Targeting the tumour microenvironment with an enzyme-responsive drug delivery system for the efficient therapy of breast and pancreatic cancers

Abstract

The development of novel therapeutic strategies allowing the destruction of tumour cells while sparing healthy tissues is one of the main challenges of cancer chemotherapy. Here, we report on the design and antitumour activity of a low-molecular-weight drug delivery system programmed for the selective release of the potent monomethylauristatin E in the tumour microenvironment of solid tumours. After intravenous administration, this compound binds covalently to plasmatic albumin through Michael addition, thereby enabling its passive accumulation in tumours where extracellular β-glucuronidase initiates the selective release of the drug. This targeting device produces outstanding therapeutic efficacy on orthotopic triple-negative mammary and pancreatic tumours in mice (50% and 33% of mice with the respective tumours cured), leading to impressive reduction or even disappearance of tumours without inducing side effects.

Fig. 1. The principle of tumour targeting with the β-glucuronidase-responsive drug delivery system 1. (a) In the blood, prodrug 1 binds to circulating albumin (step 1). The resulting macromolecule 2 accumulates passively in malignant tissues (step 2) where the cleavage of the glucuronide by extracellular β-glucuronidase triggers the release of MMAE (step 3). (b) The maleimide-bearing side chain of prodrug 1 reacts with the thiol at the cysteine 34 position of albumin through Michael addition (step 1). Hydrolysis of the glucuronide trigger by β-glucuronidase induces the release of MMAE via a 1,6-elimination mechanism followed by a spontaneous decarboxylation (step 3).

Scheme 1. Synthesis of the glucuronide prodrug 1. (a) 4-Nitrophenyl chloroformate, CH₂Cl₂, pyridine, 0 °C to rt, 1 h, quantitative; (b) MMAE, HOBt, DMF/pyridine, rt, 16 h, 94%; (c) O-(2-aminoethyl)-O′-(2-azidoethyl)nonaethylene glycol, Cu(MeCN)₄PF₆, CH₂Cl₂, rt, 20 h, 77%; (d) LiOH, H₂O/MeOH, (e) 7, DMSO, rt, 12 h, 33% (2 steps) after preparative-reverse phase HPLC (purity > 95%).

Fig. 2. The glucuronide prodrug 1 binds covalently with HSA and efficiently releases the MMAE in the presence of β-glucuronidase. (a) Disappearance of 1 over time when placed in the presence of HSA at 37 °C. (b) Kinetics of MMAE release from 2 in the presence of β-glucuronidase (133 U mL^–1). (c) Antiproliferative activity of MMAE and 1 with or without β-glucuronidase after 3 days of treatment. Each point shows the mean ± s.e.m. from 3 independent experiments in triplicate. (d) Mean body weights of mice treated with a single i.v. injection of 1 at 1, 2, 4, 8 or 12 mg kg^–1 at day 0. Each point shows the mean ± s.e.m. from 3 mice.

Fig. 3. Antitumour activity of the β-glucuronidase-responsive albumin-binding prodrug 1 in mice with subcutaneous KB xenografts. (A) Structures of the β-galactosidase-responsive folate-MMAE conjugate 8 and the glucuronide prodrug of MMAE 9. (B) Relative quantities of MMAE measured at day 21 in KB tumours of mice treated with two i.v. injections on days 7 and 14 of free MMAE (0.50 mg kg^–1 per injection), 1, 8 and 9 (0.77 mg kg^–1 per injection of MMAE equivalents). Each bar shows the mean ± s.e.m. from 4 independent tumours. **P < 0.01 and ***P < 0.001; one-way analysis of variance with the Bonferroni post-test. (C) Representative volumes determined by 3D echography imaging (scale bar: 5 mm) of KB xenografts post-implantation at days 7, 14, 28 and 35 when treated with vehicle, MMAE, 1, 8 and 9 (i.v. injection at days 7, 14, 21 and 28). (D) Tumour growth over time under therapy with vehicle, MMAE, 1, 8 and 9. Each point shows the mean ± s.e.m. from 8 tumour volumes. **P < 0.01 and ***P < 0.001; two-way analysis of variance with the Bonferroni post-test. (E) Mean body weights of each group of mice. Each point shows the mean ± s.e.m. from 8 mice.

Fig. 4. Antitumour activity of prodrug 1 on MDA-MB-231 and MIA PaCa2 orthotopic models. (a) MDA-MB-231 tumour growth inhibition under therapy with vehicle, MMAE and 1. Each point shows the mean ± s.e.m. from 6 tumour volumes. **P < 0.01 and ***P < 0.001; two-way analysis of variance with the Bonferroni post-test. (b) Tumour volumes at day 50 of mice bearing MDA-MB-231 xenografts treated with vehicle, MMAE and 1. (c) Mean body weights of each group of mice bearing MDA-MB-231 xenografts. Each point shows the mean ± s.e.m. from 6 mice. (d) MIA PaCa2 tumour growth inhibition under therapy with vehicle, MMAE and 1. Each point shows the mean ± s.e.m. from 6 tumour volumes. **P < 0.01 and ***P < 0.001; two-way analysis of variance with the Bonferroni post-test. (e) Mean body weights of each group of mice bearing MIA PaCa2 xenografts. Each point shows the mean ± s.e.m. from 6 mice. (f) Tumour volumes at day 70 of mice bearing MIA PaCa2 xenografts treated with vehicle, MMAE and 1. (g) Tumour volumes of highly hypoxic MIA PaCa2 xenografts in mice treated with vehicle and 1. (h) Representative volumes determined by 3D echography imaging (scale bar: 5 mm) of highly hypoxic MIA PaCa2 xenografts in mice treated with 1. Each point shows the mean ± s.e.m. from 5 tumour volumes. (i) Survival study comparing mice with 2.5–3.5 cm³ orthotopic MIA PaCa-2 tumours treated with prodrug 1 or untreated.