Highly Selective Molecular Recognition of Biologically Active Substances Using Liquid Phase Separation

The development of new chiral stationary phases has been very important in the a ccurate analysis of drug enantiomers and their metabolites in biological samples during drug discovery and development. New chiral stationary phases have been d eveloped using conalbumin and flavoprotein from chicken egg whites, which have b een applied to a broad range of drug enantiomers. The application and characteri zation of these two chiral columns for high-performance liquid chromatograp hy have been documented. Both specific and non-specific interactions, based on the silica gel surface and linker moiety, influenced retention and chiral separa tion of solutes. Interactions between drug enantiomers and proteins, as a pseudo chiral stationary phase, were investigated with affinity capillary electrophore sis, in order to avoid the effects of non-specific interactions. The chiral dis crimination region for ketoprofen on the flavoprotein surface was concluded to c onsist of an α-helix structure. Studies with chemically modified flavoprot ein indicated that two types of interactions at the chiral discrimination region were required for chiral separation: a π-π interaction between a tryptophan residue and the aromatic ring of ketoprofen, and an ionic interaction between th e carboxyl group of ketoprofen and an amino and carboxyl group of the protein. I n the bod y, drugs and biologically active substances having a carboxyl group have been kn own to transform various metabolites such as acyl glucuronide. The acyl adenylat e has also been noted as a chemically active intermediate of coenzyme A ligation . Both the acyl adenylate and the acyl glucuronide produced protein adducts by r eacting with nucleophilic groups such as amino groups on protein molecules. To c haracterize both active intermediates and protein adducts, analytical techniques conferring highly selective molecular recognition, such as high-performance li quid chromatography and mass spectrometry, were required.

Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of E3024, a Novel and Selective Dipeptidyl Peptidase-IV Inhibitor, in Healthy Japanese Male Subjects: Rash Development in Men and Its Possible Mechanism

E3024 (3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-dihydro-4H-imidazo[4,5-d]pyridazin-4-one tosylate) is a dipeptidyl peptidase-IV (DPP-IV) inhibitor that was expected to be an antidiabetic agent. Its safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) were investigated in a randomized, double-blind, placebo-controlled, ascending single-dose study in 48 healthy Japanese male subjects. Fasted subjects were orally administered E3024 (5, 10, 20, 40, or 80 mg) or placebo. E3024 was rapidly absorbed, with tmax values ranging between 0.33 and 3 h after dosing. The mean t1/2 ranged from 5.34 to 11.68 h. AUC0-inf and Cmax increased dose-proportionately. PK-PD relationship of E3024 was evaluated by using an Imax model, indicating that plasma E3024 concentrations and inhibitory effects of plasma DPP-IV activity were well correlated. The IC50 value was calculated as 33.7 ng/mL, which was consistent with in vitro data. Thus, E3024 showed a good PK profile and inhibited DPP-IV dose-dependently. Of 30 subjects administered E3024, 12 (40%) experienced adverse events (AEs). Dose escalation to 160 mg was abandoned owing to undesired subjective/objective findings in 4 of 6 subjects receiving 40 mg and 5 of 6 subjects receiving 80 mg. The most prominent AE was rash, but there were no serious AEs or deaths. The maximum tolerated dose was considered to be 20 mg. We hypothesized that histamine was a cause of the rash induction, and examined blood histamine levels of normal Fischer rats treated with E3024. Blood histamine levels were increased significantly by E3024 at 500 mg/kg (p < 0.001), but not by vildagliptin or valine-pyrrolidide (DPP-IV inhibitors) at the same dose. No blood histamine increases were observed in genetically mast cell-deficient Ws/Ws rats treated with E3024 at 500 mg/kg. In in vitro assays, E3024 induced histamine release from normal rat peritoneal mast cells in a concentration-dependent manner, but not from basophils. The structure-activity relationship study suggested that a piperazine group N-linked to the 2-position of the 5,6-membered fused heterocyclic rings was a key structural element for triggering histamine release.

Synthesis and BK channel-opening activity of novel N-acylhydrazone derivatives from dehydroabietic acid

A series of hydrazone and N-acylhydrazone derivatives of dehydroabietic acid were synthesized and evaluated for BK channel-opening activities in an assay system of CHO-K1 cells expressing hBKa channels.The assay results indicated that the activities of the investigated compounds were influenced by the physicochemical properties of the substituent at hydrazone moiety.