This review briefly explains concept, methodology, and historical background of passive targeting of drug. Historically, it was believed that active targeting using monoclonal antibodies was a more selective and efficient targeting methodology than passive targeting. In contrast, all the approved targeting systems for anticancer drugs are of passive targeting. Authors emphasize significance of passive targeting, and point out that the passive targeting aspect is also important and must be carefully considered for potent active targeting system design because passive pharmacokinetic processes are included to a substantial degree in active targeting systems.

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Passive targeting strategies utilizing macromolecular drug carriers were reviewed from the viewpoint of pharznacokinetics. Although active targeting using target-specific carriers is a very attractive approach, successful results are limited. A variety of problems should be overcome to achieve not only active targeting but also passive targeting. Pharmacokinetic analysis based on a clearance concept has demonstrated that in vivo behavior of macromolecules after systemic injection depends on their physicochemical properties such as molecular weight and electric charge. Based on the findings, passive targeting systems for antitumor agents were developed. Prolonged retention of protein drugs in the blood circulation was also possible by chemical modification with macromolecular carriers including polyethylene glycol. The success in passive targeting approach will facilitate the development of active drug targeting systems.

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Passive targeting with liposomal drug carrier systems was reviewed. The current status of the passive targeting by the pnlyethyleneglycol coated liposome (PEG-liposome) were described in this review. Newly developed liposomes, containing either monosialoganglioside G_M1 or amphipathic polyethylene glycol (PEG) derivatives, are not readily taken up by the macrophages in the RES and hence stay in the circulation for a relatively long period of time. Particularly, PEG is useful because of its ease of preparation, relatively low cast, controllability of molecular weight and linkability to lipids or protein including the antibody by a variety of methods as compared with G_M1 molecules. So many recent studies have focused on the use of liposomes with surface associated PEG. The presence of PEG reduces binding of serum protein, i.e. opsonins marking the lipnsome for clearance by MPS. Pharmacokinetic analysis and therapeutic studies with tumor bearing mice revealed that PEG-liposomes with an average diameter of 100-200 nm were accumulated efficiently in tumor tissue. Due to the capillary permeability of the endothelial barrier in newly vascularized tumors is significantly greater than that of normal tissues, PEG-liposomes could extravasate from blood circulation to tumor tissue. Results from clinical studies with doxorubicin encapsulated into PEG-liposomes (DOXIL^®) in AIDS-related Kaposi's sarcoma revealed an increased therapeutic efficacy compared to free-drug. Solid tumors generally possess the following pathophysiological characteristics : (a) hypervasculature, (b) incomplete vascular architecture, (c) secretion of vascular permeability factors that stimulate extravasation within the cancer, (d) little drainage (lack of lymph vessel) of macromolecules and particles, which results in their long-term retention in tumor tissue. These characteristics of solid tumors are the basis of the so-called EPR effect (enhanced permeability and retention effect). Thus, the permeability of the endothelial barrier in newly vascularized tumors is increased compared with that of healthy tissues. PEG-liposome can take advantage of the EPR effect for efficient targeting binding in the tumor. The localization of PEG-liposomes into the interstitial space between tumor cells by a process of extravasatiun from tumor vessels (EPR effect) was revealed by the electron microscopic observations. Immunoliposomes for the treatment of solid tumor should satisfy a number of requirements aimed at maximum targeting effect of immunoliposome administered systemically in the bloodstream. Antigen binding site of the liposome-cunjugated antibody must be accessible for unperturbed interaction with antigen on the surface of target cells. The blood clearance of immunoliposomes must be minimized in comparison with rate of extravasation in the tumor. As described above, PEG-liposomes offer the development of immunoliposomes with both long survival times in circulation and target recongnition being retained in vivo. A new type of long-circulating immunoliposome, which was PEG-immunoliposome attached antibody at the distal end of PEG chain, so called the pendant type immunoliposome, was designed. To assist extravasation, the liposomes were of uniform, small size (100-130 nm). Elimination of immunogenic effect of Fc portion and of the increased RES clearance through specific recognition by MPS cells carrying Fc receptor, was achieved by using Fab' fragment instead of the whole antibody.

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Passive targeting with microparticulate drug carrier systems is reviewed. Passive targeting characters of microparticulates such as microspheres, emulsions, and polymeric micelles are determined by their particle size and surface chemical structures. Microparticulates larger than blood capillaries are known to be stuck in capillaries and this phenomenon is utilized in chemoembolization. Particles smaller than ca. 200 nm in diameter can circulate in bloodstream for long time periods by avoiding reticuloendothelial uptake if these particles are large (>ca. 4 nm) enough to avoid the renal excretion. Furthermore, particle surface must be hydrophilic for avoidance of the reticulocndothclial uptake. Present status of three passive targeting particulate carriers is summarized. Microspheres (They are called nanospheres when their diameters are smaller than 1 μm.) are mainly used in chemoembolization with diameters larger than capillaries. A therapy with prostaglandin E1-containing emulsion was approved in Japan in 1988. Emulsions are mainly used for solubilization of hardly water-soluble or water-insoluble drug and for local injection such as arterial infusion. A polymeric micelle is an alternative type drug carrier that is an assembly of polymers with heterogeneous structures, typically block copolymers. Polymeric micelles inherently pos.sess several benefits as drug carriers, e. g., high structural stability, very small size (<100nm), and large drug incorporation capacity. A polymeric micelle system containing an anticancer drug, adriamycin, showed much enhanced in vivo antitumor activity with selective accumulation in solid tumor sites. This is a successful example of passive targeting to solid tumors utilizing the enhanced permeability and retention effect (EPR effect) of tumor vascularture. For passive targeting, deep understanding of vascular endothelial cell structures that are responsible for permeation of particulates is important. However, these structures have not been analyzed enough to present quantitative estimation of particulate permeation. The hypertension chemotherapy using angiotensin II is a successful drug delivery methodology. Angiotensin II increased blood flow selectively at solid tumor sites. This therapy was approved in Japan against stomach tumor using conventional low molecular weight anticancer drugs. Research is further carried out for combination use of angiotensin II with a macromolecular drug carrier system.

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For patients with infectious diseases including AIDS or those with cancer, induction of protective immunity, which can eliminate the cells involved in the respective pathologic conditions, is essential. In particular, induction of cytotoxic T lymphocytes (CTL) becomes important in such patients, since CTL palys a central role in protective immunity. It was reported that endogenous antigens in the cytoplasm are generally presented in association with class I major histocompatibility complex (MHC) molecules, while exogenous antigens enter the endosomal pathway by endocytosis, then presented in association with class II MHC molecules. In recent years, attention has been focused on component vaccines due to their higher safety. However, since component vaccines are exogenous antigens, most component vaccines are endocytosed after administration, then presented in association with class II rather than class I MHC. That is, it is a major drawback of component vaccines that the efficiency of antigen presentation in association with class I MHC molecules, which is essential for CTL induction, is markedly low. Moreover, when short synthetic peptides that contain CTL epitopes are used as CTL vaccines, there is the risk that these peptides may undergo degradation in endosomes during the usual pathway of antigen presentation via endocytosis even when antigen molecules ascaped from endosomes into the cytoplasm. Therefore, antigen presentation via class I MHC molecules is little expected when such vaccines are administered alone. To design effective component vaccines for CTL induction, it is essential to develop a novel technique considering intracellular kinetics of antigens ; that is, an antigen delivery system that directly introduces into the cytoplasm and then efficiently directs into the class I MHC presentation pathway. To date, we have been developing fusogenic liposomes whose membrane fusion capacity was identical to that of Sendai virus. Fusogenic liposomes facilitate direct and efficient induction of inclusion substances into the cytoplasm by membrane fusion without accompanying cytotoxicity. Therefore, we applied fusogenic liposomes to a component vaccine and attempted to solve the above problems. Antigens induced via fusogenic liposomes were presented to the antigen specific CTLs in conjunction with class I MHC molecules in vitro more efficiently than the efficiency of antigen presentation via plane liposomes. In addition, antigen presentation by class I MHC, which was increased by fusogenic liposomes, was not inhibited by endocytosis inhibitors, but was dependent on the activity of envelop proteins derived from Sendai virus. Furthermore, reflecting the above results, when mice were immunized with antigens induced via plane liposomes, antigen-specific CTL was negligibly induced. However, when mice were immunized with antigens induced via fuscgenic liposomes, antigen-specific CTL was induced more potently than in mice immunized with complete Freund's adjuvant (CFA). This observation suggested that when antigen molecules were directly induced into the cytoplasm via fusogenic liposomes and recognized as endogenous antigens, these antigens were efficiently transferred to the class I MHC presentation pathway. Therefore, it is necessary to consider both systemic and intracellular kinetics of antigens to obtain the effects of vaccination efficiently, which suggested the importance of DDS techniques rather than immunomodulator functions, which are to be conferred on CFA, in the development of vaccines. The potential of the present system for clinical use and future of the development of CTL vaccine delivery system is discussed.

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History of vaccine development was presented. Introduction of gene technology since early 1980's was described together with its advantages and problems, and success of Hbs vaccine by gene technology was described. For further success, development of more efficient and safe immunoadjuvant has been awaited. As new approaches, development of DNA vaccines and mucosal vaccines have been studied. Difference of mechanism of immune response by live viral vaccine and killed or subunit vaccine was described. Live virus infect immunocompetent cells and viral antigen produced in them (endogeneous protein) prime cytotoxic lymphocyte (CTL, CD8) together with MHC class I antigen, while foreign protein is presented to CD4 lymphocyte together with MHC class II antigen after digestion by lysozyme. DNA vaccine seems to mimic live viral vaccine in priming CTL. However, long-term safety must be considered carefully for general use, particularly for children. Benefit/Risk and Cost/Effectiveness ratios should be taken into consideration for development of vaccines, and pathogenesis of diseases should also be well understood for evaluation of the vaccine. Live varicella vaccine has widely been used for prevention of chickenpox, and it is expected that vaccination of children would reduce the incidence of zoster in their later life. Immunization trial of the elderly with varicella vaccine to prevent zoster, particularly post herpetic neuralgia, is being done in a large scale in USA.

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Pirenoxine ophthalmic solution was developed based on the quinoid theory which is one of the theories for development mechanism of senile cataracts, and its usefulness for treatment of cataracts has been proved. Since pirenoxine is unstable in aqueous solution, conventional products are required the reconstitution procedure of tablet (or granule) with accompanied solvent before use. Therefore, there are some problems with respect to good patient's compliance. Under these circumstances, one bottle type and stable pirenoxine ophthalmic product has been desired. Our concepts for designing new pirenoxine ophthalmic product are as follows, (1) To be one bottle type product, (2) To stabilize pirenoxine in aqueous media for three years at room temperature, (3) To keep its bioequivalence to conventional products. Pirenoxine showed very low solubility in acidic aqueous solution and good stability in aqueous suspension (pH 3.4-4.0) for three years at room temperature in our study. On the other hand, pirenoxine showed good solubility in neutral aqueous solution. We applied these unique properties and developed a new pirenoxine ophthalmic product, Kary Uni^® ophthalmic suspension. As no buffer agent is contained in Kary Uni^® ophthalmic suspension, the pH of the solution is elevated to the physicological pH of tears immediately after instillation, and pirenoxine microparticles rapidly dissolve in tears. As a result, the bioequivalence to conventional products can be obtained. Kary Uni^® is a one bottle type ophthalmic suspension in ready to use, without the reconstitution procedure required for conventional products and is especially evaluated as a new type DDS pharmaceutical product taking advantage of the transformation on the living environment of ocular surface.

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非イオン性物質であるマンニトールのヘアレスラット摘出皮膚透過性に及ぼすエレクトロポレーションの効果を,陽陰の電極の位置,パルス頻度および電極材料をかえて測定した.まず,Ag電極(陽極)を角質層側(donor)に,Ag/AgCl電極(陰極)を真皮側(receiver)に配置したときと,この逆に電極を配置したときのマンニトールの皮膚透過性を比較した.いずれの電極配置でもエレクトロポレーション群では,非通電(コントロール)群にくらべてマンニトールの透過は著しく促進されたが,両エレクトロポレーション群のマンニトール透過に有意差はみられなかった.このことより,イオントフォレシスで報告されているような,convective flowによる促進効果は生じないことが示唆された.また,パルス発生装置のコンデンサー容量を1から25μFとすると透過速度が大きくなり,容量の増加に基づく通電時間の増加が透過促進に関係していることが示唆された.また,同様にパルス頻度を多くすると,通電時間の増加が原因と思われるマンニトールの透過促進が観察された.さらに,マンニトールの皮膚透過性に及ぼす電極位置の影響について試験した.皮膚を挟んで電極を配置した場合と陽極,陰極とも皮膚角質層側にセットした場台で,エレクトロポレーション効果に差はなかった.また,分極(Pt)電極と非分極(Ag/AgCl)電極を用いた場合を比較した結果,どちらの場合もマンニトールの透過はほぼ等しく,エレクトロポレーションでは電極の分極はほとんど引き起こらないか,もしくは促進効果に影響がないものと思われた.以上より,エレクトロポレーションによる薬物透過促進では,電極の位置,材料に影響されない点でイオントフォレシスと大きく異なるが,電圧の適用時間に大きく左右されることではイオントフォレシスと同様であることが明らかとなった.

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The influence of additives in topical formulation (cataplasm) on delivering piroxicam (Px) to local tissue underlying the application site was investigated. The additives were chosen from the group of fatty acids, their esters, carbohydrates, cyclic alcohols or polyhydric alcohols. In vivo experiments were carried out using guinea-pigs. Also, the solubility of Px in those additives was determined and compared with the plasma concentration at 8 hours after application of cataplasm containing corresponding additives. Those cataplasms containing high Px solubility additives showed relatively high plasma concentration, whereas those containing relatively low Px solubility resulted in lower plasma concentration. The inorganic/organic ratio of additives which showed the higher plasma concentration as well as the higher Px solubility was located in between 0.5 and 2.0 in organic conceptual diagram. The Px concentration in plasma and in muscle beneath the application was measured with the formulation containing 20% of propylene glycol (PG) and additives. Diisopropyl adipate (DIA), diethyl sebacate and triacetin showed relatively high plasma concentration at 8 hours after application. The ratio of muscle/plasma concentration by DIA was found to be the highest among them. The influences of the amount of DIA and PG on Px in plasma, viable skin, cutaneous muscle and muscle were investigated. DIA and PG increased the Px delivery to the local tissues with concentration dependent manner.

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筆者らは,合成糖蛋白質結合型リポソームを調製し,これを用いて膜面上での糖蛋白質とレクチンとの多価性相互作用に関する知見を得るとともに,ターゲティングキャリアとしての可能性を検討している.これまでの研究により,単糖および二糖と蛋白質(BSA)とリポソームとの化学的結合法ならびに反応条件を確立し,新規な合成穂蛋白質・リポソーム複合体の調製法を開発した.本研究では,さらに,酵素合成法を利用して糖残基を伸長し,オリゴ糖鎖を有するリポソーム複合体を合成してその特性を調べた.まず,既報の化学的手法によりN-アセチルグルコサミン-BSA結合リポソームを調製した.つぎに,3種の糖転移酵素を触媒として,糖残基(ガラクトース,シアル酸およびフコース)を付加することにより,シアリルラクトサミンやルイスX糖鎖結合型の合成糖蛋白質・リポソーム複合体が合成できた.これらの新規リポソームは,マイクロプレート表面に固定化されたレクチンと強い多価性の相互作用を示すとともに,担癌マウスを用いた体内動態解析で,糖鎖構造特異的な臓器移行性を有することが示唆された.

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One of the key technologies for regenerations of damaged and lost tissues is the controlled release of biologically active growth factors. The present study was undertaken to investigate sorption and desorption of various biologically active growth factors from hydrogels prepared through glutaraldehyde crosslinking of gelatin with isoelectric points (IEPs) of 5.0 and 9.0, which are named “acidic” and “basic” gelatins, respectively, because of their electric feature. In the present study, basic fibroblast growth factor (bFGF), transforming growth factor-β1 (TGF-/β1), vascular endothelial growth factor (VEGF), and bone morphogenetic protein-2 (BMP-2) were employed as growth factors, because their expected biological functions seem to be closely related to clinical applications. Basic bFGF and TGF-β1 were well sorbed with time to the acidic gelatin hydrogel, while less sorption was observed for the basic gelatin hydrogel. This can be explained in terms of the electrostatic interaction between the basic growth factors and acidic gelatin. However, VEGF and BMP-2, though their IEPs are higher than 7.0, were sorbed to the acidic gelatin hydrogel to a less extent than the two other factors. An in vivo experiment revealed that the hydrogels were degraded with time. The growth factors were retained in the body for a longer time period with their increasing sorption in vitro to the acidic gelatin hydrogel. A correlation of the in vivo retention between the growth factor and hydrogel suggested that the basic growth factor ionically complexed to the acidic gelatin hydrogel was released in vivo as a result of hydrogel degradation.

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The effects of dexamethasone palmitate incorporated into lipid emulsion(D-lipo) on foam cell formation and atherosclerosis model mice were examined. The cholesterol ester(CE) concentration in derived macrophages from mouse peritoneal cavity was increased markedly by oxidized LDL. The CE increment was associated with conversion of macrophages to foam cell. D-lipo significantly inhibited the CE increment on macrophages induced by oxidized LDL. The inhibition effect of D-lipo on foam cell formation was similar with that of dexamethasone phosphate as control. The atherogenic mouse was induced by the maintaining with an atherogenic diet for 14 weeks. D-lipo or dexamethasone phosphate was intravenously administered to mouse once a week from 8 to 14 weeks. The cholesterol concentration in serum and aortic CE deposition on the atherogenic mouse were significantly increased as compared to those of the control mouse. Although the increment of cholesterol concentration in serum was not changed, CE accumulation in the aorta of atherogenic mouse was inhibited by D-lipo(150 and 750 nmol/kg). Dexamethasone phosphate(750 nmol/kg) did not affect to cholesterol concentration in serum and CE accumulation in the aorta of atherogenic mouse. These findings suggest that D-lipo is useful as an anti-atherosclerosis.

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The pharmacological effect of lecithinized superoxide dismutase(PC-SOD) was evaluated using rat acute renal failure(ARF) model induced by ischemia-reperfusion. When PC-SOD was injected intravenously to rats 1 hr prior to ischemia, the increased plasma creatinine and BUN levels at 24 hr after reperfusion were significantly reduced. Treatment of PC-SOD immediately after reperfusion also reduced both of the increased ones. In while, inactivated PC-SOD(apo PC-SOD) did not show such effects at all. Unmodified SOD(U SOD) decreased the elevated plasma creatinine and BUN levels, but there was no significant difference. Renal SOD activity in ischemic ARF rats was significantly decreased, while renal myeloperoxidase(MPO) activity, a parameter of local infiltration of neutrophils was increased. The distribution study in ischemic ARF rats revealed that [^3H] PC-SOD persisted much longer in the blood stream, while fewer amounts were detected in the kidney organ than those of [^3H] U-SOD. The elevated renal MPO activity was significantly suppressed by PC-SOD. These results suggest that PC-SOD protected kidney injury in ischemic ARF rats by scavenging O_2^- on the cell membrane followed by inhibiting neutrophils infiltration.

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