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.

Stimulation of bone marrow by administration of excessive doses of recombinant human erythropoietin.

Recombinant human erythropoietin (rhEPO) was administered intravenously to Beagle dogs in daily doses of 100, 500 and 3000 units/kg/day for 3 months. The high dose was more than 200-fold the therapeutic human maintenance dose. Such excessive rhEPO doses elicited extreme erythropoiesis. There was a dose-dependent stimulation of bone marrow fibroblasts, leading to bone marrow fibrosis in some of the high dose animals. The extent of myelofibrosis was intra- and interindividually different in various bones. Sternebrae proved to be practical for morphometric studies. The point-counting method was used for measurement. The portion of fatty tissue, sinusoids and fibrous tissue in the medullary space as well as the number of blood vessels and megakaryocytes were calculated. Such experimental conditions are not of relevance in human patients whose rhEPO therapy is interrupted as soon as their PCV reaches 35 vol%. Experimentally induced myelofibrosis should therefore not be considered as a risk in patients receiving therapeutic doses of rhEPO.

Automatic image analysis as a valuable aid for the objective assessment of the enlargement of hepatocytes in toxicological studies.

Evaluation of histopathological slides is a subjective science; objectivity is needed, especially with minimal alterations. Quantitation using measurement procedures is of special value in toxicological studies. The present study tests a method in which the hepatocytic nuclei were measured per area of liver tissue, excluding the sinusoidal space. Different grades of hepatocellular enlargement were induced by different dose levels of a test compound (combination of two diuretics). The method demonstrates the well-established dose dependence of liver cell enlargement and permits differentiation between slight drug-induced enlargement and the normal variation in cell size. Special reference is made to avoid measurement artefacts.

Teratologic studies on the Himalayan rabbit: new aspects of thalidomide-induced teratogenesis.

The aim of our study was to determine the period of maximum sensitivity for the induction of characteristic malformations with thalidomide (TH) in Himalayan rabbits. TH was administered orally in different doses (50, 100, 150 and 200 mg/kg) four times at 24-h intervals starting at 192 h of gestation. The malformations affected various organs: renal defects (dysplasia) and limb anomalies (dysmelia)--which had never occurred spontaneously in this strain--appeared as dose-dependent effects of the drug. By administering single doses of TH (200 and 300 mg/kg body wt) between hours 192 and 264 of gestation, we discovered the different periods of maximum sensitivity for induction of renal dysplasia (clearly prior to the 220th h of gestation) and dysmelia (between hours 230 and 240 of gestation). The types of limb malformations that we observed in the rabbit were identical to those produced in man following the intake of TH. Three doses of TH (300 mg/kg each) given between hours 222 and 228 of gestation produced characteristic limb malformations in 9 of 11 litters treated. These results make it possible to conduct in vivo experiments on a readily available laboratory animal with minor drug exposure of the gravid dam and under avoidance of toxic side effects.

Artifacts in dilution pharmacokinetic models caused by adherent bacteria.

Liquid cultures of a Pseudomonas aeruginosa strain in Mueller-Hinton broth diluted at rates higher than the bacterial growth rate showed the expected decrease in CFU only for 1 to 2 h. Later the CFU started to increase. This phenomenon can be explained by a hypothesis that assumes that the bacteria multiply in two different compartments. From the first compartment, which comprises bacteria homogeneously distributed in the broth, cells are eliminated at a rate that is dependent on the dilution and growth rates. Concomitantly, the second compartment is formed as a nondilutable adherent population on the surface of the culture vessel. Eventually, only cells stemming from that population appeared in the medium and were subsequently diluted. This hypothesis can be described mathematically by a linear combination of two exponential functions. The calculated values fit the experimental data well. Because similar CFU versus time curves were also found with other strains, care should be taken in interpreting results of experiments performed in liquid cultures and evaluated in terms of CFU. One should bear in mind that within a liquid culture an adherent population may exist, which differs in size according to selective influences (dilution, addition of antibiotics, etc.). This may give rise to artificial and unexpected results.

Differences in the nitrite ion formation and the toxicological findings between isosorbide dinitrate and isosorbide-5-mononitrate.

In the metabolism of isosorbide dinitrate (ISDN), fast denitration with the formation of nitrite ions and a mononitrate plays an important role. In contrast, the denitration of isosorbide-5-mononitrate (IS-5-MN) to isosorbide is very slow. Accordingly po administration of high doses of ISDN (92.5 and 236 mg/kg) in conscious dogs led to maximum nitrite concentrations in the blood of 0.9 and 3.3 mg/liter, respectively. In contrast, with equimolar doses of IS-5-MN (75 and 191 mg/kg) we were able to detect nitrite ions reliably only at the higher dose and this gave a maximum blood concentration of 0.4 mg/liter. The rise in nitrite ion concentration is followed by the formation of methemoglobin. As is known from the literature, there is a rise in the activity of alkaline phosphatase in the serum of rabbits in addition to methemoglobin formation following repeated administration of sodium nitrite. So we have specifically investigated whether this is also the case following ISDN and IS-5-MN administration. On po administration of 236 mg/kg ISDN/day to dogs, there was a continuous rise in alkaline phosphatase from about the 20th day onward which we did not observe after the equimolar dose of IS-5-MN (191 mg/kg). NaNO2, 35 mg/kg po, led to a comparable maximal rise in methemoglobin to that obtained with 236 mg/kg ISDN. Repeated po administration of 35 mg/kg NaNO2/day also caused a rise in alkaline phosphatase. It is concluded that the formation of nitrite ions from ISDN is the reason for the rise in methemoglobin and alkaline phosphatase. The lower formation of nitrite ions from IS-5-MN can also be of clinical importance, at least in certain cases.