Neutrophil Elastase-Activatable Prodrugs Based on an Alkoxyamine Platform to Deliver Alkyl Radicals Cytotoxic to Tumor Cells

Abstract

Current chemotherapies suffer low specificity and sometimes drug resistance. Neutrophil elastase activity in cancer is associated with poor prognosis and metastasis settlement. More generally, tumors harbor various and persistent protease activities unseen in healthy tissues. In an attempt to be more specific, we designed prodrugs that are activatable by neutrophil elastase. Upon activation, these alkoxyamine-based drugs release cytotoxic alkyl radicals that act randomly to prevent drug resistance. As a result, U87 glioblastoma cells displayed high level caspase 3/7 activation during the first hour of exposure in the presence of human neutrophil elastase and the prodrug in vitro. The apoptosis process and cell death occurred between 24 and 48 h after exposure with a half lethal concentration of 150 μM. These prodrugs are versatile and easy to synthetize and can be adapted to many enzymes.

Scheme 1. General Scheme for Elastase-Activated Alkoxyamines

Scheme 2. Synthesis of the Prodrugs 1a to 4a

Scheme 3. Expected Reaction Paths of the Prodrugs in the Presence of HNE

Figure 1

HPLC traces showing purity of the native alkoxyamine (red trace) and the release of the free peptide in the presence of HNE (blue trace).

Figure 2

Monitoring of the release of TEMPO and DBNO by EPR in the presence (red curves) or absence (blue curves) of HNE (20 nM) at 37 °C assessing the enzymatic activation and the subsequent homolysis of the alkoxyamines.

Figure 3

Acceleration of TEMPO release in the presence of a free radical scavenger: (A) EPR spectra of a solution of suc-AAPV-anilide-TEMPO with or without HNE (1.6 μM). The enzymatic reaction was stopped by adding galvinoxyl in THF 1/1 at 1 min (blue line), 2 min (red line) and 10 min (black line) incubation at 37 °C. A control stopped at 10 min incubation without HNE is shown in green. In this control, only galvinoxyl is detected. (B) Plot of the released TEMPO versus time in the presence of galvinoxyl (black line) compared to the kinetics without galvinoxyl (red line with HNE (0.68 μM), blue line without HNE).

Figure 4

Viability of U87 cells in the presence of the prodrugs ± HNE. (A) Dose response study of the viability of U87 cells against suc-AAPV-anilide-TEMPO, suc-AAPV-anilide-DBNO, suc-AAPV-phenol-TEMPO, and suc-AAPV-phenol-DBNO in the presence (red symbols) or absence (blue symbols) of added HNE (0.68 μM). (B) Dose response study of the viability of U87 cells against suc-AAPV-anilide-TEMPO at 24 and 72 h in the presence (red symbols) or absence (blue symbols) of HNE (0.68 μM).

Figure 5

Effect of the elastase inhibitor eglin C on U87 cells rescue at 48 h. Conditions are identical to those in Figure 4 (prodrug only: blue symbols; prodrug in the presence of HNE (0.68 μM): red symbol) except for the addition of eglin C (10 μM) (prodrug in the presence of eglin C: green symbols; prodrug in the presence of eglin C and HNE (0.68 μM): black symbols).

Figure 6

Viability of U87 cells after 48 h in the presence of suc-AAPV-anilide-TEMPO or suc-AAPV-phenol-TEMPO that were beforehand incubated 24 h with or without HNE (0.68 μM) to test the effect of free radicals exhaustion(red symbols with HNE; blue symbols without HNE).

Figure 7

Rescue of U87 cells viability at 48 h by increasing addition of troxerutin (0, 50, 100, and 150 mM), a free radical scavenger. Cells were treated with (A) suc-AAPV-anilide-TEMPO or (B) suc-AAPV-phenol-TEMPO with (red symbols) or without HNE (0.68 μM) (blue symbols).

Figure 8

Timeline of cell death mechanisms with suc-AAPV-anilide-TEMPO. Cells were treated with 75 μM (cyan line), 150 μM (green line), and 250 μM (red line) alkoxyamine in the presence of HNE (0.68 μM). Signals without HNE are blue line for 75 μM, yellow line for 150 μM, and orange line for 250 μM of alkoxyamine. Doxorubicin treatment is a positive control of apoptosis (black columns). The negative control, HNE treatment without alkoxyamine, is represented by gray columns. The viability control which is only cells is represented by white columns.