Formation of spherical micelles by the novel platelet activating factor receptor antagonist, E5880.

E5880, a novel platelet activating factor receptor antagonist, was dispersed in water for the preparation of injectable formulation and the physicochemical properties of the micelles were characterized. The critical concentration for formation of micelles was 0.12 mM. Using the area per molecule results, the critical packing parameter was calculated and showed that the structure was composed of spherical micelles and the number of the molecules per micelle was 88. The diameter of a micelle was 8.1 nm. The fluidity and micropolarity around the hydrocarbon region of the micelles were evaluated and compared to the surfactants, stearyltrimethylammmonium chloride and cetyltrimethylammonium chloride.

Optimization of the formulation and characterization of the physicochemical properties of the novel platelet-activating factor receptor antagonist E5880.

The injectable formulation of E5880, a novel platelet-activating factor (PAF) receptor antagonist, was determined from the study of pH stability, the selection of excipient, and the relationship between moisture and stability. The physicochemical properties of E5880 in the optimized formulation (0.6 mg/ml of E5880, 0.1% [4.8 mM] citric acid, 10% lactose, pH 2.8) were characterized. The critical micelle concentration of E5880 in the buffer was 0.1 mg/ml, and the structure was of spherical micelles. The micellar size was 5.6 nm and did not change before and after lyophilization and storage. The number of the molecules per micelle was 40. The micropolarity around the hydrocarbon region of the micelle was similar to that of butanol.

A temperature study on critical micellization concentration of the novel platelet-activating factor receptor antagonist E5880 in water by electric conductivity measurements.

To clarify the behavior of the novel platelet-activating factor (PAF) receptor antagonist E5880 in aqueous solution, electric conductivity was measured at different temperatures (every 5 degrees C), ranging from 15 degrees C to 50 degrees C. Critical micellization concentration (CMC) of E5880 was dependent on the temperature; at 30 degrees C, the CMC value was smallest (0.143 mM). Below that temperature, the enthalpy for formation of the micelle (delta Hm0) was positive, and the formation of micelles was endothermic; above that temperature, delta Hm0 was negative, and the formation of micelles was exothermic.

Characterization of the physicochemical properties of the micelles of platelet-activating factor (C18:0).

The purpose of this study was to clarify the physicochemical properties of the micelles of platelet-activating factor (PAF; C18:0). The critical micelle concentration (CMC) of PAF (C18:0) was determined (0.20 microM) using fluorescence techniques. The fluidity and the micropolarity of the PAF (C18:0) micelle were similar to those of the micelle of stearoyl lysophosphatidylcholine.

Synthesis and application of neoglycolipids for liposome modification.

We synthesized various glycolipid derivatives and examined the in vivo behaviors of liposomes modified with these novel glycolipid derivatives. Gal-t-psa (1,¿8-(2-hexadecyloctadecanoylamido)-3,6-dioxaoctyl¿-beta-D- galactoside), Lac-t-psa (3, 8-(2-hexadecyloctadecanoylamido)-3,6-dioxaoctyl beta-D-lactoside) and GalNAc-t-psa (4, 8-(2-hexadecyloctadecanoylamido)-3,6-dioxaoctyl 2-acetamido-beta-D-galactopyranoside) modified liposomes were recognized by the liver. Lac-t-psa (3) modified liposome was accumulated to the highest degree, followed by GalNAc-t-psa (4) modified liposome and then Gal-t-psa (1) modified liposome. The intrahepatic distributions of Gal-t-psa (1), GalNAc-t-psa (4), Glc-t-psa (2, 8-(2-hexadecyloctadecanoylamido)-3,6-dioxaoctyl beta-D-glucopyranoside) and Lac-t-psa (3) modified liposomes were investigated. GalNAc-t-psa (4) and Lac-t-psa (3) modified liposome were accumulated to greater extents than Gal-t-psa (1) modified liposome in hepatic parenchymal cells. The intrahepatic distribution of these liposomes showed that Lac-t-psa (3) and GalNAc-t-psa (4) were preferable to Gal-t-psa (1) for the selective delivery of liposomes to hepatic parenchymal cells.

Hepatic accumulation of glutamic acid branched neogalactosyllipid modified liposomes.

We synthesized branched type galactosyllipid derivatives for liposome modification for the targeting of asialoglycoprotein receptors on the surface of liver cells. Galactose was coupled to the alpha- and gamma-carboxyl groups of glutamic acid via a triethyleneglycol spacer, then this glutamic moiety was bound to the lipid anchor. Ricinus communis agglutinin (RCA120) induced the agglutination of liposomes modified with mono-, bi- and tri-antennary neogalactosyllipid. With the bi- or tri-antennary derivatives, agglutination was observed at fewer galactosyl residues on the liposomes. We examined the effect of the branching structure in vivo. The difference in accumulation of liposomes between non-branched type neogalactosyllipid and branched type neogalactosyllipid was not large. Liver accumulation of liposomes depended on the galactosyl residues. The number of galactosyl residues was more effective for accumulation in the liver than for branching. We studied the effect of asialofetuin preinjection on the hepatic accumulation of neogalactosyllipid modified liposomes. Hepatic accumulation of liposomes was inhibited by preinjection of asialofetuin. The effect of preinjection was almost equal among the ligands. These results show that the saccharide density on the liposome surface seemed to be a more important factor than the branching structure of the ligand for liver targeting.

In vivo behavior of liposomes modified with a novel galactosyllipid derivative.

We studied the in vivo behavior of galactosyllipid-modified liposomes for targeting the asialoglycoprotein receptor present on the surface of liver parenchymal cells. We examined the effects of the lipid composition of liposomes on the in vivo behavior of galactosyllipid-modified liposomes, and found good accumulation in the liver of liposomes of a high cholesterol content and liposomes made of lipids of a high gel-liquid crystalline phase transition temperature. The amount of modification with the galactosyllipid derivative required for effective targeting to the liver was found to be more than 5% of the total lipids. The concentration of galactosyllipid-modified liposomes was lower than that of control liposomes in all the organs except for the liver, showing high selectivity of galactosyllipid-modified liposomes for the liver. Hepatic accumulation of liposomes was inhibited by preinjection of asialofetuin. This result suggests that hepatic accumulation of ¿8-(2-hexadecyloctadecanoylamido)-3,6-dioxaoctyl¿-beta-D-gal actoside (Gal-t-psa) liposome was involved with the asialoglycoprotein receptor in the liver. Therefore, it was concluded that our neogalactolipid-modified liposomes are useful for selective delivery to the liver.

Establishment and characterization of immortalized human coronary endothelial cells.

We established an immortalized cell line from endothelial cells derived from a human coronary artery, isolated at autopsy from 76-year-old male, by transfecting the cells with origin-minus simian virus 40 DNA. These cells showed SV40 T antigen in the nuclei and Ulex europaeus I agglutinin and factor VIII-related antigen, as endothelial cell markers, in their cytoplasm. This cell line synthesized prostacyclin, tissue-type plasminogen activator (tPA), and plasminogen activator inhibitor-1 (PAI-1) as well as produced the proform of matrix metalloproteinase 1, which was activated by cultivating the cells with plasminogen. These findings reveal that this immortalized endothelial cell line retains characteristics of human coronary endothelial cells, indicating that this cell line is useful for studying atherogenesis of the coronary artery.

Syntheses of novel galactosyl ligands for liposomes and the influence of the spacer on accumulation in the rat liver.

We modified the surface of liposomes with galactosyl ligands. At first we determined whether or not the galactosyl moiety was exposed on the liposomes. We then investigated the effect of the ligands on the hepatic accumulation of liposomes in rats. We introduced an oligoethylene glycol moiety as a spacer. Among the various ligands tested, those with a tri- or tetraethylene glycol moiety as a spacer caused the greatest accumulation of liposomes in the liver. Liposomes bearing ligands with a tri- or tetraethylene glycol moiety as a spacer, were aggregated by Ricinus communis agglutinin. On the other hand, those modified with ligands with a mono- or diethylene glycol spacer did not clearly agglutinate. These results show the importance of a spacer between the homing device and the ligand anchor.

Effect of angiotensin II and thromboxane A2 on the production of matrix metalloproteinase by human aortic smooth muscle cells.

Angiotensin II (AGII) and thromboxane A2 (TXA2), potent vasoconstrictors, augmented the production of the precursor of tissue procollagenase/promatrixmetalloproteinase-1 (proMMP-1) and DNA synthesis in cultured human aortic smooth muscle cells (SMC) significantly compared with that in untreated SMC. Moreover, AGII and TXA2 stimulated hydrolysis of phosphoinositides and subsequent formation of inositol triphosphate (IP3), leading to an increase in the intracellular free Ca2+ concentration. These results suggest that the production of proMMP-1 increased by AGII and TXA2 in intimal SMC in relation to cell proliferation plays a role in arterial reconstruction in vascular diseases.

Relationship between the anchor structure of the galactosyl ligand for liposome modification and accumulation in the liver.

Liposomes which have been modified with (8-hexadecanoylamido-3,6-dioxaoctyl)-beta-D-galactose (Gal-t-pa), a straight chain palmitoyl derivative, and are composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol (CH), and dicetyl phosphate (DCP) at a ratio of 10:10:1, showed the same accumulation in the liver as the control liposome. Also, liposomes which have been modified with [8-(2-hexadecyloctadecanoylamido)-3,6-dioxaoctyl]-beta-D-gal actoside (Gal-t-psa) showed remarkable accumulation in the liver. The accumulation of liposomes modified with galactose derivatives in the rat liver differed markedly according to the anchor structure. To clarify the cause of this finding, we produced [3H]inulin entrapped [14C]Gal-t-pa modified double label liposomes and evaluated changes in their rat plasma concentration, distribution in the organs, and the in vitro interaction with rat plasma. [14C]Gal-t-pa on the liposome surface bound to serum albumin and was released, resulting in no accumulation in the liver. In addition, sialic acid palmitoyl derivatives and glucuronic acid palmitoyl derivatives behaved similarly. As with the galactose derivatives, they also bound to serum albumin, being released from liposomes. These results suggest that adequate attention should be paid to the anchor structure of the ligand, in order to incorporate a recognition element into liposomes for transport to cells.

Development-related changes in matrix metalloproteinase expression in human aortic smooth muscle cells.

BACKGROUND: It is known that extracellular matrix-degrading enzymes play an important role in tissue remodeling and that large amounts of structural proteins including types I, III, IV, and V collagens and elastin are produced by smooth muscle cells (SMC) in the arterial wall. We have recently shown that matrix metalloproteinases (MMPs) produced by human aortic medial smooth muscle cells are closely related to the proliferation of the cells, leading to the formation of atherosclerotic plaques, characteristic of intimal remodeling. For a better understanding of the mechanism of atherogenesis, therefore, it is important to clarify the relationship between the production of matrix-degrading enzymes and artery development. EXPERIMENTAL DESIGN: In vivo or in vitro synthesis of MMPs by SMC was analyzed by immunohistochemistry and immunoblotting. Elastase activity in the culture medium was also estimated. RESULTS: Production of proMMP 1, 2, and 3 was detected in cultured SMC isolated from the aortas of both neonates and fetuses; in medial SMC cultured from young individuals, production of proMMP-1 and -3 was extremely decreased, but was apparent in intimal SMC. Immunohistochemical observation indicated that in the media of fetal or neonatal aorta, SMC synthesized large amounts of the three proMMPs; in aortas from older individuals, proMMP-2, but not proMMP-1 and -3, was detected in the media, and relatively large amounts of proMMP-1, -2, and -3 were produced by SMC in the slightly thickened intima. Assay of elastase activity in the culture medium gave results similar to those for MMPs. CONCLUSIONS: We conclude that the production of proMMP-1 and -3 is associated with phenotypic modulation of SMC to a "synthetic" state, and that the ability of SMC to produce MMPs plays an important role in the development and/or aging of the human aorta through remodeling of the extracellular matrix; furthermore elastase is also involved in these processes in the arterial wall.

Syntheses of novel galactosyl ligands for liposomes and their accumulation in the rat liver.

The effects of liposomes, bearing galactosyl ligands on their surface, on their clearance from circulation and on tissue distribution were studied in rats. The ligands were obtained through coupling 2,3,4,6-tetraacetyl-(2-[2-(2-aminoethoxy)ethoxy]ethyl)-beta- D-galactoside p-toluenesulfonate with hydrophobic anchors, followed by deprotection. We introduced mid-chain or long, branched or non-branched alipathic chains, and cholesterol as anchors. Among various anchors tested, that with a dihexadecyl branch caused the greatest accumulation of liposomes in the liver. On the other hand, liposome bearing these ligands were aggregated by Ricinus communis agglutin. This showed that these ligands were similarly incorporated to liposomes. These results show the importance of the balance between lipophilicity and the structure of ligands.

Effect of linoleic acid hydroperoxide on production of matrix metalloproteinases by human aortic endothelial and smooth muscle cells.

The effect of linoleic acid hydroperoxide on the production of proforms of matrix metalloproteinase-1, -2, and -3 (proMMP-1, -2, and -3) in cultured human arterial endothelial and smooth muscle cells was investigated. Upon cultivation of the endothelial cells in the absence of the hydroperoxide, only proMMP-1 was detected, and its amount was increased by cultivation with the hydroperoxide. In the cultures of the intimal smooth muscle cells in the absence of the hydroperoxide, a large amount of proMMP-2 and small amounts of proMMP-1 and -3 were detected, and the hydroperoxide treatment increased remarkably the amounts of proMMP-1 and -3, but rather decreased the amount of proMMP-2. In the cultures of the medial smooth muscle cells, the same tendency was observed. However, the amounts of these proenzymes found in the intimal smooth muscle cells exceeded those found in the medial smooth muscle cells, both in the absence and in the presence of the hydroperoxide. Possible involvement of these phenomena in atherogenesis was discussed.

Immortalization of human aortic smooth muscle cells with origin-minus simian virus 40 DNA.

We established two cell lines of human smooth muscle cells (SMC) by transfection of cells from the aortic intima and aortic media with origin-minus simian virus 40 (ori-minus SV40) DNA. Ori-minus SV40 DNA very efficiently immortalized human smooth muscle cells in culture. Proteins that these cell lines produced included type I, III, IV, and V collagens, fibronectin, and human matrix metalloproteinases (MMP)-1 (tissue collagenase), -2 ("type IV collagenase"), and -3 (stromelysin). The protein production in these cell lines generally mimicked that of normal SMC, but the immortalization stimulated the cell line of medial SMC to produce excessive MMP-2 and to secrete MMP-9 (92-kDa gelatinase). However, since these cell lines did not show a fully malignant phenotype, we concluded that, in addition to the degradation of extracellular matrix macromolecules, including basement membrane components by MMP-2, -3, and/or -9, some additional factors must be involved for the malignancy of fully transformed cells and that these immortalized human aortic SMC, which share many characteristics with normal SMC, will prove useful to study the role(s) of metalloproteinases in atherosclerosis.

Rectal absorption of E-2078 (dynorphin analogue peptide) in rats.

The plasma levels in rats of a dynorphin analogue peptide (E-2078) after rectal administration have been studied. The bioavailabilities of E-2078 after rectal administration, subcutaneous injection, intramuscular injection, and oral administration were 21.6, 67.8, 67.1, 0.7%, respectively. The effect of dose, pH, osmolarity and viscosity on the rectal absorption of E-2078 were studied. A sigmoid relationship between the dose and the AUC was observed on rectal administration, while there was a linear relationship on intramuscular administration. The AUC was increased in acidic solution and highly viscous solution. The osmolarity did not affect the absorption of E-2078. In microscopic studies, E-2078 caused little or no damage to the rectal mucosa in rats.

[Differential effect of sodium bicarbonate on disposition of sulfadimethoxine and sulfisoxazole in rabbits].

The orally co-administered sodium bicarbonate significantly enhanced the blood concentration of sulfadimethoxine at the early stage after oral administration to rabbits, by increasing its intestinal absorption. On the other hand, the sodium bicarbonate significantly reduced the blood concentration of sulfisoxazole at the elimination phase after oral administration to rabbits, by increasing its urinary excretion. The fact that sodium bicarbonate exhibits different effects in the disposition of these two sulfonamides is an interesting example to gain a better understanding for the complexity of drug interaction.