Pyrogen testing of lipidic parenterals with a novel in vitro test--application of the IPT based on cryopreserved human whole blood.

The European Pharmacopoeia has made the testing of small volume parenterals (< 15) obligatory since 2004. This concerns many formulations, e.g. vitamins, steroids and hormones, many of which are applied intramuscularly using a lipidic carrier. Lipopolysaccharides, the best established endotoxins from Gram-negative bacteria, bind strongly to lipophilic substances, which mask them in Limulus amebocyte lysate assays. End-product testing, therefore, can only be carried out in rabbit pyrogen tests. This will lead to a pronounced increase in animal experiments if no alternative procedures become available. We have described a novel in vitro pyrogen test (IPT) based on human whole blood, which has recently been validated in a collaborative study including the European Pharmacopoeia. Here, the utility of the IPT for lipophilic substances and lipid-containing end-products was assessed. For a variety of lipids commonly added to formulations of injectable endproducts, namely peanut oil, sesame oil, miglyol and paraffin, a protocol which allows interference-free testing was established applying the pharmacopoeial criterion of 50 to 200% retrieval of an LPS spike. Furthermore, end-product testing for three sample formulations was possible. In all, a method could be established which allows the determination of given or calculated ELC (endotoxin limit concentrations) according to Pharmacopoeia. It is concluded that monocytes do react to lipid-bound LPS, indicating that immune responses to contaminated endproducts must be anticipated, and that the IPT is suitable for endproduct control of these formulations.

[A new application for the human whole blood test: development of an assay to assess the health risk of air-borne microbial contaminations]. / Beispiel eines neuen Einsatzbereichs des humanen Vollbluttests: Entwicklung eines Messverfahrens zur Abschätzung der gesundheitlichen Gefährdung durch luftgetragene mikrobielle Verunreinigungen.

The pathogenic properties of environmental microorganisms as well as pyrogens as fragments of those bacteria (especially endotoxins) for humans is increasingly recognised. Various clinical syndromes are described after contact with airborne microbial contaminants via the respiratory tract: Sick-building-syndrome, humidifier lung (a form of hypersensitive pneumonitis), "Monday sickness" etc. Air-conditioning and ventilation systems intensify this problem as well as storage of compost within the household which represents a considerable source of airborne pollutants. In 1995 a new method for the detection of pyrogenic (fever-inducing) hazardous substances was described by Hartung and Wendel. This whole blood assay utilises the natural reaction of the immune system in order to detect a broad spectrum of pyrogens very sensitively in the relevant species. Injectable drugs are the main area of application in which this innovative test has already proven effective and is currently validated for inclusion into European Pharmacopoeia. In co-operation with the FU Berlin we could demonstrate in ventilation systems in animal stables that the whole blood pyrogen test can also detect airborne environmental microorganisms very sensitively. The filtration technique for collection of these germs is an established method for air-conditioning and ventilation systems. In co-operation with the FU Berlin (Institut für Tier-und Umwelthygiene) and the filter producer Sartorius this method is currently developed for the detection of airborne contaminations.

Detection of endotoxins and other pyrogens using human whole blood.

When cells of the immune system, i.e. primarily blood monocytes and macrophages, come into contact with pyrogens (fever-inducing contaminations) they release mediators transmitting the fever reaction through the organism to the thermoregulatory centres of the brain. The new test discussed here exploits this reaction for the detection of pyrogens: human whole blood taken from healthy volunteers is incubated in the presence of the test sample. If there is pyrogen contamination, the endogenous pyrogen interleukin-1 is released, which is then determined by ELISA. According to the pharmacopoeia, the rabbit pyrogen test determines the fever reaction following injection of a test sample. In comparison, the new whole blood assay is more sensitive, less expensive and determines the reaction of the targeted species. Compared to the well established in vitro alternative, i.e. the limulus amebocyte lysate assay (LAL), the new blood assay is not restricted to endotoxins of gram-negative bacteria, it is not affected by endotoxin-binding blood proteins and it reflects the potency of different endotoxin preparations in mammals. Here, interim results of the ongoing optimization and pre-validation are reported and the present state of the evaluation for biological and pharmaceutical drugs are presented.

New Applications of the Human Whole Blood Pyrogen Assay (PyroCheck).

The absence of pyrogens in injectable drugs is an indispensable safety control because contaminants causing fever pose a life-threatening risk to the patient resulting in the worst case in death by shock. When fever- inducing agents, i.e.pyrogens, come into contact with the immunocompetent cells in blood, these cells release mediators which transmit the fever signal to the thermoregulatory centre of the brain. The Phamocopoeia lists currently two test systems for pyrogenicity: 1. The in vivo rabbit pyrogen test which measures the fever reaction following injection of the sample to the animals. 2. The in vitro Limulus Amebocyte Lysate assay (LAL) which measures the coagulation in a lysate prepared from the blood of the horseshoe crab specifically initiated by endotoxins, i.e. cell wall components from Gram-negative bacteria. The new test presented here (PyroCheck) exploits the reaction of monocytes/macrophages for the detection of pyrogens: human whole blood taken from healthy volunteers is incubated in the presence of the test sample in any form, be it a solution, a powder or even solid material. Pyrogenic contaminations initiate the release of the "endogenous pyrogen" Interleukin-1beta determined by ELISA after a fixed incubation time. The technology presently listed in the Pharmacopoeia is limited to parenteralia (rabbit test: biologicals and pharmaceuticals, LAL: predominantly pharmaceuticals). In the EU Medical Devices Directive from 1995 the rabbit pyrogen test for medical products is in some cases requested. (in some cases LAL of an eluate from the device). However, pyrogen-testing needs to cover also innovative high-tech products such as medical devices (implants, medical plastic materials, dialysis machines), cellular therapies and species-specific agents (e.g. recombinant proteins). Here we report that the human blood test PyroCheck is suitable for testing filters in air quality control as well as for assessing medical devices and biocompatibility (dialysate fluid), i.e. that it can be extended to a wide spectrum of applications.

Radial astrocytes: toxic effects induced by antiepileptic drug in the developing rat hippocampus in vitro.

Neuron-glia relationships are crucial for differentiation of both glial and neuronal cells. Interference with these intricate cell interactions could affect regular neuroembryogenesis. In order to analyse potential developmental neurotoxic effects of therapeutically administered antiepileptics such as valproate, we employed organotypic cultures of the rat hippocampus. In these cultures thin tissue slices were continuously rotated between the gas and medium phases, which greatly improved oxygen and nutrient accessibility. This resulted in long-term preservation of the native cytoarchitecture. Exposure of organotypically cultured hippocampi to valproate hampered, in a dose-dependent manner, regular formation of the pyramidal cell layer. Most interestingly, radial astrocytes, which comprise a transient cell population during distinct developmental periods, were selectively affected even by low doses of valproate, but not by structurally related non-teratogenic isomer 2-ethyl-4-methyl-pentanoic acid. The xenobiotic effect did not represent a general gliotoxic insult, since neither the glutathione quotient as determined by HPLC, nor the DNA content, nor the total amount of glial fibrillary acidic protein evaluated by ELISA were significantly altered. Instead, the morphology of astrocytes proved to be the most sensitive index of intoxication with the orientation of radial astrocytes being most affected as revealed by immunofluorescence. In contrast to radial astrocytes, other astrocytic populations proved to be fairly resistent. The data indicate that developmentally regulated cell polarity of astrocytes is a target of therapeutically relevant xenobiotics. This could in turn disturb neuronal differentiation and normal histogenesis.

Drug metabolism in in vitro organotypic and cellular models of mammalian central nervous system: activities of membrane-bound epoxide hydrolase and NADPH-cytochrome P-450 (c) reductase.

The membrane-bound form of epoxide hydrolase and NADPH-cytochrome P-450 (c) reductase are two important enzymes involved in the bioactivation/bioinactivation balance of cerebral tissue. In vivo, the developmental profiles and regional localizations of these two enzymes were investigated in the rat. The regional distribution study showed that they are ubiquitously present among the major brain structures. Both enzyme activities were present in the brain prior to birth, and hence tissue from early developmental stages is suitable to develop in vitro cellular or organotypic models for toxicity studies involving these metabolic pathways. Because various neurotoxicological effects can be dependent on spatio-temporally regulated cell-cell interactions, we aimed to employ organotypic tissue cultures in which the cytoarchitecture was well preserved. In such cultures, the temporal expression profiles of epoxide hydrolase and NADPH cytochrome(c) P-450 reductase reflected the in vivo situation. The technically less demanding pure neuronal and glial cell cultures were also investigated. Detoxification of benzopyrene-4,5-epoxide and superoxide production arising from the reductive metabolism of various drugs were determined in all three systems. The results indicate that though organotypic culture is a good model for the metabolic pathways studied, less complicated single cell cultures can also represent appropriate model systems, providing that the expression of the enzymes involved has been first established in these systems. NADPH-cytochrome P-450 reductase-dependent metabolism is active in both neuronal and glial cells, whereas the detoxification of reactive epoxides occurs mainly in glia.

A novel organotypic long-term culture of the rat hippocampus on substrate-integrated multielectrode arrays.

Spatiotemporally coordinated activity of neural networks is crucial for brain functioning. To understand the basis of physiological information processing and pathological states, simultaneous multisite long-term recording is a prerequisite. In a multidisciplinary approach we developed a novel system of organotypically cultured rat hippocampal slices on a planar 60-microelectrode array (MEA). This biohybrid system allowed cultivation for 4 weeks. Methods known from semiconductor production were employed to fabricate and characterize the MEA. Simultaneous extracellular recording of local field potentials (LFPs) and spike activity at 60 sites under sterile conditions allowed the analysis of network activity with high spatiotemporal resolution. To our knowledge this is the first realization of hippocampus cultured organotypically on multi-microelectrode arrays for simultaneous recording and electrical stimulation. This biohybrid system promises to become a powerful tool for drug discovery and for the analysis of neural networks, of synaptic plasticity, and of pathophysiological conditions such as ischemia and epilepsy.