This review deals with the current status of newly developed pendant-type PEG-immunoliposomes (Type C), carrying monoclonal antibodies or their fragments (Fab') at the distal ends of the PEG chains. In terms of target binding of Type C, two different anatomical compartments are considered. They are mouse lung endothelium as a readily accessible site via the intravascular route and the implanted solid tumor as a much less accessibel target site erached via extravasation. Distearoyl phosphatidylethanolamine derivatives of PEG with a carboxyl group (DSPE-PEG-COOH) and dipalmitoyl phosphatidylethanolamine derivatived of PEG with a maleimidyl group (DPPE-PEG-Mal) at the PEG terminus were newly synthesized. Small unilamellar liposomes (90-130 nm in diameter) were prepared from phosphatidylcholine and cholesterol (2 : 1, m/m) containing 6 mol% of DSPE-PEG-COOH or DPPE-PEG-Mal. For targeting to the vascular endothelial surface in the lung, 34A antibody, which is highly specific to mouse pulmonary endothelial cells, was conjugated to PEG-liposomes (34A-Type C). The degree of lung binding of 34A-Type C in BALB/c mouse was significantly higher than that of 34A-Type A, which is an ordinary type of immunoliposome (without PEG derivatives). For targeting to solid tumor tissue, 21B2 antibody (anti-human CEA) and its Fab' fragment were used. The targeting ability of Fab'-Type C was examined by using CEA-positive human gastric cancer strain MKN-45 cells inoculated into BALB/c nu/nu mice. Fab'-Type C showed low RES uptake and a long circulation time, and enhanced accumulation of the liposomes in the solid tumor was seen. The small Fab'-Type C predominantly passed through the leaky tumor endothelium by passive convective transport. These studies offer important insights into the potential of Type C liposomes for target-specific drug delivery.
Lentinus edodes (shiitake) produces three base non- specific and acid ribonucleases, RNase Le2, RNase Le37 and RNase Le45. The primary structures of the former two RNases, having molecular masses about 24 and 37 kDa, respectively, have been elucidated to be members of the RNase T2 family. The latter two are excreted from mycelia into the medium. In this report, we estimated the primary structure of RNase Le45 using the following experimental evidence. (i) The partial amino acid sequence of RNase Le45 determined that up to about 60% of total protein was identical with that of RNase Le37. (ii) The amino acid composition of RNase Le45 was identical to that of RNase Le37. (iii) The elution profiles on HPLC of lysylendopeptidase and Staphylococcus aureus V8 protease digests of RCM-RNase Le45 (reduced and S-carboxymethylated RNase Le45) were very similar to those of RNase Le37, except for the absence of C-terminus peptide which contained O-glycosylated peptides. However, RNase Le45 contained about 70 residues of mannose and 4 residues of hexosamine. These values were more than twice those of RNase Le37. (iv) RNase Le45 was immunologically indistinguishable from RNase Le37. (v) After treatment with both glycosidase EndoH and α-mannosidase, RNases Le37 and Le45 gave complex bands by slab-gel electrophoresis. However, one of the major bands with the highest mobility from RNase Le45 and Le37 showed the molecular mass of 29 kDa in common, which is slightly larger than that of RNase Le2 containing no carbohydrate. These results indicated that RNase Le45 is an enzyme which is a heavily glycosylated species of RNase Le37.
The contribution of mannose 6-phosphate (Man 6-P)-dependent and -independent systems to lysosomal targeting of cathepsin H, a lysosomal cysteine cysteine protease, was investigated by metabolic labeling with [32P]phosphate and [35S]methionine/cysteine in primary cultures of rat hepatocytes. Metabolic labeling experiments with [32P]phosphate revealed that only the proform of cathepsin H acquired Man 6-P residues on its high mannose type oligosacchardie, and that most of the phosphorylated procathepsin H was secreted into the medium without having undergone significant intracellular dephosphorylation. Thus, acquisition of Man 6-P residues did not correlate with targeting of cathepsin H to lysosomes. Pulse-chase experiments with [35S]methionine/cysteine showed that only a about 10% of the newly synthesized cathepsin H was secreted as a proform while the remainder was retained intracellularly in processed mature form. These results indicate that the majority of newly synthesized cathepsin H is targeted to lysosomes by a Man 6-P-independent mechanism, at least in rat hepatocytes.
We investigated the expression of sulfotransferases (STs) in the human colon carcinoma cell line, Caco-2, which is widely used as a human intestine model for orally administered drugs. By reverse transcription-polymerase chain reaction (RT-PCR), we could detect mRNA expressions of phenol STs (P-ST), hydroxysteroid ST (HS-ST) and estrogen ST (E-ST). Treatment with 1, 25-dihydroxyvitamine D3, which induces CYP3A4 expression in Caco-2 cells, did not affect the ST expression of the cells. We detected significant P-ST activity toward 2-naphthol and dopamine in the cell extract. We also observed detectable HS-ST activity toward dehydroepiandrosterone (DHEA), but no E-ST activity toward β-estradiol was observed in the extract. Analyses by RT-PCR and separation of P-ST activities on ion-exchange column chromatography showed that the thermolabile monoamine phenol sulfotransferase (M-PST) is dominant in Caco-2 cells.
Non-steroidal anti-inflammatory drugs (NSAIDs) have cancer preventive and tumor regressive effects in the human colon, perhaps due to their capability to induce apoptosis of the colon cancer cells. Here, we report that indomethacin induced the expression of Nur77 which has been implicated in activation-induced apoptosis of T-lymphocytes, in a colon cancer cell line, HCT-15. The transcript- and protein-level, the transcriptional activity of Nur77 promoter, and the DNA binding of Nur77 were significantly induced following indomethacin treatment. Among the two potential trans-acting factors that activate Nur77-promoter, indomethacin induced DNA binding and reporter gene activity of AP-1, but not that of related serum response factor (RSRS), suggesting that the transcriptional induction of Nur77 may be mediated through activation of AP-1. Further, we showed that alltrans-RA repressed the induction of Nur77 as well as the apoptosis-induced by indomethacin, providing evidence that transcriptional induction of Nur77 may be an important mechanism by which indomethacin induces apoptosis in colon cancer cells.
We evaluated whether a novel dual inhibitor of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE), SA7060, (S)-2-[3-[(S)-2-(butoxycarbonyl)-2-hydroxyethyl]-3-isobutylureido]-3-(2-naphtyl) propionic acid, prevents deoxycorticosterone acetate (DOCA)-salt-induced hypertension and related organ damage, such as cardiovascular hypertrophy, renal dysfunction and renal tissue injury in rats. The effectiveness was compared with candoxatril and enalapril, which are a selective NEP and ACE inhibitor, respectively. During DOCA-salt treatment for 4 weeks, the rats were given SA7060, candoxatril, enalapril or vehicle, once daily by gavage. The 4-weeks treatment with DOCA and salt produced progressive increases in systolic blood presure. Daily administration of SA7060, candoxatril or enalapril significantly suppressed the development of hypertension induced by DOCA and salt, although the effect of enalapril was less potent at 4-weeks of the treatment period. In vehicletreated DOCA-salt rats, decreases in creatinine clearance and increases in urinary excretion of protein and blood urea nitrogen were observed. This funcitonal damage was improved most efficiently by the treatment with SA7060. There were significant increases in urinary excretions of atrial natriuretic peptide and cyclic GMP in SA7060- or candoxatril-treated animals. Histopathological examination of the kidney in DOCA-salt rats revealed tubular, glomerular and vascular lesions, all of which were improved in animals given SA7060 or candoxatril. When the vascular hypertrophy of the aorta was evaluated, there were significant increases in wall thickness, wall area and the wall-to-lumen ratio in vechicle-treated DOCA-salt rats compared with the sharm rats. The development of vascular hypertrophy was suppressed by the treatment with SA7060, candoxatril or enalapril. Our findings indicate that SA7060 efficiently prevents DOCA-salt-induced hypertension and related tissue injury, mainly by inhibiting NEP. Thus, SA7060 may be useful for treatment of both renin-dependent and renin-independent hypertensive subjects, although further studies examining efficiency in a renin-dependent hypertensive model are needed.
The correlation between the steady-state level of inducible nitric oxide synthase (iNOS) mRNA and skin tumors induced following treatment with 12-o-tetradecanoylphorbol-13-acetate (TPA) was investigated in transgenic TG-AC mice, which carry the v-Ha-ras oncogene fused to the promoter of the mouse embryonic α-like, ξ-globin gene. In animals treated with TPA (2.5 μg×2/week, for 2 weeks), the increase of iNOS mRNA wa locally confined only to the regions of papillomas, but not to the skin tissues surrounding the papillomas. However, the tissues surrounding the papillomas showed only a minor increase in the steady-state level of iNOS mRNA. These data suggest that iNOS gene expressions may underlie tumorigenesis during TPA promotion in TG-AC mice.
Rhaponticin and chrysophanol 8-o-β-D-glucopyranoside isolated from the rhizomes of Rheum undulatum (Family Polygonaceae) are metabolized to rhapontigenin and chrysophanol, respectively, by human intestinal microflora. Most intestinal bacterial isolated from human feces catalyzed these metabolic pathways. Among rhaponticin and chrysophanol 8-o-β-D-glucopyranoside and their metabolites, rhapontigenin had the most potent inhibitory activity on a hyaluronidase, a histamine release from mast cell and passive coutaneous anaphylaxis (PCA) PCA reaction. The inhibitory activity of rhapontigenin was more potent than that of disodium cromoglycate, one of the commercial anti-allergic drugs. These results suggest that rhaponticin in the rhizomes of R. undulatum is a prodrug that has an extensitve anti-allergic property.
In the course of our research on the oligoglycosidic constitutents of Turkish Astragalus species, we have isolated a number of cycloartane-type triterpene glycosides. The current study examines the immunostimulatory effects of nineteen of these cycloartane-type compounds using a transcription-based bioassay for Nuclear Factor kappa B (NF-κB) activation in a human macrophage/monocyte cell line, THP-1. All compounds were inactive at 100 μg/ml except astragaloside I which increased NF-κB directed luciferase expression to levels about 65% as compared with maximal stimulation by E. coli lipopolysaccharide (LPS) at 10 μg/ml. None of the compounds were active at low dosage levels (0.1 μg/ml) in combination with 50 ng/ml LPS. Astragaloside I also increased mRNA expression of the inflammatory cytokines interleukin-1β(IL-1β) and tumor necrosis factor-α (TNF-α) as measured using reverse transcriptase-polymerase chain reaction (RT)-PCR. Based on these results it is clear that certain structural features are required for immunostimulation of cycloartane-type triterpene glycosides.
Apparent membrane permeation coefficients (Papp) of poorly water-soluble drugs such as indomethacin (IDM) and triamterene (TAT) were obtained by the chamber method using an isolated rat intestinal tissue after solubilization of the drugs by additives. For the additives, sodium deoxycholate (DOC), polyethylene glycol 600 (PEG 600), dimethylsulfoxide (DMSO), ethanol (EtOH), propylene glycol (PG), and rat bile were examined. Their concentrations were determined in ranges considered to be appropriate from the results of in vivo experiments and physiological findings. From the correspondence between this membrane permeability and in vivo bioavailability, we evaluated the validity of our in vitro experiment. On the basis of these evaluations, it was shown that 5% DMSO and 10% PEG 600, which did not affect the membrane integrity, were most appropriate additives for chamber experiments. Papp of IDM was greater than that of TAT, indicating that the order corresponded with that of in vivo bioavailability after oral administration of their PEG 600 solutions. Accordingly, it was concluded that Papp obtained by our in vitro system can be used to assess the in vivo bioavailability.
The effects of a series of fatty acids on the percutaneous penetration of ozagrel (OZ), a selective thromboxane A2 synthetase inhibitor, through rat skin and the mechanism by which fatty acids enhance the skin penetration of OZ were examined in vitro. Lauric acid, at the fatty acid : OZ molar ratio of 2 : 1, was the most potent agent as far as increasing the skin penetration was concerned, with a flux 24-fold higher than that without fatty acid. A molar ratio of 3 : 1 also produced a large enhancing effect, comparable with that of a molar ratio of 2 : 1. When the gel formulation with lauric aicd (molar ratio of 2 : 1) was applied to the skin for 6 h, the amount of drug penetrating into the skin was significantly increased compared with that after the formulations without lauric acid and with capric and palmitic acids. However, lauric acid did not change the apparent partition coefficient of OZ between n-heptane and phosphate buffer (pH 7.4). The 13C-NMR spectra of OZ was also unaffected by the addition of lauric acid, indicating that a complex or ion pair with lauric acid was not formed. A possible mechanism for the enhancing effect is the increased incorporation of lauric aicd with OZ into the bulk lipid phase of the stratum corneum, where the fatty acid would act as a co-penetrant enhancing passage through the stratum corneum.
Rat skin permeability after treatment by electroporation (newly developed frog type electrode, 100 V, 10 pulses), oleic acid/propylene glycol (PG) and a combination of both were investigated using Fourier transformed infrared attenuated total reflectance (FT-IR/ATR) analysis. Electroporation immediately disordered the stratum corneum lipid structure up to a certain threshold level. This action lasted throughout the experiment. This may be attributed to the formation of long lifetime of metastable lipid structures, which may allow molecules to pass to the inside of the stratum corneum due to the electroporation-induced fluidized lipid membranes. Electroporation also altered the protein structure of the stratum corneum. When elevtroporation was combined with 0.05 M oleic acid/PG, uptake of oleic acid and PG into the stratum corneum was remarkably accelerate compared to the application of only 0.05 M oleic acid/PG to the skin. This indicates that electroporation enables oleic acid and PG to penetrate the stratum corneum easily by disrupting the structure of the latter. PG transfer into the dermis from the epidermis was accelerated, not because of the direct action of electroporation on the dermis, but because electroporation induced the rapidly disordering action of oleic acid on the stratum corneum. Lipid-soluble indomethacin permeated the skin more rapidly when the skin was treated with electroporation plus oleic acid/PG than with 0.05 M oleic acid/PG in vitro.
The present study was designed to establish the significance of carrier-mediated transport in the intestinal absorption of monocarboxylic acids by examining the stereoselectivity of transepithelial transport of chiral monocarboxylic acids. The transport of L- and D-lactic acids was examined in vitro using rat intestinal tissue sheets and in situ by means of intra-jejunal administration, followed by measurement of the plasma concentration. Both the absorptive and secretory transport of L-[14C]lactic acid across the intestinal epithelial tissues of rats was significantly greater than that of the D-isomer. The secretory transport of the L-isomer was significantly greater than the absorptive transport, implying ne transport in the secretory direction. When L- and D-[14C]lactic acids wre administered to the rat jejunum, the absorption ratio of the L-isomer was lower than that of the D-isomer at 15 min after administration. The concentration-dependence of absorption for both L- and D-[14C]lactic acids indicated the involvement of both saturable and nonsaturable processes. The saturable process showed a higher affinity and lower capacity for L-lactic acid compared with the D-isomer, while no significant difference between the isomers was observed in the nonsaturable process. The absorption of L-lactic acid was inhibited by chiral 2-hydroxymonocarboxylic acids in a stereoselective manner. Chiral monocarboxylic acids were shown to cross the intestinal epithelial tissues and to be absorbed in a stereoselective manner after oral administration, suggesting the involvement of specific carrier-mediated transport mechanism(s) in their intestinal absorption in vivo.
A physiological pharmacokinetic model describing the absorption and disposition of topically applied drugs was proposed, and the effect of various pharmacokinetic and physiological parameters on the drug delivery into the targeted muscle was simulated. The proposed model consists of vehicle, and stratum corneum, viable epidermis and muscle below the application and referene sites, and plasma, each joined with transfer clearance and plasma flow. Indomethacin concentrations in tissues and plasma after topical application to rats could be explained by the model. Most indomethacin delivered into the underlying muscle was via direct penetration. The model simulation showed that the increase in plasma clearance and clearance between viable skin and muscle, and the decrease in application area and plasma flow rate into viable skin and muscle would promote the targeting efficacy of topically applied drugs to the underlying muscle.
Sparassis crispa is an edible mushroom recently cultivable in Japan. Polysaccharide fractions were prepared from the cultured S. crispa by repeated extraction with hot water (SCHWE), cold NaOH (SCCA), and then hot NaOH (SCHA). HWE was further separated by 1 volume (SCHWE1v) or 4 volumes (SCHWE4v) of ethanol-precipitable fractions. By chemical, enzymic, and NMR analyses, the primary structures of SCHWE1v, SCCA, and SCHA were 6-branched 1, 3-β-glucan, having one branch in approximately every third mainchain unit. All of these fractions showed antitumor activity to the solid form of Sarcoma 180 in ICR mice with strong vascular dilation and hemorrhage reaction. These fractions also showed enhanced hematopoietic response to cyclophosphamide induced leukopenic mice following intraperitoneal or peroral administration.
Some 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which are used as hypolipidemic drugs, have been reported to have the potential to reduce the oxidizability of plasma low-density lipoprotein (LDL) when they are administered in vivo. Their in vivo mechanism is believed to be closely related to their hypolipidemic action based on the HMG-CoA reductase inhibitory activity. We hypothesized that some type of HMG-CoA reductase inhibitor has additional mechanism inhibiting LDL oxidation in vivo due not to its hypolipidemic action but to its direct antioxidative effect based on its unique chemical structure. We directly compared in vitro the antioxidative effects of well-konwn HMG-CoA reductase inhibitors (fluvastatin, pravastatin, simvastatin, cerivastatin and atorvastatin) on the hydrogen peroxide-induced oxidative destruction of hemin and LDL. Fluvastatin but not the others showed the inhibitory effect on this system. Its effect was dose-dependent and almost as strong as the natural antioxidants, α-tocopherol and ascorbic acid. Further, M2, which is a hydroxylated metabolite of fluvastatin, showed stronger antioxidative activity than did fluvastatin. We suggest that among these HMG-CoA reductase inhibitors, fluvastatin especially has an ability to retard the LDL oxidation which is based on not only its hypolipidemic action but also its direct antioxidative effect.
Two angiotensin-I-converting enzyme (ACE) inhibitory peptides were isolated from a tryptic hydrolysate of human serum albumin (HSA). The peptides were identified by sequencing and other analyses as Ala-Trp and the nonapeptide Ala-Phe-Lys-Ala-Trp-Ala-Val-Ala-Arg (human albutensin A), corresponding to f(213-214) and f(210-218) of HSA, respectively. Synthetic versions of both peptides had previously been shown to have ACE inhibitory activity. The present results are the first to show that these peptides have a potential natural origin in humans. Additional studies were done to define the inhibitory properties of these peptides, as they had not been previously reported. The dipeptide and nonapeptide showed dose-dependent inhibition of ACE, with IC50 values of 12 and 1.7 μmol/l, respectively. Lineweaver-Burk plots suggested that Ala-Trp is a competitive inhibitor, and that human albutensin A is a noncompetitive inhibitor.
In activated macrophages the inducible form of nitric oxide synthase (i-NOS) generates high amounts of the toxic mediator, nitric oxide (NO) which contributes to the circulatory failure associated with septic shock. Two polyacetylenes were isolated from the medicinal plant Angelica gigas and their structures were elucidated as octadeca-1, 9-dien-4, 6-diyn-3, 8, 18-triol (1) and 18-acetoxy-octadeca-1, 9-dien-4, 6-diyn-3, 8-diol (2) by spectroscopic method. These polyacetylenes and their peracetate, 3, 8, 18-triacetoxy-octadeca-1, 9-dien-4, 6-diyn (3) inhibited the production of NO in LPS-activated RAW 264.7 cells by suppressing the i-NOS enzyme expression. These new inhibitors of i-NOS expression may have potential in the treatment of endotoxemia and inflammation accompanied by the overproduction of NO.
Anti-herpes simplex virus type 1 (HSV-1) activity of oleanane-tpe triterpenoidal saponins obtained from some fabaceous plants were examined. Among sophoradiol glycosides, the order of potency was kaikasaponins III>I»sophoradiol monoglucurnoide. It was suggested that the trisaccharide group showed greater action than the disaccharide group. Neither the monoglucuronide of sophordadiol nor that of soyasapogenol B showed activity. Among the trisaccharide group of soyasapogenol B, the order of activity was azukisaponin V>soyasaponin II>astragaloside VIII»soyasaponin I. Therefore, the saponin having a glucosyl unit in the central sugar moiety seemed to show greater action. In comparison with the activities for a group having the same trisacchardie, the potency of the sapogenol moieties was found to be in the order of soyasapogenol E>sophoradiol»soyasapogenol B. Hence, the carbonyl group at C-22 would be more effective than the hydroxyl group in anti-HSV-1 activity, while the hydroxyl group at C-24 could reduce the activity.
A crude methanol extract of Picrorhizae Rhizoma, the dried underground parts of Picrorhiza scrophulariiflora PENNELL, has been shown to potentiate the nerve growth factor (NGF)-induced neurite outgrowth from PC12D cells. The methanol extract was partitioned between ethyl acetate and water. The NGF-potentiating activity was observed in the ethyl acetate fraction. The ethxl acetate solubles were fractionated by silica gel chromatography, monitoring the NGF-potentiating activity to give two iridoid glycosides, picrosides I and II. Picrosides did not exhibit neurotrophic activity but caused a marked enhancement of the NGF-mediated neurite outgrowth from PC12D cells. The pharmacological data suggest that picrosides I and II enhance neurite outgrowth from PC12D cells, probably by amplifying a step in the NGF-receptor-mediated intracellular signaling pathway.
The binding site of 7-hydroxystaurosporine (UCN-01) on α1-acid glycoprotein (AGP) was studied by fluorescence and ultracentrifugation experiments. Three ligands, propranolol, warfarin and progesterone were employed as marker ligands and quinaldine red was employed as a fluorescent probe. The persence of UCN-01, propranolol, warfarin and progesterone resulted in a significant quenching of the fluorescence of quinaldine red, when bound to AGP, depending upon the potency of the binding to AGP. The construction of Klotz plots indicated that the displacement effects of propranolol, warfarin and progesterone on UCN-01-AGP binding were competitive in nature. These data suggest that the binding site of UCN-01 on the AGP partly overlaps and binding site for basic drugs, acidic drugs, as well as steroid hormones.