Antivascular Activity Research Articles

Dehydro-α-lapachone, a plant product with antivascular activity

Antivascular agents have become a standard of treatment for many malignancies. However, most of them target the VEGF pathway and lead to refractoriness. To improve the diversity of options for antivascular therapy, we applied a high-throughput screen for small molecules targeting cell adhesion. We then assayed the resulting antiadhesion hits in a transgenic zebrafish line with endothelial expression of EGFP (Tg(fli1:EGFP)(y1)) to identify nontoxic molecules with antivascular activity selective to neovasculature. This screen identified dehydro-α-lapachone (DAL), a natural plant product. We found that DAL inhibits vessel regeneration, interferes with vessel anastomosis, and limits plexus formation in zebrafish. Furthermore, DAL induces vascular pruning and growth delay in orthotopic mammary tumors in mice. We show that DAL targets cell adhesion by promoting ubiquitination of the Rho-GTPase Rac1, which is frequently up-regulated in many different cancers.

Abstract 640: The Akt inhibitor Perifosine strongly enhances the antitumor and antivascular activity of CD34+ cells engineered to express membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

Abstract Introduction. We have previously demonstrated that adenovirus-transduced CD34+ cells expressing membrane-bound (m)TRAIL (CD34-TRAIL+ cells) exert potent antitumor activity against a variety of hematopoietic tumors by targeting both tumor cells and tumor vasculature (Blood, 115:2231-40, 2010). Recently, we have identified non-Hodgkin lymphoma cell lines which are resistant to the in vivo antitumor activity of mTRAIL. Perifosine has been shown to increase the toxicity of soluble TRAIL against cancer cell lines by inhibiting the PI3K/Akt pathway as well as enhancing pro-apoptotic TRAIL receptors. We therefore investigated the efficacy of Perifosine in modulating the antitumor activity of CD34-TRAIL+ cells using as model systems the TRAIL-resistant SU-DHL-4V and the TRAIL-sensitive KMS-11 cell lines. Methods and Results. In vitro, Perifosine significantly enhanced the cytotoxic activity of CD34-TRAIL+ cells against both SU-DHL-4V and KMS-11 cell lines by increasing the expression of TRAIL receptors and down-modulating phospho-Akt, cFLIP and Mcl-1 expression. In vivo, in NOD/SCID mice bearing subcutaneous nodules, Perifosine significantly increased the antitumor activity of CD34-TRAIL+ cells against both tumor types. In fact, CD34-TRAIL+ cells used as single agent exerted a limited if any activity against TRAIL-resistant SU-DHL-4V nodules, whereas, when combined with Perifosine, transduced cells reduced SU-DHL-4V growth by 43% (p< .001) over controls. TRAIL-sensitive KMS-11 nodules were reduced by 39% (p< .001) following treatment with CD34-TRAIL+ cells, and addition of Perifosine further reduced tumor growth by 65% (p<.001) over controls. Upon in vivo treatment with Perifosine, confocal microscopy analysis revealed a strong down-modulation of phospho-Akt expression by tumor cells and tumor endothelial cells (TECs), and flow cytometry analysis of TECs revealed a strong induction of TRAIL-R2 expression. In particular, Perifosine induced de novo TRAIL-R2 expression by TECs enriched from SU-DHL-4V tumors (41.6 ± 5.7% vs 4.0 ± 4.0%, p<.05), and enhanced TRAIL-R2 expression by TECs from KMS-11 tumors (81.3 ± 7.6% vs 41.7 ± 8.2%, p<.05). Increased levels of endothelial TRAIL-R2 was associated with a significant increase of (i) tumor specific CD34-TRAIL+ cells-induced vascular damage, (ii) tumor hemorragic necrosis and (iii) tumor apoptosis. Conclusions. Our results demonstrate that: (i) TRAIL-R2 expression by TECs correlates with the in vivo antivascular activity of CD34-TRAIL+ cells; (ii) Perifosine potentiates the antitumor activity of TRAIL-armed CD34+ cells and is able to overcome in vivo resistance to mTRAIL by inducing TRAIL-R2 expression on tumor endothelial cells. These results may open new perspectives in view of clinical applications of CD34-TRAIL+ cells. 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 640. doi:10.1158/1538-7445.AM2011-640

Abstract 5309: Antivascular activities of novel tubulin binding agent BPR0L075

Abstract BPR0L075, 6-methoxy-3-(3′,4′,5′-trimethoxy-benzoyl)-1H-indole, is a novel tubulin binding agent that inhibits tubulin polymerization through binding to the colchicine binding site. We evaluated the antiangiogenic and antivascular properties of BPR0L075 in vitro and in vivo. Human umbilical vein endothelial cells (HUVEC) were treated with BPR0L075, and effects on endothelial cell morphology, migration, and inhibition of tube formation on extracellular matrix were determined in vitro. The BPR0L075 induced inhibition of neovasculature formation was evaluated by rat aortic ring ex vivo assay. Using a validated dynamic bioluminescence imaging method, we assessed acute vascular disrupting effects of BPR0L075 on luciferase-expressing MCF7 human breast mammary fat pad xenografts. The disruption of tumor vascular perfusion after BPR0L075 treatment was confirmed using the perfusion marker Hoechst 33342 and CD31 staining by fluorescence microscopy. Exposure of BPR0L075 (10 nM) inhibits the endothelial cell migration (within 24 hr) and tube formation on Matrigel (3-6 hr). BPR0L075 treatment also blocked angiogenesis in rat aortic ring assay. A single dose of BPR0L075 induced rapid temporary tumor vascular shutdown evidenced by rapid and reproducible decrease of light emission from luciferase-expressing breast tumors. Histological staining using Hoechst dye and anti-CD31 antibody revealed a time-dependent reduction of tumor perfusion after 2, 4, and 24h of BPR0L075 treatment. Taken together, our results show that the novel tubulin-binding agent BPR0L075 alters endothelial cell tubule formation, prevents cell migration, and inhibits the formation of neovessels. These properties of BPR0L075 may ultimately contribute to the loss of tumor blood vessel integrity causing rapid tumor vascular shutdown in experimental breast tumors. 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 5309. doi:10.1158/1538-7445.AM2011-5309

Antitumor effect of the vascular-disrupting agent ZD6126 in a murine renal cell carcinoma model

ZD6126 is a vascular-disrupting agent that affects the endothelial tubulin cytoskeleton causing selective occlusion of tumor vasculature and extensive tumor cell necrosis. The present study evaluated the antitumor and antivascular activities of ZD6126 in the clinically relevant murine renal cell carcinoma (RENCA) model and also evaluated biological response to therapy using color Doppler imaging as biomarker. Mice were implanted with RENCA tumor cells (day 0) and established tumors were treated with ZD6126 (100 mg/kg i.p.) or vehicle with repeated intermittent doses on day 10, 14 and 18. ZD6126 treatment led to a significant reduction in tumor size and was associated with extensive tumor necrosis and a reduction in tumor blood flow versus controls. MVD increased with intermittent treatment (day 10, 14 and 18). In an additional study, animals were treated at day 19 and quantitative three-dimensional microvascular corrosion casting was performed to enable detailed assessment of the tumor vascular architecture. Corrosion casting showed that tumor vessel architecture is affected by treatment, whereas pre-existing vessels in control tissues are practically not affected. Inter-vessel and inter-branch distances as well as vessel diameters are influenced by treatment. In conclusion, ZD6126 showed potent antitumor efficacy in the RENCA model and our data suggest that decrease in tumor blood flow may be a useful surrogate marker of treatment effect.

Efficacy and Safety/Toxicity Study of Recombinant Vaccinia Virus JX-594 in Two Immunocompetent Animal Models of Glioma

The purpose of this study was to investigate the oncolytic potential of the recombinant, granulocyte macrophage colony-stimulating factor (GM-CSF)-expressing vaccinia virus (VV) JX-594 in experimental malignant glioma (MGs) in vitro and in immunocompetent rodent models. We have found that JX-594 killed all MG cell lines tested in vitro. Intratumoral (i.t.) administration of JX-594 significantly inhibited tumor growth and prolonged survival in rats-bearing RG2 intracranial (i.c.) tumors and mice-bearing GL261 brain tumors. Combination therapy with JX-594 and rapamycin significantly increased viral replication and further prolonged survival in both immunocompetent i.c. MG models with several animals considered “cured” (three out of seven rats >120 days, terminated experiment). JX-594 infected and killed brain tumor-initiating cells (BTICs) from patient samples grown ex vivo, and did so more efficiently than other oncolytic viruses MYXV, Reovirus type-3, and VSVΔM51. Additional safety/toxicity studies in nontumor-bearing rodents treated with a supratherapeutic dose of JX-594 demonstrated GM-CSF-dependent inflammation and necrosis. These results suggest that i.c. administered JX-594 triggers a predictable GM-CSF-mediated inflammation in murine models. Before proceeding to clinical trials, JX-594 should be evaluated in the brains of nonhuman primates and optimized for the viral doses, delivery routes as well as the combination agents (e.g., mTOR inhibitors).

462 Micro-environmental influences on cancer stem cells

To investigate the early effects of a vascular disrupting agent (VDA) in ectopic and orthotopic tumors by using macromolecular contrast media (MMCM)-enhanced magnetic resonance imaging (MMCM-MRI).The MMCM-MRI of ectopic and orthotopic MCA205 murine fibrosarcomas was performed using the intravascular contrast agent albumin-(gadopentetate dimeglumine)35. Change in longitudinal relaxation rate (ΔR1) was measured 24 hours after treatment with 5,6-dimethylxanthenone-4-acetic acid (DMXAA; 30 mg/kg) and used to compute tumor vascular volume and permeability. Correlative histologic and immunohistochemical evaluation was carried out, along with measurement of tumor necrosis factor α and vascular endothelial growth factor levels in whole tumor extracts using the enzyme-linked immunosorbent assay.Orthotopic tumors showed higher vascular volume (p < 0.05) than ectopic tumors before treatment. Twenty-four hours after DMXAA treatment, a significant (p < 0.0001), but differential, decrease in ΔR1 (70% in ectopic and 50% in orthotopic tumors) was observed compared with baseline estimates. Consistent with this observation, greater levels of tumor necrosis factor α, an important mediator of the antivascular activity of DMXAA, were measured in ectopic tumors 3 hours posttreatment compared with orthotopic tumors (p < 0.05). Immunohistochemical (CD31) and histologic (hematoxylin and eosin) sections of ectopic and orthotopic tumors showed highly tumor-selective vascular damage after treatment with the presence of viable surrounding normal tissue.The MMCM-MRI provided early quantitative estimates of change in tumor perfusion after VDA treatment that showed good correlation with cytokine induction. Differences in the response of ectopic and orthotopic tumors highlight the influence of the host microenvironment in modulating the activity of VDAs.

The roles of phytochemicals in bronchial asthma.

Despite gaps in our knowledge of how phytochemicals interfere with cellular functions, several natural plant products are utilized to prevent or treat a wide range of diseases. Identification of an agent with therapeutic potential requires multiple steps involving in vitro studies, efficacy and toxicity studies in animal models, and then human clinical trials. This review provides a brief introduction on natural products that may help to treat and/or prevent bronchial asthma and describes our current understanding of their molecular mechanisms based on various in vitro, in vivo, and clinical studies. We focus on the anti-inflammatory and anti-vascular actions of the plant products and other roles beyond the anti-oxidative effects.

TU‐D‐201C‐03: Monitoring Cancer Treatment with Contrast‐enhanced Ultrasound Imaging

There is growing interest in evaluating cancer therapies that target tumor vasculature. However, assessing these therapies has been difficult because the conventional approach of measuring tumor size does not indicate microvascular activity. Furthermore, cancer microvessels re‐grow between treatments, requiring continual evaluation during the course of therapy.The goal of our studies is to develop techniques that allow longitudinal imaging of tumor microvascular flow. Towards this goal, techniques that employ Doppler or grayscale ultrasound with microbubble contrast agent to visualize blood flow through microvessels will be presented. The metrics used for evaluating vascular changes induced by vascular targeting therapies will be reviewed and their efficacy demonstrated in mice tumors. The antivascular action (decrease in tumor microvascular flow) of three different treatments (combretastatin, ionizing radiation and sonication) will be discussed.Before antivascular therapy, contrast agent perfusing vascular tumors is seen in the images as a uniform flush of color filling the entire lesion. After treatment, the images show that contrast medium does not enter several regions of the tumor, indicating that these parts of the tumor are now avascular. The decrease in area enhanced by the contrast agent and the rate of inflow of contrast agent in the image plane are useful endpoints for assessing cancer treatments affecting tumor microvascular flow.Learning Objectives:1. Understand the techniques of contrast‐enhanced ultrasound imaging2. Understand the issues related to microbubble disruption and ultrasound exposure3. Understand the issues associated with the use of contrast‐enhanced imaging for monitoring vascular targeted therapies

Synthesis and Antitumor Activity of 1,5-Disubstituted 1,2,4-Triazoles as Cis-Restricted Combretastatin Analogues

A series of 1-aryl-5-(3',4',5'-trimethoxyphenyl) derivatives and their related 1-(3',4',5'-trimethoxyphenyl)-5-aryl-1,2,4-triazoles, designed as cis-restricted combretastatin analogues, were synthesized and evaluated for antiproliferative activity, inhibitory effects on tubulin polymerization, cell cycle effects, and apoptosis induction. Their activity was greater than, or comparable with, that of the reference compound CA-4. Flow cytometry studies showed that HeLa and Jurkat cells treated with the most active compounds 4l and 4o were arrested in the G2/M phase of the cell cycle in a concentration dependent manner. This effect was accompanied by apoptosis of the cells, mitochondrial depolarization, generation of reactive oxygen species, activation of caspase-3, and PARP cleavage. Compound 4l was also shown to have potential antivascular activity, since it induced endothelial cell shape change in vitro and disrupted the sprouting of endothelial cells in the chick aortic ring assay.

Evaluating antivascular effects and antitumoral activity in patients with hepatocellular carcinoma treated with JX-594, a targeted multimechanistic oncolytic poxvirus, prior to sorafenib therapy.

e14564 Background: JX-594 is a first-in-class targeted oncolytic poxvirus designed to selectively replicate in and destroy cancer cells with epidermal growth factor receptor (EGFR)/ ras pathway activation. Direct oncolysis plus granulocyte macrophage, colony stimulating factor (GM-CSF) expression stimulates acute tumor vascular shutdown and antitumoral immunity. JX-594 has been shown to be well tolerated by intravenous (IV) infusion and intratumoral (IT) injection and JX-594 is being developed as a novel therapy for patients with refractory hepatocellular carcinoma (HCC). Sorafenib is an antiangiogenic agent approved for patients with advanced HCC. Complementary antitumor effects are predicted with these two agents due to acute vascular disrupting effects of JX-594 and chronic antiangiogenic effects of sorafenib. Methods: Patients with confirmed treatment-refractory HCC were treated with JX-594 followed by standard sorafenib therapy. A subset of patients had previously received and failed sorafenib therap...

Selective inhibition of proliferating endothelial cells: A phase I study of the novel organoarsenical compound GSAO in patients with advanced solid tumors.

TPS167 Background: 4-(N-(S- glutathionylacetyl)amino)phenylarsonous acid (GSAO) is a new water-soluble organoarsenical that selectively inhibits proliferating endothelial cells (EC) in vitro and angiogenesis and tumor growth in human tumor xenografts in vivo. The prodrug GSAO is cleaved by γ-glutamyl transpeptidase (γGT), which is commonly overexpressed on the surface of cancer cells before entering surrounding ECs where it interacts with its mitochondrial target, adenine nucleotide translocase (ANT). ECs are specifically sensitive to GSAO due to their low expression of the multidrug resistance-associated proteins 1 and 2 (MRP 1/2), whereas cancer cells are less sensitive because of higher MRP 1/2 expression. This phase I dose escalation study aims to establish the maximum tolerated dose (MTD) of GSAO, to assess the safety and toxicity profile and to identify the dose limiting toxicity (DLT). Methods: Patients with solid cancers refractory to standard therapy are eligible for the study. Three patients are recruited per dose level with a planned expansion to 6 patients if a DLT occurs. The MTD is the highest dose at which 1 of 6 patients in a cohort experience DLT. Intravenous infusions of GSAO are administered on days 1-5 and 8-12 in a 21-day cycle. Radiological response is assessed after 2 cycles. The pharmacokinetics of GSAO are evaluated by measuring plasma levels using liquid chromatography/mass spectrometry. Dynamic Contrast Enhanced-Magnetic Resonance Imaging (DCE-MRI) is used to determine blood flow parameters (Ktrans, ve, vp, IAUC60). Plasma markers of angiogenesis (VEGF, VEGFR1, VEGFR2 and CD105), cell death (Cytokeratin 18 [CK18] fragments) and apoptosis (CK18asp396) are measured in samples before and after GSAO administration. Tumor tissue γGT, measurement of circulating ECs, and protein phosphorylation (Hic-5) in blood samples serve as exploratory biomarkers. To date 18 patients have been recruited into 5 dose levels (1.3 mg/m2/day–9.3 mg/m2/day). GSAO has been well tolerated with no observed DLTs. Preliminary evidence of antivascular activity was detected by DCE-MRI and soluble biomarkers. Recruitment is ongoing. No significant financial relationships to disclose.

Structure–activity relationships of indole compounds derived from combretastatin A4: Synthesis and biological screening of 5-phenylpyrrolo[3,4- a]carbazole-1,3-diones as potential antivascular agents

A series of 5-(3′,4′,5′-trimethoxyphenyl)pyrrolo[3,4- a]carbazole-1,3(2 H,10 H)-diones was designed as cis-restricted analogues of 3-aroylindoles, arylthioindoles and 3-benzylidoneindolin-2-ones derived from combretastatin A4 (CA-4). Starting from various indoles, compounds were synthesized by means of a convenient two-step procedure involving a one-pot multicomponent reaction as key step. Intermediate tetrahydro[3,4- a]carbazoles and their corresponding carbazoles were submitted to biological screening tests involved in antivascular action, including the cytotoxicity against murine B16 melanoma cells, the rounding up of endothelial cells (EA.hy 926) and the inhibition of tubulin polymerization. Of the 31 compounds screened, those bearing a methoxy group at the 8-position endowed significant biological activities. A carbazole compound 30 was identified as a promising candidate for further development of novel vascular targeting agents.

Abstract 852: Prognostic value of circulating endothelial and progenitor cells in advanced cancer patients enrolled in phase 1 in association with clinical prognostic scores

Abstract Background New surrogate biomarkers such as circulating endothelial cell (CEC) and progenitor cell (CEP) have been proposed to monitor anti-vascular drug activity. During anti-angiogenic therapy, dramatic changes in both CEC and CEP levels were observed. Moreover, these biomarkers may also be used as a candidate prognostic and predictive biomarker in patients with advanced cancer. Methods Medical charts of patients treated in one centre and enrolled into five Phase I trials with anti-vascular therapies between October 2005 and April 2009 were reviewed for clinical characteristics and validated clinical prognostic factors (albumin, lactate dehydrogenase, number of metastatic sites)(Arkenau et al, JCO 2009). Overall 70 patients had CEC or CEP evaluation at baseline. CECs were measured in one ml erythocyte lysed whole blood by four color flow cytometry (FCM) and identified by a CD45-CD31+CD146+ 7-amino-actinomycin (7AAD)- phenotype… CEPs were measured in 10ml blood after progenitor cell enrichment and were identified by FCM as extremely rare events with a CD45dimCD34+VEGFR2+7AAD- phenotype. We studied the impact of CEC and CEP values on the overall survival (OS) adjusted on the clinical prognostic score. Results The median age was 50 years (range, 21 to 78 years). Median baseline CEC value was 12/ml (range, 0 to 154). The CEPs were measured in 57 patients; the median CEP value was 1.3% of hematopoietic stem cells, with range from 0% to 32.5% (absolute counts: 12/ml, range, 0 to 392/ml). 31/57 had CEP values greater than 1% of HSC. Median follow-up was 23 months. The risk of death was significantly increased in patients with prognostic score greater than 0 as compared to patients with score equal to 0 (Median OS 9.2 versus 24.2 respectively) as well as in patients with increased CEP (&amp;gt;1%) as compared to those with lower values (Median OS 9.0 versus 13.3). Both factors remained significant in multivariate analysis (HR=3.9, 95%CI, 0.95-16.6, p=0.02 and HR=2.1, 95%CI, 1.0-4.3, p=0.04, respectively). We did not observe any relationship between the CEC level and the outcome. Conclusion This analysis suggests that a prognostic score based on CEP level combined with clinical characteristics could be a helpful tool to determine the prognosis of patients with advanced cancers enrolled into phase 1 trials. Further studies are requested to confirm these findings. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 852.

Abstract LB-368: III-tubulin knockdown in endothelial cells increases the antivascular activity of chemotherapeutic drugs

Abstract The microtubule cytoskeleton plays a crucial role in tumor angiogenesis, as evidenced by the potent anti-vascular properties of microtubule-targeting anticancer agents (MTAs). Accumulating evidence shows that the expression level of β-tubulin isotypes influences drug response and βIII-tubulin in particular has been involved in cancer cell resistance to MTAs as well as other classes of chemotherapeutic agents (1). However, the role of β-tubulin isotypes in the context of tumor angiogenesis and anti-vascular therapy remains unknown. Comparative western blot analysis revealed that human vascular endothelial cells express relatively high levels of βII- and βIII-tubulin. Using siRNA gene silencing, we effectively suppressed the expression levels of βII-and βIII-tubulin isotypes in vascular endothelial cells, by 90% and 80%, respectively. Functional analysis showed that βII-tubulin expression did not significantly influence either the capacity of endothelial cells to form vascular structures on Matrigel or the anti-vascular properties of MTAs. In contrast, knocking down the expression of βIII-tubulin in endothelial cells significantly inhibited the formation of capillary-like structures on Matrigel by 18 ± 3 % (p&amp;lt;0.001). Furthermore, the anti-angiogenic activity of the microtubule-stabilizing agent, paclitaxel, and the microtubule-depolymerizing agents, vinblastine and 2-methoxyestradiol (2-ME2), was significantly increased between 1.6 and 2.5-fold (p&amp;lt;0.05) in βIII-tubulin knockdown endothelial cells, as compared to control siRNA and βII-tubulin knockdown cells. Similarly, the vascular-disrupting activity of ENMD-1198, a new analog of 2-ME2, was also increased by 2.2-fold (p&amp;lt;0.05) in βIII-tubulin knockdown endothelial cells. More broadly, βIII-tubulin knockdown in endothelial cells also increased the anti-angiogenic activity of other classes of chemotherapeutic drugs, such as topoisomerase inhibitor etoposide (18 ± 1%, p&amp;lt;0.01) and alkylating agent cyclophosphamide (35 ± 5%, p&amp;lt;0.05). Altogether these results strongly suggest that βIII-tubulin may play a role in tumor angiogenesis and influence the anti-vascular activity of chemotherapeutic drugs. Further investigations are currently underway to unravel the underlying mechanisms.

Disrupting established tumor blood vessels

The unique characteristics of tumor vasculature represent an attractive target that may be exploited by vascular-targeting anticancer agents. A promising strategy involves the selective disruption of established tumor blood vessels by tumor-vascular disrupting agents (tumor-VDAs), which exhibit antivascular activity, resulting in inhibition of tumor blood flow and extensive necrosis within the tumor core. The tumor-VDA class can be subdivided into flavonoid compounds, which are related to flavone acetic acid, and tubulin-binding compounds. ASA404, of the flavonoid class, is the most advanced tumor-VDA in clinical development and has been evaluated preclinically and in several phase 1 and phase 2 studies. Preclinical studies have demonstrated the selective apoptosis of tumor endothelial cells and the inhibition of tumor blood flow. Synergistic activity was observed with ASA404 and with several chemotherapeutic agents, particularly taxanes. In clinical trials, compared with chemotherapy alone, ASA404 was tolerated well and produced improved activity in patients with nonsmall cell lung cancer when combined with paclitaxel and carboplatin. Phase 3 clinical trials are ongoing. Selectively targeting established tumor vasculature with tumor-VDAs represents a promising and innovative approach to improving the efficacy of standard anticancer therapies.

Antiangiogenic Activities of 2,5-Dimethyl-Celecoxib on the Tumor Vasculature

Our laboratory has previously shown that a novel compound, 2,5-dimethyl-celecoxib (DMC), which is structurally similar to the cyclooxygenase-2 (COX-2) inhibitor celecoxib but lacks the COX-2-inhibitory function, mimics the antitumor effects of celecoxib. Most studies on DMC, however, focused on its effects on tumor cells. Here, we investigated the activities of DMC as an antiangiogenic agent in both in vitro and in vivo systems. Using primary cultures of human glioma specimens, we found that DMC treatment was cytotoxic to tumor-associated brain endothelial cells (TuBEC), which was mediated through the endoplasmic reticulum stress pathway. In contrast, confluent cultures of quiescent human BEC did not undergo cell death. DMC potently suppressed the proliferation and migration of the TuBEC. DMC caused no apparent effects on the secretion of vascular endothelial growth factor and interleukin-8 but inhibited the secretion of endothelin-1 in tumor-associated EC. DMC treatment of glioma xenografts in mice resulted in smaller tumors with a pronounced reduction in microvessel density compared with untreated mice. In vitro and in vivo analyses confirmed that DMC has antivascular activity. Considering that DMC targets both tumor cells and tumor-associated ECs, this agent is a promising anticancer drug.

Disruption of angiogenesis and tumor growth with an orally active drug that stabilizes the inactive state of PDGFRβ/B-RAF

Kinases are known to regulate fundamental processes in cancer including tumor proliferation, metastasis, neovascularization, and chemoresistance. Accordingly, kinase inhibitors have been a major focus of drug development, and several kinase inhibitors are now approved for various cancer indications. Typically, kinase inhibitors are selected via high-throughput screening using catalytic kinase domains at low ATP concentration, and this process often yields ATP mimetics that lack specificity and/or function poorly in cells where ATP levels are high. Molecules targeting the allosteric site in the inactive kinase conformation (type II inhibitors) provide an alternative for developing selective inhibitors that are physiologically active. By applying a rational design approach using a constrained amino-triazole scaffold predicted to stabilize kinases in the inactive state, we generated a series of selective type II inhibitors of PDGFRbeta and B-RAF, important targets for pericyte recruitment and endothelial cell survival, respectively. These molecules were designed in silico and screened for antivascular activity in both cell-based models and a Tg(fli1-EGFP) zebrafish embryogenesis model. Dual inhibition of PDGFRbeta and B-RAF cellular signaling demonstrated synergistic antiangiogenic activity in both zebrafish and murine models of angiogenesis, and a combination of previously characterized PDGFRbeta and RAF inhibitors validated the synergy. Our lead compound was selected as an orally active molecule with favorable pharmacokinetic properties which demonstrated target inhibition in vivo leading to suppression of murine orthotopic tumors in both the kidney and pancreas.

Antivascular and anticancer activity of dihalogenated A-ring analogues of combretastatin A-4

The generally accepted view is that the 3,4,5-trimethoxy-substituted aromatic A-ring of combretastatin A-4 (CA-4) and its analogues should be conserved in order to maintain biological activity through enforcing an active molecular conformation. Contrary to this, we have found that substituting the larger meta-methoxy groups of CA-4 with smaller halogen atoms results in compounds that are equipotent or more potent than CA-4 itself in vitro.

Preliminary Results of a Phase II Trial with the Multikinase Inhibitor Sorafenib in Heavily Pretreated Patients with Relapsed/Refractory Non-Hodgkin Lymphoma (NHL).

Abstract 1658Poster Board I-684 IntroductionNHL patients with refractory disease or relapsing after autologous or allogeneic stem cell transplant (SCT) have very poor prognosis with currently available salvage chemotherapy. Sorafenib (Nexavar, BAY43-9006, Bayer) is an oral multikinase inhibitor approved by FDA for the treatment of renal cell carcinoma and hepatocellular carcinoma. Sorafenib exerts a broad range of antiproliferative, antiangiogenic, and proapoptotic effects against a variety of nonhematological tumors through the inhibition of the RAF/MEK/ERK pathway, the receptor tyrosine kinases c-kit, Flt3, RET, as well as the proangiogenic vascular endothelial growth factor receptors (VEGFRs), and platelet-derived growth factor receptor-β (PDGFR-β). Several lines of evidence suggest that Sorafenib might have a significant clinical impact in the treatment of malignant lymphomas by overcoming the cytoprotective effects of Bcl-XL, ERK, and Mcl-1 and eventually targeting additional signalling pathways relevant to lymphomagenesis. Our preclinical data demonstrating a marked cytotoxic activity of Sorafenib against NHL cell lines in vitro and in vivo in xenograft models, established the rationale for this currently ongoing phase II study aimed to determine safety and activity of Sorafenib in relapsed/refractory NHL. MethodsBetween March 2008 and May 2009, 21 patients (16 males and 5 females; median age, 65 years; range, 29-74 years) with relapsed/refractory diffuse large B cell lymphoma (DLBCL, n = 11), follicular lymphoma (FL, n =4), mantle cell lymphoma (MCL, n =2), lymphoplasmacitoid lymphoma (LPL, n =1), chronic lymphocytic leukemia (CLL, n =2), and peripheral T-cell lymphoma (PTCL, n =1) who have failed second- or subsequent-line salvage chemo-radiotherapy were enrolled in this phase II trial. Prior to study entry, patients received a median of 4 (range 2 - 7) lines of treatment, including autologous SCT in 15 (71%) and an additional allogeneic SCT in 5 (24%) cases. At study entry, 7 (33%) patients had relapsed and 14 (67%) refractory disease. Eligibility criteria included absence of any available treatment options of proven efficacy, at least one target lesion ≥2 cm, ECOG performance status of 0-1, and adequate bone marrow, liver and renal functions. Sorafenib (400 mg BID, per os) was administered continuously until disease progression or appearance of clinical significant toxicity probably related to study drug. Tumor responses were assessed according to the revised response criteria for malignant lymphoma of the International Working Group. NCI CTCAE v3.0 was used for toxicity assessment. ResultsTo date, 21 patients received a median of 3 months (range, 1 – 11) of Sorafenib therapy. All patients are evaluable for toxicity and response, and 1 patient is still on therapy. Overall, therapy was well tolerated without significant adverse events. The most common drug-related non-hematological toxicities were grade 1-2 mucositis (14%), diarrhea (24%), hand-foot syndrome (24%), anorexia (29%), and fatigue (29%). Grade 3-4 hand-foot syndrome occurred in 19% of patients. Hematological toxicities included grade 1-2 neutropenia (10%) and thrombocytopenia (24%). Grade 3-4 neutropenia and thrombocytopenia were observed in 14%, and 24% of patients, respectively. Best response to Sorafenib included 1 (5%) complete remission (CR) occurring in the patient with LPL, and 1 (5%) partial remission (PR) in a patient with cutaneous DLBCL, for an overall response rate (ORR) of 10%. In both patients, response duration was 6 months. In addition, 9 (42%) patients achieved stable disease (SD) for a median of 3 months (range, 2 – 10), with 3 (14%) patients achieving SD for ≥6 months, while 10 (48%) patients progressed. Upon Sorafenib treatment, an extensive necrosis involving the central area of the tumor associated with a nearly complete disappearance of tumor vascularization was documented by computed tomography and contrast-enhanced ultrasound in two DLBCL and one FL patients bearing latero-cervical or abdominal lymphoid masses. ConclusionsSorafenib as a single agent was well tolerated. Despite limited clinical activity (10% ORR), disease stabilization was experienced by 42% of patients. The potent antiangiogenic activity of Sorafenib in NHL patients bearing highly vascularized lymphoid masses suggests that further research should focus on combinations of Sorafenib with molecularly targeted agents eventually exerting antivascular activities. DisclosuresOff Label Use: The multikinase inhibitor Sorafenib has been used in a phase II trial in patients with relapsed/refractory NHL.

Combretastatin-like chalcones as inhibitors of microtubule polymerization. Part 1: Synthesis and biological evaluation of antivascular activity

The α-methyl chalcone SD400 is a potent inhibitor of tubulin assembly and possesses potent anticancer activity. Various chalcone analogues were synthesized and evaluated for their cell growth inhibitory properties against the K562 human chronic myelogenous leukemia cell line (SD400, IC 50 0.21 nM; combretastatin A4 CA4, IC 50 2.0 nM). Cell cycle analysis by flow cytometry indicated that these agents are antimitotic (SD400, 83% of the cells are in G 2/M phase; CA4 90%). They inhibit tubulin assembly at low concentration (SD400, IC 50 0.46 μM; CA4, 0.10 μM) and compete with [ 3H]colchicine for binding to tubulin (8% [ 3H]colchicine remained bound to tubulin after competition with SD400 or CA4). Upon treatment with SD400, remarkable cell shape changes were elicited in HUVEC cells, consistent with vasculature damaging activity.