P-032 - (Poster #32) The Multimodal Molecule: A Comprehensive, Evidence-based Hierarchical Synthesis of Acetaminophen Mechanisms of Analgesic Action

Background: Pain is a complex, multidimensional phenomenon meditated by numerous mechanisms including nociceptive, inflammatory, and modulatory signals and the search for effective, safe, and well-tolerated analgesic regimens continues to be a global health priority. Acetaminophen, one of the most commonly used analgesics, is a mainstay in treating mild to moderate pain across a wide range of pain conditions especially in patients with comorbidities where other classes of analgesics may be contraindicated. Even as the pleiotropic nature of acetaminophen’s mechanism of action (MoA) is broadly recognized, a systems-level integration of these mechanisms into models of pain perception and modulation and interpretation of their relative clinical significance is currently limited. Mechanistic reviews have largely focused on cataloguing acetaminophen’s metabolites and cellular targets, often presenting them as independent or competing observations. While still pharmacologically valuable, these reviews have been limited in their assessment of biological plausibility, integration into higher order systems of pain perception, and their clinical relevance, and thus had limited ability to translate the observations into clinically meaningful appreciation of acetaminophen’s biological action. A framework is needed to evaluate the evidence supporting each mechanism and its contribution to overall acetaminophen-mediated analgesia.

Purpose/Objectives: This review comprehensively evaluated major proposed mechanisms of acetaminophen-mediated analgesic action based on their efficacy evidence level, biological relevance and clinical significance. We conducted a systematic literature review to identify studies of acetaminophen’s MoA, developed and employed a novel weight of evidence framework based on evidence strength, consensus and relevance supporting each identified mechanism, the formation and fate of major metabolites, as well as their interactions with molecular targets.

Methods: A PubMed search for studies investigating acetaminophen’s MoA used the terms ((paracetamol OR acetaminophen) AND ("mechanism of action" OR "mode of action")) and was limited to primary publications, in English through April 2025. Three large language models (ChatGPT 4o, Claude 3.5 Sonnet and Claude 3.7 Sonnet) were used to conduct initial abstract screening via text extraction and semantic analysis for identification of key themes and recurring patterns across literature. This AI-assisted curation of acetaminophen’s mechanisms was manually verified against each original source publication to ensure accuracy, completeness, and appropriate interpretation of the scientific evidence. While AI tools were used to enhance the efficiency of the literature review process and summarization, authors conducted all final analyses, interpretation, and the development of the conclusions. To evaluate the evidence supporting each proposed mechanism identified, we developed and employed a structured classification framework and ordinal scoring system, assessing three key criteria, namely (i) efficacy evidence strength based on type of data, (ii) evidence consensus based on volume and agreement among studies, and (iii) biological relevance based on pharmacological plausibility and clinical relevance.

Results: Based on over 120 publications, we identified 17 different mechanisms by which acetaminophen may exert its analgesic effects. Systematic review and assessment of each mechanism using our structured classification framework identified central cyclooxygenase (COX) inhibition, serotonergic pathway modulation, TRPV1, and endocannabinoid signaling as the MoAs with the highest likelihood of mediating acetaminophen’s clinically observed analgesic effects. Among these, the mechanisms with the strongest clinical significance include selective inhibition of COX1/COX2 variants in the central nervous system (CNS). Mechanisms involving inhibition of Cav3.2 channels (a T-type voltage-gated calcium channel), activation of opioid receptors, activation of Kv7 channels (low-threshold, voltage-gated potassium channels), and modulation of some specific transient receptor potential (TRP) channels (e.g., TRPA1) were assessed to have moderate likelihood of inducing clinically relevant analgesic effects. Mechanisms involving suppression of T-cell activation, inhibition of nitric oxide (NO) synthase, the purported COX3 splice-variant, macrophage migration inhibitory factor (MIF), diacylglycerol lipase-α (DAGL- α) and lipolysis were assessed to have the lowest likelihood of inducing clinically relevant analgesic effect.
When we assessed how each of these potential mechanisms may impact central processes of pain sensing, modulation and cognition that ultimately define the perception of pain, the MoAs with the highest likelihood of inducing analgesic effects affected all three higher-order systems of pain regulation namely, (i) the afferent nociceptive transmission, (ii) central cognition and (iii) descending inhibitory signaling that collectively function to raise pain threshold and lower the pain perception.
Our results provide a composite picture of acetaminophen MoA using a novel, evidence-based hierarchical ranking of each purported mechanism for their relative contributions to the overall analgesic effect and highlight the foundational role of acetaminophen’s multimodal action in the context of acute and chronic pain management in clinical practice.
This assessment of acetaminophen’s MoA included review of English language publications indexed in PubMed identified using a relatively small number of search terms and thus may be limited by selection biases and publication bias. The proposed framework included a limited number of criteria and utilized an ordinal scoring system. Further development of the framework could include additional parameters and explore alternative ranking approaches.

Conclusions/Implications for future research and/or clinical care: We sought to address limitations of prior reviews of the MoA of acetaminophen by proposing a new framework to evaluate the evidence supporting each purported mechanism. This approach enabled an appraisal of the potential likelihood that each mechanism contributes to overall clinical analgesia elicited by acetaminophen. We were able to identify the proposed MoAs with the greatest amount of supporting evidence and found that even considering only the MoAs with highest level of evidence, acetaminophen most likely possesses pleiotropic action—providing additional insights into how acetaminophen provides meaningful pain relief which has been long established across pain conditions clinically. In addition, by contextualizing individual mechanisms within the broader architecture, we provide a cohesive model that advances both understanding and clinical utility of acetaminophen MoAs. The crosstalk between these purported MoAs and their relative contributions further underscores the potentially central role of acetaminophen in pain management as a foundational first-line choice due to a unique multimodal action distinct from other analgesic choices. Understanding the higher-order pathways and multiple potential pharmacological MoAs that mediate the analgesic activity of acetaminophen may allow investigation of additional strategies to augment acetaminophen’s efficacy, safety and use in multimodal pain management regimens.