A systematic review with meta-analysis of the role of anxiety and depression in irritable bowel syndrome onset.

BACKGROUND: It is well established that people with irritable bowel syndrome (IBS) have higher levels of anxiety and depression compared with controls. However, the role of these as risk factors is less clearly established. The aims of this systematic review were to investigate: (1) whether anxiety and/or depression predict IBS onset; (2) the size of the relative risk (RR) of anxiety versus depression in IBS onset. Subgroup analyses explored if methodological factors affected the overall findings. METHOD: Prospective cohort or case-control studies were included if they: (1) focused on the development of IBS in population-based or gastroenteritis cohorts; (2) explored the effects of anxiety and/or depression at baseline as predictors of IBS onset at a future point. In all, 11 studies were included of which eight recruited participants with a gastrointestinal infection. Meta-analyses were conducted. RESULTS: The risk of developing IBS was double for anxiety cases at baseline compared with those who were not [RR 2.38, 95% confidence interval (CI) 1.58-3.60]. Similar results were found for depression (RR 2.06, 95% CI 1.44-2.96). Anxiety and depression seemed to play a stronger role in IBS onset in individuals with a gastrointestinal infection although this could be attributed to other differences in methodology, such as use of diagnostic interviews rather than self-report. CONCLUSIONS: The findings suggest that self-reported anxiety and depression provide a twofold risk for IBS onset. There is less support for the role of anxiety or depressive disorder diagnosed using clinical interview. These findings may have implications for the development of interventions focused on IBS prevention and treatment.

Acute tumour response to the MEK1/2 inhibitor selumetinib (AZD6244, ARRY-142886) evaluated by non-invasive diffusion-weighted MRI.

BACKGROUND: Non-invasive imaging biomarkers underpin the development of molecularly targeted anti-cancer drugs. This study evaluates tumour apparent diffusion coefficient (ADC), measured by diffusion-weighted magnetic resonance imaging (DW-MRI), as a biomarker of response to the MEK1/2 inhibitor selumetinib (AZD6244, ARRY-142886) in human tumour xenografts. METHODS: Nude mice bearing human BRAF(V600D) WM266.4 melanoma or BRAF(V600E) Colo205 colon carcinoma xenografts were treated for 4 days with vehicle or selumetinib. DW-MRI was performed before and 2 h after the last dose and excised tumours analysed for levels of phospho-ERK1/2, cleaved caspase 3 (CC3) and necrosis. RESULTS: Selumetinib treatment induced tumour stasis and reduced ERK1/2 phosphorylation in both WM266.4 and Colo205 tumour xenografts. Relative to day 0, mean tumour ADC was unchanged in the control groups but was significantly increased by up to 1.6-fold in selumetinib-treated WM266.4 and Colo205 tumours. Histological analysis revealed a significant increase in necrosis in selumetinib-treated WM266.4 and Colo205 xenografts and CC3 staining in selumetinib-treated Colo205 tumours relative to controls. CONCLUSION: Changes in ADC following treatment with the MEK1/2 inhibitor selumetinib in responsive human tumour xenografts were concomitant with induction of tumour cell death. ADC may provide a useful non-invasive pharmacodynamic biomarker for early clinical assessment of response to selumetinib and other MEK-ERK1/2 signalling-targeted therapies.

Guidelines for the welfare and use of animals in cancer research.

Animal experiments remain essential to understand the fundamental mechanisms underpinning malignancy and to discover improved methods to prevent, diagnose and treat cancer. Excellent standards of animal care are fully consistent with the conduct of high quality cancer research. Here we provide updated guidelines on the welfare and use of animals in cancer research. All experiments should incorporate the 3Rs: replacement, reduction and refinement. Focusing on animal welfare, we present recommendations on all aspects of cancer research, including: study design, statistics and pilot studies; choice of tumour models (e.g., genetically engineered, orthotopic and metastatic); therapy (including drugs and radiation); imaging (covering techniques, anaesthesia and restraint); humane endpoints (including tumour burden and site); and publication of best practice.

Correction: A chemical inhibitor of PPM1D that selectively kills cells overexpressing PPM1D.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A phase I study of the nitroimidazole hypoxia marker SR4554 using 19F magnetic resonance spectroscopy.

BACKGROUND: SR4554 is a fluorine-containing 2-nitroimidazole, designed as a hypoxia marker detectable with 19F magnetic resonance spectroscopy (MRS). In an initial phase I study of SR4554, nausea/vomiting was found to be dose-limiting, and 1400 mg m(-2) was established as MTD. Preliminary MRS studies demonstrated some evidence of 19F retention in tumour. In this study we investigated higher doses of SR4554 and intratumoral localisation of the 19F MRS signal. METHODS: Patients had tumours > or = 3 cm in diameter and < or = 4 cm deep. Measurements were performed using 1H/19F surface coils and localised 19F MRS acquisition. SR4554 was administered at 1400 mg m(-2), with subsequent increase to 2600 mg m(-2) using prophylactic metoclopramide. Spectra were obtained immediately post infusion (MRS no. 1), at 16 h (MRS no. 2) and 20 h (MRS no. 3), based on the SR4554 half-life of 3.5 h determined from a previous study. 19Fluorine retention index (%) was defined as (MRS no. 2/MRS no. 1)*100. RESULTS: A total of 26 patients enrolled at: 1400 (n=16), 1800 (n=1), 2200 (n=1) and 2600 mg m(-2) (n=8). SR4554 was well tolerated and toxicities were all < or = grade 1; mean plasma elimination half-life was 3.7+/-0.9 h. SR4554 signal was seen on both unlocalised and localised MRS no. 1 in all patients. Localised 19F signals were detected at MRS no. 2 in 5 out of 9 patients and 4 out of 5 patients at MRS no. 3. The mean retention index in tumour was 13.6 (range 0.6-43.7) compared with 4.1 (range 0.6-7.3) for plasma samples taken at the same times (P=0.001) suggesting (19)F retention in tumour and, therefore, the presence of hypoxia. CONCLUSION: We have demonstrated the feasibility of using 19F MRS with SR4554 as a potential method of detecting hypoxia. Certain patients showed evidence of 19F retention in tumour, supporting further development of this technique for detection of tumour hypoxia.

Overexpression of EZH2 in multiple myeloma is associated with poor prognosis and dysregulation of cell cycle control.

Myeloma is heterogeneous at the molecular level with subgroups of patients characterised by features of epigenetic dysregulation. Outcomes for myeloma patients have improved over the past few decades except for molecularly defined high-risk patients who continue to do badly. Novel therapeutic approaches are, therefore, required. A growing number of epigenetic inhibitors are now available including EZH2 inhibitors that are in early-stage clinical trials for treatment of haematological and other cancers with EZH2 mutations or in which overexpression has been correlated with poor outcomes. For the first time, we have identified and validated a robust and independent deleterious effect of high EZH2 expression on outcomes in myeloma patients. Using two chemically distinct small-molecule inhibitors, we demonstrate a reduction in myeloma cell proliferation with EZH2 inhibition, which leads to cell cycle arrest followed by apoptosis. This is mediated via upregulation of cyclin-dependent kinase inhibitors associated with removal of the inhibitory H3K27me3 mark at their gene loci. Our results suggest that EZH2 inhibition may be a potential therapeutic strategy for the treatment of myeloma and should be investigated in clinical studies.

Hsp90 inhibitors in the clinic.

Specific inhibitors of Hsp90 have recently entered human clinical trials. At the time of writing, trials have been initiated only in metastatic cancer, although a rationale exists for using these agents in a variety of human diseases where protein (mis)folding is involved in the disease pathophysiology. Hsp90 inhibitors offer a unique anti-cancer opportunity because they provide simultaneous combinatorial blockade of multiple oncogenic pathways. The first compound in this class, 17-AAG, has completed phase I trials and phase II trials are in progress. The toxicity has been manageable and evidence of possible clinical activity has been seen in metastatic melanoma, prostate cancer and multiple myeloma. Other inhibitors with improved properties are approaching clinical trials. This chapter presents an update of the current clinical trials using Hsp90 inhibitors, focussing on the areas that will be increasingly relevant in the next 5 years.

Reductase enzyme expression across the National Cancer Institute Tumor cell line panel: correlation with sensitivity to mitomycin C and EO9.

BACKGROUND: Many antitumor drugs require metabolic activation to exert their cytotoxic or cytostatic effects. The so-called bioreductive compounds, whose conversion into active antitumor agents is catalyzed by reductase enzymes, are examples of such drugs. The identification of specific enzymes involved in the activation of these compounds is important in understanding cellular factors that may influence drug antitumor activity. PURPOSE: We measured expression levels of three different reductase enzymes-DT-diaphorase [NAD(P)H (i.e., reduced nicotinamide adenine dinucleotide, with or without phosphate): quinone oxidoreductase]; NADPH:cytochrome P-450 reductase; and NADH (i.e., reduced nicotinamide adenine dinucleotide): cytochrome-b5 reductase- in 69 cell lines (most of the National Cancer Institute [NCI] human tumor cell panel) to see if relationships could be established between the activities of these enzymes and cellular sensitivities to the bioreductive compounds mitomycin C and EO9. METHODS: For all 69 cell lines, the activity of each enzyme was determined using cellular extracts and photometric assays involving the reduction of cytochrome c. Western blot analysis was used to measure the relative amount of DT-diaphorase protein in each extract, and coupled reverse transcription and polymerase chain reactions were employed to assess DT-diaphorase and NADPH:cytochrome P-450 reductase messenger RNA (mRNA) levels in a subset of the cell lines. The cytotoxic and/or cytostatic activities of mitomycin C and EO9 toward the cell lines were determined under aerobic conditions. Relationships between enzyme activity levels and drug sensitivities were assessed by use of the COMPARE program and Pearson correlation coefficients. RESULTS: In general, DT-diaphorase activity levels were higher than those observed for the other two reductases across the entire cell line panel. Measured activities for all three enzymes varied among cell lines derived from the same tissue as well as between lines derived from different tissues; however, tissue-specific patterns of expression could be discerned. Differences in the activity levels of individual enzymes appeared to reflect differences in corresponding enzyme protein and/or mRNA levels. A relationship between enzyme activity and chemosensitivities to mitomycin C and EO9 was observed only for DT-diaphorase (Pearson correlation coefficient = .424 [two-sided P<.0005] for mitomycin C and .446 [two-sided P< or = to .0013] for EO9). CONCLUSIONS: Reductase enzyme expression is heterogeneous across human tumor cell lines, and tissue-specific patterns of expression are apparent. DT-diaphorase activity levels correlate with sensitivities to mitomycin C and EO9, supporting a role for this enzyme in the bioactivation of these anticancer compounds. IMPLICATIONS: Comparison of biochemical, molecular biological, and chemosensitivity data obtained from screening a large number of cell lines (e.g., the NCI tumor cell line panel) may facilitate investigation of factors influencing drug antitumor activity. The knowledge gained may be of value in the development of new anticancer agents or in the selection of patients to receive specific therapies.

DT-Diaphorase expression and tumor cell sensitivity to 17-allylamino, 17-demethoxygeldanamycin, an inhibitor of heat shock protein 90.

BACKGROUND: To our knowledge, 17-allylamino,17-demethoxygeldanamycin (17AAG) is the first inhibitor of heat shock protein 90 (Hsp90) to enter a phase I clinical trial in cancer. Inhibition of Hsp90, a chaperone protein (a protein that helps other proteins avoid misfolding pathways that produce inactive or aggregated states), leads to depletion of important oncogenic proteins, including Raf-1 and mutant p53 (also known as TP53). Given its ansamycin benzoquinone structure, we questioned whether the antitumor activity of 17AAG was affected by expression of the NQO1 gene, which encodes the quinone-metabolizing enzyme DT-diaphorase. METHODS: The antitumor activity of 17AAG and other Hsp90 inhibitors was determined by use of a sulforhodamine B-based cell growth inhibition assay in culture and by the arrest of xenograft tumor growth in nude mice. DT-diaphorase activity was determined by use of a spectrophotometric assay, and protein expression was determined by means of western immunoblotting. RESULTS: In two independent in vitro human tumor cell panels, we observed a positive relationship between DT-diaphorase expression level and growth inhibition by 17AAG. Stable, high-level expression of the active NQO1 gene transfected into the DT-diaphorase-deficient (by NQO1 mutation) BE human colon carcinoma cell line resulted in a 32-fold increase in 17AAG growth-inhibition activity. Increased sensitivity to 17AAG in the transfected cell line was also confirmed in xenografts. The extent of depletion of Raf-1 and mutant p53 protein confirmed that the Hsp90 inhibition mechanism was maintained in cells with high and low levels of DT-diaphorase. 17AAG was shown to be a substrate for purified human DT-diaphorase. CONCLUSION: These results suggest that the antitumor activity and possibly the toxicologic properties of 17AAG in humans may be influenced by the expression of DT-diaphorase. Careful monitoring for NQO1 polymorphism and the level of tumor DT-diaphorase activity is therefore recommended in clinical trials with 17AAG.

Multiparametric flow cytometry of the modulation of tumor cell membrane permeability by developmental antitumor ether lipid SRI 62-834 in EMT6 mouse mammary tumor and HL-60 human promyelocytic leukemia cells.

(+-)-2-[Hydroxy[tetrahydro-2-(octadecyloxy)methylfuran-2- yl]methoxyl]phosphinyloxy-N,N,N-trimethylethaniminium hydroxide, inner salt (SRI 62-834) is a tetrahydrofuran analogue of platelet activating factor (PAF) that is currently entering clinical trial. Like other ether lipids it is of interest as a membrane-active antitumor agent. Here, we have used two-color multiparameter flow cytometry to study simultaneously its effects on cell membrane permeability, intracellular pH, and cell size/structure of EMT6 mouse mammary tumor cells and HL-60 human promyelocytic leukemia cells in vitro. Concentrations as low as 1 microM up to 100 microM SRI 62-834 caused a rapid, dose-dependent increase in membrane permeability, initially towards outward efflux of the preloaded fluorescein probe bis(carboxyethyl)carboxyfluorescein (green fluorescence) and then towards influx of extracellular propidium (red fluorescence). At the same time, median cell size from light scatter was reduced with an increased coefficient of variation, and the proportion of cell debris was elevated. In vitro antitumor activity was seen over the same concentration range, as measured by tetrazolium dye reduction and cell growth curves. Neither low concentrations of PAF (50 nM) nor the potent PAF antagonist 3-[4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f][1,2,4]tria- zolo[4,3a][1,4]diazepin-2-yl]-1-(4-morpholinyl)-1-propanone (0.5-100 microM) had any influence on the membrane effects of SRI 62-834, and at higher concentrations (1-200 microM) PAF mimicked the behavior of SRI 62-834. In addition, the PAF antagonist did not modulate the cytotoxicity of SRI 62-834 or PAF. HL-60 cells were more sensitive to SRI 62-834 than were EMT6 cells in terms of both cytotoxicity and membrane permeability. However, PAF was more potent than SRI 62-834 in causing membrane permeabilization with both cell lines, whereas PAF was less active than SRI 62-834 in cytotoxicity assays. The results support a membrane-damaging role in the cytotoxicity of SRI 62-834 but suggest that additional factors are also involved. Membrane permeabilization may be related to its reported effects on protein kinase C-dependent intracellular calcium signaling but apparently does not involve a conventional PAF receptor in HL-60 or EMT6 cells.

Stimulation by localized tumor hyperthermia of reductive bioactivation of 2-nitroimidazole benznidazole in mice.

We have investigated the effects of localized tumor hyperthermia (LTH; 43.5 degrees C x 30 min) on the reductive bioactivation of the 2-nitroimidazole benznidazole in C3H mouse normal tissues and KHT tumors. Mice were allocated to one of three treatment groups: (a) unrestrained controls, (b) sham tumor treatment, and (c) LTH. Concentrations of benznidazole and its amine metabolite were determined by high-performance liquid chromatography. Conscious mice were given LTH or sham treatment 2.5 h after 2.5 mmol/kg benznidazole i.p. This gave steady-state plasma benznidazole concentrations of 120-170 micrograms/ml at 2-5 h in all three groups. Plasma amine concentrations were very low at 0.1-1 micrograms/ml in all cases. Liver benznidazole concentrations were similar to plasma but amine concentrations were 30-40-fold greater at 20-40 micrograms/g in all three groups, implicating the liver as a major site of reductive metabolism. Benznidazole concentrations in tumors from unrestrained mice were comparable to those in plasma and liver, with tumor/plasma ratios of 85-113%. Tumor amine concentrations were intermediate at about 2-3 micrograms/g, indicating reductive bioactivation had occurred. Sham treatment decreased tumor benznidazole concentrations by 25-50%, particularly at later times, and amine concentrations were correspondingly increased. This may be a result of sham tumor treatment at 37 degrees C, a temperature 3-4 degrees C higher than in unrestrained controls. More importantly, LTH further decreased tumor benznidazole concentrations over sham treatment, e.g., by 59% from 114 to 47 micrograms/g (P less than 0.01) immediately after heating. Amine concentrations were correspondingly elevated, e.g., by 40% from 5.1 to 8.4 micrograms/g (P less than 0.01). These results clearly show that LTH can selectively enhance the reductive bioactivation of benznidazole in KHT tumors in mice, and support a particular role for the use of bioreductive agents with heat.

Inhibition of signal transduction by the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in cytostasis and apoptosis.

17-Allylamino-17-demethoxygeldanamycin (17AAG) is a first-in-class heat shock protein 90 (Hsp90) molecular chaperone inhibitor to enter clinical trials. The downstream molecular and cellular consequences of Hsp90 inhibition are not well defined. 17AAG has shown activity against human colon cancer in cell culture and xenograft models. In this study, we demonstrated that in addition to depleting c-Raf-1 and inhibiting ERK-1/2 phosphorylation in human colon adenocarcinoma cells, 17AAG also depleted N-ras, Ki-ras, and c-Akt and inhibited phosphorylation of c-AKT: A consequence of these events was the induction of cell line-dependent cytostasis and apoptosis, although the latter did not result from dephosphorylation of proapoptotic BAD: One cell line, KM12, did not exhibit apoptosis and in contrast to the other cell lines overexpressed Bag-1, but did not express BAX: Taken together with other determinants of 17AAG sensitivity, these results should contribute to a more complete understanding of the molecular pharmacology of 17AAG, which in turn should aid the future rational clinical development and use of the drug in colon and other tumor types.

Preclinical evaluation of the fluorinated 2-nitroimidazole N-(2-hydroxy-3,3,3-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide (SR-4554) as a probe for the measurement of tumor hypoxia.

A novel probe, N-(2-hydroxy-3,3,3,-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide (SR-4554), has been used to detect tumor hypoxia noninvasively by 19F magnetic resonance spectroscopy (19F MRS). The compound was designed to undergo a hypoxia-dependent, one-electron reduction to metabolites that are selectively retained in tumors and has attractive pharmacokinetic, toxicological, and detection sensitivity properties. As a prelude to clinical studies, we report here for the first time on the ability to detect a MR signal following SR-4554 administration in various transplantable tumors and describe validation studies, consisting of a correlation between signal retention and radiobiological hypoxic fraction, and the effects of modulating the degree of hypoxia by hydralazine and carbogen breathing. SR-4554 was absorbed and then eliminated from EMT6 tumors with a half-life of 51 min following an injection of 180 mg/kg i.p. of SR-4554. Using a quantitative 19F MRS technique, the 19F retention index (19FRI; 19F signal level at 6 h/45 min) was determined for four commonly used murine tumors (EMT6, SCCVII, KHT, and RIF-1). The retention of high tumor concentrations of fluorinated probe at 6 h, despite the much lower (20-fold) concentration of parent SR-4554 detected by high-performance liquid chromatography, was consistent with the involvement of one or more nitroreduced metabolites and suggested that 19F MRS might give a quantitative measure of tumor hypoxia. In these murine tumors, 19FRI correlated with the reported radiobiological hypoxic fraction of the tumors (r = 0.988; P = 0.01). In addition, changes in tumor microenvironment were detected by 19F MRS. An increase in hypoxia induced by hydralazine treatment of RIF-1 tumor-bearing mice was associated with a 2.4-fold increase in 19FRI compared to untreated controls. In contrast, carbogen breathing by C3H mammary tumor-bearing mice produced a 6-fold decrease in the 19FRI compared to air-breathing mice. The data presented support the preclinical and clinical development of SR-4554 as a noninvasive probe for tumor hypoxia.

Decreased NADPH:cytochrome P-450 reductase activity and impaired drug activation in a mammalian cell line resistant to mitomycin C under aerobic but not hypoxic conditions.

Mitomycin C (MMC) is regarded as the prototype bioreductive alkylating agent in clinical use. To elucidate the biochemical basis of MMC resistance, we isolated a drug resistant derivative (designated CHO-MMC) of a Chinese hamster ovary cell line (CHO-K1) by exposure to progressively higher concentrations of MMC. CHO-MMC cells exhibited a 17-fold increase in resistance to MMC and were 33-fold cross-resistant to the monofunctional derivative, decarbamoyl mitomycin C. In contrast, CHO-MMC cells showed only a 2-fold level of resistance to BMY 25282, a more easily activated analogue of MMC, and exhibited parental sensitivity to MMC under radiobiologically hypoxic conditions. CHO-MMC cells showed no increased resistance to a range of DNA damaging agents including several other alkylating agents (e.g., melphalan and methyl methanesulfonate). Cross-resistance to drugs associated with the multidrug resistant phenotype (e.g., Adriamycin and vincristine) was present only at very low levels. Using a specific high performance liquid chromatography technique, we examined the rates of reduction of MMC and BMY 25282 in cell extracts from CHO-K1 and CHO-MMC cells under both aerobic (air) and hypoxic (N2) conditions. Reduction rates for both drugs were at least 30-fold faster under nitrogen than in air. Metabolism of MMC was undetectable in air but was readily detectable under nitrogen and was 2- 3-fold slower in CHO-MMC cell extracts than in CHO-K1 cell extracts. Although BMY 25282 was more readily reduced under nitrogen, no difference was detected between extracts from CHO-K1 or CHO-MMC cells in the rate of reduction of BMY 25282 under either air or nitrogen. The activity of NADPH:cytochrome P-450 (cytochrome c) reductase, an enzyme implicated in the bioreductive activation of MMC, was 3-4-fold lower in CHO-MMC cells than in the parental line. These findings suggest that the resistance of CHO-MMC cells to MMC under aerobic conditions may be due to impaired metabolic activation of the drug as a result of a decrease in NADPH:cytochrome P-450 reductase activity. This supports the view that decreased bioreductive enzyme activity may be a significant mechanism for acquired resistance to MMC in tumor cells in vivo and that more readily activated analogues may be potentially useful in overcoming this specific form of resistance.

Gene expression profiling of human colon cancer cells following inhibition of signal transduction by 17-allylamino-17-demethoxygeldanamycin, an inhibitor of the hsp90 molecular chaperone.

A number of molecular therapeutic agents, derived from exploiting our knowledge of the oncogenic pathways that are frequently deregulated in cancer, are now entering clinical trials. One of these is the novel agent 17-allylamino-17-demethoxygeldanamycin that acts to inhibit the hsp90 molecular chaperone. Treatment of four human colon cancer cell lines with iso-effective concentrations of this agent resulted in depletion of c-raf-1 and akt and inhibition of signal transduction. We have used gene expression array analysis to identify genes responsive to treatment with this drug. The expression of hsp90 client protein genes was not affected, but hsc hsp70, hsp90beta, keratin 8, keratin 18 and caveolin-1 were deregulated following treatment. These observations were consistent with inhibition of signal transduction and suggested a possible mechanism of resistance or recovery from 17-allylamino-17-demethoxygeldanamycin treatment. The results shed light on the molecular mode of action of the hsp90 inhibitors, and suggest possible molecular markers of drug action for use in hypothesis testing clinical trials. Oncogene (2000) 19, 4125 - 4133

Inhibition of phospholipid signalling and proliferation of Swiss 3T3 cells by the wortmannin analogue demethoxyviridin.

Growth factors and certain oncogenes activate a range of phospholipid-mediated signal transduction pathways resulting in cell proliferation. Demethoxyviridin (DMV), a structural analogue of wortmannin and recently reported as a potent inhibitor of phosphoinositide-3-kinase, inhibited bombesin plus insulin-stimulated increase in cell number in Swiss 3T3 cells, a model of cell proliferation. The drug produced cytostatic effects at concentrations below 1 microM and cytotoxic effects at 10 microM. In intact Swiss 3T3 cells DMV inhibited insulin-stimulated PI 3- and 4-kinases and bombesin-stimulated phospholipases C, D and A2 in the nanomolar range. DMV also inhibited bombesin-stimulated tyrosine phosphorylation of a range of proteins at nM concentrations. This study shows that DMV inhibited multiple stimulated signalling pathways which lead to increased Swiss 3T3 cell proliferation. A stable analogue of DMV may have chemotherapeutic potential.

Phase I and pharmacokinetic study of tirapazamine (SR 4233) administered every three weeks.

Tirapazamine (SR 4233; 3-amino-1,2,4-benzotriazine-1,4-di-N-oxide) is a bioreductive agent exhibiting up to 200 x greater toxicity for hypoxic cells as compared to oxygenated cells. In murine studies, a selective increase in tumor kill was observed when tirapazamine was coadministered with other agents, notably cisplatin. A Phase I study of single-agent tirapazamine administered i.v. every 3 weeks was conducted to determine the toxicity of a schedule for use with systemic chemotherapy. A total of 28 patients were given 50 courses of tirapazamine at doses ranging from 36-450 mg/m2. No tumor responses were observed. Reversible deafness and tinnitus were dose-limiting, with ototoxicity observed in 1 of 6 patients treated at 330 mg/m2, 1 of 4 patients treated at 390 mg/m2, and 3 of 3 patients treated at 450 mg/m2. Muscle cramps, nausea, and vomiting were also observed. Pharmacokinetic studies revealed a greater than dose-proportional increase in the area under the plasma concentration x time curve (AUCs) of the two major metabolites. Patients who developed ototoxicity generally showed higher plasma AUC values for the parent drug and metabolites. The mean plasma tirapazamine AUC at 330 mg/m2 was 1026.5 microgram/ml x min (range 863. 8-1252.3), but no pharmacokinetic data are available for the solitary patient who developed otoxicity at this dose level. These AUC values were in the (estimated) range required for therapeutic effect in murine studies. Ototoxicity was not observed when the AUC of tirapazamine was equal to or less than 1252 microgram/ml x min. The dose of 330 mg/m2 was therefore chosen as an appropriate level for combination chemotherapy studies.

Preclinical antitumor activity and pharmacodynamic studies with the farnesyl protein transferase inhibitor R115777 in human breast cancer.

Antitumor and pharmacodynamic studies were performed in MCF-7 human breast cancer cells and companion xenografts with the farnesyl protein transferase inhibitor, R115777, presently undergoing Phase II clinical trials, including in breast cancer. R115777 inhibited growth of MCF-7 cells in vitro with an IC(50) of 0.31 +/- 0.25 microM. Exposure of MCF-7 cells to increasing concentrations of R115777 for 24 h resulted in the inhibition of protein farnesylation, as indicated by the appearance of prelamin A at concentrations >1 microM. After continuous exposure to 2 microM R115777, prelamin A levels peaked at 2 h post drug exposure and remained high for up to 72 h. R115777 administered p.o. twice daily for 10 consecutive days to mice bearing established s.c. MCF-7 xenografts induced tumor inhibition at a dose of 25 mg/kg [percentage of treated versus control (% T/C) = 63% at day 21]. Greater inhibition was observed at doses of 50 mg/kg (% T/C at day 21 = 38%) or 100 mg/kg (% T/C at day 21 = 43%). The antitumor effect appeared to be mainly cytostatic with little evidence of tumor shrinkage to less than the starting volume. Tumor response correlated with an increase in the appearance of prelamin A, but no changes in the prenylation of lamin B, heat shock protein 40, or N-Ras were detectable. In addition, significant increases in apoptotic index and p21(WAF1/CIP1) expression were observed, concomitant with a decrease in proliferation as measured by Ki-67 staining. An increase in prelamin A was also observed in peripheral blood lymphocytes in a breast cancer patient who responded to R115777. These data show that R115777 possesses preclinical antitumor activity against human breast cancer and that the appearance of prelamin A may provide a sensitive and convenient pharmacodynamic marker of inhibition of prenylation and/or response.

Scoring a bull's-eye against cancer genome targets.

New anticancer drugs are increasingly targeted to specific abnormalities in the sequence and expression of a series of genes that operate in a stepwise, combinatorial manner to drive the progression of human cancer. These new-generation molecular therapeutics are expected to be more effective and less toxic than the broadly antiproliferative cytotoxic drugs of the previous era, which still dominate medical treatment of cancer today. Molecular and genomic technologies, particularly the availability of the human genome sequence and the ongoing sequencing of cancer genomes, are now having a major impact on target discovery and validation. Over the past year, the progress of three novel anticancer agents in particular (Herceptin, Glivec and Iressa) has exemplified the potential utility of innovative molecular therapeutics in the clinic. Drugs acting on a range of new genome-based molecular targets are now in preclinical and clinical development.

New drug targets for genomic cancer therapy: successes, limitations, opportunities and future challenges.

Cancer drug therapy is undergoing a major transition from the previous pregenomic cytotoxic era to the new postgenomic era. Future mechanism-based therapeutic agents will increasingly be designed to act on molecular targets that are causally involved in the malignant progression of human cancers. Such agents are predicted to show greater therapeutic selectivity for cancer versus normal cells. New cancer drug targets are identified and validated in various ways. The determination of the normal human genome sequence, followed by that of multiple cancer genomes, is accelerating target discovery. Other new technologies, particularly high throughput screening, combinatorial chemistry and gene expression microarrays, are increasing the speed and efficiency of drug development. Examples of new molecular therapeutics showing promising activity in the clinic include Herceptin, Glivec and Iressa. However, many challenges remain as we test the vision of individualised combinatorial genome-based therapy, using drugs targeted to every significant molecular abnormality in cancer.