Report and recommendations of the workshop of the European Centre for the Validation of Alternative Methods for Drug-Induced Cardiotoxicity.

Cardiotoxicity is among the leading reasons for drug attrition and is therefore a core subject in non-clinical and clinical safety testing of new drugs. European Centre for the Validation of Alternative Methods held in March 2008 a workshop on "Alternative Methods for Drug-Induced Cardiotoxicity" in order to promote acceptance of alternative methods reducing, refining or replacing the use of laboratory animals in this field. This review reports the outcome of the workshop. The participants identified the major clinical manifestations, which are sensitive to conventional drugs, to be arrhythmias, contractility toxicity, ischaemia toxicity, secondary cardiotoxicity and valve toxicity. They gave an overview of the current use of alternative tests in cardiac safety assessments. Moreover, they elaborated on new cardiotoxicological endpoints for which alternative tests can have an impact and provided recommendations on how to cover them.

The effect of sterilization processes on the bioadhesive properties and surface chemistry of a plasma-polymerized polyethylene glycol film: XPS characterization and L929 cell proliferation tests.

The influence of several sterilization processes (autoclaving, gamma-ray irradiation, ethylene oxide exposure and Ar/H(2) low pressure plasma treatment) on the surface chemistry and the bioadhesive properties of thin films (thickness approximately 20 nm) of plasma-polymerized diethylene glycol dimethyl ether has been studied. X-ray photoelectron spectroscopy (XPS) analysis and cell proliferation tests were used to characterize the surfaces. The XPS results revealed in all cases a change in the surface chemistry of the layer after sterilization, whereas the conservation of non-bioadhesive properties of the coating depends on the type of sterilization process. In particular, the low pressure plasma-based sterilization technique leads to a loss of the non-bioadhesive properties of the plasma coating, whereas the coatings are resistant to the other standard decontamination techniques. This property makes them suitable for biomedical applications, provided that an appropriate sterilization process is selected.

Removal of immune-stimulatory components from surfaces by plasma discharges.

Immune-stimulating microbiological components like lipopolysaccharide (LPS), lipoteichoic acid (LTA) and zymosan bound onto surfaces lead to severe problems when brought in contact with the organism via surgical instruments or implants. We have shown, in recent studies, that it is possible to detect different immune-stimulating components directly on the surface, via an indirect detection method, using human whole-blood and the monocyte reaction to measure the inflammatory mediator release (IL-1beta) by ELISA. With regard to the inactivation of pyrogenic substances, we present a method based on the application of a low-pressure microwave plasma discharge working at low temperatures. We found a fast (10 s to a few minutes) removal rate of the immune-stimulating competence for LPS, LTA and zymosan. To mimic the bacterial cell-wall, LPS in combination with muramyl dipeptide was employed and the decreasing rate of the inflammatory signal did not differ from pure LPS.

An in vitro pyrogen safety test for immune-stimulating components on surfaces.

Due to the discovery of novel surgical techniques and new materials medical devices are increasingly used. Contact of these biomaterials with human tissue or blood commonly leads to inflammation of varying degrees, sometimes resulting in severe health problems. Possible causes are limited biocompatibility or pyrogenic contamination of the material. We adapted the recently validated in vitro pyrogen test (IPT), based on human whole blood cytokine release, to test the safety of biomaterials. Human whole blood is brought into direct contact with the surface of the test material and the release of the pro-inflammatory cytokine IL-1beta is measured. This procedure represents a human-relevant assay allowing the detection of pyrogens of different origins, e.g. Gram-negative (lipopolysaccharide, LPS) or Gram-positive (lipoteichoic acid, LTA), peptidoglycan (cell wall components of most bacteria) and fungal zymosan by direct material contact. The sensitivity of the test system allows a starting concentration of 10 pg/ml for LPS, 10 ng/ml for zymosan and 1 microg/ml for LTA and peptidoglycan from different strains. Furthermore, we have shown that the test for solid materials can be carried out with cryo-preserved blood, which results in an even lower detection limit.

Fouling and non-fouling surfaces produced by plasma polymerization of ethylene oxide monomer.

This paper presents the results of plasma polymerization using diethylene glycol dimethyl ether as a precursor in a capacitively coupled radio frequency system. The chemical structure of the coatings was characterized using several analysis techniques (X-ray photoelectron spectroscopy, Fourier transform-infrared spectroscopy, ellipsometry), while the biological response of these coatings has been tested by protein adsorption and cell culture experiments. The modulation of the input plasma power controls the concentration of polyethylene oxide groups in the coatings and allows the production of films with opposite protein and cell repellent properties. The study of the stability of these coatings in different media (water, acetone, phosphate-buffered saline) reveals that these films could be involved in classical lift-off processes for the production of patterned surfaces.