Protides of 5-Fluorotroxacitabine Display Potent Anti-Proliferative Properties and Circumvent Deoxycytidine Kinase-Mediated Resistance Associated with Cytotoxic Cytidine Analogues; A Novel Approach for Acute Myeloid Leukemia
Bibliographic record
Abstract
Abstract The cytotoxic nucleoside cytarabine forms the backbone of AML induction and consolidation therapies, but is associated with severe toxicities that preclude its use in patients unable to tolerate aggressive chemotherapy. Options for patients that do not respond to cytarabine, or relapse post-treatment, are limited. Elderly patients and those with relapsed/refractory AML would particularly benefit from the availability of new agents to develop treatment regimens that provide increased efficacy and tolerability compared to cytarabine, and that have a decreased susceptibility to mechanisms of cytarabine resistance, such as decreased deoxycytidine kinase (dCK) and/or upregulation of cytidine deaminase (CDA). Our preclinical evaluation of potential new anti-proliferative chemotherapeutics identified 5-fluorotroxacitabine (5FTRX), a chain-terminating cytidine-based L-nucleoside, as having promising anti-proliferative activity against AML cell lines, and resistance to degradation by CDA. To understand potential mechanisms of resistance to 5FTRX, we selected a population of THP1 (THP1-R) cells resistant to 5FTRX. THP1-R cells were 66-fold resistant to 5FTRX and cross resistant to cytarabine (35-fold) with CC50 values for both nucleosides >50 μM. We discovered that THP1-R cells had decreased levels of dCK (>95% by Western blot), the kinase responsible for the phosphorylation of cytidine and cytidine analogues such as troxacitabine and cytarabine to their corresponding monophosphates. Confirming the importance of dCK in the activation of 5FTRX and cytarabine, chemical inhibition of dCK also rendered THP1 cells >90-fold resistant to 5FTRX and cytarabine. To develop molecules that overcome resistance to both high CDA and low dCK, we used protide technology to construct nucleotide monophosphate prodrugs of 5FTRX, including one potent example, MV806. MV806 was not dependent upon dCK as it maintained similar efficacy in THP1-R cells with low dCK and against THP1 cells treated with the selective dCK inhibitor. We tested MV806 and 5FTRX in a panel of AML cell lines (n=7). MV806 was more potent than 5FTRX with CC50 values ranging from 0.0020-0.19 μM, compared to 0.057-1.2 μM for 5FTRX. MV806 also demonstrated CC50s <0.1 μM against selected T- and B-cell lymphoma cell lines (e.g. MOLT4 and RAJI). Increased in vitro potency of this prodrug compared to 5FTRX correlated with elevated intracellular triphosphate levels in AML cells; MV806 generated 5-fold more triphosphate than 5FTRX in MV4-11 cells. We also tested MV806 in combination with doxorubicin or azacytidine in two AML cell lines (MV4-11 and THP-1). In both tested cell lines, strong synergy was observed (Bliss independence analysis synergy volumes >100), demonstrating future opportunities for clinical combinations. Finally, we showed that MV806 had DMPK profiles suitable for preclinical and clinical development. Leading protides were highly soluble, had a predicted half-life of >6h in human blood and demonstrated IC50 values >1 μM against major CYP isoforms (2A6, 2C9, 2D6, 3A4) with no evidence of time-dependent inhibition at 1 μM. To conclude, we used protide technology to directly deliver the active monophosphate species of 5FTRX intracellularly and thereby overcome resistance to cytarabine due to down-regulation of dCK and increased CDA expression. Taken together, our findings support the further development of protides of 5FTRX for the treatment of AML, including AML patients with reduced sensitivity to cytarabine through high CDA expression and/or low dCK expression. Disclosures Pinho: Medivir AB: Employment, Equity Ownership. Kylefjord:Medivir AB: Employment, Equity Ownership. Rraklli:Medivir AB: Employment. Rydergård:Medivir AB: Employment, Equity Ownership. Rizoska:Medivir AB: Employment, Equity Ownership. Eneroth:Medivir AB: Employment, Equity Ownership. Bylund:Medivir AB: Employment, Equity Ownership. Moses:Medivir AB: Employment, Equity Ownership. Norin:Medivir AB: Employment, Equity Ownership. Bethell:Medivir AB: Employment, Equity Ownership. Schimmer:Otsuka Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy; Medivir AB: Research Funding. Albertella:Medivir AB: Employment, Equity Ownership. Targett-Adams:Medivir AB: Employment, Equity Ownership.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".