Calcein assay: a high-throughput method to assess P-gp inhibition.

Transporter mediated drug-drug interactions (tDDI) mediated by ABCB1 have been shown to be clinically relevant. Hence, the assessment of the ABCB1 tDDI potential early in the drug development process has gained interest. We have evaluated the Calcein assay as a means of assessing the ABCB1 tDDI that is amenable to high throughout and compared it with the monolayer efflux assay. We found the Calcein assay, when performed in K562MDR cells using the protocol originally published more sensitive than digoxin transport inhibition in MDCKII-MDR1 cells. Application of the Calcein assay to cell lines containing different amounts of ABCB1, yielded IC(50) values that varied 10-100-fold. The differences observed for IC(50) values for the same compounds were in the following rank order: IC(50, MDCKII-MDR1) >IC(50, K562MDR)>IC(50, hCMEC/D3). Higher IC(50) values were obtained in cells with higher ABCB1 expression. The Calcein assay is a high-throughput alternative to digoxin transport inhibition as it appears to have a comparable selectivity but higher sensitivity than previously published digoxin transport inhibition in MDCKII-MDR1 cells. In addition, it can be performed in a barrier-specific manner highlighting the dependence of ABCB1 IC(50) values on different ABCB1 expression levels.

Leflunomide and its metabolite A771726 are high affinity substrates of BCRP: implications for drug resistance.

BACKGROUND: Earlier publications have suggested a possible role for the efflux transporter breast cancer resistance protein (BCRP) in acquired resistance to disease-modifying antirheumatic drugs (DMARDs) such as leflunomide and its metabolite A771726 (teriflunomide). However, there is no direct evidence that BCRP interacts with these drugs. OBJECTIVES: To characterise the interaction between BCRP transporter and leflunomide and its active metabolite A771726, with emphasis on the nature of the interaction (substrate or inhibitor) and the kinetic characterisation of the interactions. METHODS: Different in vitro membrane-based methods (ATPase and vesicular transport assay) using BCRP-HAM-Sf9 membrane preparations and cellular assays (Hoechst assay and cytotoxicity assay) were performed on PLB985-BCRP and HEK293-BCRP cell lines overexpressing BCRP. RESULTS: In all assays used, an interaction between the investigated drugs and BCRP was detected. In the vesicular transport assay, both leflunomide and its metabolite inhibited BCRP-mediated methotrexate transport. Both compounds are likely substrates of BCRP as shown by the vanadate-sensitive ATPase assay. In line with the membrane assays, leflunomide and A771726 inhibited BCRP-mediated Hoechst efflux from PLB985-BCRP cells. In the cytotoxicity assay, overexpression of BCRP conferred 20.6-fold and 7.5-fold resistance to HEK293 cells against leflunomide and A771726, respectively. The resistance could be reversed by Ko134, a specific inhibitor of BCRP. CONCLUSION: Based on these results, BCRP could play an important role in the resistance to leflunomide and A771726 via interactions with these drugs. BCRP may also mediate drug-drug interactions when leflunomide is administered with other BCRP substrate drugs such as methotrexate.

Activation of Raf/ERK1/2 MAP kinase pathway is involved in GM-CSF-induced proliferation and survival but not in erythropoietin-induced differentiation of TF-1 cells.

The involvement of MAPK pathways in differentiation, proliferation and survival was investigated by comparing Epo and GM-CSF signalling in human factor-dependent myeloerythroid TF-1 cells with abnormal Epo-R. GM-CSF withdrawal induced cell-cycle arrest and apoptosis accompanied by increased caspase-3 activity, DNA degradation and reduced expression of the antiapoptotic Bcl-2 and Bcl-xl proteins. Readministration of GM-CSF but not Epo reversed these processes and induced proliferation. The GM-CSF promoted cell survival and proliferation correlated with MEK-1 dependent ERK1/2, Elk-1 and CREB phosphorylation and Egr-1, c-Fos expression as well as with increased STAT-5, AP-1, c-Myb and NF-kappaB DNA-binding. In contrast, Epo failed to activate the Raf-1/ERK1/2 MAPK pathway or to induce Egr-1 and/or c-Fos expression, while it induced erythroid differentiation in GM-CSF-deprived cells. In addition, the Epo-induced haemoglobin production was inhibited in the presence of GM-CSF. These results demonstrate that the activation of MAPK cascade is not necessary for Epo-induced haemoglobin production in TF-1 cells and suggest a negative cross-talk between the signalling of GM-CSF-stimulated cell proliferation and Epo-induced erythroid differentiation.

Effect of testosterone on fracture healing in hypophysectomized rats.

The effect of hypophysectomy and hypophysectomy + testosterone treatment on fracture healing was investigated on 60 Wistar rats (30 day old). The aim of the experiment was to elucidate whether in callus formation testosterone acts directly on the cells (peripheral effect), or its effect is mediated through the hypophysis (central effect). Changes induced by hypophysectomy take place in the course of callus formation due primarily to the impairment of the enzyme system of the cells. The effect of hypophysectomy can be attenuated with testosterone which, despite hypophysectomy, stimulates fracture healing. It may be concluded that testosterone exerts a direct peripheral effect on the callus cells, presumably on their enzyme system.

[Myocardial infarct and diabetes mellitus: incidence, management and prognosis]. / Myocardialis infarctus és diabetes mellitus: elöfordulás, kezelés, prognózis.

The authors analyse the data of the Myocardial and Diabetes Register, where 2436 diabetic patients (pts) and 1448 pts with acute myocardial infarction (AMI) were registered between 1st of January, 1992 and 31st of December 1994. In the history of diabetic patients previous AMI was present in 14.4% of the cases. The 21.6% of the AMI pts had diabetes mellitus as well. According to the type of diabetes (IDDM and NIDDM) the prevalence of AMI in the history of the registered persons was significantly different: among pts with NIDDM the previous AMI was found 14.8% of the pts and only 2% of pts with IDDM (p = 0.012). The clinical picture of AMI was also different of AMI pts with and without diabetes: chest pain suggesting AMI was present 10.9% of pts with proved AMI and diabetes mellitus, and 86.2% of pts with AMI without diabetes (p < 0.0001). The Streptokinase treatment was more common among AMI pts without diabetes (18.2% versus 12.5% p = 0.022). The hospital lethality was significantly higher among AMI pts with diabetes (42.8% versus 29.4% (p < 0.0001). The poorer prognosis was independent of age.