Lysosomal trapping of 4-dimethylamino-1-{3-(1-methyl-1H-imidazole-2-yl)propanoyl}piperidine, a hydrophilic and weakly basic amine, in human aortic vascular smooth muscle cells.

Some weakly basic compounds lead to cell death accompanied by cellular vacuolation. The novel analgesic agent, 4-dimethylamino-1-{3-(1-methyl-1H-imidazole-2-yl)propanoyl}piperidine (DMIP), is a hydrophilic and weakly basic compound that induces vacuolation in the vascular smooth muscle cells in dogs. Here, we investigated the vacuolation mechanism and the potential cytotoxicity of DMIP using human aortic vascular smooth muscle cells. When cells were treated with DMIP (0.1, 0.3, and 1 mM) for 6, 24, and 48 h, clear cytoplasmic vacuolation was observed at 1 mM after 24 and 48 h, along with an increase in the intracellular DMIP concentration. The vacuolation and intracellular DMIP were markedly reduced by bafilomycin A1, a vacuolar H+-ATPase inhibitor. The late endosome marker Rab7 and lysosome marker LAMP-2 were highly expressed but the early endosome marker Rab5 and autophagosome marker LC3 were not expressed specifically on the vacuolar membranes. These results suggested that the most vacuoles were enlarged late endosomes/lysosomes, resulting from the accumulation of DMIP by ion trapping. Moreover, DMIP did not affect lysosomal membrane integrity and was less cytotoxic than chloroquine, an inducer of phospholipidosis. The current study provides further insight into the mechanisms of vacuolation and lysosomal trapping induced by the hydrophilic and weakly basic amine DMIP.

Cytoplasmic Vacuolation and Tapetal Changes Induced by a Novel Analgesic Agent in Beagle Dogs.

Drug-induced unique cytoplasmic vacuolation was found in the subchronic oral toxicity study of 4-dimethylamino-1-{3-(1-methyl-1H-imidazole-2-yl)propanoyl}piperidine (DMIP), a potential therapeutic agent for neuropathic pain, in beagle dogs. In the first study, DMIP was administered at a dose of 250, 500, or 1,000 mg/kg/day once daily for 14 days. Discoloration of tapetum lucidum accompanied by tapetal swelling was observed at ≥250 mg/kg/day. The tapetal swelling was correlated to the light microscopic observation of cytoplasmic vacuolation in tapetal cells, and similar vacuolation was observed in several other tissues, including the coronary artery and aortal arch, in a dose-dependent manner. Immunohistochemistry for lysosomal-associated membrane protein 2 indicated that the vacuoles were enlarged lysosomes. However, the nature of these vacuoles was different from that of phospholipidosis because no lamellar bodies were observed. In the second study, DMIP was administered at a dose of 10, 50, or 250 mg/kg/day once daily for 14 days followed by a 14-day recovery period. Tapetal changes and systemic vacuolation were not observed at ≤50 mg/kg/day, and vacuolation observed at 250 mg/kg/day was reversible. A few reports have described the enlargement of lysosomes not attributable to phospholipid accumulation. Our findings provide further information about the toxicological implications of drug-induced lysosomal swelling.

Discovery of novel 7-membered cyclic amide derivatives that inhibit 11beta-hydroxysteroid dehydrogenase type 1.

A series of novel 5-trans-hydroxyadamantan-2-yl-5,6,7,8-tetrahydropyrazolo[4,3-c]azepin-4(1H)-ones that inhibit 11beta-hydroxysteroid dehydrogenase type 1 are described. We discovered these 7-membered cyclic amide derivatives by introducing a distinctive linker through pharmacophore analysis of known ligands included in X-ray co-crystal structures. Further optimization using docking studies led to highly potent inhibitors 15b and 27, which furthermore showed the potent efficacy in in vivo studies.