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A sensitive and specific analytical method for a potent antitumor agent, TZT-1027, in plasma has been developed using liquid chromatography–mass spectrometry (LC–MS) with [2H4]TZT-1027 as an internal standard (I.S.). A plasma sample was purified by solid-phase extraction on a C18 cartridge, followed by solvent extraction with diethyl ether. The extract was then injected into the LC–MS system. Chromatography was carried out on a C18 reversed-phase column using acetonitrile–0.05% trifluoroacetic acid (TFA) (55:45) as a mobile phase. Mass spectrometric analysis was performed in atmospheric pressure chemical ionization (APCI) mode with positive ion detection, and the protonated molecular ions ([M+H]+) of TZT-1027 and I.S. were monitored to allow quantitation. The method was applied to the determination of TZT-1027 in human, monkey, dog, rat and mouse plasma. As far as the sample preparation was concerned, good recoveries (73.5–99.1%) were obtained. The calibration curves were linear over the range of 0.25–100 ng per 1 ml of human, dog and rat plasma, per 0.5 ml of monkey plasma, and per 0.1 ml of mouse plasma. From the intra- and inter-day accuracy and precision, the present method satisfies the accepted criteria for bioanalytical method validation. TZT-1027 was stable when stored below −15°C for 6 months in human plasma and for 3 weeks in plasma from other species. TZT-1027 was also stable in plasma through at least three freeze–thaw cycles.

The pharmacokinetics, distribution, and excretion of radioactivity and the main metabolites, M-1, M-2 and M-3, were studied in male rat after a single intravenous administration of 14C-labeled roxatidine acetate hydrochloride (TZU) at a dose of 10 mg/kg. The concentrations of these three metabolites in plasma, tissues and urine were simultaneously quantified with LC/MS-SIM using corresponding deuterium-labeled compounds as internal standards.1) The total radioactivity in the blood decreased with the half-lives of 0.38 and 3.82 hr in α-phase and β-phase, respectively. The plasma concentration of M-1, the active metabolite, decreased with the half-lives of 0.17 hr in α-phase and 0.77 hr in β-phase.2) At 15 min after administration, a high radioactivity was found in the liver, kidney, stomach, small intestine, parotid and submaxillary glands. At 72 hr after administration, the radioactivity was less than 0.1% of the dose or below the detection limit in any tissues.3) The metabolites in tissues were qualitatively analyzed by radio-HPLC. The radioactivity of the M-1 fraction was the highest in the brain, stomach, small intestine and kidney, then followed by that of the M-2 and M-3, the total radioactivity mainly consisted of those three fractions.4) The concentrations of three metabolites were quantified with LC/MS-SIM in the brain, lung, stomach, small intestine, liver, kidney and testis. All these metabolites disappeared rapidly, the half-lives were below 3.4 hr, in every tissue except for the testis. Although the radioactivity in the testis slowly decreased with the half-life of 10.5 hr, it accounted for M-2 and M-3, because they disappeared similarly to the radioactivity.5) In the target organs, stomach and small intestine, the concentration of M-1 was the highest, confirming that TZU has an advantage as an H2-receptor antagonist.6) Only in the liver, the level of the phenolic metabolite, M-3, was the highest of the three metabolites. This finding indicated that the metabolite might be mainly produced through oxidative dealkylation in liver.7) Approximately 87 and 8 % of the dosed radioactivity were excreted within 48 hr after administration in the urine and feces, respectively. The main metabolite in the urine was M-3, consisting of 29% of the dose, and about half of that was in the conjugated form. In feces, the excretion ratios of these three metabolites were almost equal, 0.3% of the dose.8) About 20% of the dosed radioactivity was excreted into bile in billiary fistular rats. The radioactivities in the M-1 and M-2 fractions were each 0.3% of the dose. The M-3 fraction comprised about 8%, almost all of which was in the conjugated form.

1. 1. TZC-5665 is a novel pyridazinone derivative with vasodilatory and β-adrenergic blocking activities and type III phosphodiesterase inhibitory action. 2. 2. In healthy volunteers, TZC-5665 was rapidly absorbed and immediately metabolized. Its main metabolite, M-2, remained at a higher concentration in plasma. Orally administered TZC-5665 reduced end-diastolic left ventricular volume (20.16 ml) and exhibited a tendency to increase ejection fraction (0.04). 3. 3. In dogs, M-2 dose-dependently increased cardiac contractility and reduced both preload and afterload. These effects appeared more potent in the failed heart than in the normal heart. At the same dose (30 μg/kg), the effects of M-2 seem to be more potent than those of milrinone. 4. 4. We concluded that TZC-5665 is a useful medication for treating patients with chronic congestive heart failure (CHF) because of the positive inotropic and vasodilating effects due to its active metabolite in addition to its own β-adrenergic blocking actions.

State analysis of low-sulfated chondroitin 4-sulfate (LSC) in human urine and serum was performed by the use of high performance liquid chromatography and Western blot analysis. It was revealed that the most amount of LSC in urine is present as urinary trypsin inhibitor and a small amount (about 10% of total LSC) is as an LSC chain. The LSC in serum is mainly present as a proteoglycan such as inter-alpha-trypsin inhibitor (ITI), with a molecular weight of 212 kDa, but a small amount of LSC-proteoglycans having molecular weights of 128 and 38 kDa were also observed on SDS-PAGE. Those two compounds may be fragments of ITI, or one of the compounds (128 kDa) may be pre-alpha-trypsin inhibitor which was found by Enghild et al. (J. Biol. Chem., 264, 15975 (1989)).