Enhanced tumor retention of NTSR1-targeted agents by employing a hydrophilic cysteine cathepsin inhibitor

Abstract

We explored the approach of using an analog of E-64, a well-known and hydrophilic cysteine cathepsin (CC) inhibitor, as a potent cysteine cathepsin-trapping agent (CCTA) to improve the tumor retention of low-molecular-weight, receptor-targeted radiopharmaceuticals. The synthesized hydrophilic CCTA-incorporated, NTSR1-targeted agents demonstrated a substantial increase in cellular retention upon uptake into the NTRS1-positive HT-29 human colon cancer cell line. Similarly, biodistribution studies using HT-29 xenograft mice revealed a significant and substantial increase in tumor retention for the CCTA-incorporated, NTSR1-targeted agent. The intracellular trapping mechanism of the CCTA-incorporated agents by macromolecular adduct formation was confirmed using multiple in vitro and in vivo techniques. Furthermore, utilization of the more hydrophilic CCTA greatly increased the hydrophilicity of the resulting NTSR1-targeted constructs leading to substantial decreases in most non-target tissues in contrast to our previously reported dipeptidyl acyloxymethyl ketone (AOMK) constructs. This work further confirms that the CCTA trapping approach can make significant improvements in the clinical potential of NTSR1-and other receptor-targeted radiopharmaceuticals.

Keywords: Cysteine cathepsin inhibitor; E−64 analogue; NTSR1; Radiopharmaceutical; Trapping agent; Tumor retention.

Fig. 1.

Structures of the cysteine cathepsin inhibitors and peptidic conjugates studied in our previous and present work

Fig. 2.

The RP-HPLC profiles of radiolabeling NE1a (A) and NE1b (B) by ¹⁷⁷LuCl₃. The stability and purity of ¹⁷⁷Lu-NE1a (C) and ¹⁷⁷Lu-NE1a (D) in human serum at 37 °C at various time points.

Fig. 3.

The hyperbolic regression of pseudo-first-order rate constants (k_obs) (t = 0-60 min) by CatB (A) and CatL(B) versus the concentrations of NE1a ([C]). The substrates for CatB and CatL were Z-Arg-Arg-AMC and Z-Phe-Arg-AMC, respectively. The substrate concentration was from 1 to 500 nM. The K_i and k_i/K_i determined from k_obs = k_i[C]/(Ki + [C]). (C) A dose-response curve showing the inhibition of NTSR1 receptor binding of ¹⁷⁷Lu-N1 by NE1a and NE1b. Values are means ± SD (n = 3).

Fig. 4.

The internalized and surface-bound ¹⁷⁷Lu-NE1a (A) and ¹⁷⁷Lu-NE1b (B) by HT-29 cells. (C) Efflux profiles of the internalized ¹⁷⁷Lu-NE1a and ¹⁷⁷Lu-NE1b over 24 h in HT-29 cells. Values are means ± SD (n = 3).

Fig. 5.

(A) Representative confocal microscopy images of the efflux of ^natEu labeled NE1a and NE1b (red) form HT-29 cells. Cell endolysosomal compartments were stained with Lysotracker (green). Scale bar = 50 μm. (B) Time-dependent fluorescence intensity of ^natEu per cell as quantified from the confocal images. (C) Co-localization efficiencies of ^natEu (red) overlapping with Lysotracker® (green). All the analysis was performed in 6 random images and were presented as mean ± SD. **p < 0.01, ***p < 0.001, NS = not significant.

Fig. 6.

The autoradiography of the SDS-PAGE showing the CatB and CatL binding with the ¹⁷⁷Lu-NE1a and ¹⁷⁷Lu-NE1b in the presence or absence of the cysteine proteases inhibitor E-64 (A) and the NTSR1 ligand N1 (B). (C). Autoradiographic image of an SDS-PAGE gel examining the time-dependent retention of CC-conjugate adducts in HT-29 cells after pre-incubation with ¹⁷⁷Lu-NE1a for 4 h.

Fig. 7.

(A) The autoradiography of SDS-PAGE of the tumor, liver, and kidney samples at 72 h p.i. of ¹⁷⁷Lu-NE1a and ¹⁷⁷Lu-NE1b in mice. The radioconjugates were injected to the mice at a dose of 800 μCi (29.6 MBq) /mouse. (B) Percentage of the macromolecules associated radioactivity (Mw > 10 kDa) in tumor, liver, and kidney samples at 72 h after administration of the radioconjugates (n=3). *p < 0.05, **p < 0.01, ***p < 0.001, NS = not significant.

Scheme 1.

Synthetic Procedures for 1a and 1b^{a a}Reagents and conditions: (a) (i) EDCI, NHS, DMF, 0°C, 2-3h (ii) 2-azidoethanamine, DIEA,DMF, r.t., overnight; (b) (i) 20% piperidine in DMF, r.t., 30min; (ii) COMU, Fmoc-Leu-OH, DIEA, DMF, r.t., 2h; (c) (i) 20% piperidine in DMF, r.t., 30min; (ii) COMU, (+/−)-trans-oxirane-2,3-dicarboxylic acid, DMF, DIEA, r.t., overnight; (c) (i) 20% piperidine in DMF, r.t., 30min; succinic anhydride, TEA, DMF, 50°C, overnight; (e) 90% TFA in DCM, r.t., 2h.

Scheme 2.

Synthetic Procedures for NE1a and NE1b^{a a}Reagents and conditions: (a) CuSO₄, ascorbic acid, TEA, H2O:n-Butanol:DMF (1:1:2), r.t., 1h; (b) (i) DOTA-NHS, DIEA, DMF, r.t., overnight; (ii) 90% TFA in DCM, r.t., 3h.