas (38). The necessary intracellular enzymatic cleavage of pro-IL-1β might provide an additional control mechanism of a mediator which is crucial in the genesis of pyrogenic reactions.
These events in the mammalian organism provided the idea for the principle of the whole blood test. When in contact with a pyrogen, the monocyte, a
SUMMARIZING DISCUSSION
subfraction of the white blood cells, reacts with the production of proinflammatory cytokines, one of which is IL-1β. By using the whole blood as a reagent, the monocyte is left in its natural environmant with all components necessary for a reaction that is as close to the in vivo situation as possible.
IL-1β was chosen as a readout since it is a very stable monomer, which survives repeated freezing-thawing processes, something that might be necessary during the establishment of the system and for research purposes, and since it is highly regulated, which might prevent or reduce false-positive reactions or basal levels in unstimulated samples. Furthermore, IL-1β has been recognized as the most potent cytokine when injected in vivo (47, 190-193) and appears to play a crucial, if not the crucial role in the pathogenesis of fever.
In order to make the new IPT available on a large scale and therefore a true alternative to animal experiments, all reagents had to be standardized and certified. A major issue was the certification and standardization of the human blood. Freshly drawn human blood cannot be stored for more than 4 hours until the IL-1 formation declines, and is difficult to measure. Individual differences if not in the sensitivity, but the amount of cytokine produced can make interpretation of the results difficult. Furthermore, a risk of infection for the user with e.g. hepatitis cannot be excluded easily. In the first part of this work, a protocol for pooling and freezing whole blood was developed which maintains all the characteristics of the fresh blood. The cryopreservation of isolated blood cells has been successfully performed since the 1960s for transfusion and research purposes (73, 194-197). By freezing the entire blood without any isolation procedures, the monocyte is maintained in its natural environment with all the known and unknown factors that might influence the response, thus maintaining their reactivity and specificity with regard to pyrogen testing. An important plasma protein is for example the LPS binding protein (LBP) which in the blood of healthy donors presents the LPS to the CD14 receptor (162, 163).
While red blood cells require very high freezing rates in order to remain intact, the white blood cells are best frozen at rates of 1-2°C/min. This is due to the process of exosmosis of intracellular water which depends on parameters like cell surface/volume ratio and membrane permeability and which was described by Mazur in 1963, 1965 and 1977 (198-200). Our protocol therefore had to
SUMMARIZING DISCUSSION
tolerate the complete destruction of the erythrocytes with the risk, that the reaction of the monocytes would be influenced by the cell detritus. This was, at least in our experiments, never the case. The procedure involved clinical, endotoxin-free DMSO at a 10% final concentration in the blood (v/v ratio) and a computer-controlled freezing process using liquid nitrogen. For higher standardization and in order to level out individual differences in the response, the blood of 5 donors was pooled and frozen together. The frozen blood proved to be a highly sensitive and robust reagent with a very high inter-lot comparability. The possibility of pretesting the donors in question for infectious agents eliminates the abovementioned health hazard for the personnel.
In order to make the IPT available for routine application, the assay using cryopreserved blood, allowing storage at -80°C as compared to liquid nitrogen as well as an improved variation of the fresh blood was validated in an international collaborative study which followed the procedure in which the fresh blood had been validated previously in detail. The validation included three laboratories working under good laboratory practice (GLP) with 10 substances and altogether 50 blinded endotoxin spikes at or around the pyrogenic threshold of 0.5 EU/ml (16). The IPT achieved sensitivities around 90% and specificities around 80%. The cryopreserved blood described in part one had a lower sensitivity of 80% with at the same time the best specificity.
Compared to the former study (48), the whole blood assay could be improved regarding consumer-friendlyness as well as performance. Based on this outcome, the inclusion of the IPT into the Pharmacoopoeias should be possible.
In the last part of this work, the validated assay using the cryopreserved blood was adapted to suit a special application. So far, only hydrophilic substances mainly for intravenous administration had been used in the test. A change in regulation in 2004 by European Pharmacopoeia made the testing of so-called small volume parenterals (SVP) obligatory that had so far not been subjected to pyrogen testing. Suddenly, several lipophilic parenterals, e.g. with fat-soluble vitamins or hormones, for intramuscular and subcutaneous application were
SUMMARIZING DISCUSSION
concerned. Testing lipophilic substances in the rabbit by intravenous injection into the ear vein which is the accepted procedure, is extremely dangerous since the insoluble miniscule drops can clot capillaries in vital organs such as the kidneys. Apart from the viewpoint of animal protection, the outcome of such an experiment must be doubtful. The LAL, on the other hand, is impeded by the fact that the pyrogenic portion, the lipid A, is masked by lipid substances, lipidic parenterals (122), lipoproteins (121, 168), and liposomes (173) and therefore no longer accessible to the components of the coagulation cascades, leading to an underestimation of the pyrogenic contamination. It was therefore a pressing question whether such substances can be controlled by the IPT. For this purpose, the standard protocol was modified and different pure oils as well as three endproducts were measured. It turned out that all products can be controlled, although the pyrogenic threshold of 0.5 EU/ml for hydrophilic substances could not be maintained in this product group. Still, since the drugs are given at a very small volume, a higher contamination can be tolerated which will predictably not cause any adverse reactions in the recipient.
Taken together, with the new assay based on human whole blood, a standardized, reliable and highly sensitive method based on the human fever reactions and measuring all relevant pyrogens is now available for widespread use. We hope that the replacement of the rabbit pyrogen test by this assay is therefore only a question of time, simultaneously maintaining and even exceeding the already high level of safety for patients receiving parenterals.
ZUSAMMENFASSUNG
9 Summary
The detection of bacterial contaminations of all origins in parenterals has been recognized as an important issue. In 1943, the in vivo rabbit pyrogen test was introduced into the US Pharmacopoeia and has been used since then. In the 70s, another pyrogen test, the limulus amoebocyte lysate test (LAL) was introduced which uses the lysates of blood cells of the horseshoe crab and cannot measure a variety of common pyrogens nor distinguish between the potencies of given pyrogens in the mammal. The immune system is able to respond to pathogens with production and secretion of cytokines. In 1995, a novel in vitro alternative based on human whole blood which uses this reaction for the detection of all possible kinds of microbial contaminations has been developed. Its reliability and wide spectrum of application possibilities make it a promising candidate for entirely replacing the rabbit test.
•
As a first step, the human whole blood test using freshly drawn human whole blood was validated in an international collaborative study.• The most critical reagent of the new test, the human whole blood, was made available by developing a protocol for pooling and cryopreserving the fresh blood, at the same time maintaining the characteristics of the fresh blood regarding the reactivity towards all relevant pyrogens.
• This blood was then validated in an international study in order to be able to include the assay into Pharmacopoeias.
• With this validated procedure, the applications of the test were extended from hydrophilic to lipophilic parenterals meeting new requirements of European Pharmacopoeia.
With all these measures, the new pyrogen assay based on whole blood which measures all relevant pyrogens inducing a fever reaction is ready for routine use. This work adds to the replacement of an important and widely used animal test while at the same time maintaining, even superceding the existing safety standards and extending the possibilitites of applications beyond those of the rabbit test and even the LAL.
ZUSAMMENFASSUNG
10 Zusammenfassung
Die Detektion von bakteriellen Kontaminationen jeglicher Herkunft in Parenteralia ist als wichtiges Problem erkannt. 1943 wurde der in vivo Kaninchenpyrogentest in die US Pharmacopoe eingeführt und wird seither verwendet. In den 1970er Jahren wurde ein weiterer Pyrogentest, der Limulus Amoebozyten Lysattest (LAL) eingeführt, der das Lysat der Blutzellen der Hufeisenkrabbe einsetzt und der weder alle relevanten Pyrogene messen noch zwischen ihrer jeweiligen biologischen Potenz im Säugetier unterscheiden kann. Das Immunsystem antwortet auf Pathogene mit der Produktion und Sekretion von Zytokinen. 1995 wurde eine in vitro Alternative mit humanem Vollblut entwickelt, die diese Reaktion zur Detektion aller möglichen mikrobiellen Kontaminationen nutzt. Ihre Zuverlässigkeit und das weite Spektrum an Anwendungsmöglichkeiten machen sie zu einem vielversprechenden Kandidaten, den Kaninchentest vollständig zu ersetzen.
•
Als ein erster Schritt wurde der Test, basierend auf frisch abgenommenem humanem Vollblut, in einer internationalen Studie validiert.• Der wohl kritischste Bestandteil des Tests, das menschliche Vollblut, wurde verfügbar gemacht, indem ein Protokoll zum Mischen und Einfrieren von frischem Blut entwickelt wurde. Gleichzeitig blieben die Eigenschaften des frischen Blutes in Bezug auf die Reaktivität gegenüber allen relevanten Pyrogenen erhalten.
• Parallel zu dem Frischblut wurde dieses Blut in einer internationalen Studie validiert, um den Test in die Pharmakopoen einführen zu können.
• Mit dieser validierten Methode wurden die Anwendungen für den Test erweitert, um den neuen Anforderungen der Europäischen Pharmacopoe Genüge zu tun: von hydrophilen zu lipophilen Parenteralia.
Damit ist der neue Pyrogentest auf Vollblutbasis, der alle relevanten fiebererzeugenden Pyrogene mißt, zum routinemäßigen Einsatz bereit. Die vorliegende Arbeit trägt dazu bei, einen wichtigen und viel genutzten Tierversuch zu ersetzen, gleichzeitig die bestehenden Sicherheitsstandards beizubehalten bzw. zu übertreffen und die Anwendungsmöglichkeiten über die des Kaninchentests und sogar des LAL hinaus auszuweiten.
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