Isamu Horikoshi

Preparation and characterization of dextran magnetite-incorporated thermosensitive liposomes: an on-line flow system for quantifying magnetic responsiveness.

AbstractPurpose. Dextran magnetite (DM)-incorporated thermosensitive liposomes, namely thermosensitive magnetoliposomes (TMs), were prepared and characterized in order to investigate their possibility for magnetic drug targeting. Methods. TMs containing calcein were prepared at various DM concentrations by reverse-phase evaporation of dipalmitoylphosphatidylcholine (DPPC). They were evaluated for their physicochemical properties including size, DM capture, magnetite distribution within liposomes, and temperature-dependent calcein release. Moreover, a novel on-line flow apparatus with a sample injector, a coil of tubing placed in an electromagnet, and a fluorescence detector was developed for quantifying the magnetic responsiveness of TMs. This device allowed us a real-time measurement of percentage holding of TMs by magnetic field. Results. Due to water-soluble property of DM, higher contents of magnetite up to 490 mg per mmol DPPC were successfully incorporated into the liposomes with DM than with conventional magnetite (Fe3O4). Thermosensitivity and lipid integrity of TMs were not influenced by inclusion of DM. Using the on-line flow system, percentage holding of TMs by magnetic field was shown to vary with several factors; it increases as the magnetic field strength increases, the fluid flow rate decreases, the magnetite content increases, and the liposome concentration increases. Typically, at 490 mg incorporated magnetite per mmol DPPC, 0.5 ml/min-fluid flow rate, and high magnetic field strength (≥10 kiloGauss), approximately 100% of TMs were found to be held. Conclusions. The TMs were suggested to be useful in future cancer treatment by magnetic targeting combined with drug release in response to hyperthermia.

In vitro and in vivo evaluation of taxol release from poly(lactic-co-glycolic acid) microspheres containing isopropyl myristate and degradation of the microspheres

Abstract This study describes the release and degradation profiles of taxol-loaded poly(lactic-co-glycolic acid) microspheres containing isopropyl myristate (IPM), namely, Taxol-IPM-PLGA-MS, in vitro and in vivo. Incorporation of IPM into the PLGA microspheres effectively increased the release of taxol from the microspheres in vitro. The molecular weight (Mol. Wt.) and copolymer ratio of lactic acid (LA) and glycolic acid (GA) displayed obvious influences on the characteristics of the microspheres and their in vitro release kinetics. A large number of micropores could be observed on the surface of the microspheres and the number of these pores tended to increase with the increment of Mol. Wt. and LA content of PLGA. This trend was in accordance with the rate of drug release from the microspheres in vitro. Moreover, the degradation of PLGA matrix in vitro was retarded by the incorporation of IPM to a less extent compared with its effect on the drug release. Therefore, the drug diffusion in the matrix was shown to be an important factor dominating the release rate, while the degradation rate of the polymer is a moderate factor in this case. Consistent with the in vitro results, the taxol concentrations in the lung after intravenous injection of the Taxol-IPM-PLGA-MS were 2–4 times higher over one week, as compared with those after injection of the microspheres without IPM. In conclusion, the Taxol-IPM-PLGA-MS may have great advantage in cancer chemotherapy after targeted delivery to a tumor site by chemo-embolization.

Microdialysis assessment of microfibrous collagen containing a P-glycoprotein-mediated transport inhibitor, cyclosporine A, for local delivery of etoposide.

AbstractPurpose. This study was designed to assess a local drug delivery system of an anticancer agent, etoposide (VP-16), using microfibrous collagen as a drug carrier. For this objective, the microdialysis method was utilized to investigate the local pharmacokinetics of VP-16. Methods. Microfibrous collagen sheets (CS) containing 20 mg/kg of VP-16 with and without 40 mg/kg of cyclosporine A (CyA) were prepared and applied on the liver surface of rats. VP-16 concentrations in the liver extracellular fluid (ECF) were monitored by a microdialysis method. Results. The local application of CS containing VP-16 resulted in a relatively long maintenance of drug concentrations in the liver ECF, with very low concentrations in plasma. The inclusion of CyA in the CS resulted in 2-fold and 3-fold increases of the AUC and MRT values of VP-16 in the liver ECF, respectively. The liver ECF-to-plasma AUC ratios of VP-16 were 32–39 and 0.17 with local CS application and iv administration, respectively, indicating a remarkable advantage of the local drug delivery system. A pharmacokinetic interaction experiment suggested that the observed increase of the liver ECF concentrations of VP-16 with CyA resulted from inhibition of the biliary excretion of VP-16 by CyA. Conclusions. We found that the local delivery of the CS containing CyA on the liver surface is advantageous in terms of the extent and duration of liver ECF drug concentrations, when CyA was included in the CS. The effect of CyA was probably derived from the inhibition of P-glycoprotein-mediated biliary excretion of VP-16 by CyA. The usefulness of the microdialysis technique for the assessment of the local drug delivery system was also demonstrated.

Magnetic targeting of thermosensittve magnetoliposomes to mouse livers in an in situ on-line perfusion system

We recently reported the preparation and in vitro targeting of dextran magnetite (DM)-incorporated thermosensitive liposomes, namely thermosensitive magnetoliposomes (TMs) [Viroonchatapan et al. Pharm. Res. 12 1176-1183 (1995)]. The current study was designed to determine whether these novel liposomes can be targeted to the mouse liver with the aid of an extracorporeal magnet. An on-line liver perfusion system consisting primarily of a sample injector, permanent magnets, and a fluorescence detector was established for a real-time measurement of targeting efficiency of TMs containing calcein as a fluorescent marker. Normal and reticuloendothelial system (RES)-blocked livers from mice were used for the perfusion experiments. In the RES-blocked livers, percentage holdings of TMs were 73-80% and 26-45% in the presence and absence of magnetic field, respectively, indicating an efficient targeting of TMs with a targeting advantage index (TAI) of 1.6-3.1. On the other hand, TAI in the normal livers was found to be 1.1-1.4 and less than that in the RES-blocked livers, suggesting a role of RES uptake of TMs. The effects of DM concentrations in TM suspensions on the percentage holding of TMs were shown to be minor. Liposome concentration dependence was observed for hepatic uptake of TMs, possibly because of the saturation of phagocytosis by Kupffer cells. The present results suggest that TMs would be useful in future cancer treatment by magnetic targeting combined with drug release in response to hyperthermia.

Release of 5-fluorouracil from thermosensitive magnetoliposomes induced by an electromagnetic field

Abstract Thermosensitive magnetoliposomes (TMs) have been proposed by the authors as a new drug carrier for magnetic targeting (Viroonchatapan et al., Pharm. Res. 12 (1995) 1176–1183; Viroonchatapan et al., Life Sci. 58 (1996) 2251–2261). The present study was carried out to investigate the properties of selective heating and thermosensitive drug release of TMs caused by an electromagnetic field. TMs containing 5-fluorouracil (5-FU) were prepared by reverse-phase evaporation. They were selectively heated by a 500-kHz electromagnetic field in a temperature-controlling unit kept at 37°C, and the properties of single and multiple release of 5-FU from TMs were examined. The results showed that the temperature of TMs could be effectively elevated to 42°C and maintained at this temperature against a cooling effect of the temperature-controlling unit, which mimics an in vivo situation where temperature rise in the site of TM delivery would be hindered by blood flow and surrounding tissues. The release of 5-FU from TMs in response to electromagnetic field-generated heat was accomplished. Moreover, the drug release could be executed several times by multiple exposure to the field. In conclusion, this paper presents for the first time a demonstration of both single and multiple thermosensitive drug release from TMs, induced by an electromagnetic field. It is suggested that TMs would be useful in future cancer treatment by magnetic targeting combined with drug release in response to electromagnetic induced hyperthermia.

Importance of receptor-mediated endocytosis in peptide delivery and targeting: kinetic aspects

Abstract Receptor-mediated endocytosis (RME) is a general mechanism by which eukaryotic cells internalize peptide hormones, growth factors, cytokines, plasma glycoproteins, lysosomal enzymes, toxins and viruses concomitantly with their receptors on the cell surface. Receptor-mediated drug targeting is considered to show promise as a drug delivery system (DDS). The success of RME-utilizing drug targeting is dependent not only on the in vitro efficacy but on the in vivo behavior of the drug-polypeptide complex. In fact, there has not been much progress from the “natural” targeting of glycoproteins and LDL to hepatocytes or macrophage lineages, due to unfavorable effects on the pharmacokinetics and pharmacodynamics (PK/PD) of polypeptides. In this review, we considered the kinetic modelling of polypeptide RME in isolated or cultured cells in vitro, in perfused organs and in the whole body. Typical RME analyses are described to estimate the rate constants for polypeptide-receptor internalization, the degradation of internalized polypeptide and receptor externalization, based upon the “receptor recycling” model. The kinetic RME analysis enables not only the non-linear clearance and distribution in the liver to be predicted, but also the surface-bound and internalized polypeptide in the target cell, which is important for the intracellular efficacy of drug-polypeptide complex. In particular, the kinetics of down-regulation and subsequent recovery of surface receptors were precisely elucidated, because if the receptors are once down-regulated, the next dosage should be given only after the surface receptors are restored. The therapeutic efficacy/toxicity of a drug-carrier conjugate should be assessed from its microscopic pharmacology based on the RME mechanisms, in conjunction with macroscopic pharmacokinetic modelling.

Mechanism of promotion of lymphatic drug absorption by milk fat globule membrane

Abstract A soybean oil emulsion containing vitamin D3, which was prepared using MFGM as an emulsifier, was administered to rats. The percentage of vitamin D 3 recovered in lymph over 12 h was 19.2%. This was reduced to 2.05% when rats were treated with colchicine, a chylomicron synthesis inhibitor, and further to only 0.27% when pancreatic ducts were ligated. When each of these MFGM micro-dispersions (micro-emulsions or mixed micelles of 40–150 nm diameter), i.e., without taurocholate (TC) or pancreatic lipase (PL); with TC alone; and with both TC and PL, was administered to pancreatic duct-ligated rats, the recovered percentages of vitamin D 3 in lymph were 3.45, 10.6 and 20.4%, respectively. These results suggest that pancreatic lipases and bile salts are critical factors for the absorption of vitamin D 3 in MFGM dosage forms and the promotive effect of MFGM takes place in the lumen of the intestine rather than the epithelial cells of the intestine. The particle morphology and physical characteristics of MFGM micro-dispersion were also analyzed by electron microscopy and electron spin resonance (ESR) spectroscopy. The results suggest that the micro-dispersion was a mixed micelle when TC was present, and as an emulsion when TC was absent. Furthermore, although the size and form of the mixed micelles with and without PL were very similar, the membrane fluidity evaluated from the ESR experiment for the micelles with PL was higher than that for the micelles without PL. This suggests that PL plays an important role in modifying micelle characteristics. It is concluded that mixed micelle formation by MFGM and bile salts in the lumen is a dominant mechanism of promotion of lymphatic drug absorption by MFGM.

Antitumor activity of selective hyperthermia in tumor-bearing rats using thermosensitive magnetoliposomes as a new hyperthermic material

AbstractThe antitumor activity of dextran magnetite (DM)-incorporating thermosensitive liposomes, namely thermosensitive magnetoliposomes (TMs), as a new hyperthermic material was examined in rats bearing AH60C tumors. TMs were injected at an iron concentration of 15 mg per cm3 tumor into AH60C tumors placed subcutaneously in the backs of Donryu rats. After injection, the whole body of the rat (treated group) was exposed to a 500-kHz electromagnetic field generated by inductive heating. The treated group was further divided into once- and twice-treated groups in order to examine whether repetitive hyperthermia is possible after a single TM injection. A control group also received a TM injection but was not treated with hyperthermia. In the heated groups, there was a marked temperature rise inside the tumor up to 42°C within 7 min, but surrounding tissues were not heated. The inhibition of the growth of AH60C tumor in the treated groups was significantly greater than in the control group (P <. 01). Histolo...

Sulphate conjugation enhances reversible binding of drug to human serum albumin.

Abstract— Reversible binding of model compounds, their conjugated metabolites (sulphates and glucuronides), and also derivatives of the compounds, to human serum albumin (HSA) has been examined using an ultrafiltration method. p‐Nitrophenol (P‐NP), α‐naphthol (α‐NA) and β‐naphthol (β‐NA) were used as model compounds. Reversible binding of 500 μm p‐NP sulphate to 4% HSA (96·6 ± 0·35%, mean ± s.d. n = 3) was significantly higher (P < 0·001), whereas reversible binding of p‐NP glucuronide to 4% HSA (33·3 ± 9·82%) was much lower (P < 0·001) than that of 500 μm p‐NP (90·9 ± 0·60%). Reversible binding of 500 μm p‐NP glucopyranoside to 4% HSA (25·8 ±2·82%) was comparable with that of the glucuronide, with which it is structurally similar. In contrast, reversible binding of 500 μm p‐NP phosphate, an anionic compound like p‐NP sulphate, to 4% HSA (61·4 ± 5·28%) was significantly lower than that of p‐NP (P < 0·001). Similar results were observed in reversible binding of sulphates of α‐NA and β‐NA. Significant differences of dissociation constants for HSA binding were observed between the parent compound (α‐ or β‐NA) and its sulphate conjugate (P < 0·005 for α‐NA and α‐NA sulphate, P < 0·001 for β‐NA and β‐NA sulphate), but the number of binding sites was the same. These results indicated that sulphate conjugation enhances reversible binding of a parent compound to HSA by increasing the binding affinity of the parent compound to HSA. This enhancement appeared to be advantageous for preventing random distribution of this metabolite to organs in the body.

Add-in macros for rapid and versatile calculation of non-compartmental pharmacokinetic parameters on Microsoft Excel spreadsheets

We developed a package of macro programs (named PK_MOMENT) to automatically calculate non-compartmental pharmacokinetic parameters on Microsoft Excel spreadsheets. These macros include rigorous algorithms to execute moment calculations in a comprehensive manner. An optimum number of terminal data points for infinite-time extrapolation can be calculated with one of these macros so that automatic calculation of infinite moment parameters is possible. The moment calculation with PK_MOMENT provided satisfactory results using the hybrid (mixed linear-logarithmic) trapezoidal method rather than the conventional linear trapezoidal method. The macro-aided pharmacokinetic analyses turned out to be useful in that the macro-containing cells can be easily copied and pasted to analyze other data sets and that powerful tools of Excel can be utilized. The use of our macros will be significantly time-saving for routine pharmacokinetic analyses, considering that pharmacokinetic data are usually stored in a spreadsheet format, typically with Excel.