Yoshihiko Tauchi

Efficient drug delivery to atherosclerotic lesions and the antiatherosclerotic effect by dexamethasone incorporated into liposomes in atherogenic mice

In order to confirm the efficacy of dexamethasone (DXM) incorporated into liposomes (DXM-liposomes) on atherosclerosis, drug delivery to atherosclerotic lesions and the antiatherosclerotic effect by DXM-liposomes were investigated in atherogenic mice. DXM-liposomes were prepared with egg yolk phosphatidylcholine, cholesterol and dicetylphosphate in a lipid molar ratio of 7/2/1 by the hydration method and then adjusted to three different particle sizes to clarify the influence of particle size on the drug delivery to atherosclerotic lesions and the effect on atherosclerosis. The particle sizes of DXM-liposomes were 519 nm (L500), 202 nm (L200) and 68.6 nm (L70), respectively. In both size, DXM concentration and DXM/lipid molar ratio in DXM-liposomes suspension were 1 mg DXM/ml and 0.134 mol DXM/mol total lipids, respectively. Atherogenic mice used as an experimental model develop an atherosclerotic lesion in the aorta and they were prepared by feeding an atherogenic diet for 14 weeks. The aortic pharmacokinetics of DXM-liposomes was examined by intravenous administration to atherogenic mice. The aortic uptake clearance of DXM in atherogenic mice treated with L200 was 2.6–3.2 fold greater than that in animals treated with L500, L70 or free DXM (f-DXM). Furthermore, the effects of DXM-liposomes on atherosclerosis were examined by intravenous administration to atherogenic mice once a week from 8 to 14 weeks. The antiatherosclerotic effects of DXM-liposomes were evaluated by determination of the aortic cholesterol ester (CE) level. The aortic CE level in atherogenic mice treated with L200 (55 μg DXM/kg) was significantly lower than that in animals treated with PBS. The antiatherosclerotic effect of L200 (55 μg DXM/kg) was significantly more potent than that of f-DXM (550 μg DXM/kg). These findings suggest that efficient delivery of DXM to the atherosclerotic lesions by L200 induces an excellent antiatherosclerotic effect at a lower dose. Therefore, L200 may be useful in the development of drug delivery systems for atherosclerotic therapy.

Determination of lipophilicity of two quinolone antibacterials, ciprofloxacin and grepafloxacin, in the protonation equilibrium.

The objective of this study was to compare protonation equilibrium and lipophilicity of two quinolone antibacterials, grepafloxacin (GPFX) and ciprofloxacin (CPFX), in order to give an insight into effects on the physicochemical properties by slight structural motifs. The protonation equilibrium was investigated by a spectrophotometry. Macro- and micro-dissociation constants were simultaneously determined, based on nonlinear regression analysis using the MULTI program, and then microspecies distribution could be described accordingly. Zwitterionic microspecies predominated at isoelectrical point (pI) for both drugs, and the concentration ratio of neutral to zwitterionic forms was near 4-fold greater for GPFX than that for CPFX. The apparent partition coefficient (D(O/B,pH)) versus pH profiles had the shape of a parabolic curve in an n-octanol/buffer system, and reached the maximum around pI for both, respectively. Moreover, two introduced methyl groups in GPFX increased not only intrinsic lipophilicity but also neutral microspecies fraction relative to CPFX, and D(O/B,pH) of GPFX was consequently far higher than that of CPFX. The results emphasized that there were significant differences in protonation equilibrium and lipophilicity between GPFX and CPFX, which conduced to explaining their different behavior in terms of antibacterial activities and pharmacokinetics.

Influence of Particle Size on the Distributions of Liposomes to Atherosclerotic Lesions in Mice

ABSTRACT In order to confirm the efficacy of liposomes as a drug carrier for atherosclerotic therapy, the influence of particle size on the distribution of liposomes to atherosclerotic lesions in mice was investigated. In brief, liposomes of three different particle sizes (500, 200, and 70 nm) were prepared, and the uptake of liposomes by the macrophages and foam cells in vitro and the biodistributions of liposomes administered intravenously to atherogenic mice in vivo were examined. The uptake by the macrophages and foam cells increased with the increase in particle size. Although the elimination rate from the blood circulation and the hepatic and splenic distribution increased with the increase in particle size in atherogenic mice, the aortic distribution was independent of the particle size. The aortic distribution of 200 nm liposomes was the highest in comparison with the other sizes. Surprisingly, the aortic distribution of liposomes in vivo did not correspond with the uptake by macrophages and foam cells in vitro. These results suggest that there is an optimal size for the distribution of liposomes to atherosclerotic lesions.

Protonation equilibrium and lipophilicity of olamufloxacin (HSR-903), a newly synthesized fluoroquinolone antibacterial.

This study was performed to characterize the protonation equilibrium at the molecular level and pH-dependent lipophilicity of olamufloxacin. The deprotonation fraction of the carboxyl group as a function of pH was specifically calculated at the critical wavelength 294 nm, where UV pH-dependent absorbance of olamufloxacin was independent of the ionized state of the aminopyrrolidinyl amino group but heavily depended on that of the carboxyl moiety. Accordingly, micro-protonation equilibrium could be described using a nonlinear least-squares regression program MULTI. In contrast, macro-protonation equilibrium was depicted at most wavelengths where olamufloxacin absorbance was influenced by ionized states of both proton-binding groups, results coinciding with the former. Furthermore, distribution features of four microspecies in aqueous phase were assessed. The apparent partition coefficient versus pH profile of olamufloxacin showed a parabolic curve in n-octanol/buffer system which reached peak near pH 8, agreeing with the above determined isoelectric point (pI). Ion-pair effect was observed for olamufloxacin under an acidic condition, eliciting experimental values higher than those theoretically calculated, which was similar to ciprofloxacin but not levofloxacin due to amino group type. Moreover, olamufloxacin was moderately lipophilic in comparison with other quinolones, with an apparent partition coefficient of 1.95 at pH 7.4.

Aortic drug delivery of dexamethasone palmitate incorporated into lipid microspheres and its antiatherosclerotic effect in atherogenic mice

In order to confirm the efficacy of dexamethasone (DXM) palmitate incorporated into lipid microspheres (d-lipo) on atherosclerosis, the aortic drug delivery by d-lipo and its antiatherosclerotic effect were investigated. In an in vitro uptake experiment, d-lipo or DXM was added to macrophages and foam cells, and then incubated for 1, 4, 8 and 24 h at 37°C. The uptake of drug by these cells after addition of d-lipo was higher than that of DXM at each time point. In an in vitro pharmacological experiment, the macrophages and foam cells were incubated with d-lipo or DXM for 24 h at 37°C. The inhibitory effect of d-lipo on cellular cholesterol ester (CE) accumulation in these cells was significantly more potent than that of DXM. In an in vivo pharmacokinetic experiment, d-lipo or DXM was intravenously administered to atherogenic mice, and then aorta was collected at 1, 8, and 24 h after administration. The aortic drug concentration after administration of d-lipo to atherogenic mice was higher than that of DXM at each time point. In an in vivo pharmacological experiment, d-lipo or DXM was intravenously administered to atherogenic mice once a week for 7 weeks. The inhibitory effect of d-lipo on the aortic CE accumulation in atherogenic mice was significantly more potent than that of DXM. These findings suggest that efficient drug delivery to the atherosclerotic lesions by d-lipo produces an excellent antiatherosclerotic effect at a lower dose. Therefore, d-lipo may be useful for the development of drug delivery systems for atherosclerotic therapy.

Cellular Uptake of a Dexamethasone Palmitate-low Density Lipoprotein Complex by Macrophages and Foam Cells

To evaluate the utility of a dexamethasone palmitate (DP)-low density lipoprotein (LDL) complex to transport drug into foam cells, the cellular uptake of DP-LDL complex by macrophages and foam cells was examined. The DP-LDL complex was prepared by incubation with DP and LDL, and the DP-LDL complex and murine macrophages were incubated. No cellular uptake of the DP-LDL complex by macrophages was found until 6 h after the start of incubation, but this gradually increased from 12 to 48 h. On the other hand, the cellular uptake of the oxidized DP-LDL complex was already apparent at 3 h after the start incubation, and then markedly increased until 48 h incubation along with that of the lipid emulsion (LE) containing DP (DP-LE). The cellular uptake of DP-LE by foam cells was significantly lower than that by macrophages. However, the cellular uptake of DP-LDL complex by foam cells was similar to that by macrophages. These findings suggest that the DP-LDL complex is oxidatively modified, and then incorporated into macrophages and foam cells through the scavenger receptor pathway. Since selective delivery of drugs into foam cells in the early stage of atherosclerosis is a useful protocol for antiatherosclerosis treatment, the DP-LDL complex appears to be a potentially useful drug-carrier complex for future antiatherosclerotic therapy.