Search

The design, synthesis, and biological evaluation of a series novel N1‑methyl pyrazolo[4,3-d]pyrimidines as inhibitors of tubulin polymerization and colchicine binding were described here. Synthesis of target compounds involved alkylation of the pyrazolo scaffold, which afforded two regioisomers. These were separated, characterized and identified with (1)H NMR and NOESY spectroscopy. All compounds, except 10, inhibited [(3)H] colchicine binding to tubulin, and the potent inhibition was similar to that obtained with CA-4. Compounds 9 and 11–13 strongly inhibited the polymerization of tubulin, with IC(50) values of 0.45, 0.42, 0.49 and 0.42 μM, respectively. Compounds 14–16 inhibited the polymerization of tubulin with IC(50s) near ~1 μM. Compounds 9 12, 13 and 16 inhibited MCF-7 breast cancer cell lines and circumvented βIII-tubulin mediated cancer cell resistance to taxanes and other MTAs, and compounds 9–17 circumvented Pgp-mediated drug resistance. In the standard NCI testing protocol, compound 9 exhibited excellent potency with low to sub nanomolar GI(50) values (≤10 nM) against most tumor cell lines, including several multidrug resistant phenotypes. Compound 9 was significantly (P

The design, synthesis and biological evaluation of 4-substituted 5-methyl-furo[2,3-d]pyrimidines is described. The Ullmann coupling of 5-methyl-furo[2,3-d]pyrimidine with aryl iodides was successfully optimized to synthesize these analogs. Compounds 6-10 showed single-digit nanomolar inhibition of EGFR kinase. Compounds 1 and 6-10 inhibited VEGFR-2 kinase better than or equal to sunitinib. Compounds 1 and 3-10 were more potent inhibitors of PDGFR-β kinase than sunitinib. In addition, compounds 4-11 had higher potency in the CAM angiogenesis assay than sunitinib. Compound 1 showed in vivo efficacy in an A498 renal xenograft model in mice. Multiple RTK and tubulin inhibitory attributes of 1, 4, 6 and 8 indicates that these compounds may be valuable preclinical single agents targeting multiple intracellular targets.

In an effort to optimize the structural requirements for combined cytostatic and cytotoxic effects in single agents, a series of 5-(arylthio)-9H-pyrimido[4,5-b]indole-2,4-diamines 3-7 were synthesized and evaluated as inhibitors of receptor tyrosine kinases (RTKs) as well as thymidylate synthase (TS). The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-bromo/5-chloro-9H-pyrimido[4,5-b]indole-2,4-diamine with appropriate aryl thiols. A novel four step synthetic scheme to the common intermediate was developed which is more efficient relative to the previously reported six-step sequence. Biological evaluation of these compounds indicated dual activity in RTKs and human TS (hTS). In the VEGFR-2 assay, compound 5 was equipotent to the standard compound semaxanib and was better than standard TS inhibitor pemetrexed, in the hTS assay. Compounds 3, 6 and 7 were nanomolar inhibitors of hTS and were several fold better than pemetrexed.

The efficacy of quinazoline-based antiglioma agents has been attributed to their effects on microtubule dynamics.1,2 The design, synthesis and biological evaluation of quinazolines as potent inhibitors of multiple intracellular targets, including microtubules and multiple RTKs, is described. In addition to the known ability of quinazolines 1 and 2 to cause microtubule depolymerization, they were found to be low nanomolar inhibitors of EGFR, VEGFR-2 and PDGFR-β. Low nanomolar inhibition of EGFR was observed for 1–3 and 9–10. Compounds 1 and 4 inhibited VEGFR-2 kinase with activity better than or equal to that of sunitinib. In addition, compounds 1 and 2 had similar potency to sunitinib in the CAM angiogenesis assay. Multitarget activities of compounds in the present study demonstrates that the quinazolines can affect multiple pathways and could lead to these agents having antitumor potential caused by their activity against multiple targets.

A series of methoxy naphthyl substituted cyclopenta[d]pyrimidine compounds, 4–10, were designed and synthesized to study the influence of the 3-D conformation on microtubule depolymerizing and antiproliferative activities. NOESY studies with the N,2-dimethyl-N-(6ʹ-methoxynaphthyl-1ʹ-amino)-cyclopenta[d]pyrimidin-4-amine (4) showed hindered rotation of the naphthyl ring around the cyclopenta[d]pyrimidine scaffold. In contrast, NOESY studies with N,2-dimethyl-N-(5ʹ-methoxynaphthyl-2ʹ-amino)-cyclopenta[d]pyrimidin-4-amine (5) showed free rotation of the naphthyl ring around the cyclopenta[d]pyrimidine scaffold. The rotational flexibility and conformational dissimilarity between 4 and 5 led to a significant difference in biological activities. Compound 4 is inactive while 5 is the most potent in this series with potent microtubule depolymerizing effects and low nanomolar IC(50) values in vitro against a variety of cancer cell lines. The ability of 5 to inhibit tumor growth in vivo was investigated in a U251 glioma xenograft model. The results show that 5 had better antitumor effects than the positive control temozolomide and has identified 5 as a potential preclinical candidate for further studies. The influence of conformation on the microtubule depolymerizing and antitumor activity forms the basis for the development of conformation-activity relationships for the cyclopenta[d]pyrimidine class of microtubule targeting agents.

A series of eleven conformationally restricted, 4-substituted 2,6-dimethylfuro[2,3-d]pyrimidines was designed to explore the bioactive conformation required for dual inhibition of microtubule assembly and receptor tyrosine kinases (RTKs), and their biological activities are reported. All three rotatable single bonds in the lead compound 1 were sequentially restricted to address the role of each in SAR for microtubule and RTK inhibitory effects. Compounds 2, 3, 7 and 10 showed microtubule depolymerizing activity comparable to or better than the lead 1, some with nanomolar EC50 values. While compound 8 had no effect on microtubules, 8 and 10 both showed potent RTK inhibition with nanomolar IC50s. These compounds confirm that the bioactive conformation for RTK inhibition is different from that for tubulin inhibition. The tetrahydroquinoline analog 10 showed the most potent dual tubulin and RTK inhibitory activities (low nanomolar inhibition of EGFR, VEGFR2 and PDGFR-β). Compound 10 has highly potent activity against many NCI cancer cell lines, including several chemo-resistant cell lines, and could serve as a lead for further preclinical studies.

Antiangiogenic agents (AA) are cytostatic, and their utility in cancer chemotherapy lies in their combination with cytotoxic chemotherapeutic agents. Clinical combinations of vascular endothelial growth factor receptor-2 (VEGFR2) inhibitors with antitubulin agents have been particularly successful. We have discovered a novel, potentially important analogue, that combines potent VEGFR2 inhibitory activity (comparable to that of sunitinib) with potent antitubulin activity (comparable to that of combretastatin A-4 (CA)) in a single molecule, with GI50 values of 10(-7) M across the entire NCI 60 tumor cell panel. It potently inhibited tubulin assembly and circumvented the most clinically relevant tumor resistance mechanisms (P-glycoprotein and β-III tubulin expression) to antimicrotubule agents. The compound is freely water-soluble as its HCl salt and afforded excellent antitumor activity in vivo, superior to docetaxel, sunitinib, or Temozolomide, without any toxicity.

Inhibition of receptor tyrosine kinase (RTK) signaling pathways is an important area for the development of novel anticancer agents. Numerous multikinase inhibitors (MKIs) have been recently approved for the treatment of cancer. Vascular endothelial growth factor receptor-2 (VEGFR-2) is the principal mediator of tumor angiogenesis. In an effort to develop ATP-competitive VEGFR-2 selective inhibitors the 5-chloro-N4-substituted phenyl-9H-pyrimido[4,5-b]indole-2,4-diamine scaffold was designed. The synthesis of the target compounds involved N-(4,5-dichloro-9H-pyrimido[4,5-b]indol-2-yl)-2,2-dimethylpropanamide) as a common intermediate. A nucleophilic displacement of the 4-chloro group of the common intermediate by appropriately substituted anilines afforded the target compounds. Biological evaluation indicated that compound 5 is a potent and selective VEGFR-2 inhibitor comparable to sunitinib and semaxinib.

Six novel N(4)-phenylsubstituted-6-(2-pyridin-2-ylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines and their N(2)-trimethylacetyl substituted analogs were synthesized as receptor tyrosine kinase (RTK) inhibitors. A microwave-mediated Sonogashira reaction was used as a key step for the synthesis of these compounds. Biological evaluation, in whole cell assays, showed that some analogs had remarkable inhibitory activity against a variety of RTKs and in particular cytotoxic activity against A431 tumor cells in culture. The inhibitory data against RTKs in this study demonstrated that variation of the 4-anilino substituents of these analogs dictates both potency and specificity of inhibitory activity against various RTKs. The study also supported the hypothesis that interaction of substituents on the 2-amino group with hydrophobic site-II provides an increase in potency. Compound 8 of this series was selected for evaluation in vivo in a B16-F10 syngeneic mouse tumor model and exhibited significant reduction in tumor growth rate, in tumor vascular density and in metastases to the lung compared to the control.

The utility of cytostatic antiangiogenic agents (AA) in cancer chemotherapy lies in their combination with cytotoxic chemotherapeutic agents. Clinical combinations of AA with microtubule targeting agents (MTAs) have been particularly successful. The discovery, synthesis and biological evaluations of a series of 7-benzyl-N-substituted-pyrrolo[3,2-d]pyrimidin-4-amines are reported. Novel compounds which inhibit proangiogenic receptor tyrosine kinases (RTKs) including vascular endothelial growth factor receptor-2 (VEGFR-2), platelet-derived growth factor receptor-β (PDGFR-β) and epidermal growth factor receptor (EGFR), along with microtubule targeting in single molecules are described. These compounds also inhibited blood vessel formation in the chicken chorioallantoic membrane (CAM) assay, and some potently inhibited tubulin assembly (with activity comparable to that of combretastatin A-4 (CA)). In addition, some of the analogs circumvent the most clinically relevant tumor resistance mechanisms (P-glycoprotein and β-III tubulin expression) to microtubule targeting agents (MTA). These MTAs bind at the colchicine site on tubulin. Two analogs displayed two to three digit nanomolar GI50 values across the entire NCI 60 tumor cell panel and one of these, compound 7, freely water soluble as its HCl salt, afforded excellent in vivo antitumor activity against an orthotopic triple negative 4T1 breast cancer model and was superior to doxorubicin.

With the goal of developing multitargeted receptor tyrosine kinase inhibitors that display potent inhibition against PDGFRβ and VEGFR-2 we designed and synthesized eleven N4-(3-bromophenyl)-7-(substitutedbenzyl) pyrrolo[2,3-d]pyrimidines 9a–19a. These compounds were obtained from the key intermediate N4-(3-bromophenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine 29. Various arylmethyl groups were regiospecifically attached at the N7 of 29 via sodium hydride induced alkylation with substituted arylmethyl halides. Compounds 11a and 19a were potent dual inhibitors of PDGFRβ and VEGFR-2. In a COLO-205, in vivo tumor mouse model 11a demonstrated inhibition of tumor growth, metastasis, and tumor angiogenesis that was better than or comparable to the standard compound TSU-68 (SU6668, 8).

Dandajena T. C., Ihnat M. A., Disch B., Thorpe J., Currier G. F. Hypoxia triggers a HIF-mediated differentiation of peripheral blood mononuclear cells into osteoclasts. Orthod Craniofac Res 2012;15:1–9 © John Wiley & Sons A/S Structured Abstract Background – The source and mechanisms leading to osteoclast (OC) generation during tooth movement are not clearly understood. We hypothesized that during tooth movement, OC differentiate from peripheral blood mononuclear cells (PBMNC) downstream of the global hypoxia-inducible transcription factor hypoxia-inducible factor (HIF)-1α. Objective – The objective of this study was to demonstrate up-regulation of OC growth factors from osteoblasts (OB) and subsequent conversion of PBMNC into functional OC under hypoxic stress. Material and Methods – Human primary PBMNC were cocultured with/without OB and subjected to either hypoxia (2.5% O2) or normoxia (21% O2) over 14 days. Levels of HIF, vascular endothelial growth factor (VEGF) and receptor activator for nuclear factor kappa-β ligand (RANKL) were measured. Conversion of PBMNC into OC was measured using resorption and TRAP assays. Results – Functional OC were only observed in response to hypoxia during coculture of PBMNC and OB and only after up-regulation of HIF, VEGF and RANKL in the hypoxic conditions. YC-1, a HIF inhibitor, reduced OC formation in response to hypoxia. Conclusion – Hypoxia triggers the differentiation of PBMNC into functional OC in the presence of OB in a HIF-dependent manner as would occur during orthodontic loading of the periodontal ligament space.

Comparison between a series of pyrrolo[2,3-d]pyrimidines with and without the 2-amino group is presented in order to determine the validity of our hypothesis that inclusion of this group improves potency against receptor tyrosine kinases (RTK). The 2-amino analogs were better against epidermal growth factor receptor (EGFR) and platelet derived growth factor-beta (PDGFR-beta) in whole cell inhibition assays and in the A431 cytotoxicity assay compared to the 2-desamino analogs. However, the 2-desamino analogs were more potent inhibitors against vascular endothelial growth factor-2 (VEGFR-2) than the corresponding 2-amino compounds. In addition, none of the 2-desamino compounds exhibited better anti-angiogenic activity in the chorioallantoic membrane (CAM) assay as compared to the standard and were only micromolar inhibitors. This study validates our original hypothesis that the inclusion of a 2-amino group in pyrrolo[2,3-d]pyrimidines improves multiple RTK inhibition and antiangiogenic activity.

Diabet. Med. 27, 911–917 (2010) Abstract Objective To investigate the possibility of reversing endothelial dysfunction and inflammation by glucose normalization, antioxidants and insulin per se, in different subgroups of Type 1 diabetic patients. Methods Three subgroups of Type 1 diabetic patients were studied: patients within 1 month of diagnosis (subgroup 1); patients with approximately 5 years’ disease duration and with glycated haemoglobin (HbA1c) ≤ 7.0% (subgroup 2) or > 7.0% since diagnosis (subgroup 3). Participants underwent four procedures: 2-h hyperglycaemic clamp followed by: (A) 12 h near-normalization of blood glucose, with the addition of vitamin C during the last 6 h; (B) 12-h vitamin C and near-normalization of blood glucose for the last 6 h; (C) both vitamin C and near-normalization of blood glucose for 12 h; (D) hyperglycaemic–hyperinsulinaemic clamp for 12 h, with the addition of vitamin C during the last 6 h. Results After 2 h of hyperglycaemia, markers of endothelial dysfunction, nitrotyrosine, 8-iso prostaglandin F2α, soluble intercellular adhesion molecule-1, soluble vascular adhesion molecule-1, interleukin (IL)-6 and IL-18 were increased in all the subgroups. Levels were normalized, at all time points, by treatments A, B and C in the subgroups 1 and 2. In the third subgroup, levels were normalized only by the simultaneous normalization of blood glucose and vitamin C treatment. During treatment D, the levels were improved at 6 h in all the subgroups, but normalized at 12 h only after vitamin C in subgroups 1 and 2, but not in subgroup 3. Conclusions This study suggests that different subgroups of Type 1 diabetic patients react identically to acute hyperglycaemia and insulin, but differently to glucose normalization.

To optimize dual receptor tyrosine kinase (RTK) and dihydrofolate reductase (DHFR) inhibition, the E- and Z-isomers of 5-[2-(2-methoxyphenyl)prop-1-en-1-yl]furo[2,3-d]pyrimidine-2,4-diamines (1a and 1b) were separated by HPLC and the X-ray crystal structures (2.0 and 1.4A, respectively) with mouse DHFR and NADPH as well as 1b with human DHFR (1.5A) were determined. The E- and Z-isomers adopt different binding modes when bound to mouse DHFR. A series of 2,4-diaminofuro[2,3-d]pyrimidines 2-13 were designed and synthesized using the X-ray crystal structures of 1a and 1b with DHFR to increase their DHFR inhibitory activity. Wittig reactions of appropriate 2-methoxyphenyl ketones with 2,4-diamino-6-chloromethyl furo[2,3-d]pyrimidine afforded the C8-C9 unsaturated compounds 2-7 and catalytic reduction gave the saturated 8-13. Homologation of the C9-methyl analog maintains DHFR inhibitory activity. In addition, inhibition of EGFR and PDGFR-beta were discovered for saturated C9-homologated analogs 9 and 10 that were absent in the saturated C9-methyl analogs.

Background Patients with long-term exposure to high levels of hyperglycaemia remain more susceptible to diabetes-related complications, even with subsequent lower levels of hyperglycaemia. We sought to confirm the hypothesis that exposure to continuous increased glucose results in a memory of cellular stress in isolated endothelial cells, even when switched back to normal glucose, and to investigate the ability of gliclazide to attenuate this phenomenon. Methods Human umbilical vein endothelial cells were incubated for 21 days in normal glucose (5 mmol/L), high glucose (30 mmol/L), or high glucose for 14 days followed by normal glucose for 7 days (memory condition). The effects of gliclazide (10 mu mol/L) and glibenclamide (1 mu mol/L) were evaluated in the memory condition and added to the culture media early (first 14 days), late (last 7 days), or throughout the study. Oxidative stress and cell apoptosis parameters were investigated. Results Continuous high glucose increased reactive oxygen species, 8-OHdG, nitrotyrosine, caspase-3, and reduced Bcl-2 expression. These deleterious effects were also observed in the memory condition. Gliclazide applied early or throughout the study improved all parameters. In contrast, glibenclamide showed no relevant effect on study parameters. Conclusions Our results suggest that gliclazide prevents endothelial cell apoptosis by reducing oxidative stress. The results appear to confirm the hypothesis that exposure of cells to continuous increased glucose results in a hyperglycaemic cellular memory that remains, even when cells are switched back to normal glucose. Gliclazide attenuated this cellular memory, decreasing oxidative stress and protecting vascular endothelial cells from apoptosis. Copyright (C) 2007 John Wiley & Sons, Ltd.

The preparation and characterization of porous scaffolds from chitosan-PCL blends by freeze extraction, freeze gelation and freeze drying is reported. Using freeze extraction, stable structures were obtained only from PCL, but these were not porous. No stable scaffolds were obtained using the freeze gelation process. Stable scaffolds of chitosan/PCL mixtures could not be obtained using 77% acetic acid by any of these techniques. With 25% aqueous acetic acid, stable scaffolds of chitosan/PCL mixtures were obtained by the freeze drying technique. The stability and pore morphology of freeze dried scaffolds were dependent on the relative mass ratio of chitosan and PCL. A chorioallantoic membrane assay showed that formed 3D chitosan/PCL mixtures were not toxic to vasculature.

In diabetes oxidative stress plays a key role in the pathogenesis of vascular complications, and an early step of such damage is considered the development of an endothelial dysfunction. Hyperglycemia directly promotes an endothelial dysfunction inducing process of overproduction of superoxide and consequently peroxynitrite that damages DNA and activates the nuclear enzyme poly(ADP-ribose) polymerase. This process, depleting NAD+, slowing glycolisis, ATP formation and electron transport, results in acute endothelial dysfunction in diabetic blood vessels and contributes to the development of diabetic complications. These new findings may explain why classical antioxidants, like vitamin E, that work scavenging already formed toxic oxidation products, have failed to show beneficial effects on diabetic complications, and suggest new and attractive “causal” antioxidant therapy. New, low molecular mass compounds that act as SOD or catalase mimetics or L-propionyl-carnitine and lipoic acid, that work as intracellular superoxide scavengers, improving mitochondrial function and reducing DNA damage, may be good candidates for such strategy, and preliminary studies support this hypothesis. This “causal” therapy would also be associated with other promising tools such as LY 333531, PJ34 and FP15, which block protein kinase β isoform, poly(ADP-ribose) polymerase and peroxynitrite, respectively. It is now evident that, statins, ACE inhibitors, AT-1 blockers, calcium channel blockers and thiazolinediones have a strong intracellular antioxidant activity, and it has been suggested that many of their beneficial ancillary effects are due to this property. This preventive activity against oxidative stress generation can justify a large utilization and association of this compounds for preventing complications in diabetic patients where antioxidant defences have been shown to be defective.

A long-term 'memory' of hyperglycaemic stress, even when glycaemia is normalised, has been previously reported in endothelial cells. In this report we sought to duplicate and extend this finding.HUVECs and ARPE-19 retinal cells were incubated in 5 or in 30 mmol/l glucose for 3 weeks or subjected to 1 week of normal glucose after being exposed for 2 weeks to continuous high glucose. HUVECs were also treated in this last condition with several antioxidants. Similarly, four groups of rats were studied for 3 weeks: (1) normal rats; (2) diabetic rats not treated with insulin; (3) diabetic rats treated with insulin during the last week; and (4) diabetic rats treated with insulin plus alpha-lipoic acid in the last week.In human endothelial cells and ARPE-19 retinal cells in culture, as well as in the retina of diabetic rats, levels of the following markers of high glucose stress remained induced for 1 week after levels of glucose had normalised: protein kinase C-beta, NAD(P)H oxidase subunit p47phox, BCL-2-associated X protein, 3-nitrotyrosine, fibronectin, poly(ADP-ribose) Blockade of reactive species using different approaches, i.e. the mitochondrial antioxidant alpha-lipoic acid, overexpression of uncoupling protein 2, oxypurinol, apocynin and the poly(ADP-ribose) polymerase inhibitor PJ34, interrupted the induction both of high glucose stress markers and of the fluorescent reactive oxygen species (ROS) probe CM-H(2)DCFDA in human endothelial cells. Similar results were obtained in the retina of diabetic rats with alpha-lipoic acid added to the last week of normalised glucose.These results provide proof-of-principle of a ROS-mediated cellular persistence of vascular stress after glucose normalisation.

There is much controversy in the literature regarding the role of p53 status response on hypoxia inducible factor (HIF) signaling in response to chronic relative hypoxia (CRH). The goal of this paper was to methodically examine this response in isogenically matched tumor cells. We report that p53-mutant (MUT) cells, versus p53-wild-type (WT) cells, showed decreased apoptosis, increased cell proliferation with higher basal HIF-1alpha levels in response to CRH. In addition, we found increased HIF-mediated transactivation and increased VEGF release with decreased HIF-1alpha/p53 and HIF-1alpha/MDM-2 partnering in p53-MUT versus p53-WT cells in response to CRH.

The angiogenic potential of a biomaterial is a critical factor for successful graft intake in tissue engineering. We developed a modified, rapid and reproducible chicken embryo chorioallantoic membrane (CAM) assay to evaluate the ability of biomaterials in inducing blood vessel density. Five biomaterials including one-layer porcine small intestinal submucosa (SIS), two-layer SIS, four-layer vacuum pressed (VP) SIS, polyglycolic acid (PGA) and PGA modified with poly(lactic-co-glycolic acid) (PLGA) were analyzed. A circular section (1.2 mm diameter) of each biomaterial was placed near a group of blood vessels in the CAM. Blood vessels around the biomaterials were captured with black and white images at 96 h post implantation; and the images were subjected to densitometry evaluation. One-layer SIS induced a significant increase in blood vessel density as compared to the cellulose nitrate negative control, and had the greatest increase in blood vessel density as compared to four-layer VP SIS, PGA, or PLGA modified PGA. Although two-layer SIS has enhanced physical structure for surgical manipulation, its induction in blood vessel density was significantly lower than the one-layer SIS. Stripping the SIS proteins or incubating one-layer SIS with neutralizing antibodies against basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) resulted in decreased angiogenesis. Consistent with results obtained from bladder augmentation animal models, these results confirmed that angiogenic growth factors were present in SIS and affected the angiogenic potential of biomaterials. These data also demonstrated that the CAM assay can be used to ascertain methodically the angiogenic potential of biomaterials.

High glucose, particularly in oscillating conditions, produces an increase in oxidative stress and has been shown to result in an increase in diabetic complications, retinopathy in particular. The hypothesis of this work was that chronic exposure to intermittent high glucose results in an attenuated induction of an antioxidant response as compared to continuous high glucose exposure in isolated retinal cells In this work, human retinal pericytes and ARPE-19 cells were exposed to 5 mM or 30 mM continuous high glucose or to 5 mM oscillating daily with 30 mM glucose for 14 days. Levels of antioxidant proteins and activity and levels of reactive species protein adducts were measured. We demonstrate that the induction of total cellular superoxide dismutase (SOD) activity in isolated retinal pericytes and ARPE-19 cells is significantly attenuated in response to oscillating glucose as compared to continuous high glucose. We also show that a marker of nitrosative stress, 3-nitrotyrosine and a general marker of oxidized proteins, OxyBlot™, were significantly increased in both cell types exposed to intermittent high glucose as compared to continuous high glucose. Finally, we show that levels of nitrated MnSOD were increased in response to intermittent high versus continuous high glucose and that the addition of a reactive nitrogen species scavenger to oscillating glucose resulted in significantly increased levels of SOD activity. In conclusion, our results demonstrate for the first time a link between intermittent high glucose and a decreased induction of an antioxidant response of SOD.

High grade gliomas (HGGs; grades III and IV) are the most common primary brain tumors in adults, and their malignant nature ranks them fourth in incidence of cancer death. Standard treatment for glioblastomas (GBM), involving surgical resection followed by radiation and chemotherapy with temozolomide (TMZ) and the anti-angiogenic therapy bevacizumab, have not substantially improved overall survival. New therapeutic agents are desperately needed for this devastating disease. Here we study the potential therapeutic agent AG119 in a pre-clinical model for gliomas. AG119 possesses both anti-angiogenic (RTK inhibition) and antimicrotubule cytotoxic activity in a single molecule. GL261 glioma-bearing mice were either treated with AG119, anti-VEGF (vascular endothelial growth factor) antibody, anti c-Met antibody or TMZ, and compared to untreated tumor-bearing mice. Animal survival was assessed, and tumor volumes and vascular alterations were monitored with morphological magnetic resonance imaging (MRI) and perfusion-weighted imaging, respectively. Percent survival of GL261 HGG-bearing mice treated with AG119 was significantly higher (p

Glutamate is an excitatory neurotransmitter, released by primary sensory peripheral nerve and spinal synaptic terminals during nociceptive (pain) signaling. The primary source of neurotransmitter, glutamate, is provided from its synthetic enzyme, glutaminase (GLS). Neurotransmitter glutamate is packaged into synaptic vesicles in nociceptive neurons by the vesicular glutamate transporter 2 (VGluT2). Little is known, however, what effect a surgical incision has on GLS and VGluT2 in primary afferent neurons. In this study, an aseptic, midline incision in the proximal one-third of the rat-tail was examined to determine whether sacral dorsal root ganglia innervate the area of surgical incision, utilizing the retrograde tracer Fluoro-Gold™. Subsequently, the amount of VGluT2 and GLS immunoreactivity (IR) in sacral dorsal root ganglia (DRG) was evaluated using immunofluorescence with image analysis and Western immunoblotting with density analysis. GLS messenger RNA (mRNA) changes were evaluated using real-time reverse transcriptase polymerase chain reaction (RT2-PCR). Our findings revealed that sacral-1 (S1) DRG neurons innervate the area of surgical incision. Both GLS and VGluT2-ir are elevated post-surgical incision in S1 DRG neurons for up to 72 hours, while GLS mRNA levels rapidly decreased post-incision and remain depressed for at least 96 hours. Following a surgical incision of the tail, sacral DRGs rapidly deplete their available supply of GLS mRNA and alter their production of the synthetic enzyme, GLS and the vesicular transporter, VGluT2. The rapid use of GLS mRNA and subsequent elevation of GLS protein, along with VGluT2 protein may result in both increased glutamate production and release at peripheral and central processes contributing to primary and secondary sensitization, respectively.

6-diazo-5-oxo-l-norleucine (DON) is a glutamine antagonist produced naturally by Streptomyces. It inhibits several glutamine-dependent enzyme pathways. Of particular note is its inhibitory effect on the mitochondrial enzyme, glutaminase (GLS), the primary producer of neuronal glutamate. Glutamate is an excitatory neurotransmitter released by primary sensory peripheral nerve terminals and spinal synaptic terminals during pain signaling. Previous work using the tail incision and inflammatory models of pain has demonstrated that a single application of the glutaminase inhibitor, DON, into a surgical incision or the paw of arthritic animals results in pain relief. Even though this compound shows promise as a therapeutic agent, limited data exist regarding its dermal toxicity. As a first approach, we evaluated the effect of several concentrations of DON, on the viability, mitochondrial oxidative capacity and proliferation of rat skin fibroblasts, and then examined the effect of DON after incubation with human liver microsomes on proliferation. Finally, we evaluated DON treated rat skin (tail and hind paw) for cellular necrosis, inflammation and mitotic bodies. No significant effects (p > 0.05) of DON were noted on apoptosis, necrosis, and mitochondrial activity in experiments with cultured rat skin fibroblasts. Flow cytometry revealed the absence of apoptosis in cells treated at the IC50 of 232.5 μM. Enhanced toxicity post-exposure to human microsomes was not observed when compared to DON alone. The H&E staining of the rat skin revealed no obvious pathology in the DON treatment group (10 mM). DON has no/minimal cellular toxicity in vitro on dermal fibroblasts at concentrations that effectively provide analgesia. The local application of concentrations greater than the in vitro IC50 for DON revealed no in vivo skin toxicity. These data provide results indicating zero-to-minimal cellular toxicity with DON and support the further investigation of DON as an analgesic.

Several different classes of growth factor receptors containing tyrosine kinases (RTK) are directly or indirectly involved in angiogenesis. Inhibition of these RTKs has provided a new paradigm in the treatment of tumors by restricting their growth and metastasis. We have designed, synthesized and evaluated eleven novel 2-amino-4-(3-bromoanilino)-6-substituted benzyl pyrrolo[2,3-d]pyrimidines as the first in a series of RTK inhibitors. These analogues were synthesized from appropriate α-bromomethylbenzyl ketones by cyclocondensation with 2,6-diamino-4-pyrimidone to afford the 2-amino-4-oxo-6-substituted benzyl pyrrolo[2,3-d]pyrimidines. Chlorination of the 4-position followed by displacement with 3-bromoaniline afforded the target compounds. In some instances, the 2-amino moiety of the pyrrolo[2,3-d]pyrimidines was protected prior to the chlorination and displacement followed by deprotection. The compounds were evaluated as inhibitors of vascular endothelial growth factor receptors VEGFR-2 (Flk-1, KDR) and VEGFR-1 (Flt-1); epidermal growth factor receptor (EGFR); and platelet-derived growth factor receptor-β (PDGFR-β). Selected compounds were also evaluated against the growth of A431 cells (which overexpress EGFR) in culture and as inhibitors of angiogenesis in the chicken embryo chorioallantonic membrane (CAM) assay. In each evaluation, a known standard compound was used as a comparison. Of the 11 analogues, five were more potent or equipotent as compared to standard compounds against the growth factor receptors. Two analogues showed superior inhibition of A431 cells in culture compared to the standard compounds. Three analogues were equipotent with the standard compound in the CAM assay and four of the analogues were dual inhibitors of RTKs. The structure–activity relationship for inhibition of different RTKs was quite distinct and different, and for VEGFR-2 and EGFR diametrically opposite. The inhibitory data against the RTKs in this study demonstrates that variation of the substituent(s) in the benzyl ring of these 2-amino-4-anilino 6-benzyl pyrrolo[2,3-d]pyrimidines does indeed control both the potency and specificity of inhibitory activity against RTKs.

The toxic metals arsenic(III) and chromium(VI) are considered human carcinogens, although they may act through different mechanisms. We previously showed that when administered at single low, non-overtly toxic doses, chromium, arsenic, and several other chemical carcinogens preferentially alter expression of several model inducible genes in both whole-animal and cell-culture systems. In this study, we assessed whether chromium and arsenic target specific signaling pathways within cells to selectively modulate gene expression. We examined the effects of non-cytotoxic and cytotoxic doses of arsenic(III) and chromium(VI) on nuclear binding of the transcription factors AP-1, NF-κB, Sp1, and YB-1 in human MDA-MB-435 breast cancer and rat H4IIE hepatoma cells. These transcription factors were chosen because they may regulate many inducible genes, including those previously shown to be altered by metal treatments. We report that both arsenic and chromium significantly altered nuclear binding levels of these factors to their respective cis-acting elements. However, there were qualitative and quantitative differences in these effects that were dependent on the metal, time, dose, transcription factor, and cell line. These effects may play a significant role in metal-induced alterations in gene expression. Mol. Carcinog. 25:219–229, 1999. © 1999 Wiley-Liss, Inc.

The cDNA sequences for two dioxin-inducible cytochrome P450s in chicken, CYP1A4 and CYP1A5, have recently been reported which correspond to two dioxin-inducible forms of P450 previously designated as TCDD AHH and TCDD AA , respectively. The developmental expression of CYP1A4 -associated aryl hydrocarbon (benzo[ a ]pyrene) hydroxylase (AHH) activity and its association with expression of the Ah receptor had previously been characterized in chick embryo liver. The purpose of this study was to examine the developmental regulation of the second dioxin-inducible P450 gene, CYP1A5, in chick embryo liver. A partial gene sequence for CYP1A5 indicated that the intron/exon organization of this gene was identical to that of the CYP1A1 and CYP1A2 mammalian genes and was present in a single copy in the genome. CYP1A5 mRNA was expressed basally in chick embryo liver and was highly inducible by the Ah receptor ligands, 3-methylcholanthrene, β-naphthoflavone, and 3,4,3′,4′-tetrachlorobiphenyl (TCB), but not by the phenobarbital analog, glutethimide. CYP1A5 mRNA levels were increased 40- to 50-fold within 5 h after a single TCB treatment, corresponding to a 30- to 40-fold increase in the transcription rate of the CYP1A5 gene at this time point. In contrast to a previous report that CYP1A5 mRNA expression was inducible by estradiol, we observed no effects of estradiol or dexamethasone on CYP1A5 mRNA expression, either alone or in combination with TCB. Basal and TCB-inducible CYP1A5 mRNA expression was maximal in liver at 8 days of development and remained high throughout the remainder of embryonic development. Thus, CYP1A5 appears to be regulated in a very similar manner to CYP1A4 in chick embryo liver.

Mitomycin C (MMC) is a DNA crosslinking agent that is used in cancer chemotherapy. Unlike the DNA crosslinks formed by cisplatin or psoralen, which significantly distort the DNA helix, the MMC crosslink does not significantly disturb the B-DNA helical structure. Nonetheless, MMC interstrand crosslinks and total MMC adducts are rapidly removed in vivo. We investigated whether mammalian nuclear proteins can recognize and bind to a model 23 bp DNA duplex containing a single MMC lesion. Electrophoretic mobility shift assays identified two complexes in nuclear extracts from rodent cell lines and three complexes in human cell lines, containing proteins that appeared to specifically recognize the MMC interstrand crosslink. Nuclear extracts from normal and excision repair-defective mutant Chinese hamster ovary (CHO) cell lines, from human Xeroderma Pigmentosum (XP) complementation group A and E cell lines, and a Fanconi's Anemia cell line were also examined. The UV-20 CHO line, defective in ERCC-1, was missing one of the two rodent complexes. Two of the three human complexes were also absent in the XPA human cell line and the intensity of the third complex was significantly diminished. Based on these results, a model for MMC crosslink recognition is proposed in which ERCC-1 and XPA each participate in formation of one or more multimeric complexes on the crosslinked DNA and XPA also aids in the formation, but is not a component of a higher molecularweight multimeric complex that may contain ERCC-1.

Combination chemotherapy with antiangiogenic (cytostatic) and microtubule targeting (cytotoxic) agents (MTA) have shown significant promise and several studies with such combinations have progressed to the clinic. Single agents with both antiangiogenic activities as well as cytotoxicity would circumvent pharmacokinetic problems of multiple agents, might be effective at lower doses to alleviate toxicity, and delay or prevent tumor cell resistance. We have designed, synthesized and evaluated single agents that are novel pyrrolo[3,2-d]pyrimidine MTAs that also inhibit vascular endothelial growth factor receptor-2 (VEGFR-2), epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor â (PDGFRâ). Sixteen pyrrolo[3,2-d]pyrimidine analogs were synthesized with variations on the C-2 and the 4-anilino position. The importance of various substituents was evaluated based on their abilities to cause cellular microtubule depolymerization and inhibit proliferation of MDA-MB-435 cancer cells. Compounds with N5-Me were more potent than the N5-H analogs indicating that a conformational restriction induced by N5-Me could mimic the bioactive conformation. In addition, all C2-Cl compounds afforded 2 to 10-fold more potentIC50 values than their C2-Me counterparts as illustrated by C2-Cl analog AG336 (IC50 = 18.3 ± 5.0 nM) compared to its C2-Me analog AG85 (IC50 = 193 ± 39 nM). The most active antitubulin agents were evaluated against RTKs and demonstrated good inhibition for EGFR (IC50 = 29.5 - 91.5 nM), VEGFR-2 (182.3 - 543.6 nM) and PDGFRâ (IC50 = 204.4 - 450.2 nM), thus demonstrating dual-targeting ability. Preclinical assessment of AG165 at 75 mg/kg in mice in the NCI/ADR resistant tumor model demonstrated significant reduction of tumor volume compared to paclitaxel (10 mg/kg) treated mice [p = 0.0333], indicating antitumor activity in a multidrug resistant tumor model, in addition to excellent antitumor activity in MDA-MB-435 and MDA-MB-231 xenograft models. These results demonstrate the viability of these multitargeted single agents as potential clinically active antitumor agents. Citation Format: Aleem Gangjee, Khushbu Shah, Nicholas Dybdal-Hargreaves, Susan Mooberry, Anja Bastian, Michael Ihnat. Design and biological evaluation of substituted pyrrolo[3,2-d]pyrimidines as dual acting RTK and microtubule targeting agents. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4857.

The enzyme aminopeptidase N (APN, also known as CD13) is known to play an important role in tumor proliferation, attachment, angiogenesis, and tumor invasion. In this study, we hypothesized that a radiolabeled high affinity APN inhibitor could be potentially useful for imaging APN expression in vivo. Here, we report synthesis, radiolabeling, and biological evaluation of new probestin conjugates containing a tripeptide, N,N-dimethylglycyl-l-lysinyl-l-cysteinylamide (N(3)S), chelator. New probestin conjugates were synthesized by solid-phase peptide synthesis method, purified by reversed-phase HPLC, and characterized by electrospray mass spectrometry. The conjugates were complexed with Re(V) and (99m)Tc(V) by transmetalation using corresponding Re(V) or (99m)Tc(V) gluconate synthon. The mass spectral analyses of ReO-N(3)S-Probestin conjugates were consistent with the formation of neutral Re(V)O-N(3)S complexes. Initial biological activity of ReO-N(3)S-Probestin conjugates determined by performing an in vitro APN enzyme assay using intact HT-1080 cells demonstrated higher inhibition of APN enzyme activity than bestatin. In vivo biodistribution and whole body planar imaging studies of (99m)TcO-N(3)S-PEG(2)-Probestin performed in nude mice xenografted with human fibrosarcoma tumors derived from HT-1080 cells demonstrated a tumor uptake value of 2.88 ± 0.64%ID/g with tumor-to-blood and tumor-to-muscle ratios of 4.8 and 5.3, respectively, at 1 h postinjection (p.i.). Tumors were clearly visible in whole body planar image obtained at 1 h p.i., but not when the APN was competitively blocked with a coinjection of excess nonradioactive ReO-N(3)S-PEG(2)-Probestin conjugate. These results demonstrate the feasibility of using high affinity APN inhibitor conjugates as targeting vectors for in vivo targeting of APN.

Hyaluronic acid-poly(de-co-glycolide) nanoparticles (HA-PLGA NPs) were synthesized to stabilize the porous structure of porcine small intestinal submucosa (SIS), to improve surface biocompatibility and to enhance performance in tissue regeneration. HA-PLGA NPs were characterized for size, zeta potential, surface morphology, and HA loading. Human microvascular endothelial cells responded to HA-PLGA NPs and HA-PLGA modified SIS (HA-PLGA-SIS) with elevated cell proliferation. HA-PLGA-SIS significantly enhanced neo-vascularization in an in ovo chorioallantoic membrane angiogenesis model. The angiogenic capability of the newly fabricated HA-PLGA-SIS was tested in a canine bladder augmentation model. Urinary bladder augmentation was performed in beagle dogs following hemi-cystectomy using HA-PLGA-SIS. The regenerated bladder was harvested at 10 weeks post augmentation and vascularization was evaluated using CD31 immunohistochemical staining. Bladder regenerated with HA-PLGA-SIS had significantly higher vascular ingrowth compared to unmodified SIS. This study shows that HA-PLGA NPs may represent a new approach for modifying naturally derived SIS biomaterials in regenerative medicine.

Aim Data suggest that 2h hyperglycaemia during an OGTT is less predictive for cardiovascular disease of the glucose "spike" (the difference between the baseline glucose level and the "peak" hyperglycaemia during the test). Methods A euinsulinemic hyperglycaemic clamp at 10 and 15mmol/l glucose, with or without vitamin C, was given in increasing steps in diabetic and normal subjects. Moreover, a hyperglycaemic clamp, 10mmol/l, was performed in two groups of diabetic patients with different levels of fasting glycaemia. In both the experiments flow mediated dilation and nitrotyrosine were measured. Results Glucose at 10 and 15mmol/l resulted in a concentration-dependent induction of endothelial dysfunction and oxidative stress. Vitamin C counterbalanced this effect. The increase of glycaemia to 10mmol/l induced a significant endothelial dysfunction and increased nitrotyrosine in both the groups of diabetics with different fasting glycaemia. However, when the delta (the difference between the basal value and the peak value) for endothelial function and nitrotyrosine was evaluated, patients with lower basal values showed a worse outcome. Conclusions Our data suggest that at the same level of hyperglycaemia the grading of the endothelial dysfunction is almost super imposable, but clearly worse in terms of delta from fasting glycaemia.

OBJECTIVE—To explore the possibility that oscillating glucose may outweigh A1C levels in determining the risk for cardiovascular diabetes complications. RESEARCH DESIGN AND METHODS—A euinsulinemic hyperglycemic clamp at 5, 10, and 15 mmol/l glucose was given in increasing steps as a single “spike” or oscillating between basal and high levels over 24 h in normal subjects and type 2 diabetic patients. Flow-mediated dilatation, a marker of endothelial function, and plasma 3-nitrotyrosine and 24-h urinary excretion rates of free 8-iso PGF2α, two markers of oxidative stress, were measured over 48 h postclamp. RESULTS—Glucose at two different levels (10 and 15 mmol/l) resulted in a concentration-dependent fasting blood glucose–independent induction of both endothelial dysfunction and oxidative stress in both normal and type 2 diabetic patients. Oscillating glucose between 5 and 15 mmol/l every 6 h for 24 h resulted in further significant increases in endothelial dysfunction and oxidative stress compared with either continuous 10 or 15 mmol/l glucose. CONCLUSIONS—These data suggest that oscillating glucose can have more deleterious effects than constant high glucose on endothelial function and oxidative stress, two key players in favoring cardiovascular complications in diabetes. Concomitant vitamin C infusion can reverse this impairment.

Trivalent inorganic arsenic (arsenite, arsenic trioxide, As(III)) is a primary contaminant of groundwater supplies worldwide. As(III), marketed as trisenox, is also an FDA-approved agent to treat cancer It has been previously shown by our laboratory that As(III) administered at doses lower than a therapeutic anticancer dose results in an increase in tumor formation and blood vessel density of tumors. In this work it was found that chronic administration of As(III) approaching the EPA action level of 10 ppb, given in the drinking water of mice 5 weeks prior to B16-F10 melanoma implantation, increased the growth rate of primary tumors and the number of metastases to the lung. Further, levels of arsenic in the tumor and lung were found to be much greater than those in the blood and similar to pro-angiogenic As(III) doses. Levels of hypoxia inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) surrounding the blood vessels in the tumors of the As(III)-treated mice were also found to be increased. Exposure of isolated B16-F10 tumor cells to chronic (3 or 7 day) but not acute (4 h) low-dose As(III) was found to increase HIF-1alpha expression and secretion of VEGF. Finally, coadministration of an inhibitor of HIF (YC-1) or a VEGFR-2 kinase inhibitor (SU5416) was found to antagonize the pro-angiogenic effects of low-dose As(III). Together, these results suggest that chronic exposure to low-dose As(III) could stimulate growth of tumors through a HIF-dependent stimulation of angiogenesis.

Vascular endothelial growth factor receptor-2 (VEGFR2) inhibitors lead to the reduction of angiogenesis but are cytostatic, not tumoricidal. These inhibitors have shown significant promise in the clinic when used in combination with cytotoxic agents, and several clinical trials with such combinations are in progress. We have previously reported preclinical studies on AG119, a compound that inhibits both VEGFR2 and tubulin, thereby affording antiangiogenic and cytotoxic effects in a single agent. Structural modifications of AG119 afforded AG321, a unique compound with four separate mechanisms of action in a single agent. AG321 inhibits platelet derived growth factor receptor-β (PDGFRβ) and epidermal growth factor receptor (EGFR), in addition to VEGFR2 and tubulin. AG321 is a colchicine site binding agent with potency comparable with that of combretastatin A-4 against bovine brain tubulin assembly. AG321 has potency comparable with that of erlotinib in tumor cell lines overexpressing EGFR and potency comparable with those of erlotinib, PD153035 and DMBI and in cell lines overexpressing PDGFRβ. It also has potency comparable with those of sunitinib and semaxinib in cell lines overexpressing VEGFR2. AG321 reduced tumor size (control: 15.7 ± 2.9 mm3/day; AG321 treated: 5.7 ± 0.7 mm3/day; p < 0.01 by one way ANOVA with Tukey multiple comparisons post test) and the number of lung metastases in the 4T1 triple negative mouse breast cancer allograft model and was superior to docetaxel, without overt systemic toxicity. A range of potential therapies that target EGFR are currently in development or in clinical trials for the treatment of glioblastoma. Targeting both VEGFR2 and PDGFRβ, which are two key mediators of tumor angiogenesis, increases the antiangiogenic effect even in the often intractable late stage of solid tumors. Hence, AG321, a multiple receptor tyrosine kinase (RTK) inhibitor which may also be effective against EGFR-amplified glioblastoma [AG119 reduced tumor size and vascularity in vivo in the U251 glioma model]. In summary, AG321 with antiangiogenic effects via multiple RTK inhibition and cytotoxic effects via tubulin inhibition in a single entity may delay or prevent tumor cell resistance, circumvent pharmacokinetic problems that can occur with multiple agents, avoid drug-drug interactions, be used at lower doses to alleviate toxicity and be and have reduced toxicity. Citation Format: Aleem Gangjee, Roheeth K. Pavana, Michael Ihnat, Ernest Hamel. Design and preclinical evaluation of single agents with tubulin and multiple receptor tyrosine kinase inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1622. doi:10.1158/1538-7445.AM2014-1622

Antiangiogenic agents target tumor mechanisms involved in blood vessel formation vital for tumor growth and metastases. However, these agents are mainly cytostatic and usually not tumoricidal. Several combinations of antiangiogenic and cytotoxic agents have shown significant promise in the clinic, and several clinical trials are in progress. Single agents with both antiangiogenic activities and cytotoxicity would afford agents that would circumvent pharmacokinetic problems of multiple agents, avoid drug-drug interactions, could be used at lower doses to alleviate toxicity, be devoid of overlapping toxicities, and could delay or prevent tumor cell resistance. We designed, synthesized and evaluated a novel compound AG119 that inhibits both vascular endothelial growth factor receptor-2 (VEGFR-2) affording antiangiogenic effects and tubulin for cytotoxic effects in a single agent. Structural modifications of AG119 afforded AG321 that inhibited platelet derived growth factor receptor-β (PDGFR-β) and epidermal growth factor receptor (EGFR) in addition to VEGFR-2 and tubulin. The novel compounds AG119 and AG321 were potent inhibitors (activity comparable to that of combretastatin A-4) of bovine brain tubulin assembly and of [3H]colchicine binding to tubulin, indicating that these compounds bind at the colchicine site on tubulin. Both compounds have potency comparable to those of sunitinib and semaxinib in tumor cell lines overexpressing VEGFR-2. AG321 has potency comparable to that of erlotinib in tumor cell lines overexpressing EGFR and potency comparable to those of erlotinib, DMBI and PD153035 in cell lines overexpressing PDGFR-β. Both AG119 and AG321 also inhibited angiogenesis in the chorioallantoic membrane (CAM) assay. AG321 is a unique compound with four separate mechanisms of action in a single agent including inhibition of tubulin. This is unprecedented and to our knowledge, is the first of its kind. In mice, AG119 reduced tumor size and vascularity in two flank xenograft models [the BLBC MDA-MB-435 model and U251 glioma model] and in a 4T1 triple negative breast orthotopic allograft model. In these in vivo models, the activity of AG119 was superior to those of temozolomide (U251), docetaxel and sunitinib (MDA-MB-435 and 4T1) without overt toxicity to the animals. Citation Format: Aleem Gangjee, Roheeth K. Pavana, Michael A. Ihnat, Jessica E. Thorpe, Bryan C. Disch, Anja Bastian, Lora C. Bailey-Downs, Ernest Hamel, Rouli Bai. Design and preclinical evaluation of single agents with combination chemotherapy potential. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5682. doi:10.1158/1538-7445.AM2013-5682

Agents that interfere with microtubules are important antitumor agents. Tumor angiogenic mechanisms that are vital for tumor growth and metastasis are targeted by antiangiogenic agents. Antiangiogenic agents are usually not tumoricidal but are mainly cytostatic. Combination chemotherapy with antiangiogenic and cytotoxic agents has shown significant promise, and several studies with such combinations are in progress in the clinic. Single agents with both antiangiogenic activities and cytotoxicity could afford single agents that would circumvent the pharmacokinetic problems of multiple agents, avoid drug-drug interactions, be used at lower doses to alleviate toxicity, be devoid of overlapping toxicities, and delay or prevent tumor cell resistance. We have designed, synthesized and evaluated two novel compounds, AG119 and AG425, that inhibit both vascular endothelial growth factor receptor-2 (VEGFR-2) for antiangiogenic effects and tubulin for cytotoxic effects in single agents. Both compounds have comparable potency as sunitinib and semaxinib against cell lines overexpressing VEGFR-2 and were equipotent with semaxinib on VEGFR-2 mediated endothelial cell proliferation, indicating a functional effect of these agents on VEGFR-2 inhibition (IC50: semaxanib 68.2 ± 4.2 µM, AG119: 60.2 ± 2.4 µM, AG425: 52.3 ± 3.8 µM). The novel compounds are effective and potent inhibitors (comparable to combretastatin A-4) of bovine brain tubulin assembly (IC50: combretastatin A-4 1.2 ± 0.01 µM, AG119: 3.5 µM, AG425: 1.9 ± 0.1 µM). The compounds were potent inhibitors of [3H]colchicine binding to tubulin, indicating that these compounds, particularly AG425, bind at the colchicine site. These agents cause microtubule depolymerization, arrest cells in late G2/M phase and trigger apoptotic cell death. Moreover, AG119 and AG425 are potent anticancer compounds in aggressive basal like breast cancer (BLBC) and triple negative breast cancer (TNBC) cell lines. The novel compounds showed sensitivity in taxol resistant cells, indicating that they are unlikely to be Pgp or MRP substrates [unlike taxol], and these compounds possess relative selectivity of kill on tumor versus normal cell types. In vivo these agents reduce tumor size and vascularity in two flank xenograft models [the BLBC MDA-MB-435 model and U251 glioma model] in immunodeficient mice, superior to docetaxel and sunitinib, without overt toxicity to the animals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3897. doi:1538-7445.AM2012-3897

Combinations of cytotoxic agents with antiangiogenic agents have shown significant promise in cancer treatment, and several such clinical trials are currently underway. We previously identified two compounds with a 5-(thioaryl)-9H-pyrimido[4,5-b]indole-2,4-diamine scaffold that each inhibit vascular endothelial growth factor receptor-2 (VEGFR-2) and platelet derived growth factor receptor-beta (PDGFR-β) for antiangiogenic effects and remarkably also inhibit human thymidylate synthase (hTS) for cytotoxic effects. In a COLO-205 xenograft mouse model one of these analogs demonstrated potent tumor growth inhibition, inhibition of metastasis, and antiangiogenic effects in vivo. Thus, these compounds afford combination chemotherapeutic potential in single agents. In an effort to optimize the structural requirements for antiangiogenic effects and hTS inhibition, six additional compounds were synthesized. Electron withdrawing (3,4-diClPh, 4-ClPh), electron donating (2,5-diOMePh, 4-OMePh) and bulky (1-naphthyl, 2-naphthyl) thioaryl groups in the 5-(thioaryl)-9H-pyrimido[4,5-b]indole-2,4-diamine scaffold were selected to establish optimum activity. The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-bromo-9H-pyrimido[4,5-b]indole-2,4-diamine with appropriate benzenethiols. A novel four step synthetic scheme to the common intermediate was developed which is more efficient relative to the previously reported six step synthesis. Biological evaluation of these compounds in various RTKs indicated that in the VEGFR-2 assay the 2,5-diOMePh substituted compound was equipotent to the standard SU5416 used in this assay. In the hTS assay, the 2-naphthyl, 4-OMePh, and 4-ClPh substituted compounds were nanomolar inhibitors and were 3-fold better than or equipotent to the standard TS inhibitor raltitrexed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1367. doi:10.1158/1538-7445.AM2011-1367

Bacterial cells have the ability to accumulate compatible solutes within the cytoplasm to maintain their osmolarity above that of the extracellular milieu. Glycine betaine (GB) and its biosynthetic precursor choline (Chol) are the major compatible solutes that bacteria accumulate when osmotically challenged. Different osmotically triggered active transport mechanisms have been identified for GB and Chol. In the present study we examined the bioisosteric replacement of the carboxylic group of GB with sulfonic, phosphonic or benzenesulfonamido groups. The sulfonic acid analog (sulfobetaine, compound 3) showed osmoprotectant activity equivalent to that of GB. In addition, we tested the possibility of utilizing GB/Chol transport systems to deliver cytotoxic analogs of GB into three strains of E. coli that differed in their salt resistance. We found that N1-betainyl-N4-(haloacetyl)sulfanilamides (compounds 17c-e) that are GB analogs containing alkylating side chain within their structures inhibited the bacterial growth of the tested standard and salt sensitive strains of E. coli. We also showed that the (N-methyl-cyclic ammonio)methanesulfonates (compounds 21a-c) are able to block Chol transport system in both the standard and the salt-sensitive E. coli strains used. At the concentration used (0.1 mM), none of the tested compounds showed any significant effect on the salt-resistant strain used.

RBM3 is the first RNA binding protein to be identified as a protooncogene. In previous studies, we have shown that RBM3 overexpression results in increased stability and translation of vascular endothelial growth factor (VEGF) mRNA. Furthermore, knockdown of RBM3 in HCT-116 colon cancer xenografts resulted in reduction in VEGF expression and in tumor vasculature. Similarly, curcumin the active ingredient in the spice turmeric reduced the growth of HCT-116 tumor xenografts and inhibited VEGF expression. We have now extended these observations and show that RBM3 is a critical player in tumor angiogenesis, and that curcumin inhibits tumor angiogenesis in part by suppressing RBM3 expression. Methods. RBM3 was stably transfected into NIH-3T3 mouse fibroblast cells to generate 3T3R cells. These cells were implanted on chorioallantoic membranes (CAM) in chicken embryos to determine their effect on angiogenesis. For in vitro studies, human vascular endothelial cells (HUVEC) were treated with VEGF and expression of RBM3 was determined. HUVEC-mediated capillary network formation was performed in polymerized ECMatrix. Cells were also treated with 30 µM curcumin and effects on gene expression determined by western blot analyses. Results. HUVEC cells treated with VEGF (50 ng/ml) resulted in a significant increase RBM3 expression, and high levels of capillary formation in the in vitro angiogenesis assay. SiRNA-mediated downregulation of RBM3 expression in the cells resulted in significant reduction in capillaries, which was overcome when RBM3 was ectopically expressed suggesting that RBM3 is essential for VEGF-mediated angiogenesis. HUVEC cells treated with curcumin (30 µM) treatment also demonstrated in a reduction in RBM3 expression and capillary formation, which was rescued when RBM3 was overexpressed. To determine whether RBM3 plays role in angiogenesis in vivo, 3T3 and 3T3R cells were implanted in Matrigel subcutaneously into chorioallantoic membrane of chicken embryos. 3T3R significantly increased the number of blood vessels when compared to wild type 3T3 cells. Treatment with curcumin also inhibited blood vessel formation. To further confirm these results, we determined gene expression patterns and microvessel density in HCT-116 tumor xenografts following intraperitonial administration of curcumin (200 μg/Kg/d for 21 d). Curcumin suppressed the expression of RBM3 and VEGF in the xenografts. Furthermore, there was a significant reduction in tumor microvasculature based on CD31 immunohistochemistry. Conclusion. Curcumin suppresses tumor angiogenesis in part by inhibiting the expression of RNA binding protooncogene RBM3. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1890.

PURPOSE. The pathogenesis of diabetic retinopathy (DR) is not fully understood. Clinical studies suggest that dyslipidemia is associated with the initiation and progression of DR. However, no direct evidence supports this theory. METHODS. Immunostaining of apolipoprotein B100 (ApoB100, a marker of low-density lipoprotein [LDL]), macrophages, and oxidized LDL was performed in retinal sections from four different groups of subjects: nondiabetic, type 2 diabetic without clinical retinopathy, diabetic with moderate nonproliferative diabetic retinopathy (NPDR), and diabetic with proliferative diabetic retinopathy (PDR). Apoptosis was characterized using the TUNEL assay. In addition, in cell culture studies using in vitro-modified LDL, the induction of apoptosis by heavily oxidized-glycated LDL (HOG-LDL) in human retinal capillary pericytes (HRCPs) was assessed. RESULTS. Intraretinal immunofluorescence of ApoB100 increased with the severity of DR. Macrophages were prominent only in sections from diabetic patients with PDR. Merged images revealed that ApoB100 partially colocalized with macrophages. Intraretinal oxidized LDL was absent in nondiabetic subjects but present in all three diabetic groups, increasing with the severity of DR. TUNEL-positive cells were present in retinas from diabetic subjects but absent in those from nondiabetic subjects. In cell culture, HOG-LDL induced the activation of caspase, mitochondrial dysfunction, and apoptosis in HRCPs. CONCLUSIONS. These findings suggest a potentially important role for extravasated, modified LDL in promoting DR by promoting apoptotic pericyte loss. (Invest Ophthalmol Vis Sci. 2008;49:2679‐2685) DOI:10.1167/iovs.07-1440

Trivalent inorganic arsenic (arsenite, arsenic trioxide, As[III]) is currently being used to treat hematologic tumors and is being investigated for treating solid tumors. However, low concentrations of As(III) stimulate vascular cell proliferation in cell culture, although this has not been confirmed in vivo. Therefore, the hypothesis that As(III) enhances blood vessel growth (angiogenesis) and tumorigenesis was tested in two in vivo models of angiogenesis and a model of tumor growth. In the first, arsenite caused a dose-dependent increase in vessel density in a chicken chorioallantoic-membrane (CAM) assay. The threshold As(III) concentration for this response was 0.033 microM and inhibition of vessel growth was observed at concentrations greater than 1 microM. Mouse Matrigel implants were used to test the angiogenic effects of As(III) in an adult mammalian system. Mice were injected with 0.8-80 microg/kg As(III)/day over a three-week period. During the last two weeks, Matrigel plugs were placed on the abdominal wall. Low and high doses of As(III) were synergistic with fibroblast growth factor-2 (FGF-2) in increasing vessel density in the Matrigel assay, while a middle dose had no effect. To test the effects of As(III) on tumor growth, GFP-labeled B16-F10 mouse melanoma cells were implanted in nude mice, which subsequently received biweekly injections of 0.5-5.0 mg/kg As(III). Significant tumor growth and lung metastasis was seen in all animals, with the largest tumors occurring in animals treated with lower doses of As(III). These studies support the hypothesis and indicate that induction of angiogenesis, enhanced tumor growth, and metastasis are potential dose-dependent toxic side effects of arsenic therapies.