Characterization of eltenac and novel COX-2 selective thiopheneacetic acid analogues in vitro and in vivo.

We assessed the effect of novel selective thiopheneacetic acids on cyclooxygenase isoenzymes in vitro and in vivo. Thiopheneacetic acid Eltenac and derivatives were investigated in this study. In human whole blood experiments these derivatives were potent inhibitors of COX-2 (IC(50)=0.02-0.4 microM) with less pronounced effect on COX-1 (IC(50)=0.15-5.6 microM). With COX-1/COX-2 ratios between 7.5- and 16-fold they are in the range of Celecoxib (13-fold). The parent drug Eltenac demonstrated no selectivity for COX-2. In a rat paw edema model, these compounds showed reduction of edema volume in the range of 36-45% at 10 mg/kg (Eltenac 52%, Diclofenac 51%). However, the compounds were superior to Diclofenac and Eltenac with respect to their ulcerogenic and gastrointestinal properties. Introduction of a nitrate-ester moiety to either Eltenac or a derivative did neither improve selectivity or potency in vitro, nor ulcerogenicity in vivo. Molecular modeling of selective thiopheneacetic acid derivatives to the active site of human COX-2 suggested similar binding properties as Lumiracoxib and Diclofenac. In summary, modification of Eltenac generates moderately selective COX-2 drugs in the range of Celecoxib with respect to potency and selectivity. The drugs showed potent anti-inflammatory properties and significant improvement of animal survival in a sub-chronical experimental set up. Thiopheneacetic derivatives are characterized by low pK(a) values, short microsomal half-lives and binding mode to COX-2 similar to Diclofenac and Lumiracoxib. These properties may also have an impact on the transient inhibition of COX-2-dependent prostacyclin, thereby being less associated with vascular complications.

Characterization of the dexniguldipine binding site in the multidrug resistance-related transport protein P-glycoprotein by photoaffinity labeling and mass spectrometry.

Human P-glycoprotein (P-gp), an integral membrane transport protein, is responsible for the efflux of various drugs, including cytostatics from cancer cells leading to multidrug resistance. P-gp is composed of two homologous half domains, each carrying one nucleotide binding site. The drug extrusion is ATP-dependent and can be inhibited by chemosensitizers, such as the dihydropyridine derivative dexniguldipine-HCl, through direct interaction with P-gp. To evaluate the mechanism(s) of chemosensitization and identify the binding sites of dexniguldipine-HCl, a tritium-labeled azido analog of dexniguldipine, [(3)H]B9209-005, was used as a photoaffinity probe. Using the multidrug resistant T-lymphoblastoid cell line CCRF-ADR5000, two proteins were specifically labeled in membranes by [(3)H]B9209-005. These proteins were identified by immunoprecipitation such as P-gp and its N-terminal fragment. The membranes were solubilized and the labeled P-gp proteins first isolated by lectin-chromatography and then digested with trypsin. SDS-polyacrylamide gel electrophoresisanalysis of the digest revealed a major radioactive 7-kDa fragment. The tryptic fragments were separated by high-performance liquid chromatography and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The MS results, corroborated by MALDI-MS of peptides after one step of Edman analysis, identified the radioactive 7-kDa band as the dexniguldipine-bound, tryptic P-gp peptide, 468-527. This sequence region is flanked by the Walker motifs A and B of the N-terminal ATP-binding cassette suggesting direct interaction of the chemosensitizer with the nucleotide binding site is involved in the mechanism of chemosensitization.