Synthesis, aqueous reactivity, and biological evaluation of carboxylic acid ester-functionalized platinum-acridine hybrid anticancer agents.

The synthesis of platinum-acridine hybrid agents containing carboxylic acid ester groups is described. The most active derivatives and the unmodified parent compounds showed up to 6-fold higher activity in ovarian cancer (OVCAR-3) and breast cancer (MCF-7, MDA-MB-231) cell lines than cisplatin. Inhibition of cell proliferation at nanomolar concentrations was observed in pancreatic (PANC-1) and nonsmall cell lung cancer cells (NSCLC, NCI-H460) of 80- and 150-fold, respectively. Introduction of the ester groups did not affect the cytotoxic properties of the hybrids, which form the same monofunctional-intercalative DNA adducts as the parent compounds, as demonstrated in a plasmid unwinding assay. In-line high-performance liquid chromatography and electrospray mass spectrometry (LC-ESMS) shows that the ester moieties undergo platinum-mediated hydrolysis in a chloride concentration-dependent manner to form carboxylate chelates. Potential applications of the chloride-sensitive ester hydrolysis as a self-immolative release mechanism for tumor-selective delivery of platinum-acridines are discussed.

Unusual Reactivity of a Potent Platinum-Acridine Hybrid Antitumor Agent.

The formation of unusual seven-membered, sterically overloaded chelates [Pt(en)(L/L´)](NO(3))(2) (4a/4b) from the corresponding potent hybrid antitumor agents [PtCl(en)(LH/L´H)](NO(3))(2) (3a/3b) is described, where en is ethane-1,2-diamine and L(H) and L´(H) are (protonated) N-(2-(acridin-9-ylamino)ethyl)-N-methylpropionimidamide and N-(2-(acridin-9-ylamino)ethyl)-N-methylacetimidamide, respectively. Compounds 3a and 3b inhibit H460 lung cancer cell proliferation with IC(50) values of 12 ± 2 nM and 2.8 ± 0.3 nM, respectively. The new derivative 3b proves to be not only the most cytotoxic platinum-acridine hybrid of this kind, but also one of the most potent platinum-based anticancer agents described to date. The chelates 4a and 4b do not undergo ligand substitution reactions with nucleobase nitrogen and cysteine sulfur and do not intercalate into DNA. Despite their inertness, the two chelates appear to maintain micromolar activity in H460 cells. The results are discussed in the context of potential DNA-mediated and DNA-independent cell kill mechanisms and the potential use of the chelates as prodrugs.

Probing platinum-adenine-n3 adduct formation with DNA minor-groove binding agents.

Me-lex(py/py), an adenine-N3-selective alkylating agent, and the reversible minor-groove binder netropsin were used to probe the formation of unusual minor-groove adducts by the cytotoxic hybrid agent PT-ACRAMTU ([PtCl(en)(ACRAMTU)](NO(3))(2); en = ethane-1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea). PT-ACRAMTU was found by chemical footprinting to inhibit specific Me-lex-mediated DNA cleavage at several adenine sites but not at nonspecific guanine, which is consistent with the platination of adenine-N3. In a cell proliferation assay, a significant decrease in cytotoxicity was observed for PT-ACRAMTU, when cancer cells were pretreated with netropsin, suggesting that minor-groove adducts in cellular DNA contribute to the biological activity of the hybrid agent.

Major allergen measurements: sources of variability, validation, quality assurance, and utility for laboratories, manufacturers, and clinics.

The isolation and characterization of prominent allergenic proteins or glycoproteins is an important step in the development of allergenic extracts exhibiting improved definition, consistency, and clinical utility. Quantitative analyses specific for major allergenic components currently are being performed in numerous corporate and academic laboratories but have not been validated within or across laboratories in a systematic manner. In our laboratory, validation of double-bind (sandwich) ELISA assays for a diverse group of major allergens or extract components revealed a number of critical assay variables and reagent incubation conditions that directly influenced the precision, accuracy, specificity, and robustness of these tests. Data from ELISA methods for six allergens (Dermatophagoides farinae Der f 1, Alternaria Alt a 1, dog albumin, dog Can f 1, fire ant Sol i 3, and yellow jacket venom Ves 5) showed that up to twofold differences in results were observed when analysts or microplates were varied. Analyses of dog allergens using multiple reagents and concentrations indicated that twofold variations in results also can be produced by distinct combinations of materials or incubations from different assay steps. Data from Can f 1 and egg white analyses produced up to fivefold differences in antigen concentrations based on changes in the capture antibody source (mouse monoclonal versus rabbit polyclonal) or storage buffer. These results suggest that differences in major allergen concentrations reported by different testing laboratories may be related to assay differences as well as extract variations and raise questions as to the accuracy of major allergen concentrations and therapeutic dose recommendations reported at regional and national allergy meetings. Validated double-bind ELISA methods may be well suited for consistency monitoring and standardization of extracts provided that reference materials, reagent qualifications, and interlaboratory comparability are defined precisely.