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To enhance tumor magnetic resonance imaging (MRI) signals via the selective accumulation of contrast agents, we prepared folate-modified gadolinium-lipid-based nanoparticles as MRI contrast agents. Folate-modified nanoparticles were comprised of polyethylene glycol (PEG)-lipid, gadolinium diethylenetriamine pentaacetic acid lipid, cationic cholesterol derivatives, folate-conjugated PEG-lipid, and Cy7-PEG-lipid. Folate receptor-mediated cellular nanoparticle association was examined in KB cells, which overexpress the folate receptor. The biodistribution of nanoparticles after their intravenous injection into KB tumor-bearing mice was measured. Mice were imaged through in vivo fluorescence imaging and MRI 24 h after nanoparticle injection, and the intensity enhancement of the tumor MRI signal was evaluated. Increased cellular association of folate-modified nanoparticles was inhibited by excess free folic acid, indicating that nanoparticle association was folate receptor-mediated. Irrespective of folate modification, the amount of nanoparticles in blood 24 h after injection was ca. 10% of the injected dose. Compared with non-modified nanoparticles, folate-modified nanoparticles exhibited significant accumulation in tumor tissues without altering other biodistribution, as well as enhanced tumor fluorescence and MRI signal intensity. The results support the feasibility of MRI- and in vivo fluorescence imaging-based tumor visualization using folate-modified nanoparticles and provide opportunities to develop folate targeting-based imaging applications.

Previously, we prepared cationic nanoparticles (NP and NP-N) composed of cholesteryl diamine (OH-Chol, (3S)-N-(2-(2-hydroxyethylamino)ethyl)cholesteryl-3-carboxamide) and cholesteryl triamine (OH-N-Chol, (3S)-N-(2-(2-(2-hydroxyethylamino)ethylamino)ethyl)cholesteryl-3-carboxamide), respectively, with Tween 80 for small interfering RNA (siRNA) delivery into tumor cells. In this study, we prepared NP-0.25 N composed of OH-Chol and OH-N-Chol at a molar ratio of 3/1 with Tween 80, and evaluated the transfection efficiency of plasmid DNA (pDNA) into tumor cells. NP-N exhibited lower transfection activity than NP; however, NP-0.25 N showed higher transfection activity than both NP and NP-N in various tumor cells. NP-0.25 N increased the amount of internalized pDNA by increased cellular association, and improved the escape from endosomes after clathrin-mediated endocytosis. The results of the experiments suggested that cholesteryl triamine may have potential as a helper lipid to increase the transfection for pDNA delivery by cationic cholesterol-based nanoparticles.

It was believed for a long time that mRNA is very unstable, and can not be used for therapeutic purposes. In the last decade, however, many research groups proved its transfection feasibility along with advantages and applications. Our investigation is aimed at establishing a potent and efficient mRNA delivery system. We previously reported that an inorganic-organic hybrid carrier by exploiting the advantages of inorganic nano apatite particles onto organic carrier DOTAP {N-[1-(2,3-dioleoloxy)propyl]-N,N,N-trimethyl ammonium chloride} and showed potential effect of carbonate apatite particles on each of the mRNA delivery steps in dividing and non-dividing cell. Here, we report on the development of a more efficient mRNA carrier by complexing ECM protein, fibronectin with the DOTAP-apatite carrier. The carrier showed enhanced uptake of luciferase mRNA both qualitatively and quantitatively. Accelerated cellular endocytosis rate was evaluated using labeled endosome. Finally expression of lucifearse mRNA was higher for fibronectin complexed carrier in compared to the uncoated one.

In this study, we investigated the effect of manufacturing factors such as particle size, water content and manufacturing method on the physical stability and solubility of solid dispersion formulations of a low-glass-transition-temperature (T(g)) drug. Solid dispersions were prepared from polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC) by hot melt extrusion or spray drying. Water content of solid dispersions prepared by hot melt extrusion determined by dynamic moisture sorption measurement was increased drastically with relative humidity below a certain level of particle size. The blends with a lower water content (0.8%) prepared by hot melt extrusion during storage were more stable than those with a higher water content (3.5%) prepared by spray drying, which caused rapid recrystallization. Physical stability in the hot melt blends may be attributed to reduced molecular mobility due to a higher T(g). Dissolution study revealed that solid dispersions prepared by hot melt extrusion with the smallest particle size showed decreased solubility, attributed to reduced wetting properties (surface energy), which is not predictable by the Noyes-Whitney equation. Taken together, these results indicate that the control of particle size concerned in water content or wetting properties is critical to ensuring the physical stability or enhancing solubility of low-T(g) drugs. Further, hot melt extrusion, which can reduce water content, is a suitable manufacturing method for solid dispersions of low-T(g) drugs.

The purpose of this study was establishing a solid dispersion formulation containing a low glass transition temperature (T(g)) and poorly water-soluble drug. Drug/polymer blends with differing physicochemical stabilities and oral absorption were prepared from copolyvidone (PVP-VA), polyvinylpyrrolidone (PVP) or hydroxypropylmethylcellulose (HPMC) by a hot melt extrusion. HPMC drastically increased the drug oral absorption property, while PVP-VA or PVP stabilized solid dispersions during storage by increasing the T(g) in proportion to polymer concentration. Experimental T(g) values corresponded closely with theoretical T(g) values; indeed, the T(g) values of solid dispersion with HPMC did not increase significantly compared to the T(g) value for the drug alone. A solid dispersion formulation incorporating two different polymers-HPMC and either PVP-VA or PVP-maintained increased T(g), physicochemical stability, solubility, and bioavailability of the solid dispresions owing to each polymer. These findings suggested that both oral absorption and physicochemical stability of low-T(g) drug will be improved using less amount of solid dispersion of combined two polymers than polymer alone.

Everolimus has demonstrated antitumor efficacy for various cancers as a result of its inhibition of the mammalian target of rapamycin (mTOR) signaling cascade, which activates cell growth and cell proliferation. However, the low water solubility and low bioavailability of everolimus have prevented its clinical development as an anticancer drug. Therefore, to address the unsuitable characteristic of everolimus, we attempted to prepare liposomal everolimus as a viable drug delivery system, and then evaluated the anticancer efficacy of this system against a medullary thyroid carcinoma cell line (TT cells), a breast cancer cell line (MCF-7 cells) and a small lung carcinoma cell line (NCI-H446 cells). The particle size and entrapment efficacy of liposomal everolimus was ca. 80 nm and more than 90%, respectively. Liposomal everolimus showed higher cytotoxicity against NCI-H446 cells compared with TT cells. Against NCI-H446 tumors, significant suppression of the tumor volume was observed in liposomal everolimus-treated mice by intravenous injection, compared with free everolimus-treated mice by intraperitoneal injection, at a dose of 5 mg/kg without body weight loss. This study showed that liposomal everolimus could be a powerful formulation with anticancer efficacy for some cancers.

Menthosomes, novel deformable carriers for the enhancement of transdermal delivery are introduced in this study. Meloxicam (MX)-loaded menthosomes were formulated, and their physicochemical characteristics and skin permeability were evaluated. A two-factor spherical and second-order composite experimental design was used to prepare the formulation of the menthosomes. Ten formulations of menthosomes composed of a phospholipid as the lipid bilayer carrier, cholesterol (Chol) as a stabilizer and cetylpyridinium chloride (CPC) and L-menthol as penetration enhancers were prepared. The amounts of Chol and CPC were selected as causal factors. Physicochemical characteristics (particle size, size distribution, zeta potential, elasticity and drug content) and an in vitro skin-permeation study of meloxicam-loaded menthosomes were evaluated. The concentrations of MX that permeated the skin at 2-12 h and the flux were selected as response variables. The optimal formulation was estimated using a nonlinear response-surface method incorporating thin-plate spline interpolation. The experimental values were very close to the values predicted by the computer programs in this study. A Bayesian network analysis was applied to gain a mechanistic understanding of the relationships between causal factors and response variables.

Histone deacetylase inhibitor (HDACI), suberoylanilide hydroxamic acid (SAHA), approved by the Food and Drug Administration (FDA) for the treatment of cutaneous T cell lymphoma, is a promising new treatment strategy for various cancers. In this study, we hypothesized that a liposomal formulation of HDACI might efficiently deliver HDACI into tumors. To incorporate HDACI efficiently into the liposomal membrane, we synthesized six HDACI-lipid conjugates, in which polyethylene glycol(2000) (PEG(2000))-lipid or cholesterol (Chol) was linked with a potent hydroxamic acid, HDACI, SAHA or K-182, by cleavable linkers, such as ester, carbamide and disulfide bonds. Liposomal HDACI-lipid conjugates were prepared with distearoylphosphatidylcholine (DSPC) and HDACI-Chol conjugate or with DSPC, Chol and HDACI-PEG-lipid conjugates, and their cytotoxicities were evaluated for human cervix tumor HeLa and mouse colon tumor Colon 26 cells. Among the liposomes, liposomal oleyl-PEG(2000)-SAHA conjugated with SAHA and oleyl-PEG(2000) via a carbamate linker showed higher cytotoxicity via hyperacetylation of histone H3 and induction of caspase 3/7 activity. These results suggested that liposomal HDACI-lipid conjugates may be a potential tool for cancer therapy.

In this study, to establish the conditions of a drug release assay for PEGylated liposome formulations that relates with the drug stability profile in serum in vivo, the influences of incubation temperature and serum protein in the release buffer were examined using liposomal doxorubicin (DXR). In in vitro drug release assays, a PEGylated liposomal DXR in phosphate buffered saline (PBS) at 37 degrees C showed higher drug release rate than non-PEGylated formulation although PEGylated liposomal DXR had higher stability than an equivalent non-PEGylated formulation following intravenous injection. When bovine serum albumin (BSA) and increased temperature, 50 degrees C, were used to accelerate drug release from the liposomes and to mimic in vivo result, non-PEGylated liposomal DXR showed conversely higher release than a PEGylated formulation. Since high temperature increased BSA adsorption onto liposomes, BSA may cause non-PEGylated liposomes instability more than PEGylated ones, resulting in the reverse of the drug release rate of both liposomes. This finding suggested that the conditions in the drug release assay with PEGylated liposomal DXR may be able to be set by a combination of BSA and providing additional thermal energy.

The hydrodynamic method by rapid intravenous injection of a large volume of plasmid DNA is known to be an efficient and liver-specific method of in vivo gene delivery and achieves high levels of foreign gene expression, particularly in hepatocytes. Low transgene activities have also been observed in other organs such as the spleen and lung; however, the expression profiles of mRNA and protein are still unknown. Therefore, we investigated the localization of luciferase mRNA by in situ hybridization and luciferase activity in mice after transfection of pCMV-luc encoding the luciferase gene under the control of cytomegalo virus (CMV) promoter. We found that hydrodynamic injection effectively induced mRNA expression of the transgene only in the liver although transgene activities were observed in other organs. The transgene activity observed in other organs may be due to leakage from hepatocyte gene expression by transient increase in the permeability of the hepatocyte cellular membrane caused by increased pressure by hydrodynamic injection.

As reported previously, cationic liposomes formulated with dioleoylphosphatidylethanolamine (DOPE) and N,N-methyl hydroxyethyl aminopropane carbamoyl cholesterol (MHAPC-liposomes) achieved efficient gene transfection in the mouse lung following intratracheal injection. We have studied here the role of surfactants, mannosylerythritol lipid-A (MEL-A) and polysorbate 80 (Tween 80), in affecting gene transfection of MHAPC-lipoplexes (complex with pCMV-luc DNA) in A549 cells and in the mouse lung. MEL-A increased gene transfection of MHAPC-lipoplexes significantly in vitro and slightly in the mouse lung, while Tween 80 decreased it both in vitro and in vivo. As assessed by confocal laser scanning microscopy and fluorescence imaging, MEL-A might faciliate gene dissociation from MHAPC-lipoplexes with fluorescein-labeled oligodeoxynucleotide (FITC-ODN) after internalization into the cells and retained the lipoplexes in the mouse lung for prolonged time, while Tween 80 was inefficient to deliver foreign gene into target cells and in the lung. These results demonstrated that MEL-A is advantageous to Tween 80 in the modification of cationic liposomes as gene delivery vectors in the lung.

The surface properties of cationic liposomes and lipoplexes largely determine the cellular association and gene transfection efficiency. In this study, we measured the surface properties, such as zeta potentials, surface pH and hydration levels of MHAPC- and OH-Chol-lipoplexes and their cellular association, without and with the modification of biosurfactant mannosylerythritol lipid-A (MEL-A) or Tween 80 (MHAPC=N,N-methyl hydroxyethyl aminopropane carbamoyl cholesterol; OH-Chol=cholesteryl-3beta-carboxyamindoethylene-N-hydroxyethylamine). Compared to OH-Chol-lipoplexes, the higher cellular association of MHAPC-lipoplexes correlated with the significantly higher zeta potentials, lower surface pH levels and "drier" surface, as evaluated by the generalized polarization of laurdan. Both MEL-A and Tween 80 modification of MHAPC-lipoplexes did not significantly change zeta potentials and surface pH levels, while MEL-A modification of OH-Chol-lipoplexes seriously decreased them. MEL-A hydrated the liposomal surface of MHAPC-lipoplexes but dehydrated that of OH-Chol-lipoplexes, while Tween 80 hydrated those of MHAPC- and OH-Chol-lipoplexes. In all, cationic liposomes composed of lipids with secondary and tertiary amine exhibited different surface properties and cellular associations of lipoplexes, and modification with surfactants further enlarged their difference. The strong hydration ability of Tween 80 may relate to the low cellular association of lipoplexes, while the dehydration of MEL-A-modified OH-Chol-lipoplexes seemed to compensate the negative zeta potential for the cellular association of lipoplexes.

The RNA interference (RNAi) effect is an alternative technology to antisense DNA as an experimental method of down-regulating a specific target protein. Optimal gene therapy for tumors must deliver synthetic small interfering RNA (siRNA) to tumor cells with high efficiency and minimal toxicity. Previously, we reported that cationic nanoparticles composed of cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine and Tween 80 (NP-OH) could deliver plasmid DNA with high transfection efficiency. In this study, to apply NP-OH for siRNA transfection, we optimized the charge ratio (+/-) of NP-OH/siRNA and nanoplex-forming solution, and evaluated the transfection efficiency into PC-3 cells. Positively charged nanoplex prepared in the presence of sodium chloride exhibited efficient siRNA transfer. The distribution of siRNA after transfection was strongly detected both in the cytoplasm and nucleus. Furthermore, we demonstrated that NP-OH nanoplex of bcl-2 siRNA significantly inhibited tumor growth compared with control siRNA, and the efficacy was comparable to commercial products. The results of the experiments showed that NP-OH nanoparticles have potential as a viable vector candidate for synthetic siRNA delivery.

Camptothecin (CPT) has anticancer activity. While only the lactone form of CPT is biologically active, this form exhibits poor aqueous solubility. Pharmaceutical formulation of CPT incorporated in liposomes is of significant importance to develop the therapeutic utilization of CPT. The aim of this study was to increase incorporation efficiency and stability of CPT in liposomes composed of hydrogenated soybean phosphatidylcholine, cholesterol, and oleic acid (7 : 3 : 1, molar ratio), by incorporating three kinds of artificial lipids (DBs) (DB-liposome); 4-n-(M12B), 3,5-bis(B12B) and 3,4,5-tris(dodecyloxy)benzoic acid (T12B). The interaction of CPT with DB in the state of liposomes, was examined. In DB-liposomes presenting mean diameters of 150 nm, incorporation efficiency of CPT up to 55% and final drug to lipid molar ratio up to 0.07 were obtained when the liposomes were prepared at a feeding ratio of 1/30 (w/w) CPT/total lipid. However, in the optimal formulations, incorporated DB mol% was different; T12B and D12B were incorporated about one third and half mol% of M12B, respectively. Moreover, we demonstrated that T12B stabilized CPT in liposomes significantly compared with other DBs as measured by CPT release, and by steady state fluorescence polarization degree of CPT using intrinsic fluorescence of CPT. These findings suggested that in addition of contribution of phenyl group of DB, dodecyloxy group may interact strongly with lactone ring of CPT. The capacity to contain CPT interacted with DBs may be limited in liposomes. T12B may be incorporated in the interior of the bilayers, resulting in increase of incorporation stability of CPT. This finding demonstrates a potential application of the novel liposome formulation of CPT in drug delivery.

Both polyethylenimine (PEI) polymers and cationic nanoparticles have been widely used for non-viral DNA transfection. Previously, we reported that cationic nanoparticles composed of cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine and Tween 80 (NP-OH) could deliver plasmid DNA (pDNA) with high transfection efficiency. To increase the transfection activity of NP-OH, we investigated the potential synergism of PEI and NP-OH for the transfection of DNA into human prostate tumor PC-3, human cervices tumor Hela, and human lung adenocarcinoma A549 cells. The transfection efficiency with low-molecular PEI (MW 600) was low, but that with a combination of NP-OH and PEI was higher than with NP-OH alone, being comparable to commercially available lipofectamine 2,000 and lipofectamine LTX, with very low cytotoxicity. Low-molecular weight PEI could not compact pDNA in size, but rather might help to dissociate pDNA from the complex and release pDNA from the endosome to cytoplasm by the proton sponge effect. Therefore, the combination of cationic cholesterol-based nanoparticles and a low-molecular PEI has potential as a non-viral DNA vector for gene delivery.

Previously we have described the development and applications of lipid-based nanoparticles for gene delivery vector. In an attempt to improve transfection efficiency using the cell adhesion of extracellular matrix (ECM) to DNA/lipid complex (nanoplex), the mRNA expression of integrin alpha2beta1 and CD44 in prostate cancer cells was detected as adhesion molecules for fibronectin (Fn), collagen I (Col) and laminin (Lam) using a commercially available cDNA array (GEArray) system. These ECM proteins could enhance DNA transfection activity in cells when coated on the nanoplex. Among the ECM proteins, Fn-coating nanoplexes significantly increased transfection activity 2-fold in prostate cancer PC-3 cells, and exhibited higher DNA transfection activities to PC-3 xenografts, compared with commercially available cationic polymer in vivo jetPEI. These results indicated that Fn-coating nanoplexes could facilitate efficient transfection of prostate tumor cells.

Activated macrophages are the key effector cells in rheumatoid arthritis (RA) and secrete multiple mediators of inflammation including proinflammatory cytokines. We investigated delivery of a nuclear factor kappa B (NFkappaB) decoy by folate-linked lipid-based nanoparticles (NP-F) into murine macrophages. The expression of folate receptor (FR) in RAW264.7 cells activated by lipopolysaccaride was confirmed by strong expression of FR mRNA, and association of FITC-labeled folate-BSA conjugate. When transfected via NP-F, the NFkappaB decoy was strongly detected in the cytoplasm, and an inhibitory effect on the translocation of NFkappaB into the nucleus was observed at 0.03 microM of the decoy, suggesting that NP-F effectively delivered the NFkappaB decoy into the cytoplasm. This information is of value for the design of NFkappaB decoy carrier systems targeting FR in activated macrophages in gene therapy for autoimmune diseases such as RA.

Retinoic acid (RA), a potent inducer of cell differentiation, and N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide), a potent inducer of apoptosis, are well known as anticancer agents that are administered orally to patients for leukemia, breast and prostate cancer, respectively. However, it has not been studied whether both retinoids are effective on metastatic cancer. In mice implanted with M5076 cells, murine reticulum cell sarcoma survival times were prolonged by i.v. treatment of RA and 4-HPR entrapped in liposomes containing soybean-derived sterylglucoside mixture (SG), which accumulates in liver. In contrast, free RA and 4-HPR were inactive. These results indicate that RA and 4-HPR in SG-liposomes exhibit anticancer efficacy on metastatic cancers, and may have great potential for clinical use in the treatment of various cancers.

The aim of this study was to examine the interaction of soybean-derived sterylglucoside (SG) with the human hepatoblastoma cell line HepG2 with regard to the penetration-enhancing effect of beta-sitosterol glucoside (Sit-G) to clarify the accumulation of SG-containing liposomes (SG-liposomes) to the liver in vivo. The approach was based on measurement of the association of SG-liposomes labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) in terms of asialoglycoprotein receptor (ASGP-R)-mediated endocytosis, affinity of Sit-G using lAsys and the association of FITC-dextran 4400 (FD-4) increased by Sit-G with the cells. The association of SG-liposomes was decreased by addition of asialofetuin, suggesting that SG-liposomes might be taken up via ASGP-R. Sit-G showed higher affinity with HepG2 cells than HeLa cells, and enhanced the association of FD-4 depending on the incubation time and Sit-G concentrations. Significant positive correlations were found between Sit-G and FD-4 association with the cells, indicating that Sit-G enhanced the drug penetration by distribution in cell membranes. The high degree of liver association of SG-liposomes in vivo might be related to recognition of glucose residues of SG by ASGP-R and to the high affinity and penetration-enhancing effect of Sit-G with hepatocytes.

The effects of soybean-derived sterylglucoside (SG) on the fluidity of liposomal membrane composed of dipalmitoylphosphatidylcholine (DPPC) were investigated compared with those of soybean-derived sterol (SS) and cholesterol (Ch) using an electron spin resonance spectrometer. Three kinds of liposomes were prepared in the molar ratio of DPPC/X=7/4, where X is SS, Ch or SG. The fluidity close to the polar head groups increased with an increase of temperature in the DPPC membrane containing SS, Ch and SG in the range 35 to 45 degrees C. Those near the hydrophobic end changed with an increase in temperature in liposomes containing SS, Ch and SG, which had a fluidizing effect on the DPPC membrane below the transition temperature (Tm, 41.9 degrees C) and a condensing effect over the Tm. The fluidizing effects of these compounds around 37 degrees C near the polar head group and the hydrophobic end increased in the following order: Ch < SG < or = SS and SS < Ch < SG, respectively. SG increased the fluidity of liposomal membrane dramatically above the Tm (35.4 degrees C). These results suggest that the high fluidity close to the hydrophobic end of the liposomal membranes around 37 degrees C, the decrease of Tm, and the sigmoidal nature of fluidity vs. temperature are important factors in the effectiveness of liposomes containing SG as a carrier of drugs.

Remote loading of insulin and bovine serum albumin (BSA) into neutral or positively-charged liposomes by incubation under a transmembrane pH gradient or non-pH gradient was investigated. Trapping efficiencies in several incubation conditions were compared with those of the conventional reverse-phase evaporation vesicle method (c-REV method). For neutral liposomes, insulin could not be effectively loaded into the liposomes by incubation, regardless of the incubation conditions. The trapping efficiency of insulin into positively-charged liposomes was higher than that of neutral liposomes, especially by the pH-gradient method. Insulin could be loaded into positively-charged liposomes about twofold more efficiently than by the pH-gradient method, compared with the c-REV method. Insulin distributed more on the surface of liposomes by the pH-gradient method than by the c-REV method. BSA showed significantly higher affinity to positively-charged liposomes than to neutral ones by various methods. However, the transmembrane pH-gradient method did not increase BSA loading into liposomes, compared with the c-REV and the non-pH-gradient methods. Our results suggest that the pH-gradient method, combining electrostatic interactions, may be useful for preparation of liposomal insulin and that the high hydrophobicity of BSA may not increase the remote loading of BSA into liposomes by the pH-gradient method.

The effect of shikonin (SK) on granulation tissue formation was biochemically evaluated and the biological effect of SK on cotton pellet induced granulation tissue formation and the induction of hind paw edema was compared with that of lambda-carrageenan (carrageenan) in rats. The dry weight of granulation tissue formed was increased by SK. The amounts of hemoglobin and hydroxyproline in granulation tissue were also increased by SK. These results suggest that SK has an enhancing effect on the formation of granulation tissue, accompanied by the proliferation of capillaries and the increased production of collagen in rats. SK showed mild stimulation toward swelling when injected into the hind paws of rats, as did carrageenan, while it showed a marked enhancing effect on granulation tissue formation compared with carrageenan. These results suggest that the mechanism underlying the biological effect of SK on granulation tissue formation is different from that of carrageenan, though the mild stimulation by SK might still contribute in part to the enhancing effect on granulation tissue formation.

The effect of a soybean-derived sterol mixture (SS) and a steryl glucoside mixture (SG) as enhancers of the nasal absorption of insulin in rabbits was investigated. SS consists of beta-sitosterol (Sit), campesterol (Camp), stigmasterol (Stig) and brassicasterol (Bras), and SG is a mixture of their monoglucosides. For each component of SS tested for efficacy in promoting the systemic absorption of nasally administered insulin, the following order was observed: Sit> or =Camp>Stig. This finding was in agreement with the order of the enhancers' lipophilicity. In the case of SG, the effect of beta-sitosterol beta-D-glucoside (Sit-G) was significantly greater than that of SG. The pharmacological bioavailability was 6.7% for SG and 11.3% for Sit-G in the suspension dosage forms. SG showed a greater degree of enhancement of insulin permeation through the nasal mucosa than SS. To elucidate the contribution of SG to the enhanced absorption, insulin permeation through an artificial membrane and the nasal mucosa was investigated in vitro, and the results were compared with those for SS. The findings suggest that SG and SS have some effect on nasal mucosa lipids.

A soybean-derived sterylglucoside mixture (SG) is a potential enhancer of the nasal absorption of insulin. The aim of the study was to examine the absorption of insulin given as the powder dosage form with SG and excipient and to determine the subchronic effects of SG on the morphology of rabbit nasal epithelium. The insulin powder dosage form with SG was administered to the rabbit nasal cavity for five successive days. The average bioavailability and the average pharmacological bioavailability of insulin were about 25.0 and 61.6%, respectively. The nasal mucosa was taken from the nasal cavity and side-effects were investigated using an optiphoto light microscope. The insulin powder dosage form with SG produced no signs of inflammation, erosion or squamous metaplasia. These findings indicate that SG can be considered as a safe and effective enhancer and excipient in the nasal insulin powder dosage form.

Dipalmitoylphosphatidylcholine (DPPC) and cholesterol (Ch) liposomes containing a soybean-derived sterylglucoside mixture (SG) (SG-liposomes) are effective for targeting hepatocytes in mice. We investigated uptake of SG-liposomes to hepatocytes compared with that via the galactose receptor. The association of SG-liposomes entrapping calcein was examined at 4 degrees C or 37 degrees C, changing incubation time and the SG concentration of the liposomes, and using the inhibitor on the galactose receptor in rat primary cultured hepatocytes. The amount of liposomes recovered with hepatocytes was determined by measuring the concentration of calcein and imaged with confocal laser scanning microscopy in the cultured cells. The association of SG-liposomes at 4 degrees C was significantly decreased compared with that at 37 degrees C as that of liposomes containing lactosylceramide (LC) (LC-liposomes) that were already known to be taken up by hepatocytes via the asialoglycoprotein receptor. While the association of SG-liposomes at both 4 degrees C and 37 degrees C increased with the increase of incubation time, the association of SG-liposomes at 37 degrees C was almost saturated after 1 h. The association of SG-liposomes pretreated with concanavalin A was significantly decreased (to about 40% of that without pretreatment at 37 degrees C) and was at the same level as the association of SG-liposomes and non-SG-liposomes at 4 degrees C. The association of the SG-liposomes by hepatocytes incubated for I h at 37 degrees C was almost saturated at about 2.0 nmol SG/mg protein. The association of the SG-liposomes with hepatocytes was not inhibited in the presence of LC-liposomes. The affinity of the galactose residue for hepatocytes appeared to be similar to that of the glucose residue in the liposomes, because the amount of sugar residue and the association of SG-liposomes and LC-liposomes were almost the same values. These results suggest that SG-liposomes may be associated with hepatocytes not by a galactose receptor-mediated endocytosis.

The blood clearance, tissue uptake and antitumor efficacy against liver metastasis of M5076 reticulosarcoma in mice and against primary liver cancer in rats of doxorubicin (DOX) encapsulated in two types of liposomes, with and without a soYbean-derived sterylglucoside mixture (SG), were examined. Liposomes entrapping DOX were composed of dipalmitoylphosphatidylcholine (DPPC), SG and cholesterol (Ch) at a molar ratio of 6:1:3, (SG-liposomes) and 6:0:4 (non-SG-liposomes). Pharmacokinetic analysis of drug disposition was based on the area under the curve (AUC) for liposomes up to 24 h following i.v. injection. SG-liposomes showed lower DOX concentrations in blood and higher concentrations in liver compared with non-SG-liposomes. The highest AUC of SG-liposomes in tissue was in liver, 2.4 times higher than that of the free drug. The antitumor efficacy of SG-liposomes was compared with that of free DOX and non-SG-liposomes at a dose of 5 mg DOX/kg. SG-liposomes displayed stronger antitumor activity than the free drug and non-SG-liposomes in murine reticulosarcoma M5076 tumor models and primary liver cancer models reflecting accumulation in hepatocytes. The antitumor activity of SG-liposomes in rats with primary liver cancer was significantly higher compared with free DOX and non-SG-liposomes (ILS: 92.7%).

In this study, we investigated a liposomal formulation of dipalmitoylphosphatidylcholine (DPPC) with a soybean-derived sterylglucoside mixture (SG) and cholesterol (Ch) for targeting the liver in mice. We found two distribution profiles of liposomes (reverse-phase evaporation vesicles, REV) in the liver depending on the concentration of Ch and SG in vivo; Ch tends to enhance liposomes containing 10 mol% SG accumulation in the liver, but to decrease those containing 20 mol% SG, and it prolongs the circulation in blood. Dicetylphosphate did not enhance liver targeting by liposomes with SG and Ch. More DPPC/SG/Ch-liposomes (6:1:3) were significantly taken up by cultivated hepatocytes than DPPC/SG/Ch-liposomes (6:0:4), suggesting a glucose residue. The optimal composition for the maximal liver targeting was a mixture of 0.2 micron DPPC/SG/Ch-liposomes (REV) at a molar ratio of 6:1:3. This composition of liposomes was distributed 3 times greater in hepatocytes than non-parenchymal cells 1 h after intravenous injection.

Dipalmitoylphosphatidylcholine (DPPC) liposomes with a soybean-derived sterol mixture (SS) or cholesterol (Ch) were examined as a carrier for the oral administration of insulin in rats. Four kinds of liposomes were prepared: liposomes containing SS or Ch (molar ratio of DPPC/X = 7:2 or 7:4, X = SS or Ch), respectively. The pharmacological availability was greater and the lag time for the glucose reduction was later in the order of DPPC/SS (7:4) > DPPC/SS (7:2) > DPPC/Ch (7:4) > > DPPC/Ch (7:2)-liposomes. This order appears to correspond well with that of the rigidity of the liposomal membrane. In particular, DPPC/SS (7:4)-liposomes reduced blood glucose levels for up to 21 h in rats after oral administration. The highest absolute pharmacological availability was 31.6% at a dose of 20.0 IU/kg of DPPC/SS (7:4)-liposomes among the liposomes in comparison to intravenous administration.

The mechanism of l-menthol, oleic acid and lauricidin as enhancers on percutaneous absorption was examined using diclofenac (DH) as a hydrophobic drug and sodium diclofenac (DNa) as a hydrophilic drug in in vitro diffusion experiments with two kinds of membranes: ethanol-treated and untreated silicone membranes; these were models for the lipid and pore pathways of skin. A 20% w/w ethanol-aqueous solution decreased the flux of DH but increased the flux of DNa significantly across the treated membrane compared with those fluxes across the untreated membrane, suggesting that DH penetrated by the lipid pathway and DNa by the pore pathway. The permeability of DNa through the pore pathway decreased significantly across the treated membrane with the addition of oleic acid and lauricidin. l-Menthol increased the permeability coefficients of DH and DNa more in a treated membrane than in an untreated one, showing the same tendency as in rat skin. Thus, while oleic acid and lauricidin did not increase the permeation of DNa by the pore pathway, l-menthol appeared to enhance the permeation of the drug by both the lipid and pore pathways.

The effects on Wistar rat body weight were examined after a single subcutaneous (s.c.) or oral (p.o.) administration of dipalmitoylphosphatidylcholine (DPPC) liposomes composed of soybean-derived sterols (SS) and their glucosides (SG) with or without entrapping recombinant human erythropoietin (Epo) for 1 week. Body weight increased significantly after both types of administration compared with the control groups irrespective of the existence of Epo. The neutral lipid concentration in plasma increased with the increase in body weight whereas the total contents of cholesterol and high density lipoprotein cholesterol in the plasma did not change significantly. The SS and SG suspensions following p.o. administration, however, did not alter the body weight. These findings suggest that liposomal SS and SG may be absorbed through the intestinal membrane and induce a change in the uptake of lipid, in contrast to the suspension state. SS in liposomes significantly increased body weight more than SG after p.o. administration.

Soybean-derived sterylglucoside mixture (SG) is a potentially effective absorption enhancer for the nasal absorption of insulin. Insulin is a peanut oil suspension dosage form and in a powder dosage form with SG were administered into the rabbit nasal cavity. After 0.5 or 1 h, nasal mucosa was taken from the nasal cavity and side effects were examined by an optiphoto light microscope. The insulin in the peanut oil suspension produced a histological change in the nasal epithelium mucosae, as well as mucodermal phlogistic cellular infiltration. The peanut oil suspension with SG showed side effects slightly stronger than without SG. SG alone and the insulin powder dosage form with SG produced no signs of inflammation, erosion or squamous metaplasia. The results from this study indicate that SG can be considered safe.

The effects of dose, pH and osmolarity on the intranasal absorption of a recombinant human erythropoietin (rEPO) solution were studied in male Wistar rats. The intranasal administration of rEPO was evaluated by measuring percentage circulating reticulocytes of red blood cells on a stained blood smear (smear method), and also by measuring residual circulating reticulocyte counts using a microcell counter (sysmex method). Both results suggest that rEPO solution was absorbed through the nasal mucosa of rats without enhancers after a single intranasal administration. The pharmacological availabilities of rEPO after intranasal administration compared with intravenous administration were about 7% and 4%, when estimated by smear method and sysmex method, respectively. The pharmacological activity was enhanced in low pH and hypotonic mannitol solution.

We encapsulated erythropoietin (Epo) in dipalmitoylphosphatidylcholine (DPPC) liposomes with soybean-derived sterols (SS-liposomes) and its glucoside (SG-liposomes) by reverse-phase evaporation vesicle method, and evaluated them by subcutaneous administration in rats. With 4 min of sonication, the damage to Epo activity was observed mainly in the non-encapsulated Epo in the liposomes. This study indicated that the bilayer of liposomes had the ability to protect the Epo activity, by reducing the aggregation that was caused by interaction between Epo molecules. The SG-liposomes had a higher retention of the Epo activity the SS-liposomes. 25.3% or 33.6% of activity was retained by SS-liposomes under the conditions of 4 min or 1 min of sonication, while 53.3% or 58.3% of the activity was retained by SG-liposomes under the same conditions. Shorter sonication was available to minimize the loss of the Epo activity. Epo in SG-liposomes appeared to increase the activity.

The promoting effect of d-limonene and l-menthol on the percutaneous absorption of nonionized and ionized diclofenac (DF) was investigated employing a pretreatment method. After the pretreatment of hairless rat skin with an ethanol buffer solution containing terpenes, the permeation study was performed in vitro. The permeability coefficients of nonionized (Pn) and ionized diclofenac (Pi) were calculated under the assumption that the total flux was composed of individual fluxes of nonionized and ionized DF. In the case of pretreatment with d-limonene, both Pn and Pi values were increased dramatically. However, the promoting magnitude was not affected by an extension of the pretreatment period. In contrast, when the skin was pretreated with l-menthol, the Pn and Pi values increased gradually as the pretreatment period increased. Based on the measurement of the solubility of terpenes in the pretreatment solution, the difference in promoting efficiency could arise from the difference in thermodynamic activity of the terpenes.

The permeability of liposome entrapping insulin through the nasal mucosa of rabbit has been studied and compared with the permeability of insulin solution with or without pretreatment by sodium glycocholate (GC). Insulin entrapped in liposome was not detected in the receiver cell using the diffusion cells with the nasal mucosa. On the other hand, permeability of insulin entrapped in liposome increased after the pretreatment of GC. The phospholipids which result from liposomes, were not observed in the receiver. Also, the GC remaining in the nasal mucosa was measured. Considering the mechanism of permeation of insulin entrapped in liposome through the nasal mucosa, the GC remaining in the nasal mucosa may cause the lysis of liposomes.

In previous studies on the nasal administration of human fibroblast interferon (HuIFN-beta), only rabbits have been used. Therefore, this route was investigated in mice, rats, rabbits and dogs. HuIFN-beta could be delivered across the nasal mucosa in mice, rats, rabbits and dogs, when it was mixed with sodium glycocholate as an absorption promoter. However, the pattern of the plasma HuIFN-beta concentration-time curve was different from that in rabbits. Rabbits gave the highest value of the maximum plasma HuIFN-beta concentration (Cmax), but plasma HuIFN-beta declined rapidly thereafter, upon nasal administration of the powder dosage form (8.45 x 10(2) IU/g). In rats and dogs, Cmax was lower than in rabbits, but plasma HuIFN-beta declined slowly after nasal administration of the powder or liquid dosage form (4-6 x 10(2) IU/g). These differences might be attributed to differences of absorption rate constant.

The utility of the absorption promoters, sodium glycocholate (GC-Na), ethylenediamine dihydrochloride (EDTA-2Na), sodium caprylate (Cap-Na) and sodium salicylate (Sal-Na), in the intranasal administration of human fibroblast interferon-beta (HuIFN-beta) in rabbits was investigated. The optimal amount of added EDTA-2Na, Cap-Na and Sal-Na with respect to HuIFN-beta was examined for nasal absorption in the powder dosage form. Formulations of HuIFN-beta with GC-Na showed greatly enhanced intranasal HuIFN-beta absorption, as compared to the other absorption promoters. The results of a stability study on HuIFN-beta in homogenates of nasal mucosa suggested that GC-Na behaved as a hydrolysis inhibitor in the nasal mucosa and maintained the activity of HuIFN-beta.