High-Dose Acetaminophen as a Treatment for Cancer

Round 1

Reviewer 1 Report

The paper needs addition of a figure and table for clear understanding of the information to the audience. 

The manuscript by Wu et al., entitled “High dose ………..for cancer” reviews the use of high dose of acetaminophen for treating cancers along with the use of standard drugs such as NAC.
Comments to the Author
Major Queries:
1.    There can be a table describing the use of high dose of AAP for cancer treatment with clinical trials and animal studies so that it will be easily readable.
2.    Additional of figure describing the role of AAP in cancer treatment with respect to its mechanism of action will improve the paper’s message to audiences.

Minor Queries:
1.    The in vitro and in vivo should be italicized throughout the manuscript.

Author Response

Major Queries:
1.    There can be a table describing the use of high dose of AAP for cancer treatment with clinical trials and animal studies so that it will be easily readable.

We thank the Reviewer for the suggestion.  We have added Table 1 to the manuscript to summarize the clinical trials of high dose AAP.

2.    Additional of figure describing the role of AAP in cancer treatment with respect to its mechanism of action will improve the paper’s message to audiences.

We thank the Reviewer for the suggestion, and we have added an additional Figure that summarizes AAP mechanism in cancer in and in liver.

Minor Queries:
1.    The in vitro and in vivo should be italicized throughout the manuscript.

We thank the Reviewer for this suggestion, and have made this change.

Reviewer 2 Report

High dose acetaminophen has shown promise for cancer treatment in early clinical trials, but its mechanisms were unclear. Recent preclinical studies have revealed novel free radical-independent anti-tumor mechanisms involving modulation of JAK-STAT signaling and tumor immunology. Using acetaminophen with concurrent CYP2E1 inhibitors enables much higher dosing without toxicity while preserving efficacy. With these new insights, the authors advocate for renewed clinical development of high dose acetaminophen with optimized antidote regimens as a novel cancer therapeutic approach. Additional clinical trials are warranted to validate the safety, pharmacology, and anti-tumor activity of this strategy in patients.

Here are several of the minor concerns that may need to be concisely addressed:

• The early clinical trials of high dose acetaminophen had small sample sizes and no control groups, so the efficacy remains unproven. The response rates observed require confirmation in larger controlled studies.
• The preclinical studies demonstrating anti-tumor mechanisms were conducted solely in mouse models. The effects in human tumors remain theoretical.
• The optimal antidote regimen to allow dose escalation of acetaminophen is still not definitively established. Additional preclinical studies comparing antidote combinations head-to-head are needed.
• The pharmacokinetics and pharmacodynamics of high dose acetaminophen in cancer patients requires detailed study to determine the ideal dosing schedule.
• The relative contribution of the proposed mechanisms of action - such as JAK-STAT modulation versus immune effects - to the anti-tumor efficacy remains unclear.
• The effects of high dose acetaminophen on the tumor immune microenvironment requires further characterization beyond macrophages.
• The safety profile and tolerability of concurrent antidote administration needs to be formally evaluated in clinical studies.
• Optimal cancer types, stages, and combination regimens for clinical trials are still speculative based on available data.

None.

Author Response

Here are several of the minor concerns that may need to be concisely addressed:

• The early clinical trials of high dose acetaminophen had small sample sizes and no control groups, so the efficacy remains unproven. The response rates observed require confirmation in larger controlled studies.

We thank the Reviewer for this comment, and we have addressed on line 271, "Ultimately, large clinical datasets may be needed to assess the clinical efficacy of AAP in diverse malignancies—both alone and in rational combinations with existing anti-tumor agents. " 

• The preclinical studies demonstrating anti-tumor mechanisms were conducted solely in mouse models. The effects in human tumors remain theoretical.

We thank the Reviewer for this comment and have addressed on line 272: 

"Additionally, robust correlative studies from clinical trials will be needed to evaluate the role of JAK-STAT signaling and the immune system in mediating the anti-tumor activity of high dose AAP.  "

• The optimal antidote regimen to allow dose escalation of acetaminophen is still not definitively established. Additional preclinical studies comparing antidote combinations head-to-head are needed.

We thank the Reviewer for this comment.  Please note that these experiments have been completed, see Bryan et al, JPET, 2023 Figure 1.  Thus far, we have only evaluated the most clinically validated/used AAP antidotes, namely NAC and fomepizole.  In the future, we will evaluate other antidotes as well.

• The pharmacokinetics and pharmacodynamics of high dose acetaminophen in cancer patients requires detailed study to determine the ideal dosing schedule.

We thank the Reviewer for this comment, and have addressed on line 263:

"Human trials are needed to comprehensively evaluate the safety profile, pharmacokinetics, and pharmacodynamics of high dose AAP with fomepizole-based rescue across a range of doses in patients with advanced cancer."

• The relative contribution of the proposed mechanisms of action - such as JAK-STAT modulation versus immune effects - to the anti-tumor efficacy remains unclear.

We thank the Reviewer for this comment.  We would point out that anti-tumor efficacy is lost/severely diminished in immune compromised mouse models (JPET, 2023) and more specifically in macrophage depleted models (Cancers, 2023) thus underscoring the importance of the immune system in mediating AAP anti-cancer activity.

• The effects of high dose acetaminophen on the tumor immune microenvironment requires further characterization beyond macrophages.

We thank the Reviewer for this comment, and have addressed in line 251: "Currently, our lab is comprehensively evaluating changes in the tumor immune microenvironment induced by high dose AAP using such methodologies as single cell RNA sequencing and multiplex flow cytometry."

• The safety profile and tolerability of concurrent antidote administration needs to be formally evaluated in clinical studies.

We thank the Reviewer for this comment, and have addressed in line 263:

"Human trials are needed to comprehensively evaluate the safety profile, pharmacokinetics, and pharmacodynamics of high dose AAP with fomepizole-based rescue across a range of doses in patients with advanced cancer."   

• Optimal cancer types, stages, and combination regimens for clinical trials are still speculative based on available data.

We thank the Reviewer for this comment, and have addressed in line 269:

"However, the relative efficacy of AAP in various tumor histologies—including solid tumor versus hematologic malignancies—remains to be comprehensively elucidated.  Ultimately, large clinical datasets may be needed to assess the clinical efficacy of AAP in diverse malignancies—both alone and in rational combinations with existing anti-tumor agents."