Potent and sustained inhibition of HIF-1α and downstream genes by a polyethyleneglycol-SN38 conjugate, EZN-2208, results in anti-angiogenic effects

Abstract

Topoisomerase I inhibitors down-regulate HIF-1α leading to tumor growth inhibition, but only while maintaining sustained levels of drug exposure. EZN-2208, a multi-arm 40 kDa pegylated, releasable SN38-drug conjugate, provides higher, longer lasting exposure of tumors to SN38 in contrast to SN38 that is released from CPT-11. EZN-2208 also consistently has greater antitumor activity than CPT-11 in a variety of solid and hematological tumor models. In this report, the ability of PEG-SN38 to down-regulate HIF-1α and its downstream targets, in a more potent, sustained manner compared with CPT-11 was examined. To do so, U251 glioma xenografts that stably expressed a hypoxia response element-dependent luciferase reporter gene were implanted in mice. After treatment it was found that EZN-2208 induced potent, sustained HIF-1α down-regulation (37% at 48 h and 83% at 120 h) in the tumors, whereas CPT-11 caused only minor, transient HIF-1α down-regulation. In addition, EZN-2208 down-regulated mRNA levels of HIF-1α targeted genes (MMP2, VEGF1, Glut1, Glut3 and TGFβ1). Further, western blot analyses of xenograft tumors demonstrated that EZN-2208 had significantly more effect than CPT-11 in down-regulating HIF-1α, VEGF, Glut1 and MMP2 protein levels. Significant down-regulation of HIF-1α and VEGF proteins translated to EZN-2208’s superior anti-angiogenic activity compared with CPT-11, confirmed by microvessel density reduction in a chorioallantoic membrane assay and in CD-31 immunohistochemistry studies. Additional studies done with matrigel implants devoid of tumor cells show that EZN-2208 significantly inhibits angiogenesis while CPT-11 has little or no effect. It is concluded that the superior antitumor activity of EZN-2208 compared with CPT-11 is attributed, in part, to an anti-angiogenic effect. Ongoing clinical Phase I and Phase II studies will assess safety and efficacy of EZN-2208.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-011-9209-1) contains supplementary material, which is available to authorized users.

In Vivo Topoisomerase I Inhibition Attenuates the Expression of Hypoxia-Inducible Factor 1α Target Genes and Decreases Tumor Angiogenesis

“…The molecular mechanisms that link TOP1 inhibition to the attenuation of HIF1A protein levels remain to be identified, but are not on the basis of transcriptional downregulation of the HIF1A gene. Similar observations dissociating the inhibition of HIF1A protein accumulation from mRNA modulation have been recently published for PEGylated SN-38 and irinotecan in glioblastoma xenografts (51). These results are also consistent with those reported for topotecan, another topoisomerase I inhibitor that did not affect HIF1A mRNA accumulation or protein half-life, but rather acted on the translational level (54).…”

“…These results are consistent with the downregulation of the HIF1A protein induced by the polyethylene glycol (PEG) conjugate of SN-38 in neuroblastoma and glioblastoma xenograft tumors (50,51). Also interestingly, a systematic in vitro screen of 2,000 chemically diverse compounds for specific inhibitors of the transcriptional activity of HIF1A under hypoxic conditions in a glioblastoma cell line tumor model identified four compounds, among which three were topoisomerase I inhibitors (52,53).…”

HIF-1α regulates the interaction of chronic lymphocytic leukemia cells with the tumor microenvironment

“…30 EZN-2208 is a water-soluble, polyethylene glycol conjugate of the camptothecin-11 analog SN38 with improved delivery and pharmacokinetics in xenograft models of solid tumors. 31,32 Treatment of MEC-1 cells and primary CLL cells with EZN-2208 reduced mRNA levels of HIF-1a target genes, even at concentrations that did not exert overt toxicity ( Figure 4A-D, albeit surface expression of CXCR4 did not decrease accordingly [supplemental Figure 1]), thus confirming that this compound inhibits HIF-1a function in CLL cells.…”

“…[30][31][32] Camptothecin-11 is a cytotoxic topoisomerase I inhibitor that also inhibits HIF-1a at both cytotoxic and noncytotoxic concentrations. 30 EZN-2208 is a water-soluble, polyethylene glycol conjugate of the camptothecin-11 analog SN38 with improved delivery and pharmacokinetics in xenograft models of solid tumors.…”

Chapter 8. Polymeric Nanoparticles and Cancer: Lessons Learnt from CRLX101

“…126 The SN38-bearing nanoparticle EZN-2208 (described in Section 8.2.4) has been shown to inhibit HIF-1a and its downstream targets such as VEGF1 in a U251 glioma xenograft model. 127 The nanoparticle is currently evaluated for potential HIF-1 inhibition and anti-tumour effects in combination with bevacizumab in patients with refractory tumours (ClinicalTrials.org identifier NCT01251926).…”