Biodistribution study using Egg Protein ELISA kit after administration of FITC-labeled ovalbumin solution and its double liposomes in the in situ loop method, and its implication in oral immunization.

Ovalbumin (OVA) is often used as a model antigen, and its biodistribution is important for the induction of immunization, especially oral immunization. In this study, an allergic substance-detecting kit, Egg Protein ELISA kit, was applied to the investigation of the biodistribution of fluorescein isothiocyanate-labeled ovalbumin (FITC-OVA). After FITC-OVA solution and its double liposomes were administered into the intestinal loop with one Peyer's patch, the biodistribution of FITC-OVA was examined with the Egg Protein ELISA kit. Each calibration was performed by fitting a quadratic curve to the observed ELISA response points. The ELISA response was almost the same between OVA and FITC-OVA. Similar ELISA response curves were obtained in Peyer's patch (PP) homogenate, spleen (SP) homogenate and plasma (PL). The concentration of FITC-OVA could be determined at 4 - 64 ng/ml for aqueous solution and SP homogenate and at 1 - 64 ng/ml for PP homogenate and PL. Thus, it was suggested that the ELISA kit should be useful for measurement of OVA biodistribution in an oral immunization study. After the administration of FITC-OVA solution and its double liposomes into the intestinal loop, the biodistribution of OVA-FITC in PP, SP and PL was investigated. The distributed amount was the greatest in PP. At the early time, the distributed amount in PP, SP and PL tended to be greater with FITC-OVA solution than the double liposomes. FITC-OVA was retained longer in PP with the double liposomes than FITC-OVA solution. The present results indicated that OVA could transfer well to PP and systemic circulation even with the solution dosage form in the loop method, probably because it was not exposed to harsh conditions such as a gastric fluid. Namely, it implied that the protection from gastric pH and enzyme by the double liposomes, which had been reported before, would be importantly associated with the promotion of immune induction. In addition, the double liposomes could retain OVA longer in PP, which might cause the enhancement of oral immunization.

Stability of spironolactone in rat plasma: strict temperature control of blood and plasma samples is required in rat pharmacokinetic studies.

The stability of spironolactone (SPN) in rat plasma was studied and its degradation was found to be an apparent first-order reaction. The apparent first-order rate constants (k(obs)) at 37, 23.5 and 0 degrees C were 3.543+/-0.261 (h-1, mean+/-S.D., n=3), 6.278+/-0.045 (x10(-1) h-1), and 7.336+/-0.843 (x10(-2) h-1), respectively. The half-lives were 0.20 h, 1.10 h, and 9.53 h. The degradation rate of SPN in rat plasma was markedly decreased when NaF, an esterase inhibitor, was added to the plasma, and the degradation was catalyzed by esterase in the plasma. These results indicated that not only plasma but also blood and serum samples in rat pharmacokinetic studies should be cooled to 0 degrees C, the temperature maintained, and treated as soon as possible. In pharmacokinetic studies reported previously, the temperature control of plasma, blood, and serum samples was not described. The pharmacokinetic study in rats after intravenous administration of SPN at 20 mg/kg was performed with strict temperature control of plasma and blood samples. The AUC, MRT, CL and Vd(ss) values (mean+/-S.E. of 4 rats) for SPN were 4100.8+/-212.9 ng h/ml, 0.29+/-0.01 h, 4915.7+/-248.0 ml/h/kg, and 1435.4+/-48.4 ml/kg, respectively. The AUC value was much larger than that previously reported. The AUC, MRT, Cmax and Tmax values (mean+/-S.E. of 4 rats) of canrenone, an active metabolite of SPN, after the administration of SPN were 4196.1+/-787.5 ng h/ml, 1.99+/-0.13 h, 1546.3+/-436.4 ng/ml and 1.0+/-0.0 h, respectively. This AUC value was almost identical to the value previously reported.