Underestimation of rat serum vancomycin concentrations measured by an enzyme-multiplied immunoassay technique and the strategy for its avoidance.

An enzyme-multiplied immunoassay technique (EMIT) has been widely adopted for the measurement of serum concentrations of vancomycin (VCM) in clinical practice. Because of the growing demand for its application to fundamental pharmacokinetic studies, we examined whether VCM concentrations in rat serum were accurately measured by EMIT. It was found that measured values of known amounts of VCM spiked to rat serum were markedly underestimated with a large analytical variance. When ultrafiltrated rat serum was used as the sample matrix, interference was significantly improved, and the degree of underestimation was attenuated also by diluting samples with physiological saline. These results suggest that endogenous substances of a high molecular weight in rat serum interfere with the analysis of VCM concentrations by EMIT. However, measured values of rat serum VCM concentrations by EMIT were restored to theoretical levels by exposing samples to 70°C for 3-7 min. A likely explanation for the avoidance of interference is that an appropriate thermal force eliminated the immunological function of endogenous substances falsely recognizing VCM without affecting the VCM molecule itself. Regarding serum samples collected from rats that were administered VCM, values measured by EMIT following the heat-treatment agreed well with those by the high performance liquid chromatography (HPLC) method. This is the first report showing interference by endogenous high-molecular substances in the measurement of drug concentrations in rat serum using EMIT. Our findings will contribute to the appropriate use of VCM based on evidence provided by clinical-oriented rat experiments requiring the measurement of serum VCM concentrations by EMIT.

Difference in nephrotoxicity of vancomycin administered once daily and twice daily in rats.

We compared the degree of nephrotoxicity of vancomycin (VCM) administered once daily and twice daily in rats. VCM was intraperitoneally administered once daily to rats at a dose of 400 mg/kg (VCM-1-treated) or administered at a dose of 200 mg/kg twice daily at 12-hour intervals (VCM-2-treated) for 7 consecutive days. Creatinine clearance was decreased more markedly in VCM-1 rats relative to VCM-2 rats, although there was no significant difference in renal accumulation of VCM between the two groups. Renal superoxide dismutase activity was lower in VCM-1 rats than that in VCM-2 rats. The magnitude of histological change in kidney tissue was in agreement with the degree of alterations in the abovementioned biochemical values. These results suggest that the nephrotoxic effect of once-daily VCM administration is more pronounced than that of the twice-daily treatment. Our findings provide fundamental evidence for the advantage in choosing a divided VCM administration to attenuate nephrotoxicity.

Application of terahertz absorption spectroscopy to evaluation of aging variation of medicine.

The absorption spectra of three kinds of medicines both before and after the expiration date: Amlodin OD(®) (5 mg), Basen OD(®) (0.2 mg) and Gaster D(®) (10 mg) have been measured by terahertz time domain spectroscopy (THz-TDS). All the medicines show some differences in the THz absorption spectra between medicines before and after the expiration dates. X-Ray powder diffraction (XRD) studies of all medicines suggest that the polymorph of the main effective compound is not changed before and after the expiration date. Therefore, the differences in the THz spectra between medicines before and after the expiration dates arise from aging variation of diluting agents and/or from modifications of intermolecular interaction between the effective compounds and diluting agents.

Effect of blood decrease on micafungin disposition in rats.

Micafungin (MCFG) is a novel echinocandin-class antifungal agent that extensively undergoes metabolic removal in the liver. In the present study, the influence of decreased blood volume on pharmacokinetic disposition of MCFG was examined using a rat model prepared by phlebotomy. In phlebotomized rats, hematocrit level and plasma albumin concentration were decreased by 50 and 15%, respectively. Regarding the pharmacokinetic parameters of MCFG, there were no significant differences in the total body clearance (CL(tot)) and elimination rate constant (k (e)) between control and phlebotomized rat groups. A slight increase was observed in the apparent volume of distribution at steady-state (Vd(ss)), but the degree of change was minimal. These findings demonstrate that the elimination capacity for MCFG is only slightly affected by severe anemia and moderate hypoalbuminemia, and provide experimental evidence for the preceding clinical studies suggesting that neither hematocrit level nor serum albumin concentration is a contributory factor for the metabolic clearance of MCFG.

Impact of plasma exchange on pharmacokinetic disposition of micafungin.

Change in the pharmacokinetic disposition of an antifungal agent micafungin (MCFG) by 8-hour plasma exchange (PE) with 3200 mL replacement was examined in a stem cell transplant recipient. On pharmacokinetic analysis of the time course of the serum concentrations of MCFG, it was determined that PE shortened the elimination half-life of MCFG from 16.5 hours to 6.3 hours. Total clearance (CL(tot)) was increased from 0.366 L/h to 0.932 L/h by PE. PE-dependent clearance (CL(pe)) accounted for approximately two-thirds of CL(tot), and PE was found to contribute to the removal of nearly 40% of the total body store of MCFG. It was confirmed that a significant amount of MCFG was excluded into apheresed plasma waste. In addition, adsorption of MCFG onto plasma-separating membrane was strongly suggested, because the CL(pe) exceeded the rate of plasma apheresis and MCFG concentrations in apheresed plasma were lower than those in circulating blood collected at the same time. The marked elimination of MCFG during PE can be explained by its low volume of distribution and high affinity for serum proteins. Judging from these findings as well as those of other reports, MCFG can be considered one of the drugs most susceptible to removal by PE. Our findings suggest that an increment in the regular dose of MCFG would be required at the next administration after PE.

Reduced elimination clearance of micafungin in rats with cholestatic hyperbilirubinemia.

We examined whether the pharmacokinetic disposition of micafungin (MCFG), an echinocandin class antifungal agent, is altered in hyperbilirubinemia using a rat model prepared by bile duct ligation (BDL). Serum bilirubin levels were increased depending upon the duration of BDL. The elimination rate constant and total body clearance (CL(tot)) of MCFG were reduced by 24% and 16%, respectively, after BDL for 1 h, but there was no significant change in the apparent volume of distribution at steady-state. The degree of reduction in the CL(tot) was much greater 7 days after BDL as compared with that 1 h after BDL (44% vs. 16%). However, the proportion of the biliary clearance in the CL(tot) was about 10%. This is similar to the extent of decrease in the CL(tot) by occlusion of the bile duct, demonstrating that decreased biliary excretion of MCFG makes only a minor contribution to its pharmacokinetic change. These findings suggest that the metabolic capacity of MCFG is markedly impaired in hepatic hypofunction secondary to hyperbilirubinemia, providing a fundamental explanation for the previous clinical report that there is a significant correlation between dose-adjusted plasma MCFG concentration and serum bilirubin levels.

Spironolactone exhibits direct renoprotective effects and inhibits renal renin-angiotensin-aldosterone system in diabetic rats.

Aldosterone itself has been reported to participate in mediating renal injury, and it was confirmed that the aldosterone synthase CYP11B2 gene, protein, and aldosterone production are locally present in the kidney. To test the hypothesis that a mineralocorticoid receptor antagonist might ameliorate diabetic nephropathy and the inhibition of renal CYP11B2 expression might be associated with these renoprotective effects, spironolactone (50 mg/kg/day) was administered by gavage to uninephrectomized diabetic rats for 3 weeks. Streptozotocin (55 mg/kg, i.v.) significantly increased urinary protein excretion and collagen deposition in glomerular and tubulointerstitial areas in the kidney, which were attenuated by spironolactone treatment. RT-PCR and Western blot analysis revealed that the expression of mRNA for collagen I/IV, transforming growth factor-beta, NADPH oxidase and mineralocorticoid receptor and the mineralocorticoid receptor protein in the kidney was enhanced in the uninephrectomized diabetic rat kidney and that the overexpression of these molecules was suppressed by spironolactone. Renal angiotensin converting enzyme was activated and overexpressed in diabetic rats, and spironolactone inhibited these changes. We demonstrated that spironolactone prevented the streptozotocin-induced increase in the renal CYP11B2 mRNA content. Controlling blood glucose level with insulin also attenuated the renal expression of mRNA for CYP11B2. On the other hand, the treatment of spironolactone in the present study did not affect blood glucose level or blood pressure in uninephrectomized streptozotocin-induced diabetic rats. These results suggest that spironolactone exerted renoprotective effects in uninephrectomized streptozotocin-induced diabetic rats and inhibited local renin-angiotensin-aldosterone system, such as the ACE expression and the hyperglycemia-induced overexpression of CYP11B2, in the kidney.