We investigated the selective uptake of liposomes chemically modified by polysaccharides-cholesterol derivatives (CHP) with 1-amino-lactose (lactose CHP) in a rat hepatoma cell line (AH66), two human hepatoma cell lines (HuH7 and Alexander), a human colon cancer cell line (FCC) and a human lung cancer cell line (KNS), respectively. Uptake of ³H-labeled lactose CHP-coated liposomes was 2.9 times greater than that of CHP-coated liposomes in the AH66, 4.4 times in the HuH7, 4.7 times in the Alexander, 3, 4 times in the FCC and 4.4 times in the KNS after 3-hours of incubation. In order to clarify the mechanism of this phenomenon, we also investigated the uptake of two polymers with aminolactose or lactonamide in the AH66, the Alexander and the FCC. A human hepatoma cell line (HepG2) which is proved to have the galactose receptor was also studied. One polymer was poly(vinylbenzylaminolactose-co-vinylbenzylaminomaltose) which has a 90% induction rate of aminolactose and another was poly(vinylenzyllactonamide-co-vinylbenzylmaltonamide) which has a 90% induction rate of lactonamide. The uptake of poly(vinylbenzylaminolactose-co-vinylbenzylaminomaltose) was greater than that of poly(vinylbenzyllactonamide-co-vinylbenzylmaltonamide) in the AH66, the Alexander and the FCC, although the uptakes of them were the same in the HepG2. But, adversely the uptake of poly(vinylbenzyllactonamide-co-vinylbenzylmaltonamide) was greater than that of poly(vinylbenzylaminolactose-co-vinylbenzylaminomaltose) in hepatocytes. Uptake ratios of the two polymers into hepatocytes were higher than those of cancer cells. Our results suggest that aminolactose has a higher affinity with cancer cells than lactonamide and that lactose possibly has the higher affinities with cancer cells than galactose except for the HepG2.

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Asialofetuin-labeled liposomes (AF-liposomes) have been developed as an advantageous vector for asialoglycoprotein receptor (AgpR)-mediated gene transfer to hepatic cells. Plasmid pSV2CAT DNA which encodes bacterial chloraniphenicol acetyltransferase (CAT) was almost completely associated to AF-liposomes (AF-liposome-pSV2CAT) containing N-(α-trimethylammonio-acetyl)-didodecyl-D-glutamate chloride (TMAG), and approximately two-thirds of the associated DNA was encapsulated in the internal phase. AF-liposome-pSV2CAT was efficiently incorporated into the cultured human hepatoblastoma cell line, HepG2, by the RMF and significantly high CAT activity was expressed in the cells. The cat activity in A431 and Swiss/3T3 cells transfected with AF-liposome-pSV2CAT was low and almost the same as those transfected by pSV2CAT associated with non-labeled control liposomes. After injection of AF-liposome-pSV2CAT into a portal or femoral vein of BALB/c mice, CAT activity was expressed specifically in the liver. Immunohistochemical staining revealed that the CAT was developed in a large number of parenchymal cells localizing in the periportal area. Pretreatment of the cells or animals with EDTA-encapsulated AF-liposomes increased the gene expression efficiency of AF-liposome-pSV2CAT in vitro and in vivo. AF-liposomes are capable of protecting the encapsulated plasmid DNAs from environmental degradation in circulating blood and targeting them into hepatocytes by way of AgpR.

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We evaluated the tissue distribution and antitumor effects of freeze-dried, liposome-entrapped carboplatin (lipo-CBDCA) injected via the portal vein (ipv.) to rats bearing AH 130 tumors. The liposomes consisted of egg lecithin and cholesterol. The serum concentration of platinum (Pt) was higher following the lipo-CBDCA i.p.v than following the free CBDCA given i.p.v or i.v, at 3 and 6 hours after administration. The liver concentration of Pt was increased after administration of the liposome preparation vs. free drug. The in vivo antitumor effect of lipo-CBDCA was evaluated in rats inoculated with AH 130 tumor. Administration of free CBDCA i.p.v prolonged the survival of tumor-bearing rats. Survival was further prolonged by lipo-CBDCA vs. the control group or the group receiving free CBDCA. All rats treated with lipo-CBDCA i.p.v survived, and their liver metastases were disappeared completely. No serious effects were observed in studies of liver and kidney function. These results indicate that administration of lipo-CBDCA via the portal vein may be more effective in treating liver metastases than free CBDCA, and appeared non toxic to rats.

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The purpose of this study is to evaluate quantitatively the intracellular fate of liposomes especially their degradation process in vivo. Multilamellar liposomes were double labeled with ³H-cholesteryl hexadecyl ether (³H-CHE) and with ¹²⁵I-bovin serum albumin (¹²⁵I-BSA) and the degradation of liposomes was expressed by the ratio of intact ¹²⁵I-BSA to ³H-CHE in rat liver. The effect of liposome dose on their degradation in liver was examined after intravenous administration of liposomes at 2, 60, and 200 μmol lipid/kg. The time course of liposome degradation at low dose decreased bi-phasic, indicating at least two degradation processes. A remarkable dose dependency was also observed in the hepatic degradation of liposomes as well as in the hepatic uptake of liposomes. Two kinds of kinetic models were developed to explain these phenomena, one is “Sorting model”, having heterogenous degradation processes, the other is “Traffic-Jam model”, having heterogenous intracellular transport pathways with single degradation process (k₁). The dose-dependent liposome degradation in liver was analyzed by these two models. In a Sorting model, the effect of dose was explained by a sorting ratio, f, by which liposomes were sorted into fast and slow degradation compartments. In the Traffic-Jam model, the heterogenous intracellular transport of liposomes was explained by adding a Traffic-Jam compartment to degradation compartment. The effect of dose was reflected on the fraction of liposomes into Traffic-Jam compartment and the volume of Trafic-Jam compartment indicated by k_dj/(k₁+K_dj) and k_dj/k_jd, respectively. Since both models could fit the observed data well, model discrimination was not possible based on these study. Further studies to clarify the specific mechanism of these heterogenous intracellular traffic and/or degradation processes are required for the rational strategy in the drug delivery system using liposomes as drug carriers.

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Preparation of water-in-oil-in-water (w/o/w) emulsions containing water-soluble drugs and disposition kinetics of vancomycin after administration of an aqueous solution and w/o/w emulsions were studied. We used drug solution for an internal aqueous phase, oil mixture of Lipiodol Ultra-Fluid^® and isopropyl myristate (4.5 : 5.5) containing 5 % HCO-40 for an oily phase and saline containing 5% Pluronic^® F-88 for an external aqueous phase with a composition of 1 : 4 : 5. W/o/w emulsions encapsulating water-soluble drugs such as vancomycin hydrochloride, doxorubicin hydrochloride, cytarabine and bovine serum albumin were prepared. Among two methods of preparation, the method using a homogenizer was better for preparation of the w/o/w emulsion. The particle size of the w/o/w emulsion was smaller with an increase in rotation rate of the homogenizer(40.1 μm at 2, 500 rpm and 2.81 μm at 20, 000 rpm). The drug entrapment efficiency was increased with an increase in molecular weight of drugs and decreased with an increase in rotation rate of the homogenizer. When molecular weight and the particle size were smaller, drug release was faster. Serum concentrations of vancomycin released from w/o/w emulsions after i.v injection depended on their particle sizes. The smallest w/o/w emulsion (2.81 μm) showed the highest serum concentration and the concentrations were higher than those after administration of vancomycin solution after 45 min following injection. The emulsion was found in the blood until 120 min after injection. Moreover, when the w/o/w emulsion (41.8 μm) was injected into the portal vein, the emulsion accumulated in the liver. Considering from the serum concentration profile and the existence in the blood, the w/o/w emulsion may be expected to remain in the blood and show sustained drug release.

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Transdermal iontophoretic delivery of triamcinolane acetonide (TA) and tranilast (TL) was examined using a commercially available iontophoretic system (Phoresor, Iomed, Inc.) in hairless rats. In keloid therapy, TA in a suspension form is injected intralesionally by pressure jet apparatus, but this mode of administration frequently hurts patients. TL is administered orally, but the selective distribution of TL into the restricted skin tissues such as keloids is not expected. Iontophoretic delivery has many advantages such as non-invasive drug entry into the restricted local skin and underlying tissues. A drug electrode containing 1 ml of TA solution (10 mg/1 ml in N, N-dimethylacetamide-water (7 : 3 v/v) mixture), or 1.5 ml of TL solution (8 mg/ml in ethanol-water (8 : 2 v/v) mixture) was placed on the dorsal skin of anesthetized rats and was connected to the positive pole for TA or negative pole for TL. The current density of Phoresor was set at 4 mA for TA or 2 mA for TL, and was sent in a pulsatile manner (1 min interval). The transdermal penetration of TA and TL, evaluated by determining the amount of a drug retained in the skin tissues beneath of the drug electrode, was significantly facilitated. Both TA and TL, especially TL, were retained in the restricted skin tissues for a fairly long time (about 24 h in TA and 72 h in TL) at pharmacologically effective concentrations even after removing the drug electrode. No skin damage was found by a histological observation after a 30 min-iontophoresis. These results suggest that transdermal iontophoretic delivery of TA and TL using a mixture of organic solvent and water as a drug vehicle is an effective administration mode for the therapy of human keloids and hypertrophic scars as a beneficial substitute for intralesional injection of TA or oral administration of TL.

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Calcitonin gene related-peptide (CGRP) has been known as one of the most potent vasodilator, containing in perivascular nerves innervating arteries. We previously reported that human α-CGRP (hCGRP) could dilate the spastic cerebral arteries after experimental subarachnoid hemorrhage(SAH) in rabbits. The vasodilatory effect of hCGRP, however, did not continue up to 6 hours after treatment. In the present study, we investigated a prophylactic potential of sustained higher cerebrospinal fluid (CSF) level of hCGRP at least 5 days. For this problem, hCGRP prolonged-release tablet (hCGRP tab.) was prepared. The hCGRP tab. was composed of hydroxypropylcellulose, lactose, hydrogenated oil, stearic acid and hCGRP. These materials were mixed and compressed by tabletting machine to form a tablet of 2 mm thickness and 6 mm diameter, weighing 60 mg. The release of hCGRP from the tablets was detected in vitro and in vivo using HPLC and radioimmunoassay (RIA), respectively. The tablet containing 24 μg hCGRP could slowly release approximately 80 % of its content with in the initial 5 days. The CSF level of rabbits of the hCGRP, the concentration of hCGRP in CSF before tablet implantation was below detectable limit. It was highest (23.1±18.36 nmol/l) 2 days after the implantation and remained detectable (3.08±2.67 nmol/l) until 5 days after the implantation. These results suggest that the hCGRP tab. release hCGRP over 5 days both in vitro and in vivo. This tablet may be clinically applicable in the treatment of patients with SAH against delayed vasospasm in man.

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Liquid crystal has been embedded in nylon membrane by vacuum filtration method to control the penetration rate of salbutamol sulfate. Cholesteryl oleyl carbortate(COC) which has smectic-cholesteric phase transition temperature near 18°C was used as a model liquid crystal. The results indicate the penetration rate of salbutamol sulfate through the COC-embedded membrane was dependent upon the adsorbed amount of COC in nylon membrane. At initial stage, the penetration rate of drug reduced dramatically with the increase of the adsorbed amount of COC. The adsorbed amount of COG was within 0.5 × 10^-4-3.0 × 10^-4 g mole/cm² in which salbutamol sulfate was hardly to penetrate ; when the adsorbed amount of COC was within 3.0 × 10^-4-9.0 × 10^-4 g mole/cm², the drug penetraion started again. After larger than 9.0 × 10^-4 g mole/cm² adsorption, the drug penetration stopped once more. Scanning electron microscopic (SEM) observation and water contact angle of the COC-embedded membrane might be used to explain this penetration result. The unique thermoresponsive property of COC-embedded membranes was proven in the step wise temperature change between 10°C and 25°C and made it possible to be used as thermally on-off switching membrane for stimuli-response controlled drug delivery system. The results indicate that thermo-responsive efficacy of such membrane is also depended upon the adsorbed amount of COC and the stimuli-responsive controlled membranes can be obtained by choosing an appropriate COC concentration and manufacturing processes.

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