The influence of hypoxia and energy depletion on the response of endothelial cells to the vascular disrupting agent combretastatin A-4-phosphate.

Combretastatin A-4 phosphate (CA4P) is a microtubule-disrupting tumour-selective vascular disrupting agent (VDA). CA4P activates the actin-regulating RhoA-GTPase/ ROCK pathway, which is required for full vascular disruption. While hypoxia renders tumours resistant to many conventional therapies, little is known about its influence on VDA activity. Here, we found that active RhoA and ROCK effector phospho-myosin light chain (pMLC) were downregulated in endothelial cells by severe hypoxia. CA4P failed to activate RhoA/ROCK/pMLC but its activity was restored upon reoxygenation. Hypoxia also inhibited CA4P-mediated actinomyosin contractility, VE-cadherin junction disruption and permeability rise. Glucose withdrawal downregulated pMLC, and coupled with hypoxia, reduced pMLC faster and more profoundly than hypoxia alone. Concurrent inhibition of glycolysis (2-deoxy-D-glucose, 2DG) and mitochondrial respiration (rotenone) caused profound actin filament loss, blocked RhoA/ROCK signalling and rendered microtubules  CA4P-resistant. Withdrawal of the metabolism inhibitors restored the cytoskeleton and CA4P activity. The AMP-activated kinase AMPK was investigated as a potential mediator of pMLC downregulation. Pharmacological AMPK activators that generate AMP, unlike allosteric activators, downregulated pMLC but only when combined with 2DG and/or rotenone. Altogether, our results suggest that Rho/ROCK and actinomyosin contractility are regulated by AMP/ATP levels independently of AMPK, and point to hypoxia/energy depletion as potential modifiers of CA4P response.

DNA interstrand cross-linking and in vivo antitumor activity of the extended pyrrolo[2,1-c][1,4]benzodiazepine dimer SG2057.

The pyrrolobenzodiazepines (PBDs) are naturally occurring antitumor antibiotics and a PBD dimer (SJG-136, SG2000) is in Phase II trials. SG2000 is a propyldioxy linked PBD dimer which binds sequence selectively in the minor groove of DNA forming DNA interstrand and intrastrand cross-linked adducts, and also mono-adducts depending on sequence. SG2057 is the corresponding dimer containing a pentyldioxy linkage. SG2057 has multilog differential in vitro cytotoxicity against a panel of human tumour cell lines with a mean GI(50) of 212 pM. The agent is highly efficient at producing DNA interstrand cross-links in cells which form rapidly and persist over a 48 h period. Significant antitumor activity was demonstrated in several human tumor xenograft models. Cures were obtained in a LOX-IMVI melanoma model following a single administration and dose-dependent activity, including regression responses, observed in SKOV-3 ovarian and HL-60 promyelocytic leukemia models following repeat dose schedules. In the advanced stage LS174T model, SG2057 administered either as a single dose, or in two repeat dose schedules, was superior to irinotecan. SG2057 is therefore a highly active antitumor agent, with more potent in vitro activity and superior in vivo activity to SG2000, warranting further development.

SG2285, a novel C2-aryl-substituted pyrrolobenzodiazepine dimer prodrug that cross-links DNA and exerts highly potent antitumor activity.

The pyrrolobenzodiazepines (PBD) are naturally occurring antitumor antibiotics, and a PBD dimer (SJG-136, SG2000) is in phase II trials. Many potent PBDs contain a C2-endo-exo unsaturated motif associated with the pyrrolo C-ring. The novel compound SG2202 is a PBD dimer containing this motif. SG2285 is a water-soluble prodrug of SG2202 in which two bisulfite groups inactivate the PBD N10-C11 imines. Once the bisulfites are eliminated, the imine moieties can bind covalently in the DNA minor groove, forming an interstrand cross-link. The mean in vitro cytotoxic potency of SG2285 against human tumor cell lines is GI(50) 20 pmol/L. SG2285 is highly efficient at producing DNA interstrand cross-links in cells, but they form more slowly than those produced by SG2202. Cellular sensitivity to SG2285 was primarily dependent on ERCC1 and homologous recombination repair. In primary B-cell chronic lymphocytic leukemia samples, the mean LD(50) was significantly lower than in normal age-matched B and T lymphocytes. Antitumor activity was shown in several human tumor xenograft models, including ovarian, non-small cell lung, prostate, pancreatic, and melanoma, with cures obtained in the latter model with a single dose. Further, in an advanced-stage colon model, SG2285 administered either as a single dose, or in two repeat dose schedules, was superior to irinotecan. Our findings define SG2285 as a highly active cytotoxic compound with antitumor properties desirable for further development.

Fluorescent 7-diethylaminocoumarin pyrrolobenzodiazepine conjugates: synthesis, DNA interaction, cytotoxicity and differential cellular localization.

The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a class of DNA minor groove binding agents that react covalently with guanine bases, preferably at Pu-G-Pu sites. A series of three fluorescent PBD-coumarin conjugates with different linker architectures has been synthesized to probe correlations between DNA binding affinity, cellular localization and cytotoxicity. The results show that the linker structure plays a critical role for all three parameters.

The hollow fibre model--facilitating anti-cancer pre-clinical pharmacodynamics and improving animal welfare.

We describe a modified hollow fibre assay (HFA) for investigating the potential of novel molecules as pharmaceutical agents. In particular the assay provides drug/target interaction data that can facilitate the selection of lead compounds for further evaluation in more sophisticated solid tumour models, whilst successfully implementing the 3Rs - the 'replacement' 'refinement' and 'reduction' of animals. This more ethical and rapid approach to early drug development does not compromise on the validity, sensitivity, predictivity or efficacy of preclinical evaluation. We present novel data using the standard cross-linker mitomycin C (MMC) as a positive control, and two investigational DNA interactive molecules (C1311/ SJG-136). Tumour cells were seeded in fibres and implanted into mice. Following treatment with an intraperitoneal injection, fibres were excised and cells retrieved for pharmacodynamic analysis using the comet assay/fluorescence microscopy. Microscopy results revealed nuclear uptake and localisation within cytoplasmic organelles of HT29 colorectal adenocarcinoma cells following treatment with C1311 (150 mg/kg). Following treatment with SJG-136 (0.3 mg/kg) a 27.3% (p<0.001) DNA cross-linking (s.c.) effect was observed in the HL60 acute promyelocytic leukaemia cell line. DNA cross-linking effects of 55% (i.p) and 50% (s.c.) (p<0.005) were observed in the A549 lung carcinoma cell line following administration of MMC (6 mg/kg). These data are consistent with previous activity defined using solid tumour models, and support the use of the HFA for in vivo pharmacodynamic investigation whilst significantly reducing animal numbers and the influence of tumour growth on the welfare of mice.

50 years of preclinical anticancer drug screening: empirical to target-driven approaches.

The number of anticancer agents that fail in the clinic far outweighs those considered effective, suggesting that the selection procedure for progression of molecules into the clinic requires improvement. The value of any preclinical model will ultimately depend on its ability to accurately predict clinical response. This review focuses on the major contributions of preclinical screening models to anticancer drug development over the past 50 years. Over time, a general transition has been observed from the empirical drug screening of cytotoxic agents against uncharacterized tumor models to the target-orientated drug screening of agents with defined mechanisms of action. New approaches to anticancer drug development involve the molecular characterization of models along with an appreciation of the pharmacodynamic and pharmacokinetic properties of compounds [e.g., the US National Cancer Institute (NCI) in vitro 60-cell line panel, hollow fiber assay, and s.c. xenograft]. Contributions of other potentially more clinically relevant in vivo tumor models including orthotopic, metastatic, and genetically engineered mouse models are also reviewed. Although this review concentrates on the preclinical screening efforts of the NCI, European efforts are not overlooked. Europe has played a key role in the development of new anticancer agents. The two largest academic drug development groups, the European Organisation for Research and Treatment of Cancer and Cancer Research UK, have been collaborating with the NCI in the acquisition and screening of compounds since the 1970s. As with the drug development process internationally, rational pharmacodynamic approaches have more recently been adopted by these two groups.

Characterization of the hollow fiber assay for the determination of microtubule disruption in vivo.

PURPOSE: The hollow fiber assay is used successfully as a routine in vivo screening model to quantitatively define anticancer activity by the National Cancer Institute. This study investigates whether the hollow fiber assay can be used as a short-term in vivo model to demonstrate specific pharmacodynamic end points, namely microtubule and cell cycle disruption. EXPERIMENTAL DESIGN: The growth of A549 cells was characterized within hollow fibers over 5 days in vivo at both subcutaneous (s.c.) and intraperitoneal (i.p.) sites. Drugs were administered on day 4 (i.p.). RESULTS: At 24 hours, cells were retrieved from fibers at both i.p. and s.c. sites of paclitaxel-treated (20 mg/kg) and combretastatin A1 phosphate-treated (150 mg/kg) mice. Cell cycle analysis after paclitaxel treatment revealed a mean G(2)-M phase population of 48.04% (i.p.) and 25.76% (s.c.) compared with vehicle group mice (6.78 and 5.56%, respectively; P = <0.001 and 0.005, respectively). Tumor cells retrieved from combretastatin A1 phosphate-treated mice had a mean G2-M phase population of 36.3% (i.p.) and 29.36% (s.c.) compared with cells retrieved from vehicle group mice (5.58 and 5.49%, respectively; P = <0.001). Using fluorescence and laser-confocal microscopy, paclitaxel was revealed to induce the formation of spindle asters and tubulin polymerization. Combretastatin A1 phosphate was shown to hold cells in mitosis. Changes in nuclear morphology were also observed. CONCLUSION: These data demonstrate that the hollow fiber assay can be used as a short-term in vivo model for studying the pharmacodynamic effects of both standard and novel compounds on microtubules. Evidence has also been provided to support the routine use of the in vivo hollow fiber assay for demonstrating the mechanism of action of a drug.

In vitro, in vivo, and in silico analyses of the antitumor activity of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazoles.

Phortress is a novel, potent, and selective experimental antitumor agent. Its mechanism of action involves induction of CYP1A1-catalyzed biotransformation of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) to generate electrophilic species, which covalently bind to DNA, exacting lethal damage to sensitive tumor cells, in vitro and in vivo. Herein, we investigate the effects of DNA adduct formation on cellular DNA integrity and progression through cell cycle and examine whether a relevant pharmacodynamic end point may be exploited to probe the clinical mechanism of action of Phortress and predict tumor response. Single cell gel electrophoresis (SCGE) was applied to quantify DNA damage and cell cycle analyses conducted upon 5F 203 treatment of benzothiazole-sensitive MCF-7 and inherently resistant MDA-MB-435 breast carcinoma cells. Following treatment of xenograft-bearing mice and mice possessing hollow fiber implants containing MCF-7 or MDA-MB-435 cells with Phortress (20 mg/kg, i.p., 24 hours), tumor cells and xenografts were recovered for analyses by SCGE. Dose- and time-dependent DNA single and double strand breaks occurred exclusively in sensitive cells following treatment with 5F 203 in vitro (10 nmol/L-10 micromol/L; 24-72 hours). In vivo, Phortress-sensitive and Phortress-resistant tumor cells were distinct; moreover, DNA damage in xenografts, following treatment of mice with Phortress, could be determined. Interrogation of the mechanism of action of 5F 203 in silico by self-organizing map-based cluster analyses revealed modulation of phosphatases and kinases associated with cell cycle regulation, corroborating observations of selective cell cycle perturbation by 5F 203 in sensitive cells. By conducting SCGE, tumor sensitivity to Phortress, an agent currently undergoing clinical evaluation, may be determined.