We described herein the effectiveness of hyperthermia as a promising drug delivery system, especially noticing malignant glioma thermo-chemotherapy. Previously the authors reported that the combination of thermosensitive liposome containing CDDP and localized hyperthermia using a radiofrequency antenna focused CDDP to the experimental brain tumor, providing a greater antitumor effect. Our hyperthermic treatment, above 42°C, can treat the entire tumor core of human malignant glioma for direct thermal killing. When heating, the 40°C area extended up to a wide portion, covering the area invaded by tumor cells. Therefore, a more effective thermosensitive liposome was developed for clinical use. It was designed to release adriamycin (ADR) at 40°C and above. As a result, this new thermosensitive liposome sharply released ADR at a temperature of 40°C as planned, bringing a significantly greater antitumor effect in rat tumor models. This procedure will 1) contribute to the synergistic effect of hyperthermia and antitumor drugs in regions heated above 42°C, 2) be effective in direct thermal killing, 3) help to deliver the antitumor drugs wider : to the tumor core, and also to the area invaded by tumor cells, both areas heated at 40°C and above, 4) permit application of ADR and other antitumor drugs regardless the blood-brain barrier problem. Our present hyperthermic treatment can be easily applied to the elder, and/or ill patients, even those with deep seated glioma. Taking all these advantages into consideration, we can suggest the wider effects achieved by the use of this drug delivery system.

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Arg-Gly-Asp (RGD) amino acid sequence was originally found in fibronectin, a main component of extracellular matrices, and is known as a ligand to some adhesion molecules. Synthetic peptides based on the sequence have been found to decrease metastatic colonization of metastatic tumors. To attempt stabilization and prolong the circulation time of a compound having RGD sequence, an RGD mimetic (arginyl-aminohexanoic acid, NOK) and its hydrophobized derivative (NOK-L) were synthesized. At first, the effect of NOK and liposomal NOK-L on adhesion properties of tumor cells in vitro was investigated. NOK inhibited adhesion of B 16 BL 6 murine melanoma cells to immobilized NOK-L. Liposomal NOK-L inhibited adhesion of the cells to fibronectin, suggesting that NOR mimic the function of RGD, Next, we investigated the metastasis-suppressing efficacy of NOK and liposomal NOK-L in vivo, using the models of both experimental and spontaneous metastasis of B 16 BL 6 cells. As a result, a significant decrease in lung colonization was observed in a group injected with liposomal NOK-L in both models. These results indicate that NOR has the metastasis-suppressing activity via the mechanism similar to that by RGD, and that liposomalization of NOK using NOK-L augmented the metastasis-suppressing activity. Thus, liposomal NOK may be useful as an antimetastatic agent.

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Drug delivery to specific cells by immunoliposomes represents a potentially attractive mode of therapy. However, though immunoliposomes are effective in specific binding to target cells in vitro, their targeting efficiency in vivo is relatively low. We have recently developed a new type of polyethyleneglycol (PEG)-immunoliposomes, so called a pendant type PEG-immunoliposome, which are carrying the monoclonal antibodies at the distal ends of PEG chains. Pendant type PEG-immunaliposomes showed high targetability in vivo. In this study, we have synthesized new other PEG derivatives with reactive residues at the distal terminal of PEG chains, i. e., DSPE-PEG-COOH, DPPE-PEG-Mal and DPPE-PEG-CDI. PEG-liposomes composed of ePC/CH (2:1, m/m) and 6 mol% of PEG derivative were prepared and a monoclonal IgG antibody, 34A, which is highly specific to pulmonary endothelial cells, was conjugated to the terminal of PEG-COOH-, PEG-Mal- or PEG-CDI-liposomes. PEG-liposomes without antibodies showed the prolonged circulation time and the reduced RES uptake. All 34A-PEG-immunoliposomes showed high targeting efficiency to the lung in BALB/c mice. This approach provides a simple means of conjugating antibodies or ligands directly, and should contribute to development of superior targetable drug delivery vesicles for use in diagnostics and therapy.

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We prepared the liposomes, changed the entrapped amount of adriamycin (ADR) per the amount of liposome composing lipid, and after the addition of these liposomes with the same concentration of ADR(therefore, different dose of lipid), the tumor cell uptake of ADR was examined. The high entrapped amount of ADR demonstrated the usefulness in the tumor cell uptake of the ADR liposome in vitro. The cell uptake of the liposome depended on additional amount of ADR and liposomal lipid. Next, using ADR contained liposome and irinotecan contained liposome, its usefulness on tumor cell uptake by the polyethyleneglycol (PEG) modification of the surface on the liposome in vitro examined. In both liposome, PEG modification of the surface on the liposome facilitated the initial rate of the liposome uptake into the tumor cell. We have considered that this facilitation was attributed to the lipo-hydrophilic property of PEG and the fixed aqueous layer around the liposome. Therefore, PEG modification of the surface on the liposome, prevents the adhesion of serum opsonine and avoids reticuloendothelial system, does not inhibit tumor cell uptake rather facilitates. From the results of dextran sulfate contained liposome, it is expected that these liposome passed through the membrane of the tumor cell. Therefore, a higher entrapped amount of antitumor agents in the liposome and PEG modification have been confirmed to be beneficial in the tumor cell uptake.

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In vitro skin permeation study with rats was carried out to investigate the effect of cutaneous metabolism on the penetration of lipophilic drugs in the presence or absence of an esterase inhibitor, diisopropyl fluorophosphate (DFP). After the application of butylparaben, 96% of the total penetrated amount appeared as its hydrolyzed form (p-hydroxybenzoic acid) in the receiver fluid. On the other hand, in the case of propylparaben, the amount of the metabolite penetrated achieved approximately 70% of the total. When the skin was pretreated with 1 mM DFP, the appearance of the metabolite was completely inhibited. In addition, DFP significantly decreased the total penetration (the sum of intact and hydrolyzed forms) of butylparaben, but not that of propylparaben. The analysis based on a two-layer diffusion/metabolism model revealed that partitioning of butylparaben to the nonpolar route of stratum corneum was larger than that of propylparaben, and that DFP decreased the hydrolysis of parabens without altering their penetration parameters. This means that since butylparaben rapidly penetrates the stratum corneum, primarily due to its high lipophilicity, cutaneous metabolism process would dominate the total penetration of the drug. To comprehensively understand the relationship of skin penetration of drugs with their lipophilicities and metabolic rates, a simulation study using the diffusion/metabolism model was carried out. Total skin penetration of drug is larger as it is more lipophilic, and additionally increases as drug metabolism occurs. For more lipophilic drugs, the increase in total penetration associated with the metabolism becomes larger. This suggests that prodrugs having faster bioconversion rate to parent drug are more suitable for penetration enhancement.

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In the present study, we evaluated the lung metastasis inhibitory effect and optimal dosage of N²-[(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N⁶-stearoyl-L-lysine (MDP-Lys), a lipophilic derivative of muramyl dipeptide (MDP) acting as a macrophage activator, in hamsters bearing osteosarcoma. MDP-Lys was administered either as a solution or liposomal preparation (multilamellar, about 2 μm of diameter). The time course of lung metastatic rates after transplantation of osteosarcoma to the lower extremity was estimated macroscopically and pathologically for 7 weeks. In this study, the extremity was amputated 3 weeks after transplantation. The lung metastatic rates observed were 0%, 10%, 50%, 70%, and 100%, 1, 2, 3, 5, and 7 weeks after transplantation, respectively. Based on these results, the inhibitory effect of MDP-Lys given as a solution or multilameliar liposome on lung metastasis was evaluated 7 weeks after transplantation. Hamsters, which were transplanted osteosarcoma and their extremity were amputated 3 weeks after transplantation, were divided into 3 groups. Treatments with MDP-Lys were carried out for 4 weeks after the amputation. The first group received free MDP-Lys subcutaneously as a solution (0.5, 5, 50 μg/day), the second group received liposomal MDP-Lys (2 or 20 μg, twice/week), and the third group received only empty liposome (twice/week) intravenously as a control. The inhibitory effects of MDP-Lys on lung metastasis were observed in the following two treatment groups : MDP-Lys given as a solution (50 μg/day) reduced the lung metastatic rate to 54.5%, and liposomal MDP-Lys (20 μg, twice/week) reduced to 60%. Thus, liposomal MDP-Lys showed a similar inhibitory rate to that given as a solution at a far lower dose (40 μg vs 350 μg/week). Considering that hamsters had lung metastasis at a rate of 50% before the treatment, above treatments with MDP-Lys inhibited the metastasis almost completely. The optimal dosage of MDP-Lys given as a solution would be more than 50 μg/day, and that of MDP-Lys given as a liposome would be more than 20 μg (twice per week). The greater efficacy of liposomal delivery would be due to the longer drug retention in the body. To confirm it, fluorescein was given intravenously either as a solution or multilamellar liposome (same size and composition to that of MDP-Lys), and the time course of fluorescein amount retained in the lung, liver and serum were determined. Fluorescein given as a solution was almost disappeared from the lung, liver and serum within 12 hrs. In contrast, fluorescein given as a liposome was retained in the body for about 72 hrs. Thus, it would be concluded that the greater lung metastasis inhibitory effect of liposomal MDP-Lys than that of solution would he due to the long drug retention in the body, especially in the lung.

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Lipo-PGE₁ is a drug delivery system useful for administration of small quantities of drugs. Lipo-PGE₁ efficiently delivers prostaglandin E₁ (PGE₁) to the sites of lesions, and has potent peripheral vascular dilatative and anti-platelet effects. It is very important that drugs are retained in the lipidic particles. Some studies on the retentivity of drugs in lipidic particles have been reported but ways of thinking about and experimental methods are not standardized. Therefore, we compared methods of separation of aqueous phase in Lipo-PGE₁ using a membrane filter, an ultrafilter or a dialysis tube. The dialysis method was found to be most suitable if a kinetic analysis was performed. For this analysis, diffusion theory and successive first-order reactions were used. We found that the predictions of the diffusion method agreed with the experimental values and expressed the equilibrium state. Retentivity of PGE₁ in lipidic particles was analyzed using the most suitable method. Lipo-PGE₁ exhibited high retentivity of PGE₁ in the lipidic particles and preserved its faculty as drug carrier.

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To guarantee the drug delivery function of the DDS drugs in clinical use, supplementary information closely related to the diverse clinical usage is required in addition to information listed on the Products Information. Changes in the phase-distribution of PGE₁ in lipo-PGE₁ (Palux inj. and Liple^®), a targeting-type DDS, after 10-times dilution with 6 aqueous infusions frequently prescribed for clinical treatment, i. e., 5% Otsuka Toueki (Glucose Injection JP), Otsuka Seisholcuchu (Isotonic Sodium Chioride Solution JP), Klinit^® (Xylitol Injection JP), Physiosol^®·3, Hartmann's Solution pH : 8 and Intralipos^® 10%, were examined in vitro. Total amounts of PGE₁ and the amounts of PGE₁ in the aqueous phase were determined by the ultrafiltration method to estimate phase-distribution ratios of PGE₁ after 10-times dilution. Total amounts of PGE₁ did not decrease after the dilution with each aqueous infusion. The amount of PGE₁ in the lipid emulsion phase decreased from 87% to 75, 72, 61, 47 and 20% after the dilution with Physiosol^®·3, 5% Otsuka Toueki, Klinit^®, Otsuka Seishokuchu and Hartmann's Solution pH : 8, respectively, but not with Intralipos^® 10%. The release of PGE₁ from the lipid emulsions occurred in relatively short period of time after the dilution and the phase-distribution ratios of PGE₁ were unchanged throughout the experiment for 6 h. These findings strongly suggest that the dilution with Hartmann's Solution pH : 8 and Isotonic Sodium Chloride Solution is not necessarily the recommended prescriptions for combination with lipo-PGE₁ in maintaining its drug delivery function by the pharmaceutical view points. High pH of aqueous infusion combined with lipo-PGE₁ for dilution tends to increase the release.

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