Liposomes have attracted a considerable amount of interest for potential use as a drug delivery system due to their suitable characteristics. However, the predominant uptake of liposomes by the reticuloendotherial system (RES) and a resulting rapid clearance from circulation have been a major obstacle in any attempt to deliver liposomes to cells, tissue or organs other than the RES. One of the current efforts is to overcome the above kinetic barrier by manipulating the liposome characteristics. Some attempts have been made to reduce the RES uptake by surface modification with glycolipids or synthetic polymers. Especialy, incorporation of polyctyleneglycol-lipid derivatives(PEG) in liposome led to increased blood levels of liposomes and reduced uptake by the RES. In this review, various designs of PEG containing liposomes are presented, characterization of PEG-liposomes and possible mechanisms of action are discussed. It is suggested here that the primary mechanism by which PEG molecules alter the biodistribution of liposomes in vivo involeves an inhibition of the association of blood components(opsonin ?) to liposomes, resulting in a reduced rate of clearance of liposomes from the circulation. The many advantages of long-circulating liposomes, such as PEG-liposome, make them excellent candidates as drug delivery systems for sustained drug release or drug targeting application.

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We studied the effects of test drugs on the influx and efflux of adriamycin in Ehrlich ascites carcinoma cells, by rapid and simple determinations of intracellular concentrations of adriamycin. Caffeine induced an increase in adriamycin accumulation and inhibited efflux. Biochemical modulators, aminobenzamide, ketoprofen and dipyridamole were also found to have inhibitory effect on adriamycin efflux. These test drugs had no effect on adriamycin influx from tumor cells. Caffeine amplified the inhibition of DNA biosynthesis induced by adriamycin under the efflux condition in vitro. Furthermore, caffeine significantly enhanced adriamycin concentration in the tumor in vivo. Thus, the combination of caffeine with adriamycin increased the antitumor activity of adriamycin. Therefore, the effect of caffeine on adriamycin concentration in vivo is consistent with that in vivo. These results suggest that caffeine is effective as biochemical modulator and our method in vivo is useful as the screening method of biochemical modulator.

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Basic fibroblast growth factor(bFGF)is a prototype of heparin-binding growth factor family and could play a crucial role in wound healing processes. Water-insoluble aluminum salts of cyclodextrin sulfates (Al·CyD-Suls) were prepared, and their potential use in oral formulations of bFGF intended for treatment of gastrointestinal ulcers was investigated. Adsorbates of bFGF with Al·CyrD-Suls were prepared by incubating the growth factor with a suspension of Al·CyD-Suls in water. The mitogenic activity of bFGF released from the adsorbate was almost comparable to that of the intact bFGF. Al·CyD-Suls significantly protected bFGF against proteolytic degradation by pepsin, compared with their sodium salts and other sulfated oligosaccharides tested. The in vitro release of bFGF from the Al·β-CyD-Sul adsorbate into isotonic phosphate buffer (pH 7.4) was sustained in proportion to a rise in the ratio of Al·βCyD-Sul to the growth factor. The oral administration of the adsorbate of bFGF with Al·β-CyD-Sul had the most prominent healing effects on acetic acid-induced gastric ulcers. These results suggest that Al·CyD-Suls may potentiate the healing effect of bFGF by both stabilizing and anchoring the growth factor at the site where its action is desired.

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Hepatic targeting of proteins utilizing the sugar -recognition system was investigated in mice after intravenous injection. Five proteins with different molecular weights, i.e., bovine γ-globulins(IgG), bovine serum albumin(BSA), recombinant human superoxide dismutase(SOD), soybean trypsin inhibitor(STI), and chicken egg white lysozyme(LZM) were selected and modified with 2-imino-2-methoxyethyl 1-thiogalactoside to obtain galactosylated proteins. All galactosylated proteins were dose-dependently taken up by the liver and the amounts accumulated in the liver were decreased with the increase of the administered dose. However, the hepatic clearances were greatly different among them. At doses less than 1 mg/kg, Gal-IgG, Gal-BSA, and Gal-SOD had large values of hepatic clearance close to the hepatic plasma flow (85 ml/hr) and those of Gal-STI and Gal-LZM were 40 and 20 ml/hr, respectively. Their hepatic uptake processes were analyzed by a physiologically pharmacokinetic model including the hepatic plasma flow and the saturable uptake process in the liver. This analysis based on a model revealed the low affinity to the carbohydrate receptors on hepatocytes and high glomerular filtration rate resulted the ineffective delivery of Gal-STI and Gal-LZM to the liver. These results suggested that galactosylation of proteins with molecular weight greater than that of SOD led them to be delivered to the liver at lower doses less than 1 mg/kg and that proteins with molecular weight less than that of SOD could not be effectively delivered to the liver by galactosylation because of their low affinity to the liver due to small numbers of attached galactose residues and high glomerular filtration rate.

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Various approaches have been examined to improve peptide and peotein drugs via gastrointestinal tract. In a series of investigation, we have synthesized noveI lipophilic derivatives of thyrotropin-releasing hormone, tetragastrin, and insulin by chemical modification with different carbon-chain length fatty acids. In this study, we synthesized newly lipophilic derivatives of human calcitonin(hCT) by chemical modification with short-chain length fatty acids, such as acetic acid (C₂) and caproic acid (C₆), and examined their intestinal absorption properties using an in situ closed loop method in rats and their stabilities in the intestinal mucosal homogenates. The intestinal absorption properties of acyl derivatives were estimated by calculating the pharmacological availability (PA). Their intestinal absorption and stability in the intestinal mucosal homogenates were improved by acylation in the following rank order : caproylated hCT>acetylated hCT>native hCT. Furthermore, we studied the effects of co-administered absorption enhancer, sodium glycocholate (Na-GC), on the intestinal absorption of acyl-hCT derivatives. Co-administration of Na-GC significantly improved the absorption of native hCT from the intestine. However, the promoting effect of Na-GC was found to be less pronounced for the acyl derivatives, although Na-GC enhanced their absorption by about twice. These results indicated that it may be possible to achieve remarkable absorption enhancement by hCT by using a combination of acylation and Na-GC.

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Long-circulating liposomes containing amphipathic polyethyleneglycol (PEG) have been tested for their utility as enhanced delivery system of doxorubicin(DXR) to the solid tumor. Inclusion of PEG to the small unilamellar liposomes(SUV, DSPC/CH (1 : 1, m/m), 120 nm in mean diameter) encapsulating DXR showed the increased blood level and the decreased RES uptake of SUV, and resulted in high accumulation of SUV into the colon 26 solid tumor site. Reflecting to these behavior of PEG-SUV, DXR concentration in tumor tissue was significantly increased, and effective tumor-growth retardation and increased survival time were observed. These data indicate that since the capillary permeability of the endothelial barrier in newly vascularized tumors is significantly greater than that of normal organs, SUV with prolonged circulation time could predominantly pass through leaky endothelium into tumor area by passive convective transport. DXR encapsulated in long-circulating liposomes will be much more effective for cancer chemotherapy than either the usual liposomal DXR or free DXR.

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Azetireiin, a new thyrotropin-releasing hormone (TRH)analogue, has much more potent and longer-lasting activity on the central nervous system than TRH. It is known, however, that the oral bioavailability of azetirelin is poor and less than 2% in rats and humans. This is mainly attributed to the low intestinal permeability due to the lack of lipophilicity. We have studied the effect of various absorption enhancers on the intestinal absorption of azetirelin by means of in vitro Ussing chamber and in situ closed loop experiments. Enhancers such as Na₂-EDTA, sodium glycocholatc(Na-GC), citric acid, and laurylmaltoside(LM) were used in the study. The action of these enhancers was more predominant in the large intestine than in the small intestine. LM was most effective among the enhancers studied. The enhancing activity of LM was concentration dependent and the effect of 1mM LM was still more extensive than that of 10mM of other enhancers. The release amount of membrane protein and phospholipid in the presence of these enhancers was also examined to evaluate the tissue damage. The results indicated that 1mM LM was less harmful to the large intestinal mucosa than 10mM Na₂-EDTA or Na-GC. From these results, we conclude that LM is practically safe and effective absorption enhancer for improving the large intestinal absorption of azetirelin.

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The tumor distribution and the disposition of serum albumin were investigated in mice bearing Sarcoma 180. The albumin labeled with fluoresceinisothiocyanate(FITC) were administered to the mice. The FITC-labeled albumin acylated with acid anhydrides, such as acetic anhydride, glutaric anhydride, maleic anhydride and succinic anhydride, were also administered to the mice in order to investigate the effect of chemical modification. The plasma concentration of each acylated albumin was significantly lower than that of the non-acylated albumin at 24 h after administration. The tissue distributions of the acylated albumin were also decreased compared to those of the non-acylated albumin. Especially, the accumulation of albumin in the liver and spleen was significantly reduced with the glutarylation and maleylation. Urinary excretion of albumin was significantly increased with the acylation because the degradation rates of the acylated albumins were much faster than that of non-acylated albumin. On the other hand, the acylated and non-acylated albumin were accumulated effectively in the tumor tissue in mice bearing Sarcoma 180. It was found that the tumor distribution of albumin was not impaired by the acylation. It was suggested, therefore, that the glutarylated and maleylated albumin were valuable for relative tumor-selectivity and might be utilized in the macromolecular carrier system of antitumor drugs.

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