Isao Adachi

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.

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.

α-1-C-butyl-1,4-dideoxy-1,4-imino-l-arabinitol as a second-generation iminosugar-based oral α-glucosidase inhibitor for improving postprandial hyperglycemia.

We report on the synthesis and the biological evaluation of a series of α-1-C-alkylated 1,4-dideoxy-1,4-imino-l-arabinitol (LAB) derivatives. The asymmetric synthesis of the derivatives was achieved by asymmetric allylic alkylation, ring-closing metathesis, and Negishi cross-coupling as key reactions. α-1-C-Butyl-LAB is a potent inhibitor of intestinal maltase, isomaltase, and sucrase, with IC50 values of 0.13, 4.7, and 0.032 μM, respectively. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis revealed that this compound differs from miglitol in that it does not influence oligosaccharide processing and the maturation of glycoproteins. A molecular docking study of maltase-glucoamylase suggested that the interaction modes and the orientations of α-1-C-butyl-LAB and miglitol are clearly different. Furthermore, α-1-C-butyl-LAB strongly suppressed postprandial hyperglycemia at an early phase, similar to miglitol in vivo. It is noteworthy that the effective dose was about 10-fold lower than that for miglitol. α-1-C-Butyl-LAB therefore represents a new class of promising compounds that can improve postprandial hyperglycemia.

Inhibitory Effects of Terpenoids on Multidrug Resistance-Associated Protein 2-and Breast Cancer Resistance Protein-Mediated Transport

The possibility of interactions between natural products/supplements and conventional prescription medicines is one of the most important issues in pharmacotherapeutic safety. Recently, we reported that some terpenoids such as (R)-(+)-citronellal and glycyrrhetic acid, which are present in herbal medicines, can act as inhibitors of P-glycoprotein (MDR1/ABCB1). In the present study, the effects of seven terpenoids on multidrug resistance-associated protein 2 (MRP2/ABCC2) and breast cancer resistance protein (BCRP/ABCG2)-mediated transport were investigated in vitro. Membrane vesicles were prepared from MRP2 cDNA transfected Sf9 cells derived from pupal ovarian tissue of Spodoptera frugiperda, a fall armyworm, and BCRP cDNA transfected LLC-PK1 cells derived from porcine kidney. MRP2- or BCRP-mediated efflux transport was measured as ATP-dependent accumulation of [3H]estradiol 17-β-d-glucuronide (E217βG) into membrane vesicles collected by a rapid filtration technique. The effects of (R)-(+)-citronellal, (S)-(-)-β-citronellol, α-terpinene, terpinolene, (-)-β-pinene, abietic acid, and glycyrrhetic acid on the intravesicular accumulation of [3H]E217βG were examined. Large decreases in the [3H]E217βG accumulation into vesicles from MRP2-overexpressing Sf9 cells were observed in the presence of glycyrrhetic acid and abietic acid, and their IC50 values were about 20 and 51 μM, respectively. [3H]E217βG accumulation into vesicles from BCRP-overexpressing LLC-PK1 cells was suppressed by only glycyrrhetic acid, with an IC50 value of about 39 μM. Other terpenoids used in this study did not alter the ATP-dependent accumulation of [3H]E217βG. These findings suggest that glycyrrhetic acid and abietic acid can potently inhibit MRP2- or BCRP-mediated membrane transport and may interact with their substrates in pharmacokinetic processes.

Nine of 16 Stereoisomeric Polyhydroxylated Proline Amides Are Potent β‑N‑Acetylhexosaminidase Inhibitors

All 16 stereoisomeric N-methyl 5-(hydroxymethyl)-3,4-dihydroxyproline amides have been synthesized from lactones accessible from the enantiomers of glucuronolactone. Nine stereoisomers, including all eight with a (3R)-hydroxyl configuration, are low to submicromolar inhibitors of β-N-acetylhexosaminidases. A structural correlation between the proline amides is found with the ADMDP-acetamide analogues bearing an acetamidomethylpyrrolidine motif. The proline amides are generally more potent than their ADMDP-acetamide equivalents. β-N-Acetylhexosaminidase inhibition by an azetidine ADMDP-acetamide analogue is compared to an azetidine carboxylic acid amide. None of the amides are good α-N-acetylgalactosaminidase inhibitors.

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.

3-Fluoroazetidinecarboxylic Acids and trans,trans-3,4-Difluoroproline as Peptide Scaffolds: Inhibition of Pancreatic Cancer Cell Growth by a Fluoroazetidine Iminosugar

Reverse aldol opening renders amides of 3-hydroxyazetidinecarboxylic acids (3-OH-Aze) unstable above pH 8. Aze, found in sugar beet, is mis-incorporated for proline in peptides in humans and is associated with multiple sclerosis and teratogenesis. Aze-containing peptides may be oxygenated by prolyl hydroxylases resulting in potential damage of the protein by a reverse aldol of the hydroxyazetidine; this, rather than changes in conformation, may account for the deleterious effects of Aze. This paper describes the synthesis of 3-fluoro-Aze amino acids as hydroxy-Aze analogues which are not susceptible to aldol cleavage. 4-(Azidomethyl)-3-fluoro-Aze and 3,4-difluoroproline are new peptide building blocks. trans,trans-2,4-Dihydroxy-3-fluoroazetidine, an iminosugar, inhibits the growth of pancreatic cancer cells to a similar degree as gemcitabine.

Protective Effects of Dietary 1,5-Anhydro-d-glucitol as a Blood Glucose Regulator in Diabetes and Metabolic Syndrome

1,5-Anhydro-D-glucitol (1,5-AG) is fairly widespread in food products. It is also one of the major polyols in the human body, and its concentration is homeostatically regulated. We report here on the beneficial effects of 1,5-AG in preventing hyperglycemia and its role in improving metabolic syndrome. The findings revealed that it does not affect blood glucose levels itself under normal conditions but clearly has a suppressive effect on the levels of dietary sugars, such as glucose, maltose, and sucrose. A long-term administration study revealed that feeding db/db diabetic mice 3% 1,5-AG for 8 weeks significantly decreased blood glucose levels compared to untreated mice (339 ± 30 versus 438 ± 34 mg/dL; p < 0.05). Furthermore, this treatment also significantly suppressed serum cholesterol levels (110.2 ± 18.0 versus 168.4 ± 9.8 mg/dL; p < 0.01). 1,5-AG did not inhibit intestinal α-glucosidase activities but regulated liver glucose levels via affecting both the glycogenolysis and gluconeogenesis pathways. Furthermore, the oral administration of 1,5-AG significantly increased urinary glucose excretion in hyperglycemic conditions. These results clearly suggest that dietary 1,5-AG acts as a modulator of glucose levels in hyperglycemia. 1,5-AG therefore represents a new class of promising functional sweeteners, where the daily consumption of 1,5-AG with meals could inhibit the progress of hyperglycemia and metabolic syndrome.