The basis for the blood-brain barrier in mammals is the selective transport properties of brain capillary endothelium, including the elaborate system of tight intercellular occluding junctions that occur between apposed membrane faces of these cells. This unique specialization of brain capillary endothelial cells appears late in development and has been postulated to be under the inductive influence of astrocytes in the central nervous system. To examine this astrocytic contribution to endothelial cell monolayer permeability, we employed the cocultures of bovine endothelial cells (aortic or brain capillary endothelial cells, BBEC or BAEC) with astrocytes in a double chamber system. In system 1, where astrocytes were separated from endothelial cells, a 40% reduction in L-glucose permeability of the BBEC monolayer, but not the BAEC monolayer, was observed by cocultivation with astrocytes. By contrast, in system 2, where respective endothelial cells and astrocytes layered on the upper and lower surfaces of a membrane, the permeability of both BAEC and BBEC monolayers was reduced by cocultivation with astrocytes. The obtained results suggest that primary cultured BBECs, which had been primed by astrocytes in vivo, retain a higher sensitivity to astrocytes possibly through an astrocytic soluble factor (s) to exhibit BBB-specific phenotypes, and that even BAEC from extra-neural tissues, when cultured with astrocytes in close proximity in vitro, may acquire the similar phenotypes and serve for an extensive use of BBB model in vitro.

View full abstract

Malignant glioma is one of the most lethal tumors arising in the central nervous system. The patients with malignant glioma have poor prognosis with an average survival of less than 2 years even if they receive multimodality treatment including surgery and post operative adjuvant therapy. Therefore, we need to develop a new form of therapy that can inhibit the growth and invasion of glioma cells. Here, we have evaluated the efficacy of liposomes conjugated or associated with a monoclonal antibody for malignant glioma. We prepared two types of monoclonal antibodies for the targeting therapy. One is a G-22 monoclonal antibody reactive with the CD44 that overexpresses on the surface of human glioma cells and another is a 3C10 monoclonal antibody reactive with the fusion junction of a deletion-mutant epidermal growth factor receptor (EGFR) on their cells. The deletion-mutant (Type III) was reported to be most prevalent, being approximately 17% of the patients with malignant glioma. Using these antibodies, we prepared the liposomes conjugated or associated with a monoclonal antibody (Immunoliposomes). We were successful with in vitro and in vivo transfer of anticancer drugs or biological response modifiers (BRM) or genes using the immunoliposomes. The immunoliposomes containing methotrexate, cisplatin or adriamycin have selective cytotoxicity toward glioma cells. Also, the immunoliposomes entrapping the BRM genes (ex. interferon-β, tumor necrosis factor-α) increased the growth inhibition of glioma cells much more than the liposomes with no antibody. These results suggest that liposomes conjugated or associated with a monoclonal antibody would be a useful clue for the tumor targeting of malignant gliomas.

View full abstract

A novel transdermal preparation was applied for the cancer patient who were complaining cancer pains to test the possibility of the future clinical application. Four patients with cancer pain were applied this new preparation on their chests and were examined amelioration of the cancer-related pain and the adverse effects on the skin. The patients whose pains had been controlled by MS-Contin preparation were entered in this clinical study, applying this new transdermal preparation after stoppoing the use of MS-Contin preparation for 24 hours. In most cases, amelioration of cancer pain was found and the adverse effects on the applied sites were minimal. Therefore, it seemed possible to test this preparation for the relief cancer pain clinically in the future.

View full abstract

An implantable drug delivery system consisting of morphine and polyethylene glycol (PEG) was studied for the purpose of prolonged duration of analgesia effect by the controlled slow release of morphine. Because, a long continuous administration of analgesic through catheter and the supply equipment has given great inconveniences and uncomfortableness for the patients after operations and the patients having chronic pains and cancer pains. PEG of two different molecular weights (1000 and 1500) was mixed with morphine (2.5 mg, 5.0 mg and 7.5 mg respectively in one sample) so as to have a melting point of 40°C. The mixture was molten and injected into epidural of rats. The mixture was solidified in the epidural and gradually released morphine for a certain period. The duration period of analgesia effect was tested and evaluated by tail-flick test method and the MPE values. The results showed that PEG-morphine delivery system containing morphine more than 5.0 mg had a remarkable prolongation effect for analgesia function. The system containing 5.0 mg morphine kept 6 days duration of efficacy and the system containing 7.5 mg morphine showed 13 days duration of analgesia effect. An injection of control system containing no morphine (PEG only) showed no effect. Conclusively, it was proved that a prolonged duration of analgesia effect over several days∼2 weeks was achieved with a new administration technique of PEG-morphine (more than 5.0 mg) delivery system into epidural by the liquid injection and the followed solidification in vivo.

View full abstract

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 long-circulating immunoliposomes (Type C) which can effectively bind to the designated target site in vivo. This was achieved by the use of newly synthesized DSPE-PEG-COOH to couple antibodies directly to the distal terminal of PEG chains, which allow the liposome to evade the RES uptake and achieve prolonged circulation. Liposomes were prepared from ePC and CH (2 : 1, m/m)containing 6 mol% of DSPE-PEG-COOH, and a monoclonal IgG antibody, 34A, which is highly specific to pulmonary endothelial cells, was conjugated to the carboxyl groups of DSPE-PEG-COOH to give various amount of antibody molecules per liposome. PEG-COOH-liposomes without antibodies showed prolonged circulation time and reduced RES uptake owing to the presence of PEG. The degree of lung binding of 34A-Type C was increased with increasing coupling amount of antibody. Antibody density is an important factor for target binding. We established the preparation of adriamycin (ADR) encapsulated 34A-Type C immunoliposomes. ADR encapsulation was done by employing an ammonium sulfate (AS) gradient method. PEG-COOH liposomes were prepared in 120 mM AS and attached 34A to make 34A-Type C. External medium of liposome was replaced to 120 μM AS for the creation of AS gradient between inside and outside of liposome membrane and ADR solution was added into external medium. The ADR loading efficiency was 95%. ADR level in lung after i.v. injection of ADR-34A-Type C was much higher than that of control ADR liposome and free ADR.

View full abstract

We have developed fusogenic liposomes, prepared by fusing simple liposomes and Sendai virus particles. Fusogenic liposomes can introduce their contents directly and efficiently into the cytoplasm in vitro and in vivo. In this study, we examined the characteristics of fusogenic liposomes as a gene transfer vector. Fusogenic liposomes could transfer genes into cultured cells more efficiently than cationic liposome-DNA complex. When the cells were treated with fusogenic liposomes containing pCAL2, a firefly luciferase expression plasmid, for 1-10 min, they had high luciferase activity. In contrast, the cells transfected with cationic liposome-DNA complex had little activity. In addition, fusogenic liposomes showed no cytotoxicity even at higher concentration of DNA. Furthermore, when fusogenic liposomes containing 0.75 μg of pCAL2 was i. p. injected into the mouse bearing Sarcoma-180 ascites tumor, a high level of luciferase activity was detected in Sarcoma-180 cells. In contrast, little luciferase activity was detected even when cationic liposome-DNA complex containing 40 μg of pCAL2 was administrated. These results indicates that fusogenic liposomes mediate efficient gene introduction into the animal cells in vitro and in vivo.

View full abstract

An antitumor agent irinotecan (CPT-11) is a water soluble derivative of camptothecin. It is a prodrug, and mainly metabolited to an active metabolite (SN-38) in the liver. Even if a prodrug accumulates in a target tissue, namely tumor, by liposomalization, it doesn't necessarily resulted in the increase of antitumor activity. We found that CPT-11 was clearly converted into SN-38 in the tumor in vitro, and tried liposomalization of CPT-11. The mean particle diameters of plain liposomal CPT-11 (PLCPT(11)) and polyethyleneglycol (PEG)-coated PLCPT(11) (PEG-LCPT (11)) were both about 160 nm. The trapping efficiencies were approximately 90%. After the administration of these liposomes to mice, CPT-11 and SN-38 concentrations in the blood increased by liposomalization, and the circulation time in the blood was prolonged further by PEG-modification of the liposomes (PEGylation). In the tumor, CPT-11 and SN-38 concentrations increased by liposomalization. Thus, it is considered that the prolongation of circulation time in the blood by PEGylation caused passive targeting to the tumor. We have expected an increase in the antitumor activity of CPT-11 from the change of the distribution by liposomalization and PEGylation. Because CPT-11 accumulates in the tumor by liposomalization and is converted to the active metabolite in the tumor, we have been anticipating a increase of usefulness by liposomalization of prodrug.

View full abstract

We have synthesized antisense phosphorothioate oligodeoxynucleotide (S-ODN) tageted 3' splice junction of immediate early (IE) pre-mRNA 4/5 in herpes simplex virus type 1 (HSV-1) and evaluated their antiviral activities as a novel anti-herpetic agent. The antisense S-ODN completely protected the permissive Vero cells from a cytopathic effect of HSV-1 at a lower concentration range of 0.78 to 1.56 microM in a serum-free assay system, whereas its sequence including mismatced bases of S-ODN and the sense sequence of S-ODN exhibited the protective effect at higher concentrations (=> 50 microM). The results indicated that the antisense S-ODNs targeted 3' splice junction inhibit viral growth in a sequence-specific manner. To get more informations for the usage as a therapeutic agent, the biological stability and the intracellular distribution of the antisense S-ODN were examined. In the nuclease degradation test, the antisense S-ODN had a half life of 5 to 7 hrs in culture medium containing 10% fetal calf serum at 37°C. The similar antisense phosphodiester (D) ODN was completely degraded within 60 mins. The S-ODN was shown to be considerablely more stable to nuclease degradation than D-ODN. In the same condition, the antiviral activity of the antisense S-ODN was 8 times less than that in the serum-free culture system. No antivial effect of the antisense D-ODN was detected at any concentration even in the serum-free culture system. In HSV-1 infected cells, a uniform distribution of S-ODN was observed both in nuclei and in cyto plasm at 12-hr incubation. On the other hand, in non-infected cells, S-ODN was localized into lysosome-like vesicles and such localization of S-ODN continued on and after 12-hr incubation. This indicates that a critical disadvantage of intracellular delivery to target site might be solved by viral infection. These findings suggest that the antisense S-ODN may be sufficiently worth developing as an antiherpetic therapeutic agent, and that some more devices for the improvement of nuclease-resistance and intracellular permeability could make the antisense S-ODN more effective against HSV-1 infection.

View full abstract

Saturated and unsaturated fatty acids enhanced the permeation of beraprost sodium, a chemically stable prostaglandin I₂ analogue, from a silicone oil through the skin of hairless mice in vitro. The percutaneous permeation of beraprost showed a parabolic dependence on the lipophilicity of the saturated fatty acids, where capric acid was the most potent enhancer. Of the unsaturated fatty acids tested, oleic and linoleic acids showed greater enhancing effects on the percutaneous permeation of beraprost. When a pressure sensitive silicone adhesive tape containing beraprost sodium was applied onto the shaved skin of rats in vivo, oleic and capric acids enhanced the percutaneous absorption of beraprost, the former being more effective. In particular, the topical application of beraprost sodium with oleic acid significantly inhibited the progress of the laurate-induced peripheral vascular occlusive sequelae in the ear of rabbits. Spectroscopic studies indicated that cyclodextrin derivatives (CyDs) formed inclusion complexes with beraprost sodium with efficacy increasing in the order : sulfated β-CyD (S-β-CyD)<dimethyl-β-CyD (DM-β-CyD)<hydroxypropyl-β-CyD(HP-β-CyD). When the hairless mouse skin was pretreated with oleic acid, the in vitro percutaneous permeation of beraprost from the silicone oil was sustained by the complexation with DM-β-and HP-β-CyDs, while S-β-CyD tended to increase the percutaneous permeation of the drug. Furthermore, HP-β-CyD eliminated undesirable properties of oleic acid in the silicone adhesive tape such as the reduction in the detachment force of the tape and the lability against oxidation. These results suggest that a combination of oleic acid with HP-β-CyD is particularly useful for controlling the transdermal delivery of beraprost from topical preparations intended for the systemic use.

View full abstract

We have developed a pressure-controlled colon delivery capsule (PCC) made of ethylcellulose and reported its application to a protein drug, recombinant human granulocyte colony-stimulating factor (rhG-CSF) in last year. In this report, PCC was applied to the drugs for colon diseases, and the relationship between the drug release characteristics from colon delivery systems and bioavailability (BA) was investigated using Eudragit S coated tablet as a reference solid preparation. As model drugs, 5-aminosalicylic acid (5-ASA) and tegafur (FT) were selected. With each drug, PCC and Eudragit S coated tablet were prepared, and these test preparations were administered to fasted male beagle dogs and drug concentration in the systemic circulation was measured by HPLC methods. In addition, the in vitro dissolution tests were performed according to JP XIII method. Mean AUC values of 5-ASA, 25 mg/kg, were 22. 57 for Eudragit S coated tablet and 48.09 μg·h/ml for PCC, respectively. On the other hand, mean AUG values of FT, 1 mg/kg, were 9.73 for Eudragit S coated tablet and 15.55 μg·h/ml for PCC. Mean AUC values of PCC were significantly higher than those of Eudragit S coated tablets, with two drugs. On the other hand, the in vitro dissolution test showed that the release rate of drug from tablet delayed as compared to that from PCC. As less water is present in the colon than in the small intestine, it was speculated that in the case of Eudragit S coated tablet, the solid materials inside the colon such as stools decreased both the amount and the time of drug molecules being contact to the gut wall, and drug molecules were not well dissolved by the gastrointestinal fluid, because solid preparation was used. Both the release rate from PCC and the dissolution rate of drugs were improved, because PCC can contain drug molecules as liquid state. In conclusion, PCC is thought to be safe and useful colon delivery system for the oral delivery of protein drugs and drugs for colon diseases.

View full abstract