Disruption of the vasculature supply to tumors is recognized as an effective therapeutic strategy (1
). Several colchicine-based vascular disrupting agents (VDAs) have demonstrated preclinical efficacy, but none have yet progressed through clinical trials. One reason for the lack of clinical progression of these VDAs is the prevalence of off-target effects upon the cardiovascular system (2
). A strategy to improve tumor selectivity with attendant diminution of systemic toxicity and hence improved therapeutic index is an attractive approach to the development of VDAs. Hence we have designed and developed preclinically ICT2588 as a novel approach to the selective delivery of a VDA to be activated by MT-MMP endopeptidase in the tumor microenvironment.
Elevated expression of the class-I transmembrane MMPs, particularly MT1-MMP, was demonstrated in a large number of tumor types across a panel of preclinical tumor models. Increased proteolytic activity in these tumors is supported by our previous study which demonstrated a direct relationship between MT1-MMP mRNA expression and enzyme activity (33
). Previous studies have also demonstrated a correlation between MT1-MMP expression and malignancy of different tumor types (14
). The detection of low levels of MT1-MMP protein in MCF7 xenografts, (in contrast to MCF7 cells in vitro
) indicates that MT1-MMP is also present in murine tumor stroma (18
) and tumor neovasculature (25
). Hence we rationalize that MT1-MMPs are available as a potential trigger for tumor-selective drug activation in a wide range of tumor types.
We designed and evaluated ICT2588, a C- and N-terminal modified peptide conjugate of the VDA azademethylcolchicine (ICT2552). ICT2588 possesses a peptide sequence rationalized to be specifically activated by MT1-MMP and was endcapped at its C-terminus to prevent non-specific exopeptidase degradation.
We showed conversion of ICT2588 to its active metabolite (ICT2552act
) and differential in vitro
chemosensitivity in HT1080 (high MT1-MMP) but not in MCF7 (undetectable MT1-MMP) tumor cells. MMP dependency was demonstrated by the lack of ICT2588 activation in the presence of the pan-MMP inhibitor Ilomastat. In addition, MMP selectivity was supported by cleavage of ICT2588 at the glycine-homophenylalanine bond by MT-MMPs but not secreted MMPs. Although activation by MMP-2 was also evident at the P1’- P2’ position (homophenylalanine-tyrosine), this endopeptidase is not a major contributor to ICT2588 activation since hydrolysis was not significantly diminished by an MMP-2 selective inhibitor (CTT). Furthermore, MMP-2 activation is dependent upon MT1-MMP activity (42
This therapeutic strategy relies upon specific MT-MMP activation of ICT2588 followed by subsequent non-specific exopeptidase cleavage to produce azademethylcolchicine (ICT2552), the active VDA. In our analyses of tumor metabolism of ICT2588 in vivo and ex vivo we observed only ICT2552act and no peptide-fragment metabolites of ICT2552. We interpret this to infer that MT-MMP cleavage of ICT2588 is followed by rapid exopeptidase-mediated degradation of the consequent ICT2552act-peptide fragments to produce ICT2552act.
Previous attempts to produce a MMP-activated peptide-conjugate of a chemotherapeutic were unable to produce agents that were sufficiently stable or tumor-selective in vivo
). However, our study showed stability of ICT2588 (peptide-conjugated VDA) in plasma and liver relative to rapid production of the active VDA (ICT2552act
) in tumor ex vivo
, a finding confirmed by widespread distribution and stability of the non-toxic ICT2588 in vivo
coupled to tumor specific activation to ICT2552act
. Accumulation of ICT2588 and ICT2552act
in the tumor may be due to the induction of vascular collapse and trapping of ICT2588 in the tumor microenvironment facilitating the prolonged availability of ICT2588 for metabolism by the MT1-MMP dependent angiogenic tumor neovasculature.
Formation of active VDA in the tumors in vivo
is consistent with the several-fold decrease in functional tumor vasculature and increased hemorrhagic necrosis, an observation consistent with the pharmacodynamics of other colchicine-like VDAs, e.g. ZD6126 and the combretastatins (2
). Although equimolar doses of ICT2588 and ICT2552 caused comparative vascular shutdown, the magnitude of this effect was not consistent with their respective antitumor activities. Specifically, ICT2588 (75 mg kg−1
) produced a significant two-fold greater growth delay (4.4 days (p<0.01)) compared to an equimolar dose of ICT2552 (15 mg kg−1
; 1.9 days). Furthermore, despite both the peptide-conjugated agent (ICT2588) and its authentic metabolite (ICT2552) demonstrating dose-response relationships, a significantly greater response was observed with equimolar equivalents of ICT2588. The superior efficacy of ICT2588 is likely due to the improved tumor-directed delivery and thus increased and prolonged tumor concentration of active VDA (ICT2552) in the peptide-conjugated form compared to the widely distributed and systemically active ICT2552.
Although monotherapy with ICT2588 resulted in a significant antitumor effect, a viable rim of tumor cells remained following treatment. The presence of this viable rim is consistent with previous reports following VDA treatment (1
) and is believed to be the result of these tumor cells receiving their nutrients and oxygen from the established vasculature of the surrounding normal tissues rather than the VDA sensitive tumor vasculature (4
). Consequently, the viable rim can be targeted by co-treatment with antiproliferative agents (2
). In support of this, combination of ICT2588 with Dox resulted in a significant antitumor response and more importantly tumor cures in the majority of mice. Combination of ICT2588 and Dox was superior to monotherapy irrespective of dosing and sequence schedule. The increased antitumor benefit of co-treatment with Dox, 24 h after ICT2588, is likely a result of Dox targeting the proliferative cells in the viable rim which are non-responsive to ICT2588, and the ICT2588-mediated elimination of the poorly perfused tumor areas which are likely to be inaccessible or resistant to Dox (45
). Mobilization of circulating endothelial cells into this region may also contribute, as suggested for ZD6126 and paclitaxel (45
Clinical studies show that VDAs can demonstrate efficacy below their maximum tolerated doses; an observation consistent with ICT2588. This was the case with ICT2588 and Dox in our study suggesting additive toxicity would be unlikely. The cardiovascular toxicity associated with several VDAs in clinical trial (5
) is unlikely with ICT2588 since the circulating and cardiac levels of the active agent (ICT2552) are undetectable. This suggests a greater therapeutic index for ICT2588 relative to other VDAs due to the tumor-selective release and elevated tumor concentrations of ICT2552act
, and the potential for reduced off-target toxicities. In conclusion, the significant therapeutic efficacy, in vivo
stability, mechanism of action, tumor-selective release, and potential for circumventing systemic toxicity associated with systemically active VDAs, strongly supports ICT2588 as potential therapeutic for the clinic.