MTL-CEBPA Combined with Immunotherapy or RFA Enhances Immunological Anti-Tumor Response in Preclinical Models.

The transcription factor CEBPA is a master regulator of liver homeostasis, myeloid cell differentiation and is downregulated in several oncogenic diseases. MTL-CEBPA is a small activating RNA drug which upregulates gene expression of CEBPA for treatment of hepatocellular carcinoma (HCC). We investigate whether MTL-CEBPA has immune modulatory effects by combining MTL-CEBPA with an anti-PD-1 checkpoint inhibitor (CPI) and/or radiofrequency ablation (RFA) in two preclinical models. First, mice with two flanks of HCC tumors (BNL) were treated with combinations of RFA (right flank), anti-PD-1 or MTL-CEBPA. The reduction of the left flank tumors was most pronounced in the group treated with RFA+anti-PD1+MTL-CEBPA and 7/8 animals responded. This was the only group with a significant increase in CD8+ and CD49b+/CD45+ tumor infiltrating lymphocytes (TIL). Second, a combination of anti-PD-1+MTL-CEBPA was tested in a CT26 colon cancer model and this treatment significantly reduced tumor size, modulated the tumor immune microenvironment and increased TILs. These data suggest a clinical role for combination treatment with CPIs, RFA and MTL-CEBPA through synergistic priming of the immune tumor response, enabling RFA and CPIs to have a pronounced anti-tumor effect including activity in non-treated tumors in the case of RFA.

MTL-CEBPA, a Small Activating RNA Therapeutic Upregulating C/EBP-α, in Patients with Advanced Liver Cancer: A First-in-Human, Multicenter, Open-Label, Phase I Trial.

PURPOSE: Transcription factor C/EBP-α (CCAAT/enhancer-binding protein alpha) acts as a master regulator of hepatic and myeloid functions and multiple oncogenic processes. MTL-CEBPA is a first-in-class small activating RNA oligonucleotide drug that upregulates C/EBP-α. PATIENTS AND METHODS: We conducted a phase I, open-label, dose-escalation trial of MTL-CEBPA in adults with advanced hepatocellular carcinoma (HCC) with cirrhosis, or resulting from nonalcoholic steatohepatitis or with liver metastases. Patients received intravenous MTL-CEBPA once a week for 3 weeks followed by a rest period of 1 week per treatment cycle in the dose-escalation phase (3+3 design). RESULTS: Thirty-eight participants have been treated across six dose levels (28-160 mg/m2) and three dosing schedules. Thirty-four patients were evaluable for safety endpoints at 28 days. MTL-CEBPA treatment-related adverse events were not associated with dose, and no maximum dose was reached across the three schedules evaluated. Grade 3 treatment-related adverse events occurred in nine (24%) patients. In 24 patients with HCC evaluable for efficacy, an objective tumor response was achieved in one patient [4%; partial response (PR) for over 2 years] and stable disease (SD) in 12 (50%). After discontinuation of MTL-CEBPA, seven patients were treated with tyrosine kinase inhibitors (TKIs); three patients had a complete response with one further PR and two with SD. CONCLUSIONS: MTL-CEBPA is the first saRNA in clinical trials and demonstrates an acceptable safety profile and potential synergistic efficacy with TKIs in HCC. These encouraging phase I data validate targeting of C/EBP-α and have prompted MTL-CEBPA + sorafenib combination studies in HCC.

Gene activation of CEBPA using saRNA: preclinical studies of the first in human saRNA drug candidate for liver cancer.

Liver diseases are a growing epidemic worldwide. If unresolved, liver fibrosis develops and can lead to cirrhosis and clinical decompensation. Around 5% of cirrhotic liver diseased patients develop hepatocellular carcinoma (HCC), which in its advanced stages has limited therapeutic options and negative survival outcomes. CEPBA is a master regulator of hepatic function where its expression is known to be suppressed in many forms of liver disease including HCC. Injection of MTL-CEBPA, a small activating RNA oligonucleotide therapy (CEBPA-51) formulated in liposomal nanoparticles (NOV340- SMARTICLES) upregulates hepatic CEBPA expression. Here we show how MTL-CEBPA therapy promotes disease reversal in rodent models of cirrhosis, fibrosis, hepatosteatosis, and significantly reduces tumor burden in cirrhotic HCC. Restoration of liver function markers were observed in a carbon-tetrachloride-induced rat model of fibrosis following 2 weeks of MTL-CEBPA therapy. At 14 weeks, animals showed reduction in ascites and enhanced survival rates. MTL-CEBPA reversed changes associated with hepatosteatosis in non-alcoholic methionine and cholic-deficient diet-induced steaotic liver disease. In diethylnitrosamine induced cirrhotic HCC rats, MTL-CEBPA treatment led to a significant reduction in tumor burden. The data included here and the rapid adoption of MTL-CEBPA into a Phase 1 study may lead to new therapeutic oligonucleotides for undruggable diseases.

Development and Mechanism of Small Activating RNA Targeting CEBPA, a Novel Therapeutic in Clinical Trials for Liver Cancer.

Small activating RNAs (saRNAs) are short double-stranded oligonucleotides that selectively increase gene transcription. Here, we describe the development of an saRNA that upregulates the transcription factor CCATT/enhancer binding protein alpha (CEBPA), investigate its mode of action, and describe its development into a clinical candidate. A bioinformatically directed nucleotide walk around the CEBPA gene identified an saRNA sequence that upregulates CEBPA mRNA 2.5-fold in human hepatocellular carcinoma cells. A nuclear run-on assay confirmed that this upregulation is a transcriptionally driven process. Mechanistic experiments demonstrate that Argonaute-2 (Ago2) is required for saRNA activity, with the guide strand of the saRNA shown to be associated with Ago2 and localized at the CEBPA genomic locus using RNA chromatin immunoprecipitation (ChIP) assays. The data support a sequence-specific on-target saRNA activity that leads to enhanced CEBPA mRNA transcription. Chemical modifications were introduced in the saRNA duplex to prevent activation of the innate immunity. This modified saRNA retains activation of CEBPA mRNA and downstream targets and inhibits growth of liver cancer cell lines in vitro. This novel drug has been encapsulated in a liposomal formulation for liver delivery, is currently in a phase I clinical trial for patients with liver cancer, and represents the first human study of an saRNA therapeutic.

Targeting KRAS-dependent tumors with AZD4785, a high-affinity therapeutic antisense oligonucleotide inhibitor of KRAS.

Activating mutations in KRAS underlie the pathogenesis of up to 20% of human tumors, and KRAS is one of the most frequently mutated genes in cancer. Developing therapeutics to block KRAS activity has proven difficult, and no direct inhibitor of KRAS function has entered clinical trials. We describe the preclinical evaluation of AZD4785, a high-affinity constrained ethyl-containing therapeutic antisense oligonucleotide (ASO) targeting KRAS mRNA. AZD4785 potently and selectively depleted cellular KRAS mRNA and protein, resulting in inhibition of downstream effector pathways and antiproliferative effects selectively in KRAS mutant cells. AZD4785-mediated depletion of KRAS was not associated with feedback activation of the mitogen-activated protein kinase (MAPK) pathway, which is seen with RAS-MAPK pathway inhibitors. Systemic delivery of AZD4785 to mice bearing KRAS mutant non-small cell lung cancer cell line xenografts or patient-derived xenografts resulted in inhibition of KRAS expression in tumors and antitumor activity. The safety of this approach was demonstrated in mice and monkeys with KRAS ASOs that produced robust target knockdown in a broad set of tissues without any adverse effects. Together, these data suggest that AZD4785 is an attractive therapeutic for the treatment of KRAS-driven human cancers and warrants further development.

AZD9150, a next-generation antisense oligonucleotide inhibitor of STAT3 with early evidence of clinical activity in lymphoma and lung cancer.

Next-generation sequencing technologies have greatly expanded our understanding of cancer genetics. Antisense technology is an attractive platform with the potential to translate these advances into improved cancer therapeutics, because antisense oligonucleotide (ASO) inhibitors can be designed on the basis of gene sequence information alone. Recent human clinical data have demonstrated the potent activity of systemically administered ASOs targeted to genes expressed in the liver. We describe the preclinical activity and initial clinical evaluation of a class of ASOs containing constrained ethyl modifications for targeting the gene encoding the transcription factor STAT3, a notoriously difficult protein to inhibit therapeutically. Systemic delivery of the unformulated ASO, AZD9150, decreased STAT3 expression in a broad range of preclinical cancer models and showed antitumor activity in lymphoma and lung cancer models. AZD9150 preclinical activity translated into single-agent antitumor activity in patients with highly treatment-refractory lymphoma and non-small cell lung cancer in a phase 1 dose-escalation study.

Novel neutralizing hedgehog antibody MEDI-5304 exhibits antitumor activity by inhibiting paracrine hedgehog signaling.

The hedgehog pathway has been implicated in the tumorigenesis, tumor progression, and metastasis of numerous human cancers. We generated the first fully human hedgehog antibody MEDI-5304 and characterized its antitumor activity and preclinical toxicology. MEDI-5304 bound sonic hedgehog (SHH) and Indian hedgehog (IHH) with low picomolar affinity and neutralized SHH and IHH activity in cellular mGLI1 reporter assays. The antibody inhibited transcription of hedgehog target genes and osteoblast differentiation of C3H10T1/2 cells. We evaluated the activity of MEDI-5304 in vivo in model systems that allowed us to evaluate two primary hypotheses of hedgehog function in human cancer, paracrine signaling between tumor and stromal cells and cancer stem cell (CSC) self-renewal. MEDI-5304 displayed robust pharmacodynamic effects in stromal cells that translated to antitumor efficacy as a single agent in an HT-29/MEF coimplantation model of paracrine hedgehog signaling. MEDI-5304 also improved responses to carboplatin in the HT-29/MEF model. The antibody, however, had no effect as a single agent or in combination with gemcitabine on the CSC frequency or growth of several primary pancreatic cancer explant models. These findings support the conclusion that hedgehog contributes to tumor biology via paracrine tumor-stromal signaling but not via CSC maintenance or propagation. Finally, the only safety study finding associated with MEDI-5304 was ondontodysplasia in rats. Thus, MEDI-5304 represents a potent dual hedgehog inhibitor suitable for continued development to evaluate efficacy and safety in human patients with tumors harboring elevated levels of SHH or IHH.

Challenges and key considerations of the enhanced permeability and retention effect for nanomedicine drug delivery in oncology.

Enhanced permeability of the tumor vasculature allows macromolecules to enter the tumor interstitial space, whereas the suppressed lymphatic filtration allows them to stay there. This phenomenon, enhanced permeability and retention (EPR), has been the basis of nanotechnology platforms to deliver drugs to tumors. However, progress in developing effective drugs using this approach has been hampered by heterogeneity of EPR effect in different tumors and limited experimental data from patients on effectiveness of this mechanism as related to enhanced drug accumulation. This report summarizes the workshop discussions on key issues of the EPR effect and major gaps that need to be addressed to effectively advance nanoparticle-based drug delivery.

MEDI0639: a novel therapeutic antibody targeting Dll4 modulates endothelial cell function and angiogenesis in vivo.

The Notch signaling pathway has been implicated in cell fate determination and differentiation in many tissues. Accumulating evidence points toward a pivotal role in blood vessel formation, and the importance of the Delta-like ligand (Dll) 4-Notch1 ligand-receptor interaction has been shown in both physiological and tumor angiogenesis. Disruption of this interaction leads to a reduction in tumor growth as a result of an increase in nonfunctional vasculature leading to poor perfusion of the tumor. MEDI0639 is an investigational human therapeutic antibody that targets Dll4 to inhibit the interaction between Dll4 and Notch1. The antibody cross-reacts to cynomolgus monkey but not mouse species orthologues. In vitro MEDI0639 inhibits the binding of Notch1 to Dll4, interacting via a novel epitope that has not been previously described. Binding to this epitope translates into MEDI0639 reversing Notch1-mediated suppression of human umbilical vein endothelial cell growth in vitro. MEDI0639 administration resulted in stimulation of tubule formation in a three-dimensional (3D) endothelial cell outgrowth assay, a phenotype driven by disruption of the Dll4-Notch signaling axis. In contrast, in a two-dimensional endothelial cell-fibroblast coculture model, MEDI0639 is a potent inhibitor of tubule formation. In vivo, MEDI0639 shows activity in a human endothelial cell angiogenesis assay promoting human vessel formation and reducing the number of vessels with smooth muscle actin-positive mural cells coverage. Collectively, the data show that MEDI0639 is a potent modulator of Dll4-Notch signaling pathway.

An antibody targeted to VEGFR-2 Ig domains 4-7 inhibits VEGFR-2 activation and VEGFR-2-dependent angiogenesis without affecting ligand binding.

Inhibition of VEGFR-2 signaling reduces angiogenesis and retards tumor growth. Current biotherapeutics that inhibit VEGFR-2 signaling by either sequestering VEGF ligand or inhibiting VEGF binding to VEGFR-2 may be compromised by high VEGF concentrations. Here we describe a biotherapeutic that targets VEGFR-2 signaling by binding to Ig domains 4-7 of VEGFR-2 and therefore has the potential to work independently of ligand concentration. 33C3, a fully human VEGFR-2 antibody, was generated using XenoMouse technology. To elucidate the mechanism of action of 33C3, we have used a number of competition and binding assays. We show that 33C3 binds VEGFR-2 Ig domains 4-7, has no impact on VEGF-A binding to VEGFR-2, and does not compete with an antibody that interacts at the ligand binding site. 33C3 has a high affinity for VEGFR-2 (K(D) < 1 nmol/L) and inhibits VEGF-A induced phosphorylation of VEGFR-2 with an IC(50) of 99 ± 3 ng/mL. In vitro, in a 2D angiogenesis assay, 33C3 potently inhibits both tube length and number of branch points, and endothelial tubule formation in a 3D assay. In vivo, 33C3 is a very effective inhibitor of angiogenesis in both a human endothelial angiogenesis assay and in a human skin chimera model. These data show targeting VEGFR-2 outside of the ligand binding domain results in potent inhibition of VEGFR-2 signaling and inhibition of angiogenesis in vitro and in vivo.

Dual IGF-I/II-neutralizing antibody MEDI-573 potently inhibits IGF signaling and tumor growth.

Insulin-like growth factors (IGF), IGF-I and IGF-II, are small polypeptides involved in regulating cell proliferation, survival, differentiation, and transformation. IGF activities are mediated through binding and activation of IGF-1R or insulin receptor isoform A (IR-A). The role of the IGF-1R pathway in promoting tumor growth and survival is well documented. Overexpression of IGF-II and IR-A is reported in multiple types of cancer and is proposed as a potential mechanism for cancer cells to develop resistance to IGF-1R-targeting therapy. MEDI-573 is a fully human antibody that neutralizes both IGF-I and IGF-II and inhibits IGF signaling through both the IGF-1R and IR-A pathways. Here, we show that MEDI-573 blocks the binding of IGF-I and IGF-II to IGF-1R or IR-A, leading to the inhibition of IGF-induced signaling pathways and cell proliferation. MEDI-573 significantly inhibited the in vivo growth of IGF-I- or IGF-II-driven tumors. Pharmacodynamic analysis demonstrated inhibition of IGF-1R phosphorylation in tumors in mice dosed with MEDI-573, indicating that the antitumor activity is mediated via inhibition of IGF-1R signaling pathways. Finally, MEDI-573 significantly decreased (18)F-fluorodeoxyglucose ((18)F-FDG) uptake in IGF-driven tumor models, highlighting the potential utility of (18)F-FDG-PET as a noninvasive pharmacodynamic readout for evaluating the use of MEDI-573 in the clinic. Taken together, these results demonstrate that the inhibition of IGF-I and IGF-II ligands by MEDI-573 results in potent antitumor activity and offers an effective approach to selectively target both the IGF-1R and IR-A signaling pathways.

A human monoclonal anti-ANG2 antibody leads to broad antitumor activity in combination with VEGF inhibitors and chemotherapy agents in preclinical models.

Localized angiopoietin-2 (Ang2) expression has been shown to function as a key regulator of blood vessel remodeling and tumor angiogenesis, making it an attractive candidate for antiangiogenic therapy. A fully human monoclonal antibody (3.19.3) was developed, which may have significant pharmaceutical advantages over synthetic peptide-based approaches in terms of reduced immunogenicity and increased half-life to block Ang2 function. The 3.19.3 antibody potently binds Ang2 with an equilibrium dissociation constant of 86 pmol/L, leading to inhibition of Tie2 receptor phosphorylation in cell-based assays. In preclinical models, 3.19.3 treatment blocked blood vessel formation in Matrigel plug assays and in human tumor xenografts. In vivo studies with 3.19.3 consistently showed broad antitumor activity as a single agent across a panel of diverse subcutaneous and orthotopic xenograft models. Combination studies of 3.19.3 with cytotoxic drugs or anti-vascular endothelial growth factor agents showed significant improvements in antitumor activity over single-agent treatments alone with no apparent evidence of increased toxicity. Initial pharmacokinetic profiling studies in mice and nonhuman primates suggested that 3.19.3 has a predicted human half-life of 10 to 14 days. These studies provide preclinical data for 3.19.3 as a potential new antiangiogenic therapy as a single agent or in combination with chemotherapy or vascular endothelial growth factor inhibitors for the treatment of cancer.

Acute tumor response to ZD6126 assessed by intrinsic susceptibility magnetic resonance imaging.

The effective magnetic resonance imaging (MRI) transverse relaxation rate R(2)* was investigated as an early acute marker of the response of rat GH3 prolactinomas to the vascular-targeting agent, ZD6126. Multigradient echo (MGRE) MRI was used to quantify R(2)*, which is sensitive to tissue deoxyhemoglobin levels. Tumor R(2)* was measured prior to, and either immediately for up to 35 minutes, or 24 hours following administration of 50 mg/kg ZD6126. Following MRI, tumor perfusion was assessed by Hoechst 33342 uptake. Tumor R(2)* significantly increased to 116 +/- 4% of baseline 35 minutes after challenge, consistent with an ischemic insult induced by vascular collapse. A strong positive correlation between baseline R(2)* and the subsequent increase in R(2)* measured 35 minutes after treatment was obtained, suggesting that the baseline R(2)* is prognostic for the subsequent tumor response to ZD6126. In contrast, a significant decrease in tumor R(2)* was found 24 hours after administration of ZD6126. Both the 35-minute and 24-hour R(2)* responses to ZD6126 were associated with a decrease in Hoechst 33342 uptake. Interpretation of the R(2)* response is complex, yet changes in tumor R(2)* may provide a convenient and early MRI biomarker for detecting the antitumor activity of vascular-targeting agents.

Sensitization of tumor-associated endothelial cell apoptosis by the novel vascular-targeting agent ZD6126 in combination with cisplatin.

PURPOSE: ZD6126 is a novel vascular-targeting agent that selectively disrupts the tubulin cytoskeleton of endothelial cells. In the immature vessels characteristic of tumor vasculature, this leads to endothelial cell contraction, blood vessel congestion, and, consequently, tumor cell death. ZD6126 has been shown to delay tumor growth in a range of xenograft models. The antitumor effect of ZD6126 can be increased in combination with cisplatin or radiation therapy, although the precise mechanism of this enhancement has not been demonstrated. ZD6126 treatment has also been shown to inhibit lung metastasis, and the present study has explored the potential to increase the antimetastatic effect of ZD6126 by combining with cisplatin, and the underlining mechanism has been investigated. EXPERIMENTAL DESIGN: Human lung adenocarcinoma PC14PE6 cells were injected into the tail vein of nude mice. Five weeks after injection animals were treated with ZD6126 (200 mg/kg i.p.), cisplatin (6 mg/kg i.v.), or a combination of the two agents. The animals were sacrificed 24 hours later, and the extent of lung metastases and the presence of apoptotic cells were assessed. RESULTS: Histologic analysis revealed that the ZD6126/cisplatin combination resulted in a 2 to 4-fold increase in the total number of tumor-associated apoptotic cells compared with either treatment alone. ZD6126 alone induced apoptosis of tumor-associated endothelial cells in tumors, and the extent of apoptosis was increased 2-fold in combination with cisplatin. The lung weight was significantly reduced, and the number of metastatic nodules significantly was lower in the combined treatment group than in the control group. CONCLUSIONS: These data suggest that the antimetastatic effect of the vascular-targeting agent ZD6126 can be increased by use in combination with cisplatin, which increases the incidence of endothelial cell apoptosis.

Magnetic resonance imaging measurements of the response of murine and human tumors to the vascular-targeting agent ZD6126.

PURPOSE: ZD6126 is a novel vascular targeting agent currently undergoing clinical evaluation. It acts by destabilizing the microtubulin of fragile and proliferating neoendothelial cells in tumors. The drug leads to blood vessel congestion, the selective destruction of the vasculature, and extensive necrosis in experimental tumors. The aim of the study reported here was to assess the ability of dynamic contrast enhanced magnetic resonance imaging (MRI) to measure the antivascular effects of ZD6126 in tumors. EXPERIMENTAL DESIGN: The work was carried out in mice bearing C38 colon adenocarcinoma and in patients with advanced cancers. MRI was performed before and 6 h (human tumors) or 24 h (C38 tumors) after i.v. drug administration. Contrast agent (gadolinium diethylenetriaminepentaacetate) enhancement was characterized by the initial area under the gadolinium diethylenetriaminepentaacetate uptake versus time curve (IAUC). IAUC reflects blood flow, vascular permeability, and the fraction of interstitial space. RESULTS: The median IAUC was reduced in all C38 tumors after ZD6126 administration [by 6-48% at 50 mg/kg (n = 3)], 58-91% at 100 mg/kg (n = 4), and 11-93% at 200 mg/kg (n = 6). In contrast, the administration of vehicle only led to no consistent change in median IAUC (n = 4). The ZD6126-induced changes in median IAUC appeared to be dose dependent (P = 0.045). No ZD6126-induced changes were apparent in murine muscle. Similar effects were seen in preliminary data from human tumors (11 tumors studied, 9 patients). At doses of 80 mg/m(2) and higher, the median IAUC post-ZD6126 treatment was reduced in all of the tumors studied (8 tumors, 6 patients) to 36-72% from the baseline value. There was a significant trend of increasing reductions with increasing exposure (P < 0.01). No drug-induced changes in human muscle or spleen IAUC were apparent. The reproducibility of the median IAUC parameter was investigated in patients. In 19 human tumors (measured in 19 patients) inter- and intratumor coefficients of variation were 64 and 18%. CONCLUSIONS: The contrast enhanced-MRI measured median IAUC is a useful end point for quantifying ZD6126 antivascular effects in human tumors.

Single dose of the antivascular agent, ZD6126 (N-acetylcolchinol-O-phosphate), reduces perfusion for at least 96 hours in the GH3 prolactinoma rat tumor model.

Tumor vasculature is an attractive therapeutic target as it differs structurally from normal vasculature, and the destruction of a single vessel can lead to the death of many tumor cells. The effects of antivascular drugs are frequently short term, with regrowth beginning less than 24 hours posttreatment. This study investigated the duration of the response to the vascular targeting agent, ZD6126, of the GH3 prolactinoma, in which efficacy and dose-response have previously been demonstrated. GH3 prolactinomas were grown in the flanks of eight Wistar Furth rats. All animals were treated with 50 mg/kg ZD6126. The tumors were examined with dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) 24 hours pretreatment and posttreatment, and at a single time between 48 and 96 hours posttreatment. No evidence of recovery of perfusion was observed even at the longest (96-hour) time point. Involvement of a statistician at the project planning stage and the use of DCE-MRI, which permits noninvasive quantitation of parameters related to blood flow in intact animals, allowed this highly significant result to be obtained using only eight rats.

Anti-cancer drug discovery and development summit.

The 5th Annual Anti-Cancer Drug Discovery and Development Summit brought together an international group of academic and industry scientists to discuss recent therapeutic developments in the field of oncology. The focus of the meeting was novel targeted approaches, i.e., those agents directed against targets that are overexpressed or overactive in tumour cells. It was acknowledged that cytotoxic agents will continue to play a key role in the treatment of cancer and new developments in this area were also discussed. With over 400 anticancer drugs in clinical development and a number of recent registrations, there is great optimism that significant therapeutic advances can be made.

The first international conference on vascular targeting: meeting overview.

The First International Conference on Vascular Targeting focused on vascular targeting agents (VTAs) that occlude or destroy the pre-existing blood vessels of solid tumors. The VTAs cause a rapid shutdown in the blood supply to the tumor that kills tumor cells by depriving them of oxygen and nutrients. The VTAs are distinct from antiangiogenic agents, which prevent new blood vessel formation. Two major types of VTAs are being developed for cancer: the ligand-directed VTAs that use antibodies, peptides, and growth factors to deliver toxins, procoagulants, and proapoptotic effectors to tumor endothelium, and the small molecule VTAs that do not specifically localize to tumor endothelium but exploit pathophysiological differences between tumor and normal tissue endothelia to induce acute vascular shutdown in tumors. Both approaches were described at the meeting and highlighted the variety of VTAs in preclinical development, their selectivity for tumor endothelium, their rapid antitumor effects, and the improved activity seen when combined with other anticancer approaches (antiproliferative chemotherapeutic drugs, radiation, radiolabeled antibodies, nitric oxide synthetase inhibitors, and antiangiogenic agents). Early clinical studies were summarized for the small molecule VTAs: the antitubulin drugs, combretastatin A4 phosphate (CA4P) and ZD6126, and the flavonoid, 5,6-dimethylxanthenone-4-acetic acid (DMXAA). The agents lacked the bone marrow and gastrointestinal toxicities associated with antiproliferative chemotherapy. As a marker of biological effect, blood flow reductions in tumors were measured using magnetic resonance imaging or positron emission tomography for all of the agents tested, and single-agent clinical activity was seen. These agents are now being evaluated in combined modality studies to see whether the impressive results obtained in experimental models can be translated into humans.

ZD6126: a novel small molecule vascular targeting agent.

PURPOSE: The aim of these studies was to evaluate factors that contribute to the selectivity of the novel vascular targeting agent ZD6126. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with ZD6126 phenol, and effects on morphology, detachment, and cytotoxicity (sulforhodamine-B dye incorporation) were determined. Hras5-transformed mouse 3T3 fibroblasts were implanted s.c. in athymic nude rats, and effects on the tumor were assessed after either i.v. bolus or 24-h minipump infusion of ZD6126. RESULTS: In vitro, ZD6126 phenol ( approximately 0.1 microm) rapidly (<40 min) destabilized the tubulin cytoskeleton of proliferating endothelial cells, resulting in cell shape change ("rounding up") and cell detachment at noncytotoxic drug concentrations. In vivo, in rats, an i.v. bolus dose of ZD6126 (20 mg/kg) was rapidly broken down to ZD6126 phenol, which has a short plasma elimination half-life ( approximately 1 h). Peak plasma levels of ZD6126 phenol were well above the level required to induce HUVEC morphology changes in vitro, but cytotoxic concentrations were not maintained. A single i.v. bolus dose (50 and 20 mg/kg) of ZD6126 was well tolerated and resulted in extensive central tumor necrosis in the Hras5 model. Administration of ZD6126 using a 24-h s.c. minipump resulted in decreased ( approximately 30-fold) peak plasma levels, but maintained cytotoxic drug levels over 24 h. Infusion of 50 mg/kg ZD6126 over 24 h was not tolerated. Infusion of 20 mg/kg ZD6126 resulted in increased toxicity compared with the i.v. bolus doses of ZD6126 and did not result in any increased tumor necrosis after 24 h. CONCLUSION: ZD6126 phenol induces rapid morphologic changes in HUVECs at noncytotoxic drug levels. These rapid morphologic effects combined with the rapid elimination of ZD6126 phenol contribute to the selective effects of ZD6126 on tumor vasculature at well-tolerated doses.

ZD6126: a novel vascular-targeting agent that causes selective destruction of tumor vasculature.

Physiological differences between tumor and normal vasculature provide a target for drug discovery. In particular, the immature nature of tumor vasculature may render it intrinsically sensitive to disruption by agents affecting the endothelial cell cytoskeleton, including tubulin-binding agents. In this article, we report the synthesis of a water-soluble phosphate prodrug, ZD6126, of the tubulin-binding agent N-acetylcolchinol. In vitro studies demonstrate the comparative tubulin-binding properties of the prodrug and active drug, and show the induction of pronounced, reversible changes in endothelial cell morphology at subcytotoxic doses. Neither ZD6126 nor N-acetylcolchinol showed effects on the growth of human umbilical vein endothelial cells at concentrations below 100 micro M. In contrast, changes in endothelial cell morphology were seen at much lower, noncytotoxic concentrations (0.1 micro M) of ZD6126 and more pronounced effects were seen in proliferating versus confluent endothelial cell cultures. In vivo studies were carried out using a murine tumor model (CaNT) with single administration of a dose well below the maximum tolerated dose. These studies showed a large reduction in vascular volume, induction of extensive necrosis in tumors, and a reduced tumor cell yield in a clonal excision assay, consistent with vascular rather than cytotoxic effects. A viable rim of tumor remained after single-dose administration and minimal growth delay was observed. However, well-tolerated, multiple administration regimens led to pronounced tumor-growth delay. In the human xenograft FaDu, the growth delay given by a single dose of paclitaxel was enhanced by combination with a single dose of ZD6126, and the growth delay given by the combination was greater than the sum of the growth delays from the individual treatments. These findings show that ZD6126 is a promising antivascular agent for the treatment of solid tumors.