Medically Written and Reviewed By: Vikas Londhe, M.Pharm, Pharmacology
For over a century, paracetamol (acetaminophen) has been one of the world’s most widely used analgesics and antipyretics. Despite its widespread use, its exact mechanism of action has remained unclear. It was long believed to work mainly by blocking certain enzymes called cyclooxygenases (COX), especially COX-2. These enzymes are responsible for producing prostaglandins, which are chemicals that promote pain, inflammation, and fever. By reducing prostaglandin production, paracetamol helps lower pain and fever without significantly reducing inflammation like other NSAIDs. However, it is believed that paracetamol is only effective in mild inflammations, like after tooth extraction, and it is not effective in severe inflammation that arises from rheumatoid arthritis and acute gout.
Another pathway involves the TRPV1 receptor, short for Transient Receptor Potential Vanilloid 1, which plays a role in sensing heat and pain. Some research suggests that an active metabolite of paracetamol, the fatty acid amide N-arachidonoylphenolamine (AM404), may activate TRPV1 in a way that leads to pain relief by desensitizing these pain-sensing receptors.
However, new research conducted at Hebrew University of Jerusalem and the findings are published in The Proceedings of the National Academy of Sciences (PNAS) has uncovered a previously unknown mechanism of paracetamol. The new research proposed that paracetamol acts directly at peripheral nerve endings. Its metabolite AM404 can block sodium channels in the nerves, which are essential for sending pain signals to the brain. This discovery adds a new dimension to our understanding of how paracetamol relieves pain: not just through the inhibition of certain enzymes and receptors, but also by directly inhibiting the body’s ability to send pain signals to the brain.
The Breakthrough Study
A team led by Professors Alexander Binshtok and Avi Priel from the Hebrew University of Jerusalem published these game-changing findings in the prestigious PNAS, titled “The analgesic paracetamol metabolite AM404 acts peripherally to directly inhibit sodium channels”
The key findings of the Research Includes
Local production of AM404: After oral intake of paracetamol, the body converts it to p-aminophenol in the liver, which is subsequently transformed into AM404 by fatty acid amide hydrolase (FAAH) in primary sensory neurons, essentially at the nerve endings where pain signals originate.
Inhibition of nociceptive sodium channels: AM404 directly blocks voltage-gated sodium channels Na_V1.7 and Na_V1.8, both crucial for generating action potentials in pain-sensing neurons. The blockade occurs via the local anesthetic binding site.
Peripheral analgesia: Through this localized mechanism, AM404 prevents pain signals at their source, producing potent relief in both regular and inflammatory pain models in rodents.
Researchers found that AM404, a metabolite formed from paracetamol in the body, accumulates in peripheral sensory neurons where it directly inhibits voltage-gated sodium channels Na_V1.7 and Na_V1.8. These channels are critical for the initiation and conduction of pain signals at the site of injury or inflammation. By blocking these sodium channels, AM404 effectively diminishes nociceptive signal transmission at its source, preventing pain before it even reaches the spinal cord. This peripheral action represents a fundamental shift in our understanding of how paracetamol works. It positioned paracetamol not only as a central analgesic but also as a locally acting modulator of neuronal excitability.
Results
AM404 significantly reduced sodium current amplitude in isolated dorsal root ganglion (DRG) neurons in a dose-dependent manner. The greatest effect was observed on tetrodotoxin-resistant (TTX-R) sodium currents, which are characteristic of Nav1.8, a key player in chronic and inflammatory pain. AM404 had minimal effect on potassium and calcium currents, indicating a selective action on sodium channels.
In pharmacological profiling, AM404 showed the strongest inhibition of Nav1.7 and Nav1.8, both of which are highly expressed in nociceptive (pain-sensing) neurons. Other Nav subtypes, such as Nav1.5 (cardiac) and Nav1.6 (CNS), were minimally affected, suggesting a favorable safety profile by avoiding cardiac or CNS toxicity.
In formalin-induced inflammatory pain models, peripheral injection of AM404 significantly reduced both early (neurogenic) and late (inflammatory) phases of pain behaviors (licking, flinching). In the hot plate and tail flick thermal assays, AM404 increased latency to pain response, indicating effective thermal analgesia. Systemic or central (intrathecal) administration of AM404 had less prominent effects, highlighting that peripheral action is essential for its analgesic activity.
Computational docking predicted that AM404 binds to a hydrophobic fenestration site within the channel’s domain IV S6 segment, a region known to influence channel gating and drug binding.
Implications
This research challenges the traditional view of paracetamol as a centrally acting analgesic. It highlights that peripheral mechanisms, particularly in the context of inflammatory pain, are also crucial to its analgesic action. A key finding is the active pharmacological role of AM404, a metabolite of paracetamol, which is not just a metabolic byproduct but a potent modulator contributing to its pain-relieving effects. This adds to the recognition of the importance of drug metabolites in determining therapeutic efficacy. Moreover, the study strengthens the therapeutic relevance of targeting sodium channel subtype Na_V 1.7, positioning AM404 as a promising lead compound or molecular scaffold for the development of new, non-opioid analgesics.
Broader Impact: Beyond Paracetamol
This study opens exciting new avenues in the field of pain research. It triggers a re-evaluation of some metabolites that have been silent since their discovery and are also traditionally overlooked, but may possess key pharmacological actions, suggesting that other commonly used drugs could harbor unexplored therapeutic potential through their metabolites.
Additionally, the findings strengthen the scientific rationale for targeting peripheral sodium channels, particularly in managing chronic and inflammatory pain conditions. AM404, a paracetamol metabolite, exerts analgesic effects without causing sedation or respiratory depression, positioning it as a promising foundation for developing safer, non-addictive alternatives to opioids.
Conclusion
The discovery that AM404 blocks peripheral NaV channels redefines how we understand one of the worlds’s most commonly used analgesics. By uncovering this hidden peripheral pain pathway, researchers at the Hebrew University of Jerusalem have significantly advanced the field of analgesic pharmacology. This work not only deepens our molecular understanding of paracetamol but also opens up new possibilities for developing better pain medicines and emphasizing the vital role of peripheral targets in pain relief.
References
Y Maatuf, Y. Kushnir, A.Nemirovski, et al, The analgesic paracetamol metabolite AM404 acts peripherally to directly inhibit sodium channels, Proc. Natl. Acad. Sci. U.S.A. 122 (23) e2413811122, https://doi.org/10.1073/pnas.2413811122
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Israeli study finds acetaminophen drug works by first blocking pain in nerves, The Times of Israel, https://www.timesofisrael.com/israeli-study-finds-acetaminophen-drug-works-by-first-blocking-pain-in-nerves/
New discovery: Tylenol stops pain at the nerves, before it hits the brain, ScienceDaily, https://www.sciencedaily.com/releases/2025/06/250610074247.htm#:~:text=Summary%3A,channels%20in%20pain%2Dsensing%20nerves.
Mallet C, Desmeules J, Pegahi R, Eschalier A. An Updated Review on the Metabolite (AM404)-Mediated Central Mechanism of Action of Paracetamol (Acetaminophen): Experimental Evidence and Potential Clinical Impact. J Pain Res. 2023 Mar 29;16:1081-1094. Doi: 10.2147/JPR.S393809. PMID: 37016715; PMCID: PMC10066900.
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