Glycogen synthase kinase-3 beta (GSK-3 beta) is a key target and effector of downstream insulin signalling. Using comparative protein kinase assays and molecular docking studies we characterize the emodin-derivative 4-[N-2-(aminoethyl)-amino]-emodin (L4) as a sensitive and potent inhibitor of GSK-3 beta with peculiar features. Compound L4 shows a low cytotoxic potential compared to other GSK-3 beta inhibitors determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay and cellular ATP levels. Physiologically, L4 acts as an insulin-sensitizing agent that is able to enhance hepatocellular glycogen and fatty acid biosynthesis. These functions are particularly stimulated in the presence of elevated concentrations of glucose and in synergy with the hormone action at moderate but not high insulin levels. In contrast to other low molecular weight GSK-3 beta inhibitors (SB216763 and LiCl) or Wnt-3 alpha-conditioned medium, however, L4 does not induce reporter and target genes of activated beta-catenin such as TOPflash, Axin2 and glutamine synthetase. Moreover, when present together with SB216763 or LiCl, L4 counteracts expression of TOPflash or induction of glutamine synthetase by these inhibitors. Because L4 slightly activates beta-catenin on its own, these results suggest that a downstream molecular step essential for activation of gene transcription by beta-catenin is also inhibited by L4. It is concluded that L4 represents a potent insulin-sensitizing agent favouring physiological effects of insulin mediated by GSK-3 beta inhibition but avoiding hazardous effects such as activation of beta-catenin-dependent gene expression which may lead to aberrant induction of cell proliferation and cancer.

Trans-beta-nitrostyrene (TBNS) has been reported to be a potent inhibitor of protein phosphatases PTB1 and PP2A and to display a pro-apoptotic effect even in multidrug resistant tumour cells. Here we compared the anti-tumour potential of TBNS with 5-fluorouracil (5-FU) as the standard chemotherapeutic agent for colorectal cancer in LoVo cells. Resistance to 5-FU based therapy might be a consequence of 5-FU's delayed effect requiring long-term effective concentrations in the tumour tissue. Thus, alternatives like platin containing drugs with a more rapid effect have been introduced recently. Compared to 5-FU TBNS displayed a faster cytotoxic and pro-apoptotic effect. A 50% decrease in viability was observed already after 8 h with TBNS while 5-FU displayed no significant effect before 48 h. DNA fragmentation and caspase-3 assays confirmed the more rapid apoptotic effect of TBNS. Since apoptosis affects individual cells these results about a rapidly induced apoptosis were further studied on a single cell level in microscopic assays of caspase-3 and caspase-8 activation. Adducts of trans-beta-nitrostyrene displayed an anti-tumour effect comparable to TBNS which suggests the possibility of creating adducts with optimised tissue targeting. Finally, the calculation of a drug combination index displayed a synergistic effect for the combination of TBNS and 5-FU in Lovo as well as in HT-29 and HCT116 colon cancer cells.

Purpose: Thalidomide has demonstrated clinical activity in various malignancies including androgen-independent prostate cancer. The development of novel thalidomide analogs with better activity/toxicity profiles is an ongoing research effort. Our laboratory previously reported the in vitro antiangiogenic activity of the N-substituted thalidomide analog CPS11 and the tetrafluorinated analogs CPS45 and CPS49. The current study evaluated the therapeutic potential of these analogs in the treatment of prostate cancer in vivo. Experimental Design: Severely combined immunodeficient mice bearing s.c. human prostate cancer (PC3 or 22Rv1) xenografts were treated with the analogs at their maximum tolerated doses. Tumors were then excised and processed for ELISA and CD31 immunostaining to determine the levels of various angiogenic factors and microvessel density (MVD), respectively. Results: CPS11, CPS45, and CPS49 induced prominent and modest growth inhibition in PC3 and 22Rv1 tumors, respectively. Thalidomide had no effect on tumor growth in either xenograft. Vascular endothelial growth factor and basic fibroblast growth factor levels were not significantly altered by any of the thalidomide analogs or thalidomide in both PC3 and 22Rv1 tumors. CPS45, CPS49, and thalidomide significantly reduced PC3 tumor platelet-derived growth factor (PDGF)-AA levels by 58–82% (P < 0.05). Interestingly, treatment with the analogs and thalidomide resulted in differential down-regulation (≥1.5-fold) of genes encoding PDGF and PDGF receptor isoforms as determined by DNA microarray analysis. Intratumoral MVD of 22Rv1 xenografts was significantly decreased by CPS45 and CPS49. CPS49 also reduced MVD in PC3 xenografts. Conclusions: Thalidomide analogs CPS11 and 49 are promising anti-cancer agents. PDGF signaling pathway may be a potential target for these thalidomide analogs. Detailed microarray and functional analyses are under way with the aim of elucidating the molecular mechanism(s) of action of these thalidomide analogs.

Since the pioneering work by Gossen and Bujard in 1992 demonstrating the usefulness of the Escherichia coli derived tet resistance operon for regulating gene expression a large collection of doxycycline-controlled transgenic mice has been established. Gene switching in eukaryotic tissue culture cells or mice requires administration of tetracycline, anhydrotetracycline or doxycycline to efficiently inactivate the transactivator protein tTA (TET-OFF system) or alternatively to activate the reverse transactivator protein rtTA (TET-ON system). However, the antibiotic activity of doxycycline can create an imbalance of the intestinal flora, resulting in diarrhoea and in a smaller number of animals in colitis. Previous studies reported that 4-epidoxycycline (4-ED), a hepatic metabolite of doxycycline, does not function as an antibiotic in mice. This gave us the idea that 4-ED might be useful for controlling gene expression in mice without the unwanted antibiotic side effect. To study the applicability of 4-ED for control of gene expression we used cell lines expressing the oncogene HER2 under control of tTA (TET-OFF) as well as rtTA (TET-ON). 4-ED and doxycycline were similarly efficient in switching on or -off HER2 expression. In vivo we used a conditional mouse model that allows switching off HER2 in tumor tissue. We show that (i) doxycycline, 7.5mg/ml in drinking water (used as a positive control), (ii) 4-ED, 7.5mg/ml in drinking water, (iii) 4-ED, 10mg/kg body weight, s.c., and (iv) anhydrotetracycline, 10mg/kg, s.c. (used as a second positive control), were similarly efficient. Using mice with tumor volumes of 1.6cm(3) all four schedules led to a tumor remission of more than 95% within 7 days. In conclusion, 4-ED is similarly efficient as doxycycline to control gene expression in vitro and in mice. Since 4-ED lacks the antibiotic activity of doxycycline it may help to avoid adverse side effects and selection of resistant bacteria.

Drugs containing an anthraquinone moiety such as daunorubicin (Daunoblastin) and mitoxantrone (Onkotrone) constitute some of the most powerful cytostatics. They suppress tumor growth mainly by intercalation into DNA and inhibition of topoisomerase II. and are suspected to generate free radicals leading to DNA strand scission. We established a novel strategy for obtaining new highly functionalized derivatives of emodin (1,3,8-trihydroxy-6-methyl-anthraquinone). Using emodin, DIB, and an appropriate amine as starting materials, we obtained a wide range of emodin-related structures by one-pot synthesis. Several of these derivatives showed stronger cytotoxic and cytostatic activity than emodin. In particular, compound 6 was highly effective on the HepG2 tumor cell line, but did not show any cytotoxicity on normal hepatocytes. In addition to this favorable feature, compound 6 revealed interesting binding properties to a recombinant fragment of the multi-drug-resistance transporter, pgp, and reversed the multi-drug-resistance phenotype of H4-II-E cells, thus making this compound a promising potential anti-tumor drug. (C) 2004 Elsevier Ltd. All rights reserved.

Thalidomide, 2-(2,6-dioxo-3-piperidinyl)-1H-isoindole-1,3(2H)-dione, has been shown to inhibit angiogenesis, the formation of new blood vessels from existing vasculature. As a result, there is renewed interest in this drug as a potential therapy for solid tumors. Thalidomide forms a number of metabolites and has been shown to require metabolic activation for antiangiogenic activity. A series of 39 compounds, based upon the structure of some of these metabolites, was synthesized and tested for their ability to inhibit microvessel growth in the rat aortic ring assay. The results of this testing have been used as the basis for a three-dimensional quantitative structure-activity relationship (3D-QSAR) study, utilizing comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) procedures. The best resulting CoMFA and CoMSIA models have conventional r(2) values of 0.924 and 0.996, respectively. The cross-validated q(2) values are 0.666 and 0.635, respectively. These models offer insight into the structural requirements for activity of thalidomide analogues as angiogenesis inhibitors, since there is only speculative knowledge of the target. Additionally, it appears as though there is more than one active site or mechanism of action.

Hypervalent iodine oxidation of emodin (1) with various (diacetoxyiodo)arenes 2a-f in potassium hydroxide/methanol leading to 3-aryloxy-1,8-dihydroxy-2-iodo-anthraquinones 4a-f is reported. Mechanistic studies showed reaction via iodonium ylides. (C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.

Inhibition of angiogenesis is currently perceived as one of the promising strategies in the treatment of cancer. The antiangiogenic property of thalidomide has inspired a second wave of research on this teratogenic drug. Previous studies from our group and others demonstrated that metabolites of thalidomide are responsible for the drug's pharmacological actions. On the basis of the structures of these metabolites, we synthesized 118 thalidomide analogues. Preliminary screening selected 7 of these 118 analogues for more extensive testing in the current study. In the rat aortic ring assay, all 4 analogues in the N-substituted class and 2 of the 3 analogues in the tetrafluorinated class significantly inhibited microvessel outgrowth at 12.5-200 microM. Thalidomide failed to block angiogenesis at similar concentrations. Subsequently, the effects of these analogues on human umbilical vein endothelial cell proliferation and tube formation were determined. Those analogues showing antiangiogenicity in the rat aortic ring assay also demonstrated antiproliferative action in human umbilical vein endothelial cells. Cell proliferation was not affected by thalidomide. Interestingly, all 7 analogues as well as thalidomide suppressed tube formation. Two tetrafluorinated analogues consistently showed the highest potency and efficacy in all three assays. The in vivo toxicity of representative analogues from each class was also evaluated. Taken together, our results support the further development and evaluation of novel thalidomide analogues as antiangiogenic agents.

Thalidomide has been shown to reduce the production of tumor necrosis factor-α (TNF-α), a cytokine with deleterious pathophysiologic effects in various diseases. In search of thalidomide analogues with improved TNF-α inhibiting properties, 5-ethyl-1-phenyl-5-(3,4,5,6-tetrafluorophthalimido)barbituric acid (TFBA) was found to be superior to thalidomide. Besides TNF-α, TFBA also suppressed interleukin-6 and interleukin-10 production of isolated monocytes. The possibility that TFBA exerts its action by increasing levels of cAMP via inhibition of phosphodiesterase-4 activity was excluded. TFBA had no influence on T cell proliferation; neither did it inhibit TNF-α production in peripheral blood mononuclear cells stimulated by anti-CD3 monoclonal antibody. When applied to mice treated with d-galactosamine and lipopolysaccharide, TFBA prevented a rise in serum TNF-α, had no effect on interleukin-6 levels and led to an increase in interleukin-10 production. The changes in cytokine production observed in vitro and in vivo were reflected by similar changes in the mRNA expression. Moreover, TFBA significantly reduced liver transaminase levels in d-galactosamine/lipopolysaccharide-treated mice and thus efficiently protected the animals from liver injury. Thus, according to its properties, TFBA has the potential of modulating an immune response by acting as an anti-inflammatory agent.

In order to come to conclusions relating to the optimal time of intake of zinc histidinate (Zn(His) 2 ) with respect to alimentation, the interaction of Zn(His) 2 and phytic acid as an inhibitor of zinc absorption was examined under the pH-conditions of the intestinal passage. Below a pH of 3, neither zinc phytate nor Zn(His) 2 are stable. Under the HCI-acidic conditions of the stomach with pH < 3, zinc is probably converted to ZnCl 2 . However, the typical pH range of the duodenum with values between 5.4 and 7.8 covers the maximum of the formation of unsoluble zinc phytate complexes as well as the maximum of the complex stability of Zn(His) 2 . When both compounds, phytic acid and Zn(His) 2 , are present, a buffer system of His/Zn(His) 2 is formed in the pH range of 5-7. In the ternary system of Zn 2+ /histidine/phytic acid free Zn ions can be detected over the whole range of physiological pH values, ions that would be available for absorption in vivo. At least for the supplement Zn(His)2 the conclusion can be drawn that the intake with meals will not impair zinc absorption.

The effects of Michael adducts of 6-O-palmitoyl-L-ascorbic acid (compounds 1–4) on the phosphorylation-dependent response of stimulated monocytes and neutrophils was investigated. The pyranosyl derivative 3 increased the production of tumor necrosis factor-α in human monocytes stimulated with lipopolysaccharide (LPS). Compound 3 also enhanced the release of tumor necrosis factor-α from nonstimulated monocytes. Michael adducts 1–4 inhibited the formation of reactive oxygen species in fMLP-stimulated human neutrophils as measured by luminol chemiluminescence. Treatment with 6-O-palmitoyl-L-ascorbic acid (compound 5) also led to a decreased luminescence response of neutrophils. Results are discussed with respect to the inhibitory activity of Michael adducts of ascorbic acids towards protein phosphatases PP1/PP2A.

The analysis of several impurities of the chemotherapeutic agent trimethoprim with various methods, including thin layer chromatography, gas chromatography, capillary electrophoresis as well as nuclear magnetic resonance is described. These methods were used to identify new impurities in trimethoprim batches. The main impurities were separated by column chromatography. To ensure the identity of the impurities, de novo syntheses were successfully carried out. With the methods described, it was possible to detect, separate and identify new impurities in trimethoprim batches.

A synthetic entry to derivatives of the new classes of 5-phthalimidouracils and 5-phthalimidobarbituric acids is reported. These 5-phthalimidopyrimidines as well as phthalimido-2,4-difluorobenzenes were designed as analogues of thalidomide, a well known inhibitor of TNF-α production. A preliminary in vitro investigation of the compounds as inhibitors of the TNF-α production was performed. Among the compounds of the present series, 5-ethyl-1-phenyl-5-(tetrafluorophthalimido)barbituric acid and 2-(2,4-difluorophenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3(2H)-dione were proved to be potent inhibitors. Both compounds showed inhibitory activity in the lower micromolar range on the LPS-induced TNF-α production in human monocytes.