PC-2 A liposomal formulation of HIF-1α inhibitor echinomycin inhibits growth and metastasis of experimental model of breast cancer
Authors: Wang Yin , Bailey Chris , Liu Yan , Zheng Pan , Liu Yang 
Published: April 2019

Introduction:
Hypoxia-inducible factor 1α (HIF-1α) is a transcription factor that acts as a master regulator for the expression of genes that promote tumor growth, vascularization, and metastasis, and is highly expressed in cancer tissues. HIF-1α activates transcription by binding to hypoxia response elements (HRE) in the promoter region of its target genes. HIF-1α inhibition has shown success as a therapeutic strategy in mouse models of metastatic cancer. Therefore, HIF-1α represents a prime molecular target for cancer therapy. However, no specific HIF inhibitor has been approved for clinical use.
Echinomycin is a small-molecule inhibitor of HIF-1α, which functions through its sequence-specific binding to HRE to competitively inhibit HIF-1α binding to its target genes. Targeting the DNA binding site of HIF-1α is advantageous as a strategy because it avoids the possibility of acquired drug resistance by target mutation in cancer, as there are numerous HIF targets. Before HIF-1α was identified as molecular target of echinomycin, the drug was tested in multiple phase I and phase II trials for the treatment of solid tumors. However, clinical development of echinomycin was discontinued because it was not found to be effective for patients with solid tumors that were refractory to available treatments.
Several factors could have contributed to failure of the earlier clinical trials. First, the trials were conducted in the absence of preclinical studies to identify the drug target, making it impossible to judge the in-target activity of echinomycin in vivo. Second, no methods have been developed to evaluate the drug availability in vivo. Echinomycin is extremely insoluble in water, which complicates its formulation into a suitable dosage form. In earlier clinical trials, echinomycin was formulated with Cremophor EL (CrEL). However, CrEL has been under scrutiny for its allergenic activity. In clinical trials of the conventionally-formulated echinomycin (CrEL-echinomycin), hypersensitivity reactions were observed. In addition, CrEL-echinomycin caused severe nausea and vomiting, which constituted its major dose-limiting toxicity.
Liposomes are credited for reducing toxicity and potentiating therapeutic effects in comparison to free-drug or CrEL-based drug formulations. Since liposomal formulations have been shown to improve drug delivery to solid tumors via EPR effect, we explored their potential as an enabling technology for use of echinomycin in solid tumors.

Results:
We investigated if HIF-1α protein could be effectively targeted by echinomycin, a potent HIF-1α inhibitor, using preclinical models of breast cancer growth and metastasis. We reformulated echinomycin in liposomes and compared the therapeutic index of liposomal echinomycin with the previously tested clinical formulation of echinomycin. HIF-1α expression in breast cancer cells correlated with echinomycin cytotoxicity in vitro. However, consistent with clinical observations, no significant inhibition of primary tumor growth could be observed in xenograft mouse models using the previous clinical formulation. Reformulating echinomycin in liposomes increased therapeutic index with effective in vivo <targeting of the HIF-1α pathway. Treatment with liposomal echinomycin eliminated protein expression of HIF-1α target, VEGF-A, in breast cancer cells in vivo. Our data demonstrate that, with proper formulation, echinomycin provides a safe and effective approach for in vivo targeting of HIF-1α activity to inhibit growth and metastasis of solid tumors.

Discussion:
Echinomycin is the most potent inhibitor of HIF-1α binding to DNA. Echinomycin may be advantageous over repurposing some FDA-approved compounds as HIF-1α inhibitors, which are limited by their pleiotropic drug effects. On the other hand, echinomycin has an advantage over untested HIF-1α inhibitors because safe doses have already been established in over a dozen clinical trials. Our data presented herein demonstrated that liposomal echinomycin is safer and more effective than CrEL-echinomycin, and should be better tolerated in clinical trials.
HIF-1α is overexpressed in 70% of human cancers. HIF-1α drives tumor progression because it orchestrates fundamental processes such as angiogenesis, glycolytic switch, and metastasis. It is therefore of interest to consider whether the failure of previous trials was due to improper formulation, or poor choice of targets or targeting agents. The termination of echinomycin in clinical trials immediately preceded the first clinical report identifying HIF-1α as a potential target for cancer, and nearly a decade later, echinomycin was first identified as a HIF-1α inhibitor. Therefore, while echinomycin was not effective as a putative cytotoxic therapy, it was never examined as a HIF-1α inhibitor in clinical setting. Here, we used HIF1A-addicted breast cancer cell lines to demonstrate that the previous failure was completely attributable to improper formulation. First, dose-limiting toxicities of CrEL-echinomycin may have prevented therapeutically effective doses from being reached. This is evidenced in that the maximum tolerated dose of CrEL-echinomycin was insufficient to inhibit tumor growth and eliminate metastasis in the breast cancer model. Second, the efficiency in which CrEL delivers the drug to its target is suboptimal. Although CrEL-echinomycin and liposomal echinomycin are equally effective in vitro, only liposomal echinomycin is effective in vivo. Therefore, liposomes delivered echinomycin to relevant cells more efficiently in vivo, and could potentially provide the same advantage in humans. These results are in line with the concept that liposomal formulations reduce the toxicity of drugs, while providing enhanced therapeutic effects. Liposomal echinomycin achieved in vivo targeting as evidenced by the reduction of VEGF-A protein.

Conclusions:
Previous failures in echinomycin trials for multiple cancer types with HIF-1α activation is likely due to inappropriate formulation. Liposomal nanoformulation of echinomycin allows effective targeting of HIF-1α in experimental breast cancer. Given the extensive data showing the critical function of the target in human cancer, it is of great interest to re-evaluate the potential of this new formulation in new clinical trials.