[Immunogenicity of biosimilars]. / Immunogeniciteit van biosimilars.

Biosimilars of more complex recombinant protein drugs, such as monoclonal antibodies and fusion proteins, are entering the market. The manufacturer should demonstrate that its product does not show any relevant differences in terms of quality characteristics, biological activity, safety and efficacy compared to the reference product, as outlined in EMA guidelines. This should be established with an extensive comparability exercise. One aspect that is subject to particular scrutiny is the immunogenicity of the biosimilar and the reference medicinal product. For three cases, one etanercept and two infliximab biosimilars, we describe how data are assessed and an opinion is reached by authorities. Not in all cases unanimity exists whether all remaining uncertainties on biosimilarity have been resolved satisfactorily before marketing authorisation. The Dutch Medicines Evaluation Board therefore emphasises that even after marketing authorisation, biosimilars and other biologicals should be properly monitored.

N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB): its properties and possible risks.

Abstract MBDB (N -methyl-1-(1,3-benzodioxol-5-yl)-2-aminobutane) is the alpha-ethyl homologue of MDMA (3,4-methylenedioxy-N-methylamphetamine). MBDB is metabolized and excreted similarly to MDMA: presumably, the majority of oral MBDB is excreted in urine unmetabolized.The main metabolic routes in man are thought to be O-dealkylation and subsequent methylation, sulphation and glucuronidation of the newly formed hydroxy groups. The major acute neuropharmacological effects of MBDB in the rat are an increase in serotonin release in the brain and an inhibition of serotonin and noradrenaline re-uptake. These effects compare well with those of MDMA, although the latter is more potent. MBDB may also slightly increase dopamine release and inhibit dopamine re-uptake, but to a lesser extent than MDMA. This is important, as dopamine release has been implicated in the reinforcing qualities of substances such as cocaine and amphetamine. The neuroendocrine effects of MBDB resemble those of MDMA. Both substances increase plasma ACTH, corticosterone, prolactin and renin. The neurophysiological effects of MBDB are characterized by a decrease in electrical activity throughout the brain, most notably in the alpha 2 and delta frequency bands. In contrast, hallucinogens increase the activity in the alpha 1 band, especially in the corpus striatum. In drug discrimination tests in the rat, MBDB, like MDMA, can be distinguished clearly from both stimulants and hallucinogens.The class of substances to which MBDB belongs may be named entactogens. MBDB dose-dependently increases locomotor activity and decreases exploratory behaviour in the rat and causes distress vocalization and wing extension in the newly hatched chicken. The rewarding properties of MBDB appear to be smaller than those of MDMA, as suggested by a 2.5 times weaker potency in the conditioned place preference test in rats. The main subjective effects of MBDB in man are a pleasant state of introspection, with greatly facilitated interpersonal communication and a pronounced sense of empathy and compassion between subjects. In this respect, MBDB again resembles MDMA. However, there are also differences. MBDB has a slower and more gentle onset of action than MDMA, produces less euphoria and has less stimulant properties. The few toxicological data available suggest that MBDB may cause serotonergic deficits in the brain, although the potency of MBDB to cause this neurotoxic effect is smaller than that of MDMA. Severe acute reactions in man as have been reported for MDMA have not been published for MBDB. The dependence potential of MBDB appears to be small, probably even smaller than that of MDMA. MBDB has been available at least since 1994 but its position on the synthetic drugs market is marginal. Subjective reports indicate that MBDB is less popular among users than MDMA. The reason may be that MBDB produces less euphoria than MDMA. Another possible explanation is that MBDB largely lacks the stimulant properties of MDMA.We calculated a margin of safety with a method similar to one used in the risk assessment of pharmaceuticals. The results suggest that MBDB is three times less likely to cause serotonergic brain deficits than MDMA. However, it should be noted that for both substances the margin of safety is less than one, indicating that the risk of neurotoxicity is not negligible. In animals, serotonergic brain deficits after exposure to MDMA have been linked to the degeneration of serotonergic nerve terminals.

Stereospecific in vitro embryotoxicity of l-homocysteine in pre- and post-implantation rodent embryos.

Recently a derangement of homocysteine metabolism has been suggested as a possible risk factor for neural tube defects and recurrent spontaneous abortion. To investigate a possible role of homocysteine in the aetiology of neural tube defects we tested the in vitro embryotoxicity of l-homocysteine by culturing day 10 post coitum post-implantation rat embryos in whole embryo culture (WEC) for 24 hr and day 2 post coitum pre-implantation mouse embryos for 48 hr. With an area under curve (AUC) of 6.3 mm/hr, l-homocysteine significantly reduced the percentage of mouse embryos that developed into blastocysts. In rat WEC, an AUC for l-homocysteine of 3.6 mm/hr reduced the mitotic index of the neural epithelium of the rhombencephalon and the cell density of the mesenchyme adjacent to it, while at an AUC of 7.2 mm/hr l-homocysteine reduced the total morphological score and the number of malformations was increased. Malformations most often seen were transparent rhombencephalon, no or delayed formation of forelimb buds, dysmorphogenesis of the somites, and blister formation dorso-laterally of the place of forelimb bud formation. The embryotoxicity of l-homocysteine was stereospecific since d-homocysteine caused no embryotoxic effects. Also the oxidation product l-homocystine (AUC, 72 mm/hr) and the metabolite l-methionine (AUC, 144 mm/hr) were not embryotoxic. Both stereoisomers of homocysteinethiolactone were embryotoxic at an AUC of 72 mm/hr. The results are discussed in relation to the metabolism of homocysteine and methionine and their possible role in the neurulation process.

Sex difference in Aroclor 1254 induction of rat hepatocytes: Consequences for in vitro embryotoxicity and mutagenicity of cyclophosphamide.

The sequential culture of rat hepatocytes and post-implantation rat embryos has been proposed as a model for the in vitro testing of pro-teratogens. Comparing this model with a model in which embryos and hepatocytes are cultured simultaneously a striking difference in sensitivity was noted. To address the question of whether this difference could be explained by different sex and/or Aroclor 1254 pretreatment of the rats providing the hepatocytes, an experiment was designed with four groups: male Aroclor 1254 pretreated (M(1)), male untreated, pregnant female Aroclor 1254 pretreated (F(1)) and pregnant female untreated rats. Hepatocytes were incubated in the presence of cyclophosphamide (CP) and rat embryos were cultured in the media derived from the hepatocyte culture (i.e. the sequential culture model). Additionally, the CP concentrations of the media were analysed and subsequently the media were tested in a bacterial mutagenicity test (Salmonella typhimurium TA1535). With a CP concentration of 300 mum, M(1) produced maximum embryotoxicity and mutagenicity after 4 hr of hepatocytes incubation. All other groups showed no or only a slight increase in embryotoxicity and mutagenicity for all hepatocyte incubations. M(1) was also quickest to eliminate CP from the medium. These results indicate that despite a strong increase in total cytochrome P-450 in both sexes as a result of Aroclor 1254 pretreatment, and in the absence of a significant difference in total cytochrome P-450 between M(1) and F(1), Aroclor 1254 pretreatment has a much more pronounced effect in male rats than in pregnant female rats with regard to the production of embryotoxic and mutagenic metabolites of CP.

Biotransformation of cyclophosphamide in post-implantation rat embryo culture using maternal hepatocytes in co-culture.

The post-implantation rat embryo culture technique is employed to study embryotoxic effects of xenobiotic compounds in the absence of the maternal compartment. For compounds biotransformed in vivo the embryo culture technique must be adapted in order to mimick the in vivo effects. In the present study the possibility of co-culturing metabolically active maternal hepatocytes suspended in the standard culture system with rat serum as a medium was investigated. Cyclophosphamide (CP) was used as a model compound as it needs bioactivation to display embryotoxicity. Morphologic and histologic effects were studied. Neither hepatocytes nor CP alone affected embryo development, whereas in the presence of hepatocytes embryotoxicity was observed at 30 micrograms/ml CP. Embryotoxicity was decreased in the additional presence of metyrapone, a monoxygenase inhibitor. Hepatocyte suspensions prepared via slicing or perfusion of livers were equally effective. In conclusion, co-culture of embryos and suspended hepatocytes can be performed under optimal conditions for embryo development and in the presence of biotransforming activity.

Airborne concentrations, skin contamination, and urinary metabolite excretion of polycyclic aromatic hydrocarbons among paving workers exposed to coal tar derived road tars.

The exposure of surface dressing workers to polycyclic aromatic hydrocarbons (PAH) was studied. Four different paving sites, at which coal tar-containing binders were applied, were selected as work sites with high exposure levels of PAH. Breathing zone airborne particulates, contamination of the skin with PAH, and 1-hydroxypyrene in urine of the workers involved in chip sealing were determined. Substantial concentrations of cyclohexane-soluble airborne particulate matter were found (GM = 0.2 mg/m3, n = 28). Skin contamination was determined using two different methods: with exposure pads and by hand washing. Pads were mounted on several parts of the body: wrist, elbow, neck, shoulder, and ankle. The pads located on the wrist appeared to be the most contaminated (pyrene: GM = 22 ng/1.77 cm2, n = 40). The end-of-shift hand washing showed that the hands of the workers were contaminated with PAH (pyrene: GM = 70 micrograms, n = 35). Preshift hand washing showed far lower, but detectable, quantities of PAH on workers' hands (pyrene: GM = 5 micrograms, n = 35). Enhanced levels of urinary 1-hydroxypyrene among the workers were found. The highest levels were found in the end-of-shift urine samples. Correlations between the pyrene exposure variables were studied. Significant positive correlations were found between pyrene on the wrist pad versus end-of-shift urinary 1-hydroxypyrene; between pyrene on the hands versus end-of-shift urinary 1-hydroxypyrene; and between the two different skin contamination variables.