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Overexpression of efflux transporters, in human cells, is a mechanism of resistance to drug and also to chemotherapy. We found that multidrug resistance protein-4 (MRP4) overexpression has a role in reducing aspirin action in patients after bypass surgery and, very recently, we found that aspirin enhances platelet MRP4 levels through peroxisome proliferator activated receptor-α(PPARα). In the present paper, we verified whether exposure of human embryonic kidney-293 cells (Hek-293) to aspirin modifies MRP4 gene expression and its correlation with drug elimination and cell toxicity. We first investigated the effect of high-dose aspirin in Hek-293 and we showed that aspirin is able to increase cell toxicity dose-dependently. Furthermore, aspirin effects, induced at low dose, already enhance MRP4 gene expression. Based on these findings, we compared cell viability in Hek-293, after high-dose aspirin treatment, in MRP4 overexpressing cells, either after aspirin pretreatment or in MRP4 transfected cells; in both cases, a decrease of selective aspirin cell growth inhibition was observed, in comparison with the control cultures. Altogether, these data suggest that exposing cells to low nontoxic aspirin dosages can induce gene expression alterations that may lead to the efflux transporter protein overexpression, thus increasing cellular detoxification of aspirin.

SummaryPlatelet multidrug resistance protein4 (MRP4)-overexpression has a role in reducing aspirin action. Aspirin in vivo treatment enhances platelet MRP4 expression and MRP4 mediated transport inhibition reduces platelet function and delays thrombus formation. The aim of our work was to verify whether MRP4 expression is enhanced in platelets obtained from patients under chronic aspirin treatment and whether it correlates with residual platelet reactivity. We evaluated changes on mRNA and protein-MRP4 expression and platelet aggregation in four populations: healthy volunteers (HV), aspirin-free control population (CTR), patients who started the treatment less than one month ago (ASA2 months patients). In platelets obtained from ASA>2 months patients, it was found a statistically significant MRP4 enhancement of both mRNA and protein expression compared to HV, CTR and ASA2 months patients that present high levels of platelet MRP4, have higher serum TxB2 levels and collagen-induced platelet aggregation compared to patient with low levels of MRP4 in platelets. In addition collagen induced platelet aggregation is higher in in vitro aspirinated platelets obtained from patients with high levels of MRP4 patients compared to those obtained from patients with low MRP4 levels. We can assert that, in patients under chronic aspirin treatment, platelets that present high MRP4 levels have an increase of residual platelet reactivity, which is due in part to incomplete COX-1 inhibition, and in part to COX-1–independent mechanism.

Platelet Multidrug Resistance Protein 4 (MRP4)-overexpression has a role in reducing aspirin action in patients after by-pass surgery. Aspirin induces platelet MRP4 over-expression, through megakaryocytes genomic modulation. Aim of our work was to verify whether other non-steroidal antiinflammatory drugs (NSAIDs) enhance platelet MRP4 expression and evaluate platelet function in patients who overexpressed MRP4. We evaluated MRP4-mRNA in a human megakacaryoblastic cell line (DAMI), treated with both COX-2 inhibitor (celecoxib) and traditional NSAIDs (diclofenac and naproxen). Osteoarthritis patients, who reported to take NSAIDs twice a week for at least four continuous weeks and a control population, who didn't take any drugs during the previous month, were enrolled. We evaluated platelet MRP4 amount, by both mRNA levels and protein expression (Western-Blot) and ADP induced platelet aggregation. DAMI cells treated with celecoxib, diclofenac, and naproxen showed a significant increase in MRP4-mRNA expression compared to the mock culture. Osteoarthritis patient platelets presented a higher expression of MRP4 (both at mRNA and protein levels) and an increase in ADP-induced platelet aggregation compared to the control population. NSAID treatment induced platelet MRP4 overexpression. Osteoarthritis patients, who overexpress MRP4, showed platelet hyper-reactivity. These evidences could explain in part the increased cardiovascular risk present during NSAID treatment.

Serum thromboxane-B2 (TxB2), together with arachidonic acid (AA)-induced platelet aggregation, are, at the moment, the most used tests to identify patients displaying high on-aspirin treatment platelet reactivity (HAPR). Both tests are specific for aspirin action on cyclooxygenase-1. While the correlation between serum TxB2 assay and clinical outcome is established, data are conflicting with regard to aspirin treatment and a possible association with AA-stimulated platelet markers and clinical outcome. To understand such discrepancy, we performed a retrospective study to compare both assays. We collected data from 132 patients receiving a daily dose of aspirin (100 mg/day) and data from 48 patients receiving aspirin on alternate days. All Patients who received a daily dose of aspirin were studied for AA-induced platelet aggregation together with serum TxB2 levels and AA-induced TxB2 formation was also studied in 71 patients out of entire population. Consistent with recommendations in the literature, we defined HAPR by setting a cut-off point at 3.1 ng/ml for serum levels of thromboxane B2 and 20% for AA-induced platelet aggregation. According to this cut-off point, we divided our overall population into two groups: (1) TxB23.1 ng/ml and (2) TxB23.1 ng/ml. We found low agreement between such tests to identify patients displaying HAPR. Our results show that AA-induced platelet aggregation20% identify a smaller number of HAPR patients in comparison with TxB2. A good correlation between serum TxB2 and arachidonic acid-induced TxB2 production was found (r = 0.76619).

The synthesis and the nidation inhibitory activity (indirect evidence of prolactin activity) of a new class of ergolinyl-acylureas is described. Some structure—activity relationship considerations are also reported. N -[3-(Dimethyl-amino)propyl]- N -[(ethylamino)carbonyl]-6-(2-propenyl)ergoline-8β-carboxamide (laboratory code FCE 21336; international non-proprietary name cabergoline) was the most interesting compound of the series and is now under extensive clinical evaluation in treatment of hyperprolactinemic disorders.

The treatment of methyl lysergate with mercuric acetate in methanol yields, instead of the expected 10 - methoxy - 6 - methyl - ergoline - 8 β - carboxylic acid methyl ester (2), 10 - methoxy - 8,9 - didehydro - 6 - methyl - ergoline - 8 - carboxylic acid methyl ester (3), whose structure is demonstrated. From 3, penniclavine (14) and isosetoclavine (15) were prepared according to Scheme 1.

The alpha-adrenolytic activity of 1-methyl-10-methoxydihydrolysergol 2-(3,5-dimethyl)pyrrolcarboxylate (XV, formula II) is, both in vitro and parenterally, quite similar to that of dihydroergotamine. By oral route the new compound is more active, and longer lasting than dihydroergotamine.