CONTEXT

The expression level of the cytosolic 5’-nucleotidase II (cN-II) is of crucial interest for patients treated with nucleoside analogue-based chemotherapy. Indeed, a high level of expression of cN-II mRNA in blasts is predictive of worse outcome in patients receiving cytarabine-based regimens (a well-known nucleoside analogue used to treat acute myeloid leukemia, AML). In addition, the inhibition of cN-II expression by short hairpin RNA was associated with the induction of apoptosis in human astrocytoma cells, suggesting that cN-II could be a therapeutic target in brain tumors. Finally, recent reports showed that hyperactive mutated cN-II in relapsed children with acute lymphoblastic leukemia, and treated with the anti-metabolite 6-mercaptopurine, was associated with a worse survival.
In this context, cN-II has been shown to be an attractive molecular target for the development of novel drugs circumventing resistance to cytotoxic nucleoside analogues currently used for treating leukemia and other hematological malignancies.

TECHNICAL DESCRIPTION

In light of the various structural, functional and regulatory properties of cN-II, the inventors considered cN-II as an attractive therapeutic target for developing different types of inhibitors that could interfere with protein function or regulation. Altering the enzymatic function of cN-II has an indirect effect on nucleotide pools and is supposed to enhance the therapeutic efficiency of cytotoxic nucleosides (by decreasing the competition with endogenous nucleotides). Indeed, numerous experimental evidences indicated that cN-II is interfering with anticancer treatment based on this class of therapeutics (nucleoside analogs) and therefore also governing the final outcome of patients. The main evidence is the relationship between the expression level of cN-II and the patient outcome. This feature is reinforced by the recent discovery of hyperactive mutants of cN-II that induce a similar response.
The development of potent inhibitors against cN-II has been carried out and a family of small size compounds (designed for an oral delivery) has been characterized for their capability in blocking the cN-II enzymatic function and also in sensitizing the anti-proliferative effects of some cancer drugs using preclinical models.

DEVELOPMENT STAGE

• Multi-step chemical synthesis was been optimized, all intermediates and final compounds were fully characterized. Synthesis has been carried out on mg to gram scale. In vitro evaluation towards the recombinant purified cN-II (Nucleotidase activity inhibition assays). Two different in vitro assays were used for evaluating the inhibition of compounds. A colorimetric assays based on the dosage of inorganic phosphate produced by the ·         reaction and a kinetic assay for the characterization of the inhibition mode and constant (Ki). Several compounds described in the invention have shown a potent inhibition of the cN-II activity (up to 90% of inhibition at 200 µM)
• Cytotoxic activity validated on several tumor cell lines. Direct cytotoxicity was determined using cell survival assays on a large panel of cancer cell lines, showing IC50 in low micromolar range for the most potent inhibitors. Synergy with nucleoside analogues was shown for several associations.
• Ex vivo PoC. The most potent cN-II inhibitors are also cytotoxic on human cancer cells obtained from patients with AML or CLL as shown by ex vivo incubation and cell survival analysis.
• In vivo efficacy PoC. Initial experiments in a mouse model showed an antitumoral activity of cN-II inhibitors and a slight increase in the activity of associated nucleoside analogues.

BENEFITS

The originality of the invention lies in the nature of the drug target (cytosolic and not membrane). No inhibitor of intracellular 5′-nucleotidases (cytosolic) is described to date for the treatment of human blood diseases (Acute lymphoblastic or myeloblastic leukemia and chronic lymphoid leukemia) for children and adult patients.
The unique combination of the compounds of the invention, 5′-nucleotidases inhibitors, with cytotoxic nucleoside analogs known to date, increases the effectiveness of this drug class by several mechanisms: (1) by the intrinsic inhibition cN-II inducing a shorter cell survival ; (2) by increasing the intracellular concentration of phosphorylated forms (nucleotide) of associated nucleoside analogues, these entities being responsible for its antiproliferative activity; (3) by modulating some mechanisms of cellular resistance associated with the over expression or mutation of cN-II.

INDUSTRIAL APPLICATIONS

These chemical compounds designed as inhibitors of 5′-nucleotidases can be used in the treatment of cancers, especially leukemia, alone or in combination with cytotoxic nucleoside analogs and / or nucleobases widely used in cancer chemotherapy, to potentiate their therapeutic effect. Eventually, it may be extended to other pathologies using nucleoside analogs.

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