Jephte Yao Akakpo

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.

Cisplatin is an effective chemotherapeutic drug for the treatment of bladder cancer, though cisplatin-induced nephrotoxicity (CIN) occurs in ∼20% to 30% of patients, limiting its clinical use. Evidence has shown that cytochrome P450 2E1 (CYP2E1), a drug metabolism enzyme expressed in proximal tubules, mediates the production of reactive oxygen species during cisplatin-induced injury. Previously, we showed that the repurposed drug 4-methylpyrazole (4MP) blocks CYP2E1 activity. Here, we investigated the potential protective effects of 4MP against CIN. Male and female C57BL/6J mice were treated intraperitoneally (i.p.) with a single 20 mg/kg dose of cisplatin for 3 days or 9 mg/kg/wk for 4 wk with or without 50 mg/kg 4MP as a co-treatment. Our findings revealed that acute treatment with cisplatin induced severe histological tubular damage and elevated plasma BUN and creatinine levels in male but not female mice. This difference correlated with higher basal CYP2E1 expression in the kidneys of male mice compared with female mice. We also found that cisplatin increased renal CYP2E1 activity and that inhibition of CYP2E1 with 4MP significantly reduced cisplatin-induced cell death in male mice and primary normal human kidney cells. By contrast, human bladder cancer cells do not express CYP2E1, and treatment with 4MP did not interfere with cisplatin’s anticancer effects in human bladder cancer HTB9 cells. This study highlights the critical role of CYP2E1 in CIN and suggests that its inhibition with 4MP in the kidney is a potential prophylactic therapeutic option to prevent CIN in bladder cancer patients without affecting its antineoplastic effect.

An overdose of acetaminophen (APAP) is the leading cause of drug-induced hepatotoxicity and acute liver failure in the United States. It is established that the predominant mode of hepatocyte cell death after an APAP overdose is through necrosis, and it is now recognized that this occurs through regulated pathways involving RIP kinases. These kinases, along with the pseudo-kinase MLKL, are central players in classical necroptotic cell death. Despite the skepticism regarding the role of necroptosis in APAP-induced liver injury, recent research demonstrating necroptosis-independent roles for MLKL led us to re-examine the role of this pseudo-kinase in APAP pathophysiology. Treatment of Mlkl−/− mice with a moderate (300 mg/kg) overdose of APAP resulted in an exacerbation of liver injury at 6- and 12-h post-APAP as evidenced by elevated plasma alanine aminotransferase activities, and extensive necrosis accompanied by diminished glutathione levels. Interestingly, these differences between Mlkl−/− and wild-type mice were negated at the 24-h mark, previously scrutinized by others. At 6 and 12 h post-APAP, Mlkl−/− mice exhibited augmented translocation of AIF and Endonuclease G without affecting JNK activation, suggesting enhanced mitochondrial permeability transition in the absence of MLKL. Lack of MLKL also impacted autophagy, the unfolded protein response and endoplasmic reticulum stress, with decreased levels of p62 and LC3B and increased expression of CHOP and GRP78 at 6 h post-APAP. In essence, our findings illuminate a noncanonical role for MLKL in the early phases of APAP-induced liver injury, warranting further exploration of its influence on APAP pathophysiology.

Liver injury caused by acetaminophen (APAP) overdose is the leading cause of acute liver failure in western countries. The mode of APAP‐induced cell death has been controversially discussed with ferroptosis emerging as a more recent hypothesis. Ferroptosis is characterized by ferrous iron‐catalyzed lipid peroxidation (LPO) causing cell death, which can be prevented by the lipophilic antioxidants ferrostatin‐1 and UAMC‐3203. To assess the efficacy of these ferroptosis inhibitors, we used two murine models of APAP hepatotoxicity, APAP overdose alone or in combination with FeSO₄ in fasted male C57BL/6J mice. APAP triggered severe liver injury in the absence of LPO measured as hepatic malondialdehyde (MDA) levels. In contrast, ferrous iron co‐treatment aggravated APAP‐induced liver injury and caused extensive LPO. Standard doses of ferrostatin‐1 did not affect MDA levels or the injury in both models. In contrast, UAMC‐3203 partially protected in both models and reduced LPO in the presence of ferrous iron. However, UAMC‐3203 attenuated the translocation of phospho‐JNK through downregulation of the mitochondrial anchor protein Sab resulting in reduced mitochondrial dysfunction and liver injury. Thus, APAP toxicity does not involve ferroptosis under normal conditions. The lack of effects of ferroptosis inhibitors in the pathophysiology indicates that ferroptosis signaling pathways are not relevant therapeutic targets.

Acute kidney injury (AKI) is a common complication in acetaminophen (APAP) overdose patients and can negatively impact prognosis. Unfortunately, N-acetylcysteine, which is the standard of care for the treatment of APAP hepatotoxicity does not prevent APAP-induced AKI. We have previously demonstrated the renal metabolism of APAP and identified fomepizole (4-methylpyrazole, 4MP) as a therapeutic option to prevent APAP-induced nephrotoxicity. However, the kidney has several functionally distinct regions, and the dose-dependent effects of APAP on renal response and regional specificity of APAP metabolism are unknown. These aspects were examined in this study using C57BL/6J mice treated with 300–1200 mg/kg APAP and mass spectrometry imaging (MSI) to provide spatial cues relevant to APAP metabolism and the effects of 4MP. We find that renal APAP metabolism and generation of the nonoxidative (APAP-GLUC and APAP-SULF) and oxidative metabolites (APAP-GSH, APAP-CYS, and APAP-NAC) were dose-dependently increased in the kidney. This was recapitulated on MSI which revealed that APAP overdose causes an accumulation of APAP and APAP GLUC in the inner medulla and APAP-CYS in the outer medulla of the kidney. APAP-GSH, APAP-NAC, and APAP-SULF were localized mainly to the outer medulla and the cortex where CYP2E1 expression was evident. Interestingly, APAP also induced a redistribution of reduced GSH, with an increase in oxidized GSH within the kidney cortex. 4MP ameliorated these region-specific variations in the formation of APAP metabolites in renal tissue sections. In conclusion, APAP metabolism has a distinct regional distribution within the kidney, the understanding of which provides insight into downstream mechanisms of APAP-induced nephrotoxicity.