Nitric oxide (NO)-releasing pathway of FK409 in the presence of sulfhydryl-bearing compounds.

PURPOSE: We have recently reported that degradation of FK409 with generation of NO is spontaneous and is accelerated in the presence of sulfhydryl-bearing compounds, such as L-cysteine (Cys) and glutathione (GSH). The purpose of the present study is to investigate the NO-releasing pathway of FK409 in the presence of sulfhydryl-bearing compounds. METHODS: The degradation process of FK409 in the presence of Cys or GSH was investigated by means of 1H-nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography (HPLC). RESULTS: The degradation of FK409 in the presence of Cys was dependent on concentration of Cys, and showed pH-dependency, accelerating with an increase in pH. The 1H-NMR spectra of FK409 with Cys suggested that time-dependent elimination of the hydrogen atom at the alpha-position of the nitro moiety (5-position) was accelerated by Cys in weakly alkaline solution. Cys and GSH were transformed readily, concomitant with FK409 degradation, to give their oxidized forms and probably S-nitrosothiols. CONCLUSION: The effect of sulfhydryl-bearing compounds on FK409 degradation is due to the acceleration of deprotonation of the hydrogen atom at the 5-position by thiolate anion as well as hydroxyl ion. Sulfhydryl-bearing compounds reacted with the released NO resulting in formation of disulfides via intermediate S-nitrosothiols.

A new nitric oxide (NO) releaser: spontaneous NO release from FK409.

The remarkable vasorelaxant and anti-platelet effects of FK409 have been reported to be due to nitric oxide (NO) release. The purpose of the present study is to investigate the spontaneous NO-releasing pathway of FK409 in aqueous solutions. 1H-NMR spectra of FK409 suggested that the compound underwent a time-dependent elimination of the hydrogen atom at alpha-position of the nitro moiety (at the 5-position) in weakly alkaline solutions. In addition, the degradation of FK409 monitored by HPLC showed a pH-dependency accelerating with an increase of pH. These results revealed that the first step in the degradation of FK409 might be the hydroxyl ion-dependent subtraction of the hydrogen atom at the 5-position. On the other hand, NO release from FK409 also exhibited a pH-dependency, and the velocity of NO liberation was markedly enhanced above pH 6. Furthermore, a linear relationship between the rate of FK409 degradation and that of NO formation was observed, indicating that the rate-limiting step for NO formation is the same as that for degradation. Thus, the rate-limiting process of NO formation from FK409 is due to the deprotonation reaction of the hydrogen atom at the 5-position by hydroxyl ions. The deprotonation process appears to be an essential step for both FK409 degradation and NO release. On the basis of the results, a possible kinetic scheme for NO release from FK409 is proposed.

Mechanism of optical isomerization of (S)-N-[1-(2-fluorophenyl)-3,4,6,7- tetrahydro-4-oxopyrrolo[3,2,1-jk] [1,4]-benzodiazepine-3-yl]-1H- indole-2-carboxamide (FK480) in soft capsules containing polyethylene glycol 400 and glycerol.

FK480 is a new synthetic non-peptide antagonist of cholecystokinin (CCK)-A receptors. The dosage form of FK480 is a soft capsule containing a solution of FK480 in a mixture of polyethylene glycol 400 (PEG 400) and glycerol to improve its bioavailability. Studies on the stability of this FK480 dosage form revealed that the main degradation occurred by optical isomerization at the asymmetric C-3 position of the pyrrolobenzodiazepine ring. The degradation reaction was accelerated by formic acid formed in a mixture of PEG 400 and glycerol. Addition of amino acids to the capsule solution retarded the isomerization by reacting with formic acid. Therefore, formic acid appears to accelerate optical isomerization of FK480.

Determination of the unbound concentration of hydrophobic drugs in albumin solutions by high-performance frontal analysis using a diol-silica column.

High-performance frontal analysis (HPFA) using diol-silica column allows the determination of the unbound concentration of hydrophobic drugs under protein binding equilibrium, which is often difficult by the conventional methods. After the direct injection of a drug-protein mixed solution onto a diol-silica column, the drug is eluted as a zonal peak containing a plateau region under a mild mobile phase condition (phosphate buffer, pH 7.4, ionic strength = 0.17). The unbound drug concentration was determined as the drug concentration in the plateau region. The reliability of this method was confirmed by comparison with the results obtained by the conventional ultrafiltration-HPLC method. The versatility of the HPFA method using a diol-silica column was demonstrated by developing a novel on-line HPFA-chiral HPLC system for a simple and easy determination of the unbound concentration of nilvadipine (NV) enantiomers. The direct injection of 1.33 mL of a sample solution containing 20 nM-1 microM NV and 550 microM HSA gave the plateau region due to unbound NV, which was heart-cut and transferred on-line into a preconcentration and chiral HPLC separation system. As low as a few hundred picomolar level of unbound NV enantiomers was determined by use of UV detection (at 244 nm). The binding between NV and HSA is enantioselective; (R)-NV binds more strongly than (S)-NV.