Bryan Simard

Identification, characterization and potent antitumor activity of ECO-4601, a novel peripheral benzodiazepine receptor ligand

PurposeECO-4601 is a structurally novel farnesylated dibenzodiazepinone discovered through DECIPHER® technology, Thallion’s proprietary drug discovery platform. The compound was shown to have a broad cytotoxic activity in the low micromolar range when tested in the NCI 60 cell line panel. In the work presented here, ECO-4601 was further evaluated against brain tumor cell lines. Preliminary mechanistic studies as well as in vivo antitumor evaluation were performed.MethodsSince ECO-4601 has a benzodiazepinone moiety, we first investigated if it binds the central and/or peripheral benzodiazepine receptors. ECO-4601 was tested in radioligand binding assays on benzodiazepine receptors obtained from rat hearts. The ability of ECO-4601 to inhibit the growth of CNS cancers was evaluated on a panel of mouse, rat and human glioma cell lines using a standard MTT assay. Antitumor efficacy studies were performed on gliomas (rat and human), human breast and human prostate mouse tumor xenografts. Antitumor activity and pharmacokinetic analysis of ECO-4601 was evaluated following intravenous (IV), subcutaneous (SC), and intraperitoneal (IP) bolus administrations.ResultsECO-4601 was shown to bind the peripheral but not the central benzodiazepine receptor and inhibited the growth of CNS tumor cell lines. Bolus SC and IP administration gave rise to low but sustained drug exposure, and resulted in moderate to significant antitumor activity at doses that were well tolerated. In a rat glioma (C6) xenograft model, ECO-4601 produced up to 70% tumor growth inhibition (TGI) while in a human glioma (U-87MG) xenograft, TGI was 34%. Antitumor activity was highly significant in both human hormone-independent breast (MDA-MB-231) and prostate (PC-3) xenografts, resulting in TGI of 72 and 100%, respectively. On the other hand, IV dosing was followed by rapid elimination of the drug and was ineffective.ConclusionsAntitumor efficacy of ECO-4601 appears to be associated with the exposure parameter AUC and/or sustained drug levels rather than Cmax. These in vivo data constitute a rationale for clinical studies testing prolonged continuous administration of ECO-4601.

Induction of the fibrinolytic system by cartilage extract mediates its antiangiogenic effect in mouse glioma.

Both the antiangiogenic and antitumoral activity of shark cartilage extracts (SCE) have been demonstrated in animal models and clinical trials. Studies reported that SCE induces the expression of tissue plasminogen activator gene (PLAT) in endothelial cells and increases the activity of the protein (t-PA) in vitro. The aim of this study was to demonstrate the crucial role of t-PA induction in the antiangiogenic and antitumor activity of SCE in experimental glioma. This study showed antiangiogenic and antitumoral effects of SCE in three mice glioma models (C6, HGD and GL26). Histological examination suggested perivascular proteolysis and edema as well as important intratumoral necrosis, which artefactually increased the tumor volume at high doses. Thus, the antiangiogenic effect of SCE correlated with the presence of t-PA and angiostatin in degenerating vessels. Functional in vivo experiments were conducted to modulate the plasminogen pathway. No antiangiogenic effect was observed on tumors overexpressing the plasminogen activator inhibitor-1 (PAI-1). Moreover, therapeutical effects were neutralized in mice that were cotreated with ε-aminocaproic acid (EACA, 120 mg/kg p.o.), an inhibitor that blocks the high-affinity lysine binding sites of both plasminogen and plasmin. In contrast, cotreatment with N-acetylcysteine (NAC, 7,5mg/kg i.p.), a sulfhydril donor that reduces plasmin into angiostatin or other antiangiogenic fragments, increased the benefit of SCE on mice survival. In subcutaneous models, NAC prevented the increase in tumor volume caused by high doses of cartilage extract. In conclusion, this study indicates that induction of t-PA by shark cartilage extract plays an essential role in its antiangiogenic activity, but that control of excessive proteolysis by a plasmin reductor could prevent edema and uncover the full benefit of shark cartilage extract in the treatment of intracranial tumors.

Tissue-type plasminogen activator has antiangiogenic properties without effect on tumor growth in a rat C6 glioma model.

Tissue-type plasminogen activator (tPA) plays a major role in the fibrinolytic system. According to several reports, tPA may also have antiangiogenic properties, especially in combination with a free sulfhydryl donor (FSD). In the rat C6 glioma model, in vitro and in vivo tPA synthesis by glioma cells is enhanced by differentiation therapy. To address the antiangiogenic potential of tPA in this model, tPA was overexpressed in glioma tumors by ex vivo transduction of C6 cells with a lentiviral vector encoding tPA. The transduced cells were subcutaneously implanted into nude mice. Gene transfer allowed for efficient synthesis of tPA by the C6 tumors. Although the treatment of tPA+ tumor-bearing animals with the FSD captopril generated angiostatin in situ and reduced endothelial vascularization of the tumors, it had no effect on tumor growth. Alternative mechanisms could account for this lack of effect and consequently have important implications for vascular the treatment of glioblastoma.

Shark cartilage extract induces cytokines expression and release in endothelial cells and induces E-selectin, plasminogen and t-PA genes expression through an antioxidant-sensitive mechanism.

Neovastat® is a standardized extract of marine cartilage, an avascular tissue, which contains many biologically active molecules and has multiple antiangiogenic properties. In addition to VEGFR2 and MMPs inhibition, shark cartilage extract (SCE) has recently been shown to induce tissue plasminogen activator gene (PLAT) expression in bovine endothelial cells in a TNF like manner, by inducing the typical mediators NF-κB and JNK. There is now compelling evidences that the NF-κB and JNK pathways are activated by cytokines induced generation of reactive oxygen species (ROS). We used macroarray genes expression analysis on human umbilical vein endothelial cells, to investigate if that mechanism could mediate the effect of SCE. Transcriptomic results showed that SCE induced expression of several cytokines. Their impact must be important, given that treatment of endothelial cells with the cytokine TNF-α was able to reproduce most of the effects of cartilage extract on genes expression. In addition, most of the genes, known to be inducible by NF-κB or JNK following cytokines stimulation, were less induced by SCE when endothelial cells were pretreated with the antioxidant N-Acetylcysteine (NAC), suggesting a role of ROS in endothelial cell activation by SCE. Finally, the possible effects of PLAT, PLG, SELE, IL8 and PRDX2 (those validated by q-PCR) on angiogenesis, will also be discussed.