Patricia A. Cooper

Small molecule FGF receptor inhibitors block FGFR-dependent urothelial carcinoma growth in vitro and in vivo

Background:Activating mutations of FGFR3 are frequently identified in superficial urothelial carcinoma (UC) and increased expression of FGFR1 and FGFR3 are common in both superficial and invasive UC.Methods:The effects of inhibition of receptor activity by three small molecule inhibitors (PD173074, TKI-258 and SU5402) were investigated in a panel of bladder tumour cell lines with known FGFR expression levels and FGFR3 mutation status.Results:All inhibitors prevented activation of FGFR3, and inhibited downstream MAPK pathway signalling. Response was related to FGFR3 and/or FGFR1 expression levels. Cell lines with the highest levels of FGFR expression showed the greatest response and little or no effect was measured in normal human urothelial cells or in UC cell lines with activating RAS gene mutations. In sensitive cell lines, the drugs induced cell cycle arrest and/or apoptosis. IC50 values for PD173074 and TKI-258 were in the nanomolar concentration range compared with micromolar concentrations for SU5402. PD173074 showed the greatest effects in vitro and in vivo significantly delayed the growth of subcutaneous bladder tumour xenografts.Conclusion:These results indicate that inhibition of FGFR1 and wild-type or mutant FGFR3 may represent a useful therapeutic approach in patients with both non-muscle invasive and muscle invasive UC.

Antitumour 2-(4-aminophenyl)benzothiazoles generate DNA adducts in sensitive tumour cells in vitro and in vivo.

2-(4-Aminophenyl)benzothiazoles represent a potent and highly selective class of antitumour agent. In vitro, sensitive carcinoma cells deplete 2-(4-aminophenyl)benzothiazoles from nutrient media; cytochrome P450 1A1 activity, critical for execution of antitumour activity, and protein expression are powerfully induced. 2-(4-Amino-3-methylphenyl)benzothiazole-derived covalent binding to cytochrome P450 1A1 is reduced by glutathione, suggesting 1A1-dependent production of a reactive electrophilic species. In vitro, 2-(4-aminophenyl)benzothiazole-generated DNA adducts form in sensitive tumour cells only. At concentrations >100 nM, adducts were detected in DNA of MCF-7 cells treated with 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203). 5F 203 (1 μM) led to the formation of one major and a number of minor adducts. However, treatment of cells with 10 μM 5F 203 resulted in the emergence of a new dominant adduct. Adducts accumulated steadily within DNA of MCF-7 cells exposed to 1 μM 5F 203 between 2 and 24 h. Concentrations of the lysylamide prodrug of 5F 203 (Phortress) ≥100 nM generated adducts in the DNA of sensitive MCF-7 and IGROV-1 ovarian cells. At 1 μM, one major Phortress-derived DNA adduct was detected in these two sensitive phenotypes; 10 μM Phortress led to the emergence of an additional major adduct detected in the DNA of MCF-7 cells. Inherently resistant MDA-MB-435 breast carcinoma cells incurred no DNA damage upon exposure to Phortress (⩽10 μM, 24 h). In vivo, DNA adducts accumulated within sensitive ovarian IGROV-1 and breast MCF-7 xenografts 24 h after treatment of mice with Phortress (20 mg kg−1). Moreover, Phortress-derived DNA adduct generation distinguished sensitive MCF-7 tumours from inherently resistant MDA-MB-435 xenografts implanted in opposite flanks of the same mouse.

Comparative preclinical pharmacokinetic and metabolic studies of the Combretastatin prodrugs Combretastatin A4 phosphate and A1 phosphate

Purpose: Combretastatin A4 phosphate (CA4P) and its structural analog, combretastatin A1 phosphate (CA1P), are soluble prodrugs capable of interacting with tubulin and causing rapid vascular shutdown within tumors. CA4P has completed Phase I clinical trials, but recent preclinical studies have shown that CA1P displays a greater antitumor effect than the combretastatin A4 (CA4) analog at equal doses. The aim of this study, therefore, is to compare pharmacokinetics and metabolism of the two compounds to determine whether pharmacokinetics plays a role in their differential activity. Experimental Design: NMRI mice bearing MAC29 tumors received injection with either CA4P or CA1P at a therapeutic dose of 150 mg·kg−1, and profiles of both compounds and their metabolites analyzed by a sensitive and specific liquid chromatography/mass spectroscopy method. Results: The metabolic profile of both compounds is complex, with up to 14 metabolites being detected for combretastatin A1 (CA1) in the plasma. Many of these metabolites have been identified by liquid chromatography/mass spectroscopy. Initial studies, however, focused on the active components CA4 and CA1, where plasma and tumor areas under the curve were 18.4 and 60.1 μg·h·ml−1 for CA4, and 10.4 and 13.1 μg·h·ml−1 for CA1, respectively. In vitro metabolic comparisons of the two compounds strongly suggest that CA1 is metabolized to a more reactive species than the CA4. Conclusions: Although in vitro studies suggest that variable rates of tumor-specific prodrug dephosphorylation may explain these differences in pharmacokinetics profiles, the improved antitumor activity and altered pharmacokinetic profile of CA1 may be due to the formation of a more reactive metabolite.

Organometallic osmium arene complexes with potent cancer cell cytotoxicity

Iodido osmium(II) complexes [Os(η(6)-arene)(XY)I](+) (XY = p-hydroxy or p-dimethylaminophenylazopyridine, arene = p-cymene or biphenyl) are potently cytotoxic at nanomolar concentrations toward a panel of human cancer cell lines; e.g., IC(50) = 140 nM for [Os(η(6)-bip)(azpy-NMe(2))I](+) toward A2780 ovarian cancer cells. They exhibit low toxicity and negligible deleterious effects in a colon cancer xenograft model, giving rise to the possibility of a broad therapeutic window. The most active complexes are stable and inert toward aquation. Their cytotoxic activity appears to involve redox mechanisms.

Anti-colorectal cancer activity of an organometallic osmium arene azopyridine complex

This first in vivo antitumour activity for an organometallic osmium arene complex, [Os(η6-p-cym)(4-(2-pyridylazo)-N,N-dimethylaniline)I]PF6, is reported. The complex delays the growth of HCT116 human colon cancer xenografts in mice, with negligible toxicity. Its activity appears to involve redox mechanisms and its potency towards A2780 ovarian and A549 lung cancer cells is increased significantly in combination with L-buthionine-sulfoximine.

Antitumor activity of a duocarmycin analogue rationalized to be metabolically activated by cytochrome P450 1A1 in human transitional cell carcinoma of the bladder.

We identify cytochrome P450 1A1 (CYP1A1) as a target for tumor-selective drug development in bladder cancer and describe the characterization of ICT2700, designed to be metabolized from a prodrug to a potent cytotoxin selectively by CYP1A1. Elevated CYP1A1 expression was shown in human bladder cancer relative to normal human tissues. RT112 bladder cancer cells, endogenously expressing CYP1A1, were selectively chemosensitive to ICT2700, whereas EJ138 bladder cells that do not express CYP1A1 were significantly less responsive. Introduction of CYP1A1 into EJ138 cells resulted in 75-fold increased chemosensitivity to ICT2700 relative to wild-type EJ138. Negligible chemosensitivity was observed in ICT2700 in EJ138 cells expressing CYP1A2 or with exposure of EJ138 cells to CYP1B1- or CYP3A4-generated metabolites of ICT2700. Chemosensitivity to ICT2700 was also negated in EJ138-CYP1A1 cells by the CYP1 inhibitor α-naphthoflavone. Furthermore, ICT2700 did not induce expression of the AhR-regulated CYP1 family, indicating that constitutive CYP1A1 expression is sufficient for activation of ICT2700. Consistent with the selective activity by CYP1A1 was a time and concentration-dependent increase in γ-H2AX protein expression, indicative of DNA damage, associated with the activation of ICT2700 in RT112 but not EJ138 cells. In mice-bearing CYP1A1-positive and negative isogenic tumors, ICT2700 administration resulted in an antitumor response only in the CYP1A1-expressing tumor model. This antitumor response was associated with detection of the CYP1A1-activated metabolite in tumors but not in the liver. Our findings support the further development of ICT2700 as a tumor-selective treatment for human bladder cancers. Mol Cancer Ther; 12(1); 27–37. ©2012 AACR.

Preliminary pharmacokinetic and bioanalytical studies of SJG-136 (NSC 694501), a sequence-selective pyrrolobenzodiazepine dimer DNA-cross-linking agent.

SJG-136 is a synthetic pyrrolobenzodiazepine (PBD) dimer in which two DNA-alkylating subunits are linked through an inert propanedioxy tether. Biophysical and biochemical studies of SJG-136 have shown a remarkable affinity for DNA and potent cytotoxicity in vitro. On this basis, together with its unique sequence selectivity and interstrand DNA cross-linking activity, SJG-136 has been selected for clinical trials. This study examines the pharmacological characteristics of SJG-136 and provides the first report of pharmacokinetic properties for this agent. A sensitive, selective and reproducible reversed-phase gradient LC/MS assay has been developed for detection and analysis, where a molecular ion (m/z 557.2) is detectable for the SJG-136 parent imine. Fluorescence detection (260 nm excitation, 420 nm emission) gives a limit of sensitivity of 5 nM (2.5 ng ml−1) for analysis of SJG-136 in mouse plasma. Extraction efficiencies from plasma were >65% across a range of concentrations (5–1000 nM). Following administration to mice at the MTD (i.p., 0.2 mg kg−1), high peak plasma concentrations of SJG-136 were seen (Cmax = 336 nM) at 30 min after dosing. A calculated terminal t1/2 of 0.98 h and AUC of 0.34 μM·h resulted in a clearance rate of 17.7 ml min−1 kg−1. The PBD dimer binds only moderately to proteins (65–75%), and in vitro cytotoxicity studies confirmed IC50 values of 4–30 nM with a panel of human cell lines. This finding demonstrates that plasma concentrations achieved in the mouse are substantially higher than those required to elicit an anti tumour response in vitro. This report forms an important phase in the pre-clinical characterization of the compound.

Adenovirus-mediated hypoxia-targeted gene therapy using HSV thymidine kinase and bacterial nitroreductase prodrug-activating genes in vitro and in vivo.

Hypoxia is an important factor in tumor growth. It is associated with resistance to conventional anticancer treatments. Gene therapy targeting hypoxic tumor cells therefore has the potential to enhance the efficacy of treatment of solid tumors. Transfection of a panel of tumor cell lines with plasmid constructs containing hypoxia-responsive promoter elements from the genes, vascular endothelial growth factor (VEGF) and erythropoietin, linked to the minimal cytomegalovirus (mCMV) or minimal interleukin-2 (mIL-2) promoters showed optimum hypoxia-inducible luciferase reporter gene expression with five repeats of VEGF hypoxic-response element linked to the mCMV promoter. Adenoviral vectors using this hypoxia-inducible promoter to drive therapeutic transgenes produced hypoxia-specific cell kill of HT1080 and HCT116 cells in the presence of prodrug with both herpes simplex virus thymidine kinase/ganciclovir and nitroreductase (NTR)/CB1954 prodrug-activating systems. Significant cytotoxic effects were also observed in patient-derived human ovarian cancer cells. The NTR/CB1954 system provided more readily controllable transgene expression and so was used for in vivo experiments of human HCT116 xenografts in nude mice. Subjects treated intratumorally with Ad-VEGFmCMV-NTR and intraperitoneal injection of CB1954 demonstrated a statistically significant reduction in tumor growth. Immunohistochemistry of treated xenografts showed a good correlation between transgene expression and hypoxic areas. Further investigation of these hypoxia-inducible adenoviral vectors, alone or in combination with existing modalities of cancer therapy, may aid in the future development of successful Gene-Directed Enzyme Prodrug Therapy systems, which are much needed for targeting solid tumors.

Retargeted adenoviral cancer gene therapy for tumour cells overexpressing epidermal growth factor receptor or urokinase-type plasminogen activator receptor

We have assessed the ability of bispecific fusion proteins to improve adenovirus-mediated transfer of therapeutic and marker transgenes. We constructed an expression vector that can be easily modified to synthesize a variety of fusion proteins for retargeting adenoviral gene therapy vectors to cell surface markers, which are differentially expressed between normal and cancer cells. Adenoviral transduction can be improved in a number of tumour cell lines which overexpress EGFR (epidermal growth factor receptor) or uPAR (urokinase-type plasminogen activator receptor), but which have only low levels of endogenous hCAR (human coxsackie B and adenovirus receptor) expression. Up to 40-fold improvement in β-galactosidase transgene expression was seen using an EGFR retargeting protein, and up to 16-fold using a second fusion protein targeting uPAR. In vitro, our uPAR retargeting fusion protein improved the sensitivity to adenoviral herpes simplex virus thymidine kinase/ganciclovir by an order of magnitude, whereas in vivo, our EGFR retargeting protein is able to significantly delay tumour growth in rodent animal models in a dose-dependent manner. The ‘cassette’ design of our fusion protein constructs offers a flexible method for the straightforward synthesis of multiple adenoviral retargeting proteins, directed against a variety of tumour-associated antigens, for use in clinical trials.

The microtubule depolymerizing agent CYT997 causes extensive ablation of tumor vasculature in vivo.

The orally active microtubule-disrupting agent (S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridin-3-yl)butyl)amino)pyrimidin-2-yl)phenyl)urea (CYT997), reported previously by us (Bioorg Med Chem Lett 19:4639–4642, 2009; Mol Cancer Ther 8:3036–3045, 2009), is potently cytotoxic to a variety of cancer cell lines in vitro and shows antitumor activity in vivo. In addition to its cytotoxic activity, CYT997 possesses antivascular effects on tumor vasculature. To further characterize the vascular disrupting activity of CYT997 in terms of dose and temporal effects, we studied the activity of the compound on endothelial cells in vitro and on tumor blood flow in vivo by using a variety of techniques. In vitro, CYT997 is shown to potently inhibit the proliferation of vascular endothelial growth factor-stimulated human umbilical vein endothelial cells (IC50 3.7 ± 1.8 nM) and cause significant morphological changes at 100 nM, including membrane blebbing. Using the method of corrosion casting visualized with scanning electron microscopy, a single dose of CYT997 (7.5 mg/kg i.p.) in a metastatic cancer model was shown to cause destruction of tumor microvasculature in metastatic lesions. Furthermore, repeat dosing of CYT997 at 10 mg/kg and above (intraperitoneally, b.i.d.) was shown to effectively inhibit development of liver metastases. The time and dose dependence of the antivascular effects were studied in a DLD-1 colon adenocarcinoma xenograft model using the fluorescent dye Hoechst 33342. CYT997 demonstrated rapid and dose-dependent vascular shutdown, which persists for more than 24 h after a single oral dose. Together, the data demonstrate that CYT997 possesses potent antivascular activity and support continuing development of this promising compound.