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LC-MS-based proteomics requires methods with high peak capacity and a high degree of automation, integrated with data-handling tools able to cope with the massive data produced and able to quantitatively compare them. This paper describes an off-line two-dimensional (2D) LC-MS method and its integration with software tools for data preprocessing and multivariate statistical analysis. The 2D LC-MS method was optimized in order to minimize peptide loss prior to sample injection and during the collection step after the first LC dimension, thus minimizing errors from off-column sample handling. The second dimension was run in fully automated mode, injecting onto a nanoscale LC-MS system a series of more than 100 samples, representing fractions collected in the first dimension (8 fractions/sample). As a model study, the method was applied to finding biomarkers for the antiinflammatory properties of zilpaterol, which are coupled to the beta2-adrenergic receptor. Secreted proteomes from U937 macrophages exposed to lipopolysaccharide in the presence or absence of propanolol or zilpaterol were analysed. Multivariate statistical analysis of 2D LC-MS data, based on principal component analysis, and subsequent targeted LC-MS/MS identification of peptides of interest demonstrated the applicability of the approach.

Vascular endothelial growth factor (VEGF), oncostatin M (OSM), and granulocyte chemotactic protein-2 (GCP-2/CXCL6) are up-regulated in U937 macrophages and peripheral blood macrophages exposed to LPS, beta-adrenergic receptor (beta2-AR) agonists (e.g. zilpaterol, and clenbuterol) and some other agents that induce intracellular cAMP (prostaglandin E2, forskolin, and butyryl cAMP). LPS in combination with beta2-agonists and cAMP elevating agents had an additional effect on the release of VEGF, OSM, and CXCL6. These proteins are up-regulated after 16-24 h of exposure and this is mediated by the beta2-AR, as determined by time course experiments and the use of a specific beta2-AR antagonist (ICI 118551). Beta2-AR agonists are used as bronchodilators in the treatment of asthma, but appear to have no effect on the chronic inflammation of the disease. However, the up-regulation of VEGF, OSM, and CXCL6 may have adverse effects on the inflammatory process of asthma. These mediators are involved in the recruitment of neutrophils, airway remodelling and angiogenesis, known features of chronic inflammatory diseases. We propose that the up-regulation of these proteins could play a role in the adverse effects of prolonged excessive usage of beta2-AR agonists on the airways besides the desensitization of the beta2-AR.

In this study the anti-inflammatory properties of zilpaterol, a beta2-adrenergic receptor (AR) agonist specifically developed as a growth promoter in cattle were investigated. Although zilpaterol has a different structure compared with the beta2-AR agonists known to date, it was noted that it was able to bind to both the beta2-AR (Ki = 1.1 x 10(-6)) and the beta1-AR (Ki = 1.0 x 10(-5)). Using lipopolysaccharide (LPS)-exposed U937 macrophages, the production of cyclic adenosine-3',5'-cyclic monophosphate (cAMP) and tumor necrosis factor alpha (TNF-alpha) were investigated. Zilpaterol inhibited TNF-alpha release and induced intracellular cAMP levels in a dose-dependent manner. The inhibition of TNF-alpha release and induction of cAMP production was mainly mediated via the beta2-AR, as indicated by addition of beta1- and beta2-specific antagonists. The effects of zilpaterol were investigated in LPS-treated male Wistar rats after pretreatment with zilpaterol. Zilpaterol dosed at 500 microg/kg body weight reduced the TNF-alpha plasma levels. In conclusion, zilpaterol is a beta2-adrenergic agonist and an inhibitor of TNF-alpha production induced by LPS both in vivo and in vitro.

Two-dimensional difference gel electrophoresis (DIGE) in combination with univariate (Student's t-test) and multivariate data analysis, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to study the anti-inflammatory effects of the beta(2)-adrenergic receptor (beta(2)-AR) agonist zilpaterol. U937 macrophages were exposed to the endotoxin lipopolysaccharide (LPS) to induce an inflammatory reaction, which was inhibited by the addition of zilpaterol (LZ). This inhibition was counteracted by addition of the beta(2)-AR antagonist propranolol (LZP). The extracellular proteome of the U937 cells induced by the three treatments were examined by DIGE. PCA was used as an explorative tool to investigate the clustering of the proteome dataset. Using this tool, the dataset obtained from cells treated with LPS and LZP were separated from those obtained from LZ treated cells. PLS-DA, a multivariate data analysis tool that also takes correlations between protein spots and class assignment into account, correctly classified the different extracellular proteomes and showed that many proteins were differentially expressed between the proteome of inflamed cells (LPS and LZP) and cells in which the inflammatory response was inhibited (LZ). The Student's t-test revealed 8 potential protein biomarkers, each of which was expressed at a similar level in the LPS and LZP treated cells, but differently expressed in the LZ treated cells. Two of the identified proteins, macrophage inflammatory protein-1beta (MIP-1beta) and macrophage inflammatory protein-1alpha (MIP-1alpha) are known secreted proteins. The inhibition of MIP-1beta by zilpaterol and the involvement of the beta(2)-AR and cAMP were confirmed using a specific immunoassay.

Background: 4-[3,5-Bis (trimethylsilyl) benzamido] benzoic acid (TAC-101) is a novel retinobenzoic acid derivative, which has a specific binding affinity to the retinoic acid receptors (RAR)-alpha and -beta. Apoptotic induction by TAC-101 was investigated using a rat hepatic metastatic model of rat RCN-9 colon cancer cells in vivo and FACScan analysis with the DLD-1 human colon cancer cell line in vitro., Materials and Methods: Hepatic metastatic tumors were induced using intra-portal injection of RCN-9 cells into F344 rats in vivo. TAC-101 (8 mg/kg) was orally administered for 5 consecutive days a week for 4 weeks. Subsequently, hepatic tumors were counted after laparotomy. Apoptotic index (A.I.) in the hepatic tumors was evaluated using immunohistochemistry for single-stranded DNA. The proliferative index (P.I.), Fas and Fas ligand were also immunohistochemically evaluated. Moreover, evaluation of apoptosis by TAC-101 in vitro using FACScan analysis was performed in the DLD-1 human colon cancer cell line., Results: Oral administration of TAC-101 resulted in a significant inhibition of hepatic metastasis without weight loss of the rats. TAC-101 significantly decreased P. I. but increased A. I. in the hepatic metastatic tumors. TAC-101 did not affect the expression of Fas ligand, but obviously increased the expression of Fas in the metastatic tumors. Moreover, TAC-101 induced early apoptosis in DLD-1 cells in a time-dependent manner in vitro., Conclusion: These findings suggest that TAC-101 inhibits hepatic metastasis of colon cancer and induces apoptosis partially through enhanced Fas expression.

Objectives: A novel retinobenzoic acid derivative, 4-[3,5-bis (trimethylsilyl) benzamido] benzoic acid (TAC-101), was reported to suppress the growth and invasion of human gastric cancer or hepatocellular carcinoma by induction of apoptosis. We examined the antitumor activity of TAC-101 against human ovarian carcinoma cell lines., Methods: Apoptosis of human epithelial ovarian carcinoma-derived cell lines (RMG-I, RMG-II, RTSG, RMUG-S, RMUG-L, and KF) was investigated by detecting DNA laddering and was quantified by an enzyme-linked immunosorbent assay. Inhibition of apoptosis was also examined using a caspase inhibitor. Furthermore, TAC-101 (8 mg kg(-1) day(-1) orally for 30 days) was investigated in nude mice with subcutaneous RMG-II tumors. A prominent apoptotic response to TAC-101 was observed. The antitumor effects of cisplatin (7 mg/kg intravenously on day 1) and paclitaxel (36 mg/kg intravenously on days 1 and 5) were also assessed for comparison., Results: Apoptosis occurred in all of the cell lines (except KF) in a concentration-dependent manner after exposure to TAC-101 and was markedly induced in RMG-I and RMG-II cells (derived from ovarian clear cell adenocarcinomas). A caspase inhibitor blocked the induction of apoptosis by TAC-101. The maximum inhibition of RMG-II tumor growth in nude mice by TAC-101, cisplatin, and paclitaxel was 45%, 34%, and 47%, respectively., Conclusion: Oral TAC-101 shows potential as a novel antitumor agent for ovarian carcinoma, especially ovarian clear cell adenocarcinoma.

A-285222 (A-285) is a bis-trifluoromethyl-pyrazole (BTP), a novel class of immunosuppressive agents that inhibit NFAT activity in vitro in human and non-human primate cells through a calcineurin-independent mechanism. In this preliminary study, we treated cynomolgus monkeys with different doses of A-285 for several days. Blood was collected from all animals at different times during the study. From these samples, plasma concentrations of A-285 were measured by liquid chromatography/mass spectrometry (LC/MS), and intracellular T-cell production of the cytokines IL-2, IFN-gamma, and TNF-alpha was quantified by flow cytometry using a mitogen-stimulated whole blood assay. Marked inhibition of cytokine production occurred after administration of the first dose of A-285, and this effect was comparable to that of cyclosporine. While neurological toxic side effects were seen when the plasma concentration of A-285 exceeded 4 microg/ml, at lower plasma levels the drug was well tolerated over 2 weeks and its pharmacodynamic effects were sustained throughout this time.

4-[3,5-bis(trimethylsilyl)benzamido] Benzoic acid (TAC-101) has potent antiproliferative, antiangiogenic, and antitumor effects in vitro and in vivo. These effects might be due to TAC-101 binding to retinoic acid receptor alpha (RAR-alpha) and interfering with the binding of activator protein-1 (AP-1) to DNA. However, little is known about the detailed mechanism of TAC-101 function. We investigated the mechanism of the antiangiogenic effect of TAC-101 using a rat hepatic metastatic model in vivo and DLD-1 human colon cancer cells in vitro. Liver metastases were induced by portal injection of RCN-9 rat colonic cancer cells into F344 rats. TAC-101 (8 mg/kg) was orally administered 5 days per week for 4 weeks and then hepatic tumors were immunohistochemically evaluated for microvessel density (MVD) and vascular endothelial growth factor (VEGF). TAC-101 significantly reduced both MVD and VEGF expression. Northern blot analysis and ELISA indicated that TAC-101 efficiently inhibited production of VEGF mRNA and protein in DLD-1 cells in a time- and dose-dependent manner. These findings suggest that TAC-101 may inhibit progression and metastasis in colon cancer by interfering with tumor production of VEGF.

A new series of azole derivatives of 1-triazolyl-4-trimethylsilyl-2-butanol and 1-triazolyl-5-trimethylsilyl-2-pentanol were synthesized and evaluated for fungicidal activities against rice blast, sheath blight, and powdery mildew on barley. The derivatives of 2,4-difluorobenzene exhibited high antifungal activities when applied by spray, but exhibited no fungicidal activity by submerged application.

The anti-metastatic effect of 4-[3,5-bis(trimethylsilyl)benzamido]benzoic acid (TAC-101) was investigated using our established intrahepatic metastasis model. Orthotopic implantation of a fragment of CBO140C12 hepatoma into the liver resulted in the formation of a solitary tumor nodule and its intrahepatic metastasis. Daily oral administration of TAC-101 at a dose of 8 mg/kg resulted in a significant inhibition of intrahepatic metastasis, but did not affect the growth of the tumor at the implanted site. The down-regulation of transcriptional anti-activator protein-1 (AP-1) activity by TAC-101 paralleled the inhibition of cell invasion and migration through the repression of expression of the mRNAs for urokinase-type plasminogen activator (u-PA) and its receptor (u-PAR). These findings suggest that TAC-101 may improve therapeutic efficacy for liver cancer patients to prevent intrahepatic metastasis.

Purpose: The goals of this study were to determine the safety, toxicity, and pharmacokinetics of TAC-101, a novel synthetic retinoic acid receptor-alpha (RAR-alpha) selective retinoid, in patients with advanced cancer., Patients and Methods: Twenty-nine patients at two centers received oral TAC-101 at doses ranging from 12 to 34 mg/m(2)/d. Pharmacokinetic sampling was performed on days 1 and 28., Results: The most frequent toxicities were myalgia/arthralgia, fatigue, and triglyceridemia. No dose-limiting toxicities were observed within the first 28 days up to 28 mg/m(2). However, seven of 21 patients experienced venous thromboembolic events (VTEs) during TAC-101 treatment. Eight additional patients who received 34 mg/m(2) were treated after a hypercoagulable work-up to exclude potential risk factors for VTE, and two of eight patients subsequently experienced VTEs. The maximum tolerated dose was exceeded at 34 mg/m(2)/d within the first 28 days, with one grade 3 hypertriglyceridemia, two grade 3 myalgia/arthralgia, and one grade 3 fatigue. One patient with advanced non-small-cell lung cancer had a complete response. No other responses were observed. No autoinduction of metabolism was observed with dosing over 28 days., Conclusion: This is the first human clinical study with TAC-101, a RAR-alpha selective retinoid. Musculoskeletal toxicity and hypertriglyceridemia were observed characteristics of previously studied retinoids. The recommended phase II dose is 24 mg/m(2) with this treatment schedule. Alternative treatment schedules and prospective evaluation of thrombotic risk will be investigated in subsequent studies.

The efficacy of the surface modification of natural diatomite and zeolite material by chlorosilanes is demonstrated. Chlorosilanes used were trimethylchlorosilane (TMSCI), tert-butyldimethylchlorosilane (TBDMSCI), dimethyloctadecylchlorosilane (DMODSCI), and diphenyldichlorosilane (DPDSCI) possessing different headgroups and chemical properties. Silanol groups of the diatomite and zeolite were modified by chemical reaction with the chlorosilanes resulting in a stable covalent attachment of the organosilanes to the mineral surface. The alteration of surface properties of the modified material was proved by measurements of water adsorption capacity, total organic carbon (TOC) content, and thermoanalytical data. The surface modified material showed great stability even when exposed to extremes in ionic strength, pH, and to pure organic solvents. Sorption of toluene, o-xylene, and naphthalene from water was greatly enhanced by the surface modification compared to the untreated materials which showed no measurable sorption of these compounds. The enhanced sorption was dependent on the organic carbon content as well as on chemical characteristics of the chlorosilanes used. Batch sorption experiments showed that the phenyl headgroups of DPDSCI have the best affinity for aromatic compounds. Removal from an aqueous solution of 10 mg/L of naphthalene, o-xylene, and toluene was 71%, 60%, and 30% for surface modified diatomite and 51%, 30%, and 16% for modified clinoptilolite, respectively. Sorption data were well described by the Freundlich isotherm equation, which indicated physical adsorption onto the lipophilic surface rather than partitioning into the surface organic phase. The chlorosilane modified materials have an apparent potential for application in environmental technologies such as permeable reactive barriers (PRB) or wastewater treatment.

[4-3,5-Bis(trimethylsilyl)benzamido] benzoic acido] (TAC-101), which exhibits an anti-tumor effect, can bind to retinoic acid receptors (RARs). It has retinoid-like properties, such as chemopreventive action against cancer cells. The up-regulation of connexin (Cx) expression by retinoids is well known in various epithelial cells. In this study, we investigated whether TAC-101 up-regulates gap junctional intercellular communication (GJIC) in renal epithelial cells exposed to the renal carcinogens. Madin Darby canine kidney (MDCK) cells were incubated with TAC-101 for 3 days, then briefly exposed to renal carcinogens potassium bromate (KBrO3) or dimethylnitrosamine (DMN). TAC-101 increased the expression of connexin 43 protein without affecting Cx43 phosphorylation and prevented inadequate Cx43 localisation caused by KBrO3 or DMN. Consequently, TAC-101 prevented the disruption of GJIC in MDCK cells. These data suggested that TAC-101 enhanced GJIC by up-regulating Cx43 expression and that TAC-101 might be useful for the prevention of renal cell carcinoma.

TAC-101 (4-[3,5-bis(trimethylsilyl)benzamido]benzoic acid) is a novel, synthetic retinoid that is effective against liver metastases of human gastrointestinal cancer cells such as the human stomach carcinoma line AZ-521 in animal models, and is currently in use in phase I cancer trials. However, the mechanism of its antimetastatic action is still poorly understood. Tumor metastasis depends on angiogenesis, and various retinoids have been found to exhibit antiangiogenic activity. Based on these findings we here examined the antiangiogenic effects of TAC-101. Oral administration of TAC-101 (2-8 mg/kg/day) resulted in a drastic suppression of the AZ-521 cell-induced angiogenesis in a mouse dorsal air sac assay system, compared to the vehicle alone. Immunohistochemical analysis with antibody against the endothelial marker CD31 revealed a significant reduction in microvessel density in liver metastases from animals treated with TAC-101 (8 mg/kg p.o.), compared to liver metastases from the untreated control animals. The ability of TAC-101 (8 mg/kg p.o.) to prevent experimental liver metastasis of AZ-521 cells in athymic nude mice was comparable with that of the known angiogenesis inhibitor TNP-470 (30 mg/kg s.c.). TAC-101 also affected angiogenesis in chorioallantoic membranes and some functions of endothelial cells associated with angiogenesis, whereas the retinoid failed to suppress AZ-521 cell proliferation directly. These data suggest that the TAC-101 is an orally active antiangiogenic agent and that this antiangiogenic property may contribute to its efficacy against liver metastasis of human stomach cancer cells.

Silver-ion high-performance liquid chromatography was used to fractionate a mixture of conjugated linoleic acid (CLA) isomers (as the free fatty acids, CLAFFA) in commercial CLA mixtures and biological samples. Due to the unchanged retention mechanism, it was assumed that the elution order of the isomers remained the same as that of methyl esters separated on the same column. The most abundant isomers, cis/trans 10, 12-18:2 and cis/trans 9,11-18:2, were separated better as free acids on a single column than in the methyl ester form. Quantification of the CLA standard was used as the reference profile to evaluate different methylation methods commonly used to prepare CLA methyl esters for quantitation. Acid-and base-catalyzed derivatization methods resulted in CLA intraisomerization and losses in total conjugated dienes content. Acid (HCl and BF3) methylations significantly elevated the level of trans,trans isomers and significantly reduced the cis/trans isomers. Base methylation, tetramethylguanidine/methanol, resulted in loss of trans,trans isomers, and a substantial loss of total underivatized conjugated dienes. Other catalysts such as the trimethylsilyldiazomethane produced additional peaks of unidentified artifacts. The analysis of CLAFFA appears to provide more accurate quantification of CLA isomers in commercial and biological samples.

Background: The incidence of cancers of the digestive tract has been high among all of the cancers in Japan and the western hemisphere. The poor prognosis of patients, especially those with liver metastases, has become a great challenge for the development of a new drug to cope with this problem., Materials and Methods: Mice implanted by intrasplenic injection of TMK-1, human gastric carcinoma cells, were used to examine the life-prolonging effect of TAC-101. To elucidate a mechanism of action of TAC-101, the drug-induced apoptosis was assessed by DNA ladder formation whilst the prevention of transcription factor AP-1 binding to its DNA recognition sequence was assessed by gel shift assay., Results: TAC-101 showed the life prolonging effect in a model of experimental liver metastasis of TMK-1. The antitumor effect, expressed as T/C (%), was 201, 141 and 112%, for TAC-101 (2 mg/kg), ATRA (8 mg/kg) and 5-FU (19 mg/kg), respectively. The in vitro experiments revealed that the anticancer activity of TAC-101 is related to its ability to induce apoptosis within a short period of time in TMK-1 cells and human leukemic cells, HL-60. TAC-101-induced apoptosis was suppressed by the inhibitors of proteases, specifically by Z-Val-Ala-DL-Asp-fluoromethylketone, indicating the involvement of caspase activation. TAC-101 also inhibited, in a concentration-dependent manner, the binding of AP-1 to its DNA binding sites present in the promoter region of the genes involved in the control of cell migration, invasion, and angiogenesis., Conclusion: TAC-101 may suppress liver metastasis by the induction of an apoptotic mechanism(s) in cancer cells and possibly by controlling transcriptional activity of AP-1.

To evaluate nonaromatic catechol bioisosteres, the conformationally restrained enynes 1 and enediynes 2 were synthesized via palladium-catalyzed coupling as the key reaction step. Subsequent receptor binding studies at the dopamine receptor subtypes D(1), D(2 long), D(2 short), D(3), and D(4) showed highly interesting binding profiles for the enynes 1a and 1b when compared to dopamine. At the guanine nucleotide-sensitive high-affinity binding site of the D(3) receptor, the target compound 1b (K(i) = 5.2 nM) was 10-fold more potent than dopamine but less potent at the D(2) and D(4) subtypes. In contrast to dopamine the agonists 1a and 1b showed strong selectivity for the receptors of the D(2) family (D(2)-D(4)). As far as we know, this study represents the first report on nonaromatic dopamine agonists. Comparison of molecular electrostatic potentials, derived from semiempirical molecular orbital calculations, and lipophilicity maps was performed.

The anti-tumor and anti-metastatic effects of 4-[3,5-bis(trimethylsilyl)benzamido]benzoic acid (TAC-101) were investigated using our established lung cancer model. Orthotopic implantation of Lewis lung carcinoma (LLC) cells into the lung parenchyma produced a solitary tumor nodule in the lung followed by mediastinal lymph node metastasis. Daily oral administration of TAC-101 at doses ranging from 4 to 16 mg/kg resulted in a significant inhibition of lymphatic metastasis (inhibition rate=57 to 76%), while only the dose of 16 mg/kg significantly inhibited tumor growth at the implanted sites (inhibition rate=46%). Combined treatment with cis-diamminedichloroplatinum (CDDP) and TAC-101 (8 mg/kg, p.o., daily) enhanced the anti-tumor effect of CDDP (7 mg/kg, i.v., bolus) against both the growth of implanted tumor and lymphatic metastasis. In addition, this combined treatment significantly prolonged the survival time of LLC tumor-bearing mice as compared to treatment with each agent alone. The anti-activating protein-1 (AP-1) activity of TAC-101 caused inhibition of LLC cell invasion through the repression of expression of urokinase-type plasminogen activator and its receptor. The anti-invasive activity of TAC-101 may be involved in its in vivo anti-metastatic activity. These findings suggest that TAC-101 is a novel anti-cancer agent that may improve the therapeutic modalities for lung cancer patients with metastatic disease.

We demonstrated in this study that inhibition of intra-hepatic growth of colon cancer by TAC-101 is mediated by inhibition of angiogenesis. In vitro experiments showed that TAC-101 inhibited the proliferation of murine hepatic sinusoidal endothelial (HSE) cells induced by coculture with murine colon 26-L5 (L5) cells. HSE cell proliferation was also enhanced by conditioned medium of L5 cells (CM-L5), and this enhancement of proliferation was abrogated by anti-vascular endothelial growth factor antibody. CM-L5 also induced in vitro tube formation of HSE cells on Matri-gel, and this activity of CM-L5 was abrogated by TAC-101 in a concentration-dependent manner. On the other hand, p.o. administration of TAC-101 inhibited tumor-induced angiogenesis in vivo and decreased the weights of L5 tumors in the mouse liver. Reverse transcriptase-PCR analysis using in vivo tumor tissue suggested that repression of vascular endothelial growth factor expression by TAC-101 was associated with the antiangiogenic activity. TAC-101 alone and 5-fluorouracil (5-FU)/D,L-leucovorin (LV) significantly inhibited the intrahepatic growth of L5 tumors (P = 0.002 and 0.001, respectively), whereas 5-FU alone did not (P = 0.088). When TAC-101 was administered with 5-FU/LV, marked enhancement of antitumor activity was observed (95% inhibition; P<0.001). This enhanced antitumor effect was also observed in experiments using Co-3 human colon adenocarcinoma. Concurrent treatment with TAC-101 and 5-FU/LV and sequential treatment with 5-FU/LV followed by TAC-101 resulted in significant augmentation of antitumor activity against Co-3 (overall P = 0.007 and 0.015, respectively). These findings indicate that TAC-101 inhibits tumor angiogenesis and suggest that it may be effective against hepatic metastasis of colon cancer.

Tumor-related angiogenes is expected to become an important target for improving treatment of cancer, because it plays key roles in tumor growth, invasion and metastasis. We considered that the successful development of such angiostatic treatment depended entirely upon the development of useful anti-angiogenic agents, and attempted to find novel angiogenesis inhibitors by using three in vivo assays, based on an idea of ours. As a result we have demonstrated that different types of agents with low molecular weight including microbial metabolites, cell differentiation modulators like retinoids and steroids, exhibit anti-angiogenic activity, anti-metastatic activity and/or antitumor activity. Taken these findings, an ideal anti-angiogenic agent is discussed.

We examined the in vivo anti-tumor activity of the benzoic acid derivative, TAC-101 (4-[3,5-bis(trimethylsilyl)benzamido] benzoic acid), for intrahepatic spread of JHH-7 human hepatocellular carcinoma (HCC) cells and its mechanism of action. Oral administration of TAC-101 markedly inhibited liver tumor of JHH-7 cells and prolonged the life-span of tumor-bearing mice without affecting the body weight. The life-prolonging effect of TAC-101 was more effective than that of other anti-cancer agents including CDDP, 5-FU, and CPT-11 (T/C (%) of life-span; 181 to 219, 128, 133, and 142%, respectively). In vitro, TAC-101 at the concentration of more than 10 microM showed direct cytotoxicity against JHH-7 cells caused by induction of apoptosis. Hepatocyte growth factor (HGF) enhanced the invasive ability of JHH-7 cells without affecting the cell viability. Non-cytotoxic concentrations of TAC-101 inhibited the JHH-7 invasion induced by HGF and down-regulated the expression of c-MET protein in a concentration-dependent manner. In summary, these results suggest that TAC-101 would be useful for a new class of therapeutic agents and that it may improve the prognosis of patients with liver-tumors including metastasizing tumor and HCC.

The fully methylated cyclohexyl glycosides of 2-acetamido-2-deoxy-alpha- and beta-D-hexopyranoses having the gluco, manno, and galacto configurations were each subjected to reductive-cleavage conditions using one of three promoters, namely trimethylsilyl trifluoromethanesulfonate, a mixture of trimethylsilyl methanesulfonate and boron trifluoride etherate, or boron trifluoride etherate alone. As expected, the fully methylated 1,2-trans-linked acetamido sugar derivatives were rapidly converted to their respective oxazolinium ions with all three promoters. Surprisingly, however, the fully methylated 1,2-cis-linked acetamido sugar derivatives were also converted to their respective oxazolinium ions, albeit at a much slower rate. In the latter case, evidence was obtained for anomerization to the 1,2-trans-linked isomers under reductive-cleavage conditions. Since the anomerization was relatively slow at room temperature in dichloromethane, a modified procedure was developed in which the reaction was carried out at 70 degrees C in 1,2-dichloroethane. Using the modified procedure, all 1,2-cis- and 1,2-trans-linked acetamido sugar derivatives were rapidly converted into their respective oxazolinium ions and subsequent quenching of the reactions with anhydrous methanol gave the respective 1,2-trans-linked methyl glycoside derivatives in quantitative yield. The modified procedure is recommended for the total reductive cleavage of polysaccharides comprised of acetamido sugar residues.

Retinoids, including all-trans-retinoic acid (ATRA), its isomers, and fifty synthetic retinoids (retinobenzoic acids), were tested for differentiation-inducing activity on human leukemia cell lines HL-60 and NB4. Binding activity of typical retinoids to nuclear retinoic acid receptors (RARs) was also investigated. A good linear correlation between the ED50 values of differentiation-inducing activity towards HL-60 cells and those towards NB4 cells was found. Binding activities of retinoids to RAR alpha and RAR beta also correlated well to the differentiation-inducing activities.

MDL 73,745 (2,2,2-trifluoro-1-(3-trimethylsilylphenyl) ethanone, CAS 132236(18-1) is a novel tight-binding inhibitor of acetylcholinesterase (AChE), which is in development as a potential therapeutic compound in the symptomatic treatment of Alzheimer's disease. Pharmacokinetics and pharmacodynamics of the compound were studied in the dog after single intravenous (i.v. 2 mg/kg), oral p.o. 10 mg/kg) and sub-cutaneous (s.c., 10 mg/kg) administrations of [14C]-MDL 73,745. Plasma concentrations of total radioactivity were much higher than those of parent drug after i.v., p.o. and s.c. administration, indicating extensive metabolism of the compound, although this was less after, s.c. administration than after p.o. administration. The bioavailability (F) was 34% after s.c. administration, compared with 4% after p.o. administration. The low bioavailability after p.o. administration was not due to poor drug absorption, as over 64% of the dose was absorbed. Pharmacokinetic parameters, calculated after i.v. administration, showed a terminal elimination half-life of 24 h, total body plasma clearance of around 70 ml/min/kg and apparent volume of distribution of 150 l/kg. AChE activity was almost 100% inhibited after i.v. administration, and over 80% inhibited 1 h after p.o. administration. In both cases, AChE activity returned to baseline levels by 12 h. AChE was around 80% inhibited 4 h after s.c. administration, and did not return to baseline levels until 36 h after drug administration. A combined pharmacokinetic-pharmacodynamic (PK-PD) effect model demonstrated that the extent of AChE inhibition could be correlated with plasma levels of the parent compound. As s.c. administration increased F, and led to longer AChE inhibition, transdermal (t.d.) delivery was assessed in the same animals. Patches, corresponding to a dose of 50 mg/kg, were applied to the shaved lateral abdominal skin for a period of 96 h. Sustained plasma concentrations of the parent drug were observed over the 96 h period of t.d. application. Mean (+/- SD) maximum plasma concentrations (Cmax) of 26.9 +/- 4.3 ng/ml were found 3.7 +/- 2.5 h after t.d. patch application und F was around 13%. AChE inhibition reached a maximum of 72% at 6 h after t.d. application and was still 35% at 96 h. The rate of release from the delivery system, per unit surface area, (ko) was calculated to be 7.7 micrograms/cm2. Transdermal delivery of MDL 73,745 thus decreased the important hepatic first-pass effect, and led to sustained plasma concentrations of drug, thus avoiding peaks and troughs which could lead to side-effects or poor efficacy.

Objective: To determine the pharmacokinetics, pharmacodynamics and safety of the acetylcholinesterase inhibitor zifrosilone in healthy male volunteers., Methods: Pharmacokinetics, pharmacodynamics, and tolerance of zifrosilone were studied in a double-blind, sequential, single-escalating-dose, randomized panel design. Each panel consisted of six subjects, with four subjects receiving zifrosilone (10, 30, 60, 90, 120, 150, 200, 250, and 300 mg orally) and two subjects receiving matching placebo. Serial blood samples were obtained for zifrosilone plasma concentrations and red blood cell acetylcholinesterase and butyrylcholinesterase activities. Participating subjects (n = 54) were men between the ages of 18 and 45 years. Each subject had a normal physical examination, electrocardiogram, serum chemistries, hematology, urinalysis, and test for human immunodeficiency virus at screening., Results: A greater than proportionate increase in mean plasma concentration values for area under the curve from time zero to infinity was observed over the 200 to 300 mg dose range groups. Red blood cell acetylcholinesterase showed a dose-inhibition relationship, with a mean maximum inhibition of 20.9% at 10 mg that increased to 62.1% at 300 mg. Butyrylcholinesterase activity was relatively unaffected by zifrosilone (< 20% inhibition at 300 mg). For doses > or = 200 mg, an Emax pharmacodynamic model was used to describe the relationship between zifrosilone plasma concentration and red blood cell acetylcholinesterase inhibition (Emax = 83.8%; EC50 = 0.65 ng/ml)., Conclusions: Zifrosilone showed dose-dependent pharmacokinetics after oral administration and was effective in causing selective inhibition of red blood cell acetylcholinesterase.

We postulate that the effect of cholinesterase inhibitors to ameliorate the cholinergic deficit in Alzheimer's disease is related to their ability to maintain long-lasting, non-toxic steady-state levels of acetylcholine in cortex. We investigated the effect of the cholinesterase inhibitor, MDL 73,745 (2,2,2-trifluoro-1-(3-trimethylsilylphenyl)ethanone), on the extracellular levels of acetylcholine, norepinephrine, dopamine and 5-hydroxytryptamine in the cerebral cortex of the rat by high-performance liquid chromatography coupled with electrochemical detection. The drug significantly increased acetylcholine levels above the baseline at 2 and 10 mg/kg s.c., but not at the 1 mg/kg dose. At both 2 and 10 mg/kg there was a good correlation between cholinesterase inhibition and acetylcholine increase in cortex. At the 2 and 10 mg/kg doses, the maximal cholinesterase inhibition was 64% and 77%, respectively, and the increase in acetylcholine release was 481% and 1016%, respectively. Norepinephrine and dopamine, but not 5-hydroxytryptamine levels, were also significantly increased by the 10 mg/kg dose. The increases of norepinephrine and dopamine levels reached a maximum of 124% and 370%, respectively, and continued for a period of at least 8 h. Cholinergic side-effects were most marked at the 10 mg/kg dose but were also noticeable at the 2 mg/kg dose in the form of fasciculations, tremor and splay.

Selected enzymatic systems can efficiently produce a product in the electronically excited triplet state. Earlier, only the formation of electronically excited singlet species was known. The formation of triplet species has been demonstrated with both normal substrates/metabolites and with xenobiotics, even at the cellular level. Triplet excited species have intrinsically much longer lifetimes than excited singlets, whereby they can be potentially important agents for normal and/or deleterious processes, including mutagenesis. Enzymically generated triplet species can damage DNA, even when protein coated, as in the case of the lambda-phage of Escherichia coli. Some evidence of damage by triplet species has also been reported for intact cells. Triplet excited species may produce their effects through type I and/or type II dark photosensitization, that is, the events may be started by H abstraction and/or singlet oxygen/superoxide ion production. The induction of lipid peroxidation, with concomitant clastogenic effects, appears to be of special importance.

The reaction of 2'-deoxy-5-trimethylsilyl(Tms)uridine with methanesulfonyl chloride led to the corresponding 3',5'-di-O-mesyl derivative, which was treated with lithium toluylate in DMF to give 2,3'-anhydro-1-(2-deoxy-5-O-p-toluyl-beta-D-xylofurano- syl)-5-Tms-uracil. Under these conditions 1-(2,3-dideoxy-5-O-p-toluyl-alpha-D- glycero-pent-2-enofuranosyl)-5-Tms-uracil was obtained from 1-(2-deoxy-alpha-D-ribofuranosyl)-5-Tms-uracil. Interaction of 2,3'-anhydronucleoside with LiN3 in DMF and successive deacylation with MeONa-MeOH gave 3'-azido-2',3'-dideoxy-5-Tms-uridine. Hydrogenation of this compound with 10% Pd/C in ethanol gave 3'-azido-2',3'-dideoxy-5-Tms-uridine. From 2,4,5-tris-Tms-uracil and 2,3-didehydrofurane in 1,2-dichloroethane in the presence of SnCl4 1-(2-tetrahydrofuranyl)-5-Tms-uracil was prepared. In a similar way 1-[(1,3-dioxy-2-propoxy)methyl]-5-Tms-uracil was synthesized by condensation of silylated uracil with 1,3-dibenzyloxy-2-acetoxymethylglycerol followed by the hydrogen transfer hydrogenolysis with cyclohexene--20% Pd(OH)2/C. None of the compounds exhibits cytotoxic activity against CaOv in vitro. The acycloderivative in concentration of 250 micrograms/ml has no effect on the HSV-1 and vaccinia virus replication in vitro. 3'-Azidonucleoside in dose of 100-750 mg/kg as well as 1-(2-tetrahydrofuranyl)-5-Tms-uracil in dose of 160-800 mg/kg were devoid of antitumour activity against P388 in vivo.

We have been examining a number of chemically modified mineral fibers, derived from amosite asbestos, by in vitro methods to clarify the role of the fiber surface in determining biological activity. The various fibers have identical size distributions but differ in their affinities for components of the cell membrane. They were treated with boiling toluene or chemically modified by treatment with alkyldimethylchlorosilanes (R = C8, C18) that react with free-surface hydroxyl groups to form the corresponding siloxanes. Fibers in MEM supplemented with 15% fetal calf serum were added to a suspension of V79-4 cells labeled with tritiated thymidine and the mixture was incubated. Aliquots of this mixture were spun down on a density gradient to determine the degree of cell-fiber interaction. At 37 degrees C native amosite (UICC standard) stuck to cells within 15 min of incubation, and the amount of sticking was maximum within 70 min. Decreasing the temperature decreased the amount of sticking, and at 20 degrees C no sticking was observable. The chemically modified amosite and the amosite treated with boiling toluene did not stick to the cells even after 70 min. Soaking the toluene-treated amosite with aqueous solutions at room temperature for 48 hr produced a material that had the same sticking properties as the original untreated fiber. These results indicate that the silanol content, and possibly the degree of hydration of the fiber surface, is important for a fiber to stick to a cell surface.

Several trimethylsilyl derivatives were found to be ligands of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7): trimethylsilylethyl acetate (III) and trimethylsilylmethyl acetate (V) are substrates of the enzyme, whereas trimethylsilylethanol (VIII) is a competitive inhibitor. The silicon compounds have kinetic parameters similar to those of their carbon analogues, except for trimethylsilylmethyl acetate, which is a substrate of acetylcholinesterase, whereas its carbon analogue is not susceptible to enzymic hydrolysis.

It was shown in experiments on mice that the silil derivatives of alpha-pyrrolidone, I-methyl (3-trimethylsilil) pyrrolidone-2 (1A) and 1-vinyl(3 trimethylsilil) pyrrolidone-2 (2A) in doses of 6.25-25 mg/kg exert a tranquilizing effect on emotional behavior of the animals in conflict situation and decrease shock-induced emotional reactions. At the same time IA activates the orienting response and locomotor activity of mice. In the doses that exert the tranquilizing effect the compounds under test increased the content of GABA and speeded up dopamine metabolism in the rat anterior brain. The starting products of the synthesis, 1-methylpyrrolidone-2 and trimethylsilanol given in the test doses produced no tranquilizing effect on the animals' emotional behavior and neuromediator turnover in the brain. It is suggested that the appearance of marked pharmacological activity of the silil derivatives of alpha-pyrrolidone-2 is related to a better penetration of these substances via the hematoencephalic barrier and to their effect on GABA- and dopaminergic processes in the central nervous system.

N-Trimethylsilylmethyl-N-nitrosourea (TMS-MNU), a silicon analogue of N-neopentyl-N-nitrosourea (neoPNU), was assayed for mutagenicity and/or cytotoxicity on a series of E. coli B tester strains, S. typhimurium TA100, Chinese hamster V79, and cultured murine leukemia L1210 cells. All the biological activities demonstrated in this study reveal that this nitrosourea is a biological methylating agent equivalent to N-methyl-N-nitrosourea (MNU) but definitely distinguished from all the other alkylnitrosoureas examined so far, including neoPNU (the carbon analogue of TMS-MNU). The proposed molecular mechanism is that trimethylsilylmethanediazohydroxide is produced by hydrolytic activation of TMS-MNU and undergoes a nucleophilic cleavage of the Si--CH2 chemical bond at a high rate to form methanediazohydroxide (the reactive intermediate of MNU) which, in turn, methylates the informational biopolymer leading to mutagenesis.

Suspensions of rat adrenocortical cells produce corticosterone as the major glucocorticoid. Cholesterol side-chain cleavage, the initial and rate-limiting step in the glucocorticoid biosynthetic pathway, is catalyzed by P-450scc. We have examined the effect of a variety of P-450scc inhibitors on corticosterone production by isolated rat adrenocortical cells. These inhibitors include reversible, noncovalently interacting inhibitors as well as mechanism-based inhibitors which irreversibly inactivate P-450scc in vitro. (20S)-22-nor-22-thiacholesterol and (22R)-22-aminocholesterol cause 50% inhibition of corticosterone production at 4 microM and 30 nM, respectively. Inhibition by these compounds was essentially not time-dependent. (20R)-20-(1-hexynyl)-pregn-5-en-3 beta, 20-diol and (20R)-20-(1,5-hexdiynyl)-pregn-5-en-3 beta, 20-diol at 10 microM inhibited corticosterone production in a time-dependent manner, resulting in 30% inhibition of corticosterone production during a 100-min incubation. (20S)-20-(2-trimethylsilyl ethyl)-pregn-5-en-3 beta, 20-diol inhibited in a strongly time-dependent manner. At 10 microM this compound irreversibly inhibited more than 90% of the side-chain cleavage capacity of the cell during a 40-min incubation. Cells treated with this steroid did not regain their capacity for side-chain cleavage after removal of free steroid. None of the inhibitors described above inhibited production of corticosterone by cells supplied with pregnenolone, the product of the P-450scc reaction. We suggest that the only significant effect of these compounds under these conditions is inhibition of the side-chain cleavage enzyme.

Urinary steroidal profiles were studied in five women with Cushing's syndrome and in two normal women that were chosen as controls, by means of gas chromatography and gas chromatography-mass spectrometry. Four patients presented ACTH-dependent adrenal hyperplasia and the last patient had an adrenocortical carcinoma. Steroids were analyzed in urinary extracts, as their respective trimethylsilyloximes and/or trimethylsilylderivatives. Qualitative and quantitative data about 36 urinary steroids were obtained. Three pituitary patients showed a well defined picture of "5-ene pathway" in adrenal function. The fourth patient depicted some primary deficiencies of corticosteroid biosynthesis that overshadowed most biochemical expressions of Cushing's disease. The patient with adrenal carcinoma developed a steroidal pattern resembling autonomous functioning of adrenal cortex inner zones, showing both "5-ene pathway" and "4-ene pathway" increase. This patient also had an unexpected excretion of a major metabolite of 18-hydroxycorticosterone, that did not correlate with parameters of aldosterone production. Tetrahydro-6-hydroxy-cortisol was determined in urine of two patients and original data about urinary cortoic acids in Cushing's syndrome are given. Peripheral reductive metabolism played the most important role in almost all patients. In turn, some oxidative metabolic pathways for cortisol were not specifically favoured in the cases of Cushing's disease here reported.

The use of 31P NMR to study smooth muscle is hindered by low metabolite concentrations and low tissue mass. These disadvantages can be overcome due to low rates of energy utilization and the temporal stability of smooth muscle. Smooth muscle free Mg++, ADP, and the creatine analogue beta-guanidinopropionate can be studied in ways distinctive from 31P NMR of striated muscle. The pharmacological effects of naturally occurring agents such as insulin, glucose, or norepinephrine, and of NMR markers such as phenylphosphonate or 3-(trimethylsilyl)-1-propane-sulfonate on both vascular smooth muscle and perfused smooth muscles can be measured. Given sufficient collection time, 31P NMR is as useful in assessing the behavior of smooth muscle as it is of other muscle types.

Androst-4-en-3-one analogs incorporating a trimethylsilyl or a trimethylsilylmethyl group at C-1, C-2 or C-19 were prepared and evaluated as inhibitors of aromatase. Only 10-[1-hydroxy-2-(trimethylsilyl)ethyl]estr-4-ene-3,17-dione inhibited human placental aromatase. Enzyme kinetic analysis revealed competitive inhibition [apparent dissociation constant (Ki) of 562 +/- 12 nM] associated with marginal time-dependent inhibition.

A new method for activation of glass fiber supports for immobilisation of proteins and peptides in gas-liquid phase sequencing is described. The new support offers several advantages over the presently used carrier polybrene: no precycling is required, initial yields are improved and background contamination is lower. This leads to an overall increase in detection sensitivity. The derivatisation method includes acid activation and subsequent covalent coating of glass fibers with quaternary ammonium groups thereby giving the glass surface a high binding capacity for both proteins and peptides. The activated glass has been successfully used for sequencing proteins and peptides isolated by HPLC as well as by electroelution from polyacrylamide gels.

An antimicrobial-surface kinetic test which maximizes probability of cell-to-surface contact has been developed. The measurement of rate of kill by a nonleaching antimicrobial surface is based on the number of surviving bacterial cells at specific times of exposure to various amounts of total treated surface area of test substrate. This method gives information for direct comparison of rate of kill for a variety of antimicrobial surfaces in terms of rate of kill per square centimeter of surface area. Data obtained by this method can also give valuable dose response application information as an indication of the exponential efficiency of concentration in terms of treated surface area.

In toxicological studies, the test compound FOE 3440 A, a [(3,5-dichloro-2-pyridyl)oxy]phenoxypropanoate with herbicidal properties, produced a severe increase in weight and an intensive induction of monoxygenases activity in the mouse, but not in the rat. Comparative subacute studies were performed with oral administration of 0, 5, 20 and, in some instances, 80 mg kg-1 body weight to mice, rats, hamsters, dogs and rhesus monkeys. Liver enzyme activities were measured. The evaluation of the enzyme activity results showed an unusually severe dose-related induction of the monooxygenases [7-ethoxycoumarin-O-deethylase (EOD), 7-ethoxyresorufin-O-deethylase (EOR) and aldrin epoxidase (ALD)] in the mouse and a much weaker reaction in the other species tested. This exceptional position of the mouse was also demonstrated in vitro by a cytochrome P-450 interaction (inhibition of ALD). The primary metabolite of FOE 3440 A produced a distinct inhibition of the ALD in mice liver microsomes. There were no interactions for the other species. Tests for this cytochrome P-450 interaction using microsomes from three different human livers gave no indications of an inhibition in any case. The 'phenoxypropanoic acid' moiety of FOE 3440 A is structurally similar to the pharmaceutical clofibrate, a familiar model substance for peroxisome proliferation. In order to answer the question of whether peroxisome proliferation is the second mechanism for affecting liver, the carnitine acetyl transferase activity (CA-T), a marker enzyme for peroxisome proliferation, was determined in all liver samples from the comparative species studies. The most striking result of the measurement of CA-T activity was the very large increase in the male rat in the low dose group of 5 mg kg-1. Lesser increases in the CA-T activity were measured in the female rat, the mouse, and also in the 20 mg kg-1 group of the hamster. By comparison, the changes of the activity in dog and monkey were very small. Comparative studies in mouse and hamster using a model substance described in the literature (1,4-bis[2-(3,5-dichloropyridyloxy]-benzene (TCPOBOP] indicated that the '3,5-dichloropyridyloxy' moiety of FOE 3440 A is responsible for the induction of the monooxygenases in the mouse and the 'phenoxypropanoic acid' moiety for the peroxisome proliferation in rodents.

The purpose of this study is to develop methods for determining the chemical species of lipid peroxides that occur in various types of tissue pathology. Experiments are aimed at determining the phospholipid peroxides associated with retinal degeneration as the initial test case. Phospholipid hydroperoxides are synthesized by photosensitized oxidation, chemically characterized and used to develop an effective and simplified method to identify and measure phospholipid hydroperoxides by gas chromatography-mass spectrometry (GC-MS). A sensitive reverse phase high performance liquid chromatography (HPLC) method is also presented to separate peroxidized phospholipids from phospholipids. For GC-MS, phospholipid peroxides are reduced with sodium borohydride and transesterified to form fatty acid methyl esters using a mild quaternary ammonium hydroxide catalyst. The hydroxyl groups produced by reducing the hydroperoxides are formed into trimethylsilyl ethers and GC-MS is employed (with electron ionization and negative ion chemical ionization) to identify oxidized fatty acids at the 10 ng level. Photooxidation of (palmitoyl)(linoleoyl) phosphatidylcholine yielded equal amounts of the conjugated (9 and 13 isomers) and the nonconjugated (10 and 12 isomers) linoleoyl hydroperoxides. Photooxidation of rat retina total lipids yielded oxidation products of oleolyl (18:1) esters as well as the conjugated and nonconjugated oxidation products of arachidonoyl (20:4) and docosahexaenoyl (22:6) esters virtually all of which arise from phospholipids. The nonconjugated products are of interest as indicators of photosensitized light damage in retina and other tissues. It is notable that all the possible singly oxidized products are found with the exception of the 4, 5 and 7 hydroperoxides of 22:6 and the 5 hydroperoxide of 20:4. It appears that the approach of singlet oxygen is strongly inhibited in the sterically hindered region near the phospholipid head groups.