Mitsuru Hashida

Synthesis and Pharmacokinetics of a New Liver-Specific Carrier, Glycosylated Carboxymethyl-Dextran, and Its Application to Drug Targeting

To develop a new carrier system for hepatic targeting, carboxymethyl-dextran (CMD) was modified with galactose and mannose residues (Gal-CMD, Man-CMD), and their disposition characteristics were studied in mice using 14C-labeled dextran. At a dose of 1 mg/kg, i.v.-injected Gal-CMD and Man-CMD rapidly accumulated in the liver parenchymal and nonparenchymal cells, respectively, because of their preferential uptake via carbohydrate receptors in these cells. Pharmacokinetic analysis revealed that their uptake rates were sufficiently large for selective drug targeting. Targeting of cytosine β-D-arabinoside (araC) was studied using Gal-CMD as a specific carrier to the hepatocytes. From the conjugate of araC with Gal-CMD, araC was released with a half-life of 36 hr in phosphate buffer (pH 7.4) and 23 hr in plasma. An in vivo biodistribution study demonstrated a disposition profile of the conjugated araC similar to that of the carrier, and selective delivery to hepatocytes of up to 80% of the dose was achieved. These findings suggest that glycosylated CMDs are carriers with a high affinity to liver parenchymal or nonparenchymal cells without any affinity to other tissues.

In Vivo and in Vitro Analysis of Skin Penetration Enhancement Based on a Two-Layer Diffusion Model with Polar and Nonpolar Routes in the Stratum Corneum

In vitro and in vivo skin penetration of three drugs with different lipophilicities and the enhancing effects of l-geranylazacycloheptan-2-one (GACH) were studied in rats. In vivo drug absorption profiles obtained by deconvolution of urinary excretion profiles were compared to the corresponding in vitro data obtained with a diffusion experiment. In vivo skin penetration of lipophilic butylparaben was considerably greater than that observed in vitro, while hydrophilic mannitol and acyclovir showed low penetration in both systems without GACH pretreatment. On the other hand, GACH enhanced mannitol and acyclovir penetration, especially in the in vivo system. Analysis of absorption profiles, using a two-layer skin model with polar and nonpolar routes in the stratum corneum, suggested that the diffusion length of a viable layer (viable epidermis and dermis) was shorter in vivo than in vitro and the effective area of the polar route in the stratum corneum was larger in vitro without GACH pretreatment. GACH increased the partitioning of acyclovir into the nonpolar route to the same extent in both systems. In addition, GACH increased the effective area of the polar route in vivo, probably because of enhanced water permeability; however, this effect was smaller in vitro since the stratum corneum was already hydrated even without GACH pretreatment.

Comparative Analysis of Percutaneous Absorption Enhancement by d-Limonene and Oleic Acid Based on a Skin Diffusion Model

Percutaneous absorption-enhancing effects of d-limonene and oleic acid were investigated using three model drugs with different lipophilicities in in vitro diffusion experiments with guinea pig skin. Pretreatment of the skin with d-limonene resulted in a large penetration enhancement for the lipophilic butylparaben (BP) and amphiphilic 6-mercaptopurine (6-MP) but had little effect on the hydrophilic mannitol (MT). Oleic acid caused a large effect only on 6-MP penetration. The penetration profiles were analyzed with a two-layer skin diffusion model consisting of stratum corneum with polar and nonpolar routes and viable epidermis plus dermis. Through curve-fitting, six parameters corresponding to drug diffusivity and partitioning in these three regions of the skin were obtained, and the mechanisms of enhancers were assessed in comparison with those of l-geranylazacycloheptan-2-one (GACH) reported previously. Increased penetration was caused mainly by modification of the barrier property of the nonpolar route in the stratum corneum in all cases. In the nonpolar route, d-limonene increased mainly drug diffusivity, while GACH enhanced predominately drug partitioning. On the other hand, oleic acid moderately increased both parameters.

Targeted delivery of superoxide dismutase to macrophages via mannose receptor-mediated mechanism

Human recombinant superoxide dismutase (SOD) was modified into a mannosylated form (Man-SOD), and its cellular uptake and inhibitory effect on superoxide anion release were studied in vitro, using cultured mouse peritoneal macrophages. [111In]Man-SOD was taken up by the macrophages to a great extent, whereas no significant uptake was observed for native and galactosylated SOD. The uptake of Man-SOD was inhibited significantly at a low temperature and by the presence of mannan, mannose and colchicine, demonstrating the targeted delivery of Man-SOD via mannose receptor-mediated endocytosis. Man-SOD exhibited a superior inhibitory effect on superoxide anion release from inflammatory macrophages stimulated by phorbol-myristate acetate. The present study suggested the potential of Man-SOD as a therapeutic agent for the inflammatory disease mediated by superoxide anions generated by macrophages.

Control of In Vivo Fate of Albumin Derivatives Utilizing Combined Chemical Modification

Three types of bovine serum albumin (BSA) derivatives such as lactosylated BSA (LBSA), mannosylated BSA (Man-BSA), and cationized BSA (cBSA) were synthesized and their hepatic disposition characteristics in mice were evaluated by pharmacokinetic analysis. At lower doses (< or = 1 mg/kg), LBSA and Man-BSA were very rapidly eliminated from the blood circulation due to uptake by parenchymal and nonparenchymal cells of the liver, respectively, via receptor-mediated endocytosis (Nishikawa et al., 1992; Nishida et al., 1991a, b). These uptake processes were nonlinear and the apparent hepatic uptake clearances (CLliver) were decreased at administered doses higher than 1 mg/kg, e.g. 10, 20, and 100 mg/kg. The liver accumulation of cBSA was also nonlinear, but its binding and/or uptake capacity in the liver was larger than those of LBSA and Man-BSA; i.e., CLliver decreased at doses higher than 20 mg/kg. In the next step, we modified these BSA derivatives by attaching polyethylene glycol (PEG), a modifier known to reduce the hepatic uptake and increase plasma retention, to achieve precise control of the in vivo disposition characteristics of BSA derivatives. By conjugation with PEG having a molecular weight of 10 kDa, the CLliver values of LBSA, Man-BSA, and cBSA were decreasing to one-seventh, one-fortyfifth, and one-onehundredthirtieth, respectively. However, liver accumulation of PEG modified LBSA and Man-BSA at 24 h after i.v. injection was not significantly different from unmodified BSA derivatives. These results suggest that it is possible to control the hepatic uptake of protein drugs by a combination of introduction of charge or sugar moieties and PEG conjugation.

Pharmacokinetics in design of polymeric drug delivery systems

Abstract In this paper, the utilities of the clearance concept-based pharmacokinetic analysis to study systematic disposition characteristics and targeting efficacy of various macromolecular drug carriers are demonstrated. The relationships between physicochemical and biological properties and uptake clearance values of macromolecules in various organs are discussed for designing a targeting system employing them. Based on the information about the effects of molecular size and electric charges on in vivo disposition of model macromolecules, the passive and active targeting system for an anticancer agent to a tumor is developed. Targeting efficacy of carrier systems utilizing carbohydrate-recognition mechanism were also quantitatively characterized in the same way. Thus, pharmacokinetic analysis was demonstrated to make a significant contribution in the rational design of a targeting system.

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.

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.

Targeting delivery of protein drugs by chemical modification

AbstractIn vivo disposition profiles of protein derivatives having various chemical modifications were systematically compared in mice based on the clearance concept. Proteins such as bovine γ-globulin (IgG), bovine serum albumin (BSA), superoxide dismutase (SOD), soybean trypsin inhibitor (STI), and chicken egg white lysozyme (LZM) were l)conjugated with polyethylene glycol (PEG) and dextran to increase molecular size, 2) conjugated with carboxymethyl-dextran (CMD) and diethylaminoethyl-dextran (DEAED) or coupled with diaminohexane or succinic acid to introduce electric charges, and 3) modified with galactose (Gal) and mannose (Man) moieties to bestow an affinity for receptor-mediated endocytosis in cells. By applying these modifications, in vivo disposition features of proteins were extensively changed; i.e., in the case of SOD, conjugation with CMD and PEG prolonged its circulation half-life more than 100 times but cationized SOD showed remarkable accumulation on the surface of the liver tissue. In add...