Kiyoshi Mihara

Hepatic Disposition Characteristics of Electrically Charged Macromolecules in Rat in Vivo and in the Perfused Liver

The effect of electric charge on the hepatic disposition of macromolecules was studied in the rat. Charged derivatives of dextran (T-70) and bovine serum albumin (BSA), mitomycin C–dextran conjugates (MMC-D), and lactosaminated BSA (Lac-BSA) were employed as model macromolecules. After intravenous injection, cationic macromolecules were rapidly eliminated from plasma because of their extensive hepatic uptake, while anionic and neutral macromolecules were slowly eliminated. Cationic macromolecules were recovered from parenchymal and nonparenchymal hepatic cells at a cellular uptake (per unit cell number) ratio of 1.4–3.2, while that of Lac-BSA was 14. During liver perfusion using a single-pass constant infusion mode, cationic macromolecules were continuously extracted by the liver, with extraction ratios at steady-state (Ess) ranging between 0.03 and 0.54, whereas anionic and neutral macromolecules were almost completely recovered in the outflow at steady state. The Ess for cationized BSA (Cat-BSA) and cationic MMC-Dcat were concentration dependent and decreased at low temperatures and in the presence of colchicine and cytochalasin B. The possible participation of the internalization process in the uptake of cationic macromolecules by hepatocytes was suggested.

Improvement of Therapeutic Effect of Human Recombinant Superoxide Dismutase on Ischemic Acute Renal Failure in the Rat via Cationization and Conjugation with Polyethylene Glycol

Therapeutic effect of superoxide dismutase (SOD) and three derivatives: a conjugate with polyethylene glycol (SOD-PEG2), a cationized derivative (cSOD), and a mannosylated derivative (Man-SOD), on acute renal failure induced by ischemia/reperfusion was studied in rats. SOD and derivatives were administered intravenously to the rat after nephrectomy of the right kidney and before and after 60 min occlusion of the left renal artery. At 48 hr after reperfusion, the renal function was evaluated by determining the urinary excretion rate of 14C-inulin injected intravenously. No therapeutic effect on the impaired renal function was shown in the case of low dose SOD (2600 unit/kg) treatment. In contrast, administration of cSOD which was shown to be taken up by the isolated perfused kidney from its capillary side and SOD-PEG2 which maintained high plasma concentration exhibited significant therapeutic effect, as did SOD at ten-fold higher dose (26,000 unit/kg). On the other hand, renal damage was promoted by Man-SOD. Thus, the present study demonstrated that chemical modification may improve the therapeutic effect of SOD on the ischemic acute renal failure and increased SOD concentration in the renal vascular space is an important factor for the improved effect.

Enantioselective tissue distribution of the basic drugs disopyramide, flecainide and verapamil in rats : Role of plasma protein and tissue phosphatidylserine binding

AbstractPurpose. The stereoselective distribution of three basic drugs, disopyramide (DP), flecainide (FLC) and verapamil (VP), was studied to clarify the relationships between the tissue-to-unbound plasma concentration ratio (Kpf) and drug lipophilicity and binding to phosphatidylserine (PhS), which are possible factors determining the tissue distribution of these drug enantiomers.nMethods. The drug enantiomer or racemate was administered to rats by intravenous constant infusion. Their concentrations in plasma and tissues were determined using enantioselective high-performance liquid chromatography. Plasma protein binding, and buffer-octanol and buffer-hexane containing PhS partition coefficients were also determined.nResults. The stereoselectivity of the tissue-to-plasma concentration ratio (Kp) was partly associated with that of serum protein binding. However, the Kpf value of R( + )-VP in the lung was significantly higher than that of S(−)-VP. A linear correlation was observed between the Kpf values of these drug enantiomers in brain, heart, lung and muscle, and their buffer-hexane containing PhS partition coefficients. The in vitrodata for the binding of these drugs to PhS suggest that stereoselective binding of VP to PhS may correspond to its stereoselective tissue binding.nConclusions. Our findings provide some evidence for a role of tissue PhS in the tissue distribution of basic drugs with respect to stereoselectivity of drug enantiomers distribution.

Disposition Characteristics of Protein Drugs in the Perfused Rat Kidney

The renal disposition characteristics of 111In-labeled neocarzinostatin (NCS), soybean trypsin inhibitor (STI), and superoxide dismutase (SOD) were studied in the perfused rat kidney. In a single-pass indicator dilution experiment, venous and urinary recovery profiles and tissue accumulation of proteins were determined under filtering or nonfiltering conditions. In the nonfiltering kidney perfusion experiment, no significant tissue accumulation was observed, suggesting minimal uptake from the glomerular and peritubular capillary sides. Therefore, tissue recovery corresponded to that with tubular reabsorption after glomerular filtration. The total amount of NCS or STI being filtrated through glomeruli, the sum of tissue and urinary recoveries, was similar to that of inulin, but that of SOD was about half. Similarly, the steady-state distribution volumes (Vd) of NCS and STI obtained by moment analysis of their venous outflow curves were similar to that of inulin, while the Vd value of SOD was significantly lower. These results suggest the restricted passage of SOD through the glomerular and postglomerular capillary wall. The tubular reabsorption ratio of proteins against the total filtrated amount decreased with an increase in the administered dose, suggesting nonlinearity of reabsorption. SOD had the largest reabsorption ratio. Thus, this experimental system is useful for quantitative analysis of renal disposition of proteins.

Control of the disposition profiles of proteins in the kidney via chemical modification

To construct the strategy to control the renal disposition profiles of protein drugs by chemical modification, studies were performed using the perfused rat kidney. Renal disposition processes, i.e., glomerular filtration, tubular reabsorption, and uptake from the vascular side, were quantitatively determined by single-pass indicator dilution experiments under filtering and non-filtering conditions. As the first step, the renal disposition characteristics of model protein drugs and macromolecules were evaluated. These studies clarified the relationship between physicochemical properties of macromolecules, such as molecular weight and electric charge, and their fate in the kidney in a quantitative manner. Based on these findings, an antioxidant enzyme, Superoxide dismutase (SOD), selected as a therapeutic agent for various tissue injuries including renal failure mediated by reactive oxygen species, was chemically modified. Conjugation with macromolecules, polyethylene glycol and carboxymethyl dextran, decreased glomerular filtration of SOD. Cationization enabled the enzyme to distribute to the kidney from the capillary side and to be completely reabsorbed by the tubular epithelium after glomerular filtration based on electrostatic interaction. On the other hand, glycosylation with monosaccharides, galactose and mannose, significantly reduced its tubular reabsorption and enhanced its exposure to the luminal surface. Furthermore, the mannosylated derivative accumulated in the kidney from the vascular side via a mannose-recognition mechanism. Thus, the present study demonstrates that chemical modification is useful for the control of renal disposition characteristics of protein drugs.

Stereoselective First-Pass Metabolism of Verapamil in the Small Intestine and Liver in Rats

Verapamil (VP) is used as a racemate but shows stereoselective pharmacokinetics and pharmacodynamics. It undergoes extensive first-pass metabolism. Stereoselective first-pass metabolism in the intestine and liver was investigated in vivo and in vitro to determine its impact on the disposition of VP and its main metabolite, norverapamil (NVP). VP racemate was administered to rats i.v., p.o., and via the portal vein. The formation rates of the main metabolites of the VP enantiomers were estimated in an in vitro intestinal microsomal study. The hepatic bioavailability of VP showed saturable metabolism, and the hepatic bioavailability of R-VP was higher than that of S-VP. Conversely, the intestinal bioavailability of R-VP was lower than that of S-VP, resulting in a higher systemic bioavailability of S-VP. The pharmacokinetics of the NVP enantiomers was similar. These results suggest that the stereoselectivity of the total bioavailability of VP is determined by first-pass metabolism in the small intestine and liver, and that the NVP enantiomers observed in the systemic circulation after p.o. administration of VP racemate originate mainly from the liver in rats.

Pharmacokinetics and preventive effects of targeted catalase derivatives on hydrogen peroxide-induced injury in perfused rat liver.

AbstractPurpose. To investigate the pharmacokinetics and preventive effects of liver-targeted catalase (CAT) derivatives on hepatic injury caused by reactive oxygen species.nMethods. The hepatic uptake of 111In-CAT, galactosylated (Gal-), mannosylated (Man-) and succinylated (Suc-) CAT was investigated in isolated perfused rat livers in a single-pass constant infusion mode. Then, pharmacokinetic parameters were obtained by fitting equations derived from a one-organ pharmacokinetic model to the outflow profile. Their effects in preventing hydrogen peroxide-induced injury were determined by lactate dehydrogenase (LDH) release from the perfused liver.nResults. The extraction of CAT derivatives by the liver was dose-dependent, and increased by the chemical modifications described. After being bound to the cell surface, chemically modified CAT derivatives were internalized by the liver faster than CAT. Preperfusion of a CAT derivative significantly reduced LDH release by hydrogen peroxide at least for 30 min, and Man-CAT and Suc-CAT effectively inhibited this release.nConclusions. Internalized CAT derivatives are also effective in degrading hydrogen peroxide and targeted delivery of CAT to liver nonparenchymal cells by mannosylation or succinylation is a useful method for the prevention of hepatic injury caused by reactive oxygen species.

Evaluation of Bayesian estimation of pharmacokinetic parameters

The validity of pharmacokinetic parameters estimated by the maximum a posteriori probability (MAP) Bayesian method was investigated by simulation studies. A 1-compartment model with bolus intravenous administration was used as a pharmacokinetic model, and the coefficients of variation for the parameters and residual error were set at 30% and 10%, respectively. The accuracy of the posterior modes of pharmacokinetic parameters estimated by the MAP Bayesian method was assessed by the difference between the true value and the estimated value. The results showed that the accuracy of the Bayesian estimation depended on sampling times and on the differences between the prior means and individual true parameter values. For assessing the reliability and accuracy of the Bayesian estimation, the authors suggest using the whole posterior distribution of the pharmacokinetic parameters to describe the 95th percentile range for predicted blood concentration profiles. The authors believe that the proposed procedures provide helpful information for evaluating the Bayesian estimation of pharmacokinetic profiles.