High-dose acetaminophen (AAP) with N-acetylcysteine (NAC) rescue is among the few treatments that has shown activity in phase I trials without achieving dose-limiting toxicity that has not progressed to evaluation in later line studies. While the anti-tumor effects of AAP/NAC appear not to be mediated by glutathione depletion and free radical injury, the mechanism of anti-tumor effects of AAP/NAC has not been definitively characterized. In vitro, the effects of AAP/NAC were evaluated on bone marrow derived macrophages. Effects of AAP on IL-4/STAT6 (M2) or IFN/LPS/STAT1 (M1) signaling and downstream gene and protein expression were studied. NAC reversed the AAP toxicity in the normal liver but did not reverse AAP cytotoxicity against tumor cells in vitro. AAP/NAC selectively inhibited IL-4-induced STAT6 phosphorylation but not IFN/LPS-induced STAT1 phosphorylation. Downstream, AAP/NAC inhibited IL-4 induction of M2-associated genes and proteins but did not inhibit the IFN/LPS induction of M1-associated genes and proteins. In vivo, AAP/NAC inhibited tumor growth in EF43.fgf4 and 4T1 triple-negative breast tumors. Flow cytometry of tumor-associated macrophages revealed that AAP/NAC selectively inhibited M2 polarization. The anti-tumor activity of high-dose AAP/NAC is lost in macrophage-depleted mouse syngeneic tumor models, suggesting a macrophage-dependent mechanism of action. In conclusion, our study is the first to show that high-dose AAP/NAC has profound effects on the tumor immune microenvironment that facilitates immune-mediated inhibition of tumor growth.

The use of high-dose acetaminophen (AAP) with n-acetylcysteine (NAC) rescue was studied as an anti-cancer treatment in phase I trials with promising signals of anti-tumor efficacy. Correlative analysis suggested that AAP has a free-radical-independent mechanism of anti-tumor activity—in contrast to the well-established mechanism of AAP hepatotoxicity. Subsequent “reverse translational” studies in the pre-clinical setting have identified novel mechanisms of action of high-dose AAP, including modulation of JAK-STAT signaling in both the tumor cell and the tumor immune microenvironment. Importantly, these effects are free-radical-independent and not reversed by concurrent administration of the established AAP rescue agents fomepizole and NAC. By administering high-dose AAP concurrently with fomepizole and NAC, 100-fold higher AAP levels than those of standard dosing can be achieved in mice without detected toxicity and with substantial anti-tumor efficacy against commonly used mouse models of lung and breast cancer that are resistant to standard first-line anti-cancer therapies. With these recent advances, additional clinical trials of high-dose AAP with concurrent NAC and fomepizole-based rescue are warranted.

Background/Objectives. Triple-negative breast cancer (TNBC) and pancreatic ductal adenocarcinoma (PDAC) are two of the deadliest adult cancers. High-dose acetaminophen (AAP) has anti-cancer activity when given in conjunction with CYP2E1 inhibitors to block liver toxicity. However, the administration of high doses of AAP presents practical challenges stemming from limited water solubility, making it difficult to create a concentrated treatment solution in limited volume. Propacetamol (PROP) is a water-soluble AAP pro-drug that has been used clinically as an IV AAP formulation for analgesia. Methods. We sought to investigate the pharmacokinetics, safety and anti-cancer activity of PROP in comparison to AAP using transplantable syngeneic models of TNBC and PDAC. Results. In vivo, PROP was rapidly converted to AAP within minutes of administration. PROP demonstrated enhanced effectiveness in suppressing tumor growth in the TNBC and PDAC syngeneic murine tumor models compared to AAP. The enhanced efficacy of PROP appears to stem from increased cellular uptake, leading to intracellular AAP concentrations that are 2.7-fold higher within 5 min compared to cells treated with unmodified AAP. PROP demonstrated enhanced modulation of intracellular JAK/STAT signaling, a key mediator of AAP anti-cancer activity, using both murine and humanized models. STAT3 and STAT6 inhibition by PROP led to modulation of macrophage polarization, likely contributing to its anti-TNBC and anti-PDAC activity. While parenteral administration is the preferred route of administration for PROP in anti-cancer indications, we also evaluated the safety and efficacy of oral PROP in comparison to AAP. High-dose PROP demonstrated improved safety following oral administration compared to AAP in the BALB/c murine model while maintaining anti-cancer activity in vivo. Conclusions. PROP, an AAP pro-drug, demonstrates enhanced intracellular delivery of AAP and in vivo anti-cancer activity compared to unmodified AAP, setting the stage for investigation as an anti-cancer drug across different indications.