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Triptolide γ-lactone and C-14 analogues were prepared and evaluated cytotoxity against human lung adenocarcinoma epithelial A549 cells and human colon adenocarcinoma HT-29 cells. γ-Lactone substructure and C-14 substituents affected the biological activities significantly.

The aim of this study was to elucidate the efficacy of combination therapy with irinotecan and amrubicin for lung cancer and the influence of administration schedule in a xenograft mouse model and human cancer cell culture. We investigated the antitumor activity of irinotecan and amrubicin on human small cell lung cancer cell line LX-1 inoculated in mice in vivo and the cytotoxic effect of SN-38 and amrubicinol on human lung cancer cell lines A549 and PC-6 in vitro. Combined administration of irinotecan and amrubicin in divided doses inhibited tumor growth by approximately 90%, with complete recovery observed in one case. Furthermore, combined administration in divided doses induced little loss of body weight. Combination index analysis revealed that the cell growth inhibitory effect of SN-38 combined with amrubicinol was additive, regardless of schedule or cell line. The effect of combination treatment with SN-38 and amrubicinol on cell cycle was investigated. Cell cycle showed arrest at both the S and G2/M phases. The results indicate that combination therapy with irinotecan and amrubicin can be expected to yield improved outcomes, including less toxicity, especially with divided administration.

A methanol extract of the leaves of Cephalotaxus harringtonia var. nana and its ethyl acetate (EtOAc)-soluble fraction demonstrated strong antitumor activity against A549 and HT-29 cell lines. The EtOAc-soluble fraction was purified by column chromatography and high-performance liquid chromatography (HPLC) using a reverse-phase column to yield three novel acyl flavonoids and a biflavonoid, along with 15 other known compounds that included flavonoids, biflavonoids, and other phenolics. The structures of the new compounds were elucidated using spectral data from HR-MS and NMR, including two-dimensional NMR studies, as (2R,3R)-3-O-eicosanoyltaxifolin (1), (2R,3R)-3-O-docosanoyltaxifolin (2), (2R,3R)-3-O-tetracosanoyltaxifolin (3), and 6-methyl-4′,7,7″-tri-O-methylamentoflavone (4). The isolated compounds, including the known compounds, were tested for possible antitumor activity; some of the biflavones were found to be active. The potent antitumor activity of the extract was attributed to Cephalotaxus alkaloids, such as homoharringtonine (20).

Background: The enhanced antitumor effect of paclitaxel when used with oxaliplatin in gastric cancer is reported, however the underlying biological mechanism is unknown. Methods: We tested the cytotoxic activity, apoptosis, and mitotic catastrophe of paclitaxel and oxaliplatin in MKN-28 and MKN-45 gastric cancer cell lines. The modulation of survivin expression was determined by Western blotting. Results: WST-1 assay indicated that paclitaxel plus oxaliplatin showed better cytotoxicity than paclitaxel alone, even when low concentrations of oxaliplatin were used. Flow cytometry analysis revealed significantly greater increases in apoptotic cells after treatment with paclitaxel followed by low-dose oxaliplatin (1 µM) than after any single-reagent regimen in the MKN-45 cell line. In MKN-28, a difference existed only between combination treatment and oxaliplatin treatment. Morphologic examination showed that the cells undergoing mitotic catastrophe were highest in the combination groups in the both cell lines. Downregulation of survivin expression was found by Western blotting with treatment by paclitaxel, oxaliplatin, or their combination. Conclusion: Our findings suggest that the mechanism of enhanced cytotoxicity might be through enhanced mitotic catastrophe and apoptosis, which is possibly due to chemotherapy-induced downregulation of surviving. The combination of paclitaxel and low-dose oxaliplatin should be incorporated into the design of a clinical trial.

Irinotecan hydrochloride (CPT-11) is a potent topoisomerase I inhibitor and is established and used widely as an antitumor agent. However, it sometimes causes severe side effects such as myelosuppression and diarrhea. These dose-limiting toxicities prevent the adoption of CPT-11 in aggressive chemotherapy. Thus we sought to determine in a rat model whether extending the period of infusion of CPT-11 would ameliorate the adverse reactions.CPT-11 was administered intravenously (i.v.) to rats at a dose of 60 mg/kg per day for four consecutive days as a bolus injection or as 3-, 8- or 24-h infusions, and then blood cell counts and the incidence of acute and delayed-onset diarrhea were monitored.Serious acute and delayed-onset diarrhea and marked decreases in the number of neutrophils and lymphocytes were observed in the bolus injection group. These symptoms were alleviated in the infusion groups with the degree of alleviation dependent on infusion time. In the bolus injection group, mucosal impairment of the cecal epithelium including wall thickening, edema, a decrease in the number and size of crypts, and the formation of a pseudomembrane-like substance was observed, whereas these changes were less severe in the infusion groups. Areas under the plasma concentration-time curves (AUCpla) of CPT-11 and its metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), differed little among the bolus injection group, and the 3-h and 8-h infusion groups. However, the AUCpla values of CPT-11 and SN-38 were significantly decreased and increased, respectively, in the 24-h infusion group. The maximum plasma concentrations (Cmax) of CPT-11 decreased with increasing infusion time, but those of SN-38 did not.It was confirmed that the side effects of CPT-11 were alleviated by extending the infusion time. The pharmacokinetic parameters suggested that the Cmax of CPT-11 is closely related to the incidence and severity of adverse reactions such as myelosuppression and acute and delayed-onset diarrhea.

Background. Recently, the acquisition by Helicobacter pylori of resistance to antibiotics has become a serious problem. Therefore, nonantibiotic substances are required to diminish H. pylori-induced gastric lesions. In the present study, the effects of Cladosiphon fucoidan were examined in terms of H. pylori attachment to porcine gastric mucin in vitro and Helicobacter pylori-induced gastritis in vivo. Methods. The inhibitory effect of Cladosiphon fucoidan and other polysaccharides on H. pylori attachment to porcine gastric mucin was assayed in vitro with mucin-coated microtiter plates. The effect of Cladosiphon fucoidan on H. pylori-induced gastritis was examined in vivo using Mongolian gerbils. H. pylori-inoculated gerbils were given fucoidan in drinking water. Six weeks after H. pylori-inoculation, gerbils were sacrificed for macroscopic and microscopic examination of gastric lesions and counting of viable H. pylori in the gastric mucosa. Results. Cladosiphon fucoidan inhibited the H. pylori attachment to porcine gastric mucin at pH 2.0 and 4.0. Two other sulfated polysaccharides, Fucus fucoidan and dextran sulfate sodium, also inhibited the attachment but only at pH 2.0. Inhibitory effects of these three sulfated polysaccharides were not observed at pH 7.2 and nonsulfated polysaccharides, such as mannan and dextran, exerted no influence at any pH. In the in vivo experiment, the H. pyloriinduced gastritis and the prevalence of H. pylori infected animals were markedly reduced by fucoidan in a dose-dependent manner, at doses of 0.05 and 0.5% in the drinking water. Conclusion. Cladosiphon fucoidan may deserve particular attention as a safe agent that can prevent H. pylori infection and reduce the risk of associated gastric cancer.

Aspirin has been reported to induce apoptosis in a variety of cell lines. In this study, we examined whether aspirin and sodium salicylate inhibit cell growth and induce apoptosis in rheumatoid synovial cells. Synovial cells were obtained from patients with rheumatoid arthritis, and the cells were treated with aspirin or sodium salicylate (0.1–10 mm) for 24 h. Cell proliferation and viability were assessed by 5-bromo-2′-deoxyuridine incorporation and by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay, respectively. The apoptosis of synovial cells was identified by DNA fragmentation assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay. Aspirin and sodium salicylate suppressed the proliferation (IC50 (concentration causing 50% inhibition of cell proliferation): 2.1 and 1.2 mm, respectively) and reduced the viability (IC50: 2.0 and 1.4 mm respectively) of synovial cells in a concentration-dependent manner at 0.3–10 mm. Furthermore, they induced DNA fragmentation and increased the number of TUNEL- positive synovial cells. These results suggest that aspirin and sodium salicylate can inhibit the proliferation of rheumatoid synovial cells through induction of apoptosis.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been reported to induce apoptosis in a variety of cell lines. In this study, we examined the effect of NSAIDs on the growth and apoptosis of synovial cells from patients with rheumatoid arthritis and analyzed the activation of peroxisome proliferator-activated receptor gamma (PPARgamma) as a possible mechanism of action of NSAIDs. Cell proliferation and viability were assessed from 5-bromo-2'-deoxyuridine incorporation and by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay, respectively. The apoptosis of synovial cells was identified by DNA fragmentation assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Indometacin, diclofenac, oxaprozin, and zaltoprofen reduced cell proliferation and induced apoptotic cell death in synovial cells, whereas ketoprofen and acetaminophen did not. N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methanesulfonamide (NS-398), a selective cyclooxygenase-2 inhibitor, also inhibited cell proliferation, whereas it did not cause apoptosis. Rheumatoid synovial cells expressed PPARgamma mRNA, and the PPARgamma ligands 15-deoxy-Delta(12,14)-prostaglandin J(2) and troglitazone reduced the proliferation and induced apoptosis in synovial cells. Luciferase reporter assay demonstrated that not only PPARgamma ligands but also NSAIDs, which could induce apoptosis, increased the activation of PPARgamma in synovial cells. Furthermore, the ability of NSAIDs and PPARgamma ligands to stimulate the activation of PPARgamma correlated with their ability to decrease cell viability(r = 0.92, p < 0.01) and ability to induce DNA fragmentation (r = 0.97, p < 0.001) in synovial cells. These results suggest that PPARgamma is an attractive target for induction of apoptosis in rheumatoid synovial cells and that the activation of the PPARgamma pathway is associated with the apoptotic action of NSAIDs.

Purpose: Clinically, diarrhea is the major dose-limiting toxicity of irinotecan hydrochloride (CPT-11). Using a rat model, we attempted to decrease the incidence of delayed-onset diarrhea by modifying the administration schedule of CPT-11, and studied the pharmacokinetics in this model in relation to the incidence of diarrhea. Methods: CPT-11 (total dose, 240 mg/kg) was administered intravenously (i.v.) to rats according to various schedules, and the incidence of delayed-onset diarrhea was monitored. Results: Administration of CPT-11 at a dose of 60 mg/kg once daily for four consecutive days induced severe diarrhea, while at 30 mg/kg twice daily at an interval of 9 h (daily dose 60 mg/kg) for four consecutive days alleviated the diarrheal symptoms, and at 30 or 40 mg/kg once daily for eight or six consecutive days, respectively, diarrhea was hardly induced. With the first schedule, mucosal impairment of the cecal epithelium was observed, including wall thickening, edema, decrease in crypt number and size, and formation of pseudomembrane-like substance, whereas these changes were less severe with the second schedule and were hardly observed with the other two schedules. The areas under the plasma and cecal tissue concentration-time curves (AUCpla and AUCcec), the maximum plasma concentrations (Cmax) and the biliary excretions of CPT-11 and its metabolites, 7-ethyl-10-hydroxycamptothecin (SN-38) and SN-38 glucuronide (SN-38G) in rats depended on the daily dose of CPT-11. Exceptionally, CPT-11 Cmax was significantly lower and SN-38 AUCcec was larger in the animals treated at 30 mg/kg twice daily than in those treated at 60 mg/kg once daily. Conclusion: These results suggested that the duration of exposure to both CPT-11 and SN-38 of the intestinal epithelium and CPT-11 plasma Cmax are closely related to the incidence and severity of CPT-11-induced delayed-onset diarrhea in rats.

This study demonstrates the novel actions of a non-steroidal anti-inflammatory drug aceclofenac, which is frequently used for rheumatoid arthritis and osteoarthritis. 4′-Hydroxy aceclofenac, a main metabolite of aceclofenac in humans, down-regulated the production of promatrix metalloproteinase-1/procollagenase 1 and promatrix metalloproteinase-3/prostromelysin 1 along with a decrease in their mRNAs in rabbit articular chondrocytes and synoviocytes, and interfered with the release of sulfated-glycosaminoglycans (proteoglycans) from the chondrocytes. 4′-Hydroxy aceclofenac also suppressed the proliferation of rabbit synoviocytes. In contrast, aceclofenac itself and its other metabolites, diclofenac and 4′-hydroxy declofenac, did not exert obvious actions on cellular functions. Therefore, it is suggested that the therapeutic effects of aceclofenac on rheumatoid arthritis and osteoarthrits are, at least in part, due to the novel chondroprotective effect of 4′-hydroxy aceclofenac via the suppression of promatrix metalloproteinase production and proteoglycan release. There is also evidence that inhibition of synoviocyte proliferation and the known inhibitory action on prostaglandin E2 production play a role.

The nutritional value of marine algae has long been recognized in the Orient, but in the Western world, the use of seaweeds for human consumption is limited: they exist as additives and so-called health foods. In contrast, more seaweed than ever before is being eaten in Japan, China and Korea. Seaweeds are low in fats, and contain vitamins and minerals [1,2]. They also contain a large quantity of chemically characteristic mucilaginous polysaccharides such as agar, carrageenans, alginate, laminaran, rhamnan sulfate and fucoidan. These polysaccharides differ from those in terrestrial vegetables and provide dietary fiber: the human body does not digest them [3]. These polysaccharides are used in the food and pharmaceutical industries and have recently been demonstrated to have some unique biological activities. In this paper, we review the structure and function of marine algal polysaccharides, especially the anti-ulcer effect of fucoidan.

The involvement of several cytokines in the antitumor effect induced by intrapleural (i.pl.) injection of heat-killed cells of Lactobacillus casei strain Shirota (LC 9018) in mice was investigated. Injection of LC 9018 i.pl. into Meth A fibrosarcoma (Meth A)-bearing mice not only significantly prolonged the survival of the mice, but also effectively inhibited the accumulation of malignant pleural fluid in the thoracic cavity. In the thoracic cavity of tumor-bearing mice treated with LC 9018, we observed large amounts of several cytokines including interleukin (IL)-1beta, interferon (IFN)-gamma, IL-12 and tumor necrosis factor (TNF)-alpha. Both anti-IFN-gamma and anti-IL-12 monoclonal antibody (mAb) treatments partially diminished the antitumor activity of LC 9018 in vivo, while the treatment of anti-IL-1beta mAb did not influence the survival of the mice. However, anti-TNF-alpha mAb treatment completely abolished the antitumor effect of LC 9018 in vivo, suggesting that in this model LC 9018 has a survival-prolonging effect involving certain cytokines. Moreover, i.pl. injection of mouse recombinant TNF-alpha into Meth A-bearing mice pretreated with anti-TNF-alpha mAb partially restored the survival-enhancing effect of LC 9018. These results led us to conclude that TNF-alpha induced by i.pl. injection of LC 9018 plays an important role in the antitumor effect of LC 9018 in vivo.

Arginine-vasopressin fragment 4–9 (AVP 4–9 ) has been demonstrated in animal studies to facilitate learning and memory. To clarify the mechanisms of this facilitation, we focused on the effects of AVP 4–9 on rodent cholinergic systems. AVP 4–9 (0.1 μM) enhanced the basal and the high-potassium–evoked acetylcholine (ACh) release from rat hippocampal slices (122.4 and 120.0% of control, respectively) in the presence of 1.3 mM calcium (physiological level) at 60 min after the incubation at 37°C. The AVP 4–9 -stimulated basal ACh release was inhibited by a V 1 -selective antagonist ([(β-mercapto-β,β-cyclopentamethylene propionic acid) 1 , O -methyl-Tyr 2 , Arg 8 ] vasopressin), but not by a V 2 -selective antagonist ([adamantaneacetyl 1 , O -ethyl- d -Tyr 2 , Val 4 , aminobutyryl 6 , Arg 8,9 ]-vasopressin). In addition, AVP 4–9 did not affect the basal ACh release under the calcium-free condition at 37°C or in the presence of 1.3 mM calcium at 4°C. However, AVP 4–9 facilitated the passive-avoidance response of scopolamine (a cholinergic blocker)-induced memory-deficient mice. These findings demonstrate that AVP 4–9 stimulates ACh release via mediation by V 1 -like vasopressin receptors, and shows dependence on calcium ion and temperature. The results also suggest that the mechanism of the facilitative effects of AVP 4–9 on learning and memory consist of the observed stimulation of cholinergic systems and other parallel pathways that would not be inhibited by cholinergic blocking.

We studied the inhibitory effect of Cladosiphon fucoidan on the attachment of Helicobacter pylori (H. pylori), a gastroduodenal pathogen, to human gastric cell lines. The bacterial binding in these cell lines was inhibited more by Cladosiphon fucoidan (IC50 = 16-30 mg/mL), than by the fucoidan from Fucus (IC50 > 30 mg/mL). Dextran sulfate, another sulfated polysaccharide, did not inhibit the binding at all. Pre-incubating the bacterial suspension with fucoidans reinforced the inhibitory ability of these components, and reduced the IC50 value of Cladosiphon fucoidan to approximately 1 mg/mL. However, the binding was not inhibited by pre-treatment of gastric cells with these components. It was also shown that this fucoidan blocks both Leb- and sulfatide-mediated attachment of H. pylori to gastric cells. Furthermore, fucoidan-binding proteins were found on the H. pylori cell surface by Western blot analysis. Thus, the inhibitory effect exerted by Cladosiphon fucoidan on binding between H. pylori and gastric cells might result from the coating with this component of the bacterial surface.

A structural study was carried out on a fucoidan isolated from the brown seaweed Cladosiphon okamuranus. The polysaccharide contained fucose, glucuronic acid and sulfate in a molar ratio of about 6.1 : 1.0 : 2.9. The results of Smith degradation showed that this polysaccharide has a linear backbone of 1-->3-linked alpha-fucopyranose with a half sulfate substitution at the 4-positions, and a portion of the fucose residues was O-acetylated. The data obtained from partial acid hydrolysis, a methylation analysis and NMR spectra indicated that the alpha-glucuronic acid residue is linked to the 2-positions of the fucose residues, which were not substituted by a sulfate group. These results indicated that the average structure of this fucoidan is as follows: -[(-->3Fuc-4(+/-OSO3-)alpha1-)5-->3[GlcA alpha1-->2]Fuc alpha1-]n-. (Half of each fucose residue was sulfated. One O-acetyl ester was present in every 6 fucose residues.)

The effect of Lactobacillus casei (LC) on the onset of alloxan (AXN)-induced diabetes in 7-week-old BALB/c mice were examined. It was observed that the mice given a diet containing 0.1% or 0.05% LC or orally administered LC showed significantly decreased incidence of diabetes induced by intravenous injection of AXN and that the plasma glucose level was slightly lower than that in the control group. The body weight in the LC-treated groups was higher than that in the control group, although the food intake weights were almost the same. Pathological analysis revealed that the AXN-induced disappearance of insulin-secreting beta-cells in the islets of Langerhans was strongly inhibited in the LC-treated groups. It was also shown that the serum nitric oxide level was maintained at a normal level in LC-treated mice, whereas the level in the control group was increased by AXN administration. Taken together, these findings suggest that oral administration of LC to AXN-treated BALB/c mice contributed to the reduction of diabetes and the increase in plasma glucose level.

Soybean lecithin transphosphatidylated phosphatidylserine (SB-tPS) was investigated for its effect on the impaired learning of a passive avoidance task by mice induced by scopolamine or cycloheximide. SB-tPS (240, 360, 480 mg/kg) administered orally significantly prolonged the step-through latency shortened by scopolamine. SB-tPS (240 mg/kg) administered orally also prolonged the step-through latency shortened by cycloheximide. These results suggest that the effect of SB-tPS on the impaired learning behavior may be related not only to the cholinergic system but also the serotonergic system.

Grandione, a unique isetexane diterpene dimer, was synthesized from demethylsalvicanol via the solid state hetero-Diels–Alder type dimerization reaction. By X-ray crystallography, grandione was determined to have the structure, in which the two isopropyl groups on the two monomer units are arranged syn. Grandione and some of its analogues showed a moderate cytotoxity on P388 murine leukemia cells.

The effects Lactobacillus casei YIT9108 (LC 9018) on antitumor activity and cytokine production in Meth A fibrosarcoma (Meth A)-bearing BALB/c mice were examined. Intrapleural (i.pl.) administration of LC 9018 was effective in prolonging the survival of Meth A-bearing mice, and frequently cured mice of the tumor. However, the results also indicated that the effect of LC 9018 was in part inhibited in mice treated with anti-CD3 or anti-CD8 antibody, but not affected in anti-CD4 antibody-treated mice. In contrast, LC 9018 had little effect on Meth A-bearing SCID or nude mice. These results demonstrated that CD8+ T cells participated in prolonging the survival of Meth A-bearing mice. Moreover, the examination of the production of several cytokines revealed that the production of interferon-gamma and interleukin-6 was, in particular, augmented in the exudated fluid of the thoracic cavity in BALB/c mice injected with LC 9018 i.pl. These results suggested that i.pl. administration of LC 9018 induced those cytokines which had the potential to activate the thoracic macrophages or proliferate the thoracic lymphocytes to the cytotoxic T cells. Taken together, these findings demonstrated that the prolonging effects on survival by i.pl. administration of LC 9018 depended on CD8+ T cells, and the i.pl. administration of LC 9018 into i.pl. Meth A-bearing mice induced several cytokines which participated in the subsequent immunoresponses.

A structural study was carried out on a galactose-rich polysaccharide fraction isolated from cell walls of Bifidobacterium catenulatum YIT4016 after N -acetylmuramidase digestion. The polysaccharide contained galactose and glucosamine in a molar ratio of 16.9:1.0. Data obtained by 13 C NMR spectroscopy showed that the backbone chain of this polysaccharide is composed of galactofuranose residues, while the branches consist of galactopyranosyl residues. Furthermore, the data obtained from NaIO 4 oxidation, partial methanolysis and methylation analysis indicated that this polysaccharide consists of a trisaccharide repeating unit having the following structure: [→6) βminus ; D − Galf − D − Galf (1→] n

The major fraction of rhamnogalactan was isolated from the cell wall of Bifidobacterium longum YIT4028 by treatment with N-acetylmuramidase. This polysaccharide was composed of rhamnose and galactose in a molar ratio of about 2:3. The 13C NMR spectrum indicated that it contained a pentasaccharides repeating unit. This observation, and the results of Smith degradation, partial acid hydrolysis and methylation analysis led to the conclusion that its structure is [formula: see text]

The anti-ulcer effects of bifidobacteria, lactobacilli and streptococci were examined using the acetic acid-induced gastric ulcer and ethanol-induced erosion models in rats. Bifidobacterium breve YIT4014 and 4043, and Bifidobacterium bifidum YIT4007 were administered orally, and anti-ulcer effects were confirmed for not only these organisms but also their polysaccharide fractions (PSFs). The major component of these anti-ulcer polysaccharides was rhamnose. In particular, polysaccharides in which the rhamnose content exceeded 60% were more effective in healing gastric ulcers. After administration of the PSF from B. bifidum YIT4007, the levels of epidermal growth factor and basic fibroblast growth factor increased in gastric tissues. Similar results were observed for the culture supernatant of gastric epithelial cells cultured with PSF. Furthermore, the production of 6-ketoprostaglandin F1 alpha by macrophages was also enhanced by PSF. These results indicated that these bacteria and their polysaccharides induced host repair and protective systems in the gastric ulcer model.

The antitumor effect of IHL-305, a novel pegylated liposome containing irinotecan, was investigated in human xenograft models. After subcutaneous transplantation of several human cancer cell lines (colorectal, non-small cell lung, small cell lung, prostate, ovarian and gastric cancer cells) to nude mice, IHL-305 or CPT-11 was administered intravenously 3 times at 4-day intervals. In all xenograft models tested, IHL-305 showed superior antitumor activity to that of CPT‑11 and a comparable tumor-growth-inhibitory effect at one-eighth or less of the dose of CPT-11, even against HT-29 colorectal and NCI-H460 non-small cell lung cancer cell lines, which show intrinsic resistance to CPT-11. A single injection or 2 injections of IHL-305 on several dosing schedules also resulted in a significant antitumor effect compared to that of vehicle control in a dose-dependent manner and showed comparable antitumor activity at about one-fifth the dose of the maximum tolerated dose of CPT-11. The analysis of the concentrations of irinotecan and SN-38, an active metabolite of CPT-11, in plasma and tumors revealed that irinotecan was maintained at high concentrations, and the prolonged presence of SN-38 in plasma and tumors in IHL-305 treated mice compared with CPT-11-treated mice. Therefore, the stronger tumor inhibitory effect of IHL-305, as compared to CPT-11, was associated with the difference in the concentration of irinotecan in plasma or tumors after each agent was administered and with the maintainance of a higher concentration of SN-38. These results indicate that IHL-305 demonstrated superior antitumor activity against a wide range of tumors at lower doses than CPT-11.

Three new rhazinilam-derived alkaloids, kopsiyunnanines C1, C2, and C3, and a new quebrachamine-type alkaloid, kopsiyunnanine D, which possess an unusual methoxymethyl or ethoxymethyl function, were isolated from the aerial parts of Yunnan Kopsia arborea. This is the first report of the presence of these functions in natural alkaloids. The structures and absolute configurations of the alkaloids were determined by spectroscopic methods and confirmed by semisynthesis.

The reaction of triptolide and its analogues with a fluorinating agent, that is, bis(2-methoxyethyl)aminosulfur trifluoride (Deoxo-Fluor) or (diethylamino)sulfur trifluoride (DAST), was studied. One of the fluorinated products, 14beta-dehydroxy-14beta-fluoro triptolide, was found to be more cytotoxic than the parent natural triptolide.

Isetexane diterpene analogues were semisynthesized from demethylsalvicanol isolated from Perovskia abrotanoides (Labiatae). The structure and cytotoxic activity relationships (SAR) of the natural parent diterpene, demethylsalvicanol, and its semisynthetic analogues were studied by using P388 murine leukemia cells.

Several C-ring modified analogues of a potent antileukemic diterpene, triptolide (1), were synthesized and their structure-activity relationships were studied.

Seven known abietane diterpenoids and 11-O- and 12-O-acetylcarnosic acids were isolated from a methanol extract of Perovskia abrotanoides (Labiatae). Structure and cytotoxic activity relationships (SAR) of the natural and semisynthetic analogues of the presently isolated abietane diterpenoids were studied by using P388 murine leukemia cells.

Although the influence of selective cyclooxygenase (COX)-2 inhibitors on the proliferation of colon adenocarcinoma cells have been the subject of much investigation, relatively little research has compared the effects of different COX-2 inhibitors. Celecoxib strongly suppressed the proliferation of COX-2 expressing HT-29 cells at 10–40 μM. NS-398 and nimesulide also inhibited cell proliferation, whereas rofecoxib, meloxicam, and etodolac did not. Only celecoxib induced apoptosis of HT-29 cells, as detected on the basis of DNA fragmentation, TUNEL positivity, and caspase-3/7 activation. DNA fragmentation was also increasd in COX-2 non-expressing cell lines (SW-480 and HCT-116) by exposure to celecoxib for 6–24 h. All six COX-2 inhibitors suppressed the production of prostaglandin E2 by HT-29 cells, suggesting that the pro-apoptotic effect of celecoxib was unrelated to inhibition of COX-2. Inactivation of Akt might explain the differential pro-apoptotic effect of these selective COX-2 inhibitors on colon adenocarcinoma cells.

The purpose of this study was to evaluate the neuroprotective effect of nordihydroguaiaretic acid (NDGA), an antioxidant and/or 5-lipoxygenase inhibitor, on ischemia-reperfusion injury behavioral pharmacologically and histologically in vivo. First, the antioxidant activity of NDGA was evaluated in vitro by measuring the production of thiobarbituric acid reactive substances (TBARS) in rat brain homogenate. Second, the effect of NDGA on learning and memory impairment induced by rat four-vessel occlusion transient ischemia was investigated with the Morris water-maze task. Third, the effect of NDGA on pyramidal cell loss in the hippocampus after transient ischemia was examined. NDGA inhibited the production of TBARS with an IC(50) of 0.1 microM, and significantly attenuated postischemic learning and memory impairment at 10 mg/kg. Furthermore, consecutive 4-day administration of NDGA at 10 mg/kg significantly reduced the postischemic neuronal death. NDGA was found to be potent and effective as an anti-ischemia-reperfusion injury agent in terms of behavioral pharmacology and histology. The present results suggest that NDGA has beneficial effects on behavioral deficits and histological injury caused by ischemia-reperfusion.

To elucidate the anti-ulcer potential of Cladosiphon fucoidan, anti-peptic activity, bFGF stabilizing activity and inflammatory properties of this and related substances were investigated. Anti-peptic activity was observed with this and other sulfated polysaccharides such as dextran sulfate, carrageenan, and Fucus fucoidan. However, non-sulfated polysaccharides such as mannan and dextran did not exert the anti-peptic activity. The loss of bFGF bioactivity was prevented by all sulfated polysaccharides tested except chondroitin sulfate, at pH 7.4 and at pH 4.0. At pH 2.0, only heparin protected the bFGF activity. The generation of superoxide by macrophages and PMNs was stimulated by dextran sulfate, carrageenan, and Fucus fucoidan, whereas Cladosiphon fucoidan, heparin and chondroitin did not. Dextran sulfate, carrageenan, and Fucus fucoidan also stimulated the secretion of TNFalpha from macrophages, while Cladosiphon fucoidan did not. Thus, Cladosiphon fucoidan is a sulfated polysaccharide without inflammatory action. These results suggest that Cladosiphon fucoidan is a safe substance with potential for gastric protection.

Diarylheptanoids possess potent anti-inflammatory properties. However, the mechanism of their action is not fully understood. In this study, we found that three diarylheptanoids, 1-(3, 5-dimethoxy-4-hydroxyphenyl)-7-phenylhept-1-en-3-one (YPE-01), yakuchinone B and demethyl-yakuchinone B, reduced the adhesion of both human monocytic cell line U937 and human eosinophilic cell line EoL-1 cells to tumor necrosis factor-alpha (TNF-alpha)-treated human umbilical vein endothelial cells. In addition, they suppressed interleukin-1beta- or TNF-alpha-induced expression of E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on the surface of the endothelial cells. Since YPE-01 reduced both VCAM-1 and ICAM-1 mRNA induction in TNF-alpha-stimulated endothelial cells, diarylheptanoids appeared to suppress adhesion molecule expression at the transcriptional level. Furthermore, YPE-01 suppressed both VCAM-1 and ICAM-1 mRNA induction as well as edema in 12-O-tetradecanoylphorbol 13-acetate (TPA)-inflamed mice ears in vivo. These results suggest that the anti-inflammatory action of diarylheptanoids is, at least in part, due to their suppressive effect on the surface expression of inducible adhesion molecules in endothelial cells, and subsequent leukocyte adhesion.

We investigated the effects of the prolyl endopeptidase inhibitors 1-[1-(Benzyloxycarbonyl)-L-prolyl]prolinal (Z-Pro-Prolinal) and N-benzyloxycarbonyl-thioprolyl-thioprolinal-dimethylaceta l (ZTTA) on delayed neuronal death induced by four-vessel-occlusion transient ischemia in rats. We also examined the effects of [pGlu4, Cyt6, ArgS]vasopressin (vasopressin-(4-9)) and thyrotropin-releasing hormone (TRH) on the delayed neuronal death. Furthermore, we investigated the role of vasopressin receptors in the effects of vasopressin and prolyl endopeptidase inhibitors. Z-Pro-Prolinal, vasopressin-(4-9) and TRH protected pyramidal cells in the CA1 subfield of the rat hippocampus from delayed neuronal death after 10-min ischemia. The effect of vasopressin-(4-9) was abolished by vasopressin receptor antagonists. The effect of Z-Pro-Prolinal was also abrogated by the antagonists. These results suggest that the neuroprotective effect of prolyl endopeptidase inhibitors is mediated by neuropeptides such as [Arg8]vasopressin and TRH, and indicate the involvement of vasopressin receptors in the neuroprotective effect of vasopressin-(4-9) and prolyl endopeptidase inhibitors.

Aceclofenac, 2-[(2,6-dichlorophenyl) amino] phenylacetoxyacetic acid, is a novel non-steroidal anti-inflammatory drug. We investigated the effects of aceclofenac on prostaglandin E2 production by several kinds of human cells. Aceclofenac inhibited interleukin-1beta-induced prostaglandin E2 production by human rheumatoid synovial cells, but had no inhibitory effect on cyclooxygenase-1 or cyclooxygenase-2 activities by itself. We also observed that part of the aceclofenac was converted into diclofenac, the cyclooxygenase-1 and cyclooxygenase-2 inhibitor, when aceclofenac was incubated with human rheumatoid synovial cells. Aceclofenac was also converted into diclofenac and 4'-hydroxy diclofenac by human polymorphonuclear leukocytes and monocytes. 4'-Hydroxy diclofenac suppressed prostaglandin E2 production specifically by blocking cyclooxygenase-2 activity. These findings suggested that aceclofenac can be metabolized to cyclooxygenase inhibitors (diclofenac and/or 4'-hydroxy diclofenac) by these inflammatory cells. Although detailed examinations in non-inflammatory cells remain to be studied, we concluded that aceclofenac is shown to be a new type of non-steroidal anti-inflammatory drug which is intracellulary converted into active metabolites that inhibit the prostaglandin E2 production.

The antidiabetic effects of Lactobacillus casei (LC) on a non-insulin-dependent diabetes mellitus (NIDDM) model, KK-Ay mice, were investigated. The oral administration of LC to male 4-week-old KK-Ay mice, or raising the mice on a 0.05% LC-containing diet significantly decreased the plasma glucose at 8 to 10 weeks of age compared with the control group. The body weights of the LC-treated groups were lower than those of the control group, although the food intake was nearly the same in all groups. Phenotypic analysis of spleen cell surface markers revealed that the increase in CD4+ T cells at 12 weeks was significantly inhibited by the oral treatment with LC. Cytokine production, especially that of interferon-gamma and interleukin 2, was also inhibited in the oral LC-treated group. The plasma insulin levels of the LC-treated groups were also lower than those of the control group, and the insulin binding potential of red blood cells in the LC-treated mice was augmented more than that in the control group. Taken together, these findings led us to conclude that the oral administration of LC in the NIDDM model mice, KK-Ay, was involved in the decrease in the plasma glucose level and modified the host immune responses.

The effects of a newly synthesized cationized arginine vasopressin fragment 4-9 analogue (C-AVP- 4 , 5 , 6 , 7 , 8 , 9 ) on learning and memory in rats were studied by the passive avoidance test. C-AVP- 4 , 5 , 6 , 7 , 8 , 9 and its parent peptide, arginine vasopressin fragment 4-9 (AVP- 4 , 5 , 6 , 7 , 8 , 9 ), a well known potent neuropeptide, were subcutaneously injected 1.5 hr prior to the retention test. The most effective doses of C-AVP- 4 , 5 , 6 , 7 , 8 , 9 and AVP- 4 , 5 , 6 , 7 , 8 , 9 were 8.6 × 10 −2 and 1.3 nmol/ kg, respectively. To evaluate the distribution of C-AVP- 4 , 5 , 6 , 7 , 8 , 9 in the central nervous system (CNS), apparent tissue-plasma concentration ratios (Kp, app ) of intravenously administered radioiodinated C-AVP- 4 , 5 , 6 , 7 , 8 , 9 ( 125 I-C-AVP- 4 , 5 , 6 , 7 , 8 , 9 ) in the CNS in mice were determined. At the apparent steady state of plasma concentration of 125 I-C-AVP- 4 , 5 , 6 , 7 , 8 , 9 , the Kp, app values of the 125 I-C-AVP- 4 , 5 , 6 , 7 , 8 , 9 in the cerebrum, cerebellum and spinal cord were over 12 times higher than that of the vascular space marker which slightly penetrates the BBB. Moreover, the rat cerebral homogenate converted C-AVP- 4 , 5 , 6 , 7 , 8 , 9 into its parent peptide AVP- 4 , 5 , 6 , 7 , 8 , 9 . These results suggest that the potent effects of C-AVP- 4 , 5 , 6 , 7 , 8 , 9 on learning and memory may be due to AVP- 4 , 5 , 6 , 7 , 8 , 9 generated as a result of distribution and metabolism of peripherally administered C-AVP- 4 , 5 , 6 , 7 , 8 , 9 in the CNS.

We investigated the effect of N-benzyloxycarbonyl-thioprolyl-thioprolinal-dimethylacetal (ZTTA), a novel postproline cleaving enzyme (prolyl endopeptidase, PPCE) inhibitor, on the in vitro activity of rat brain PPCE and memory impairment induced by cerebral ischemia. ZTTA noncompetitively inhibited rat brain PPCE (ki = 2.9 μM). Cerebral ischemia for 5 min increased the number of errors in a working memory task with a three-panel runway paradigm. ZTTA at 6 mg/kg, administered immediately after blood flow reperfusion, significantly reduced the increase in working memory errors expected to occur 24 h after 5 min of ischemia. The antiamnesic action of ZTTA may be ascribable to a neuroprotective effect on the central nervous system due to some neuropeptides that are substrates of PPCE in the brain.

Pharmacological studies on the antihypertensive effect of a polysaccharide-glycopeptide complex (SG-1) isolated from Lactobacillus casei were carried out by using spontaneously hypertensive rats (SHR). An antihypertensive effect of SG-1 was observed by oral, but not by intravenous or intraperitoneal administration, and the effect was attenuated by orally pre-treating with indomethacin. A single oral administration of SG-1 (20 mg/kg) decreased the peripheral vascular resistance (PR). The daily oral administration of SG-1 (10 mg/kg) for 14 days had no effect on either the urine volume or urinary electrolytes (Na+, K+, and Cl-), but it did increase the excretion of 6-keto-PGF1 alpha, a metabolite of PGI2, in the urine. Moreover, a single oral administration of SG-1 (20 mg/kg) also increased the biliary 6-keto-PGF1 alpha excretion. These results suggest that the antihypertensive effect of orally administered SG-1 resulted from an enhancement of PGI2 biosynthesis and the subsequent decrease in PR.

On considere le probleme de la commande lineaire quadratique discrete d'un systeme de grande taille a dimension croissante ou les divers sous-systemes sont relies a differents instants d'echantillonnage