How Does Acetaminophen Target Pain?

A new study from Indiana University scientists may aid the pharmaceutical industry in better understanding a popular over-the-counter pain reliever: Tylenol.

Michaela Dvorakova, a postdoctoral researcher at IU’s Gill Institute for Neuroscience and the College of Arts and Sciences’ Department of Psychological and Brain Sciences, and Gill Institute research scientist Alex Straiker published the study in Cell Reports Medicine. Their discovery, which details a previously unknown way the drug targets pain, could change how pharmacologists think about treating pain, and aid in designing safer and more effective pain medications.

The researchers found that acetaminophen inhibits an enzyme that makes one of the endogenous cannabinoids, 2-arachidonoyl glycerol, or 2-AG. Endocannabinoids are produced by the body to activate CB1 receptors, the same receptor that produces the psychoactive effects of cannabis.

Though acetaminophen, also known as Tylenol or paracetamol, is the most common pain and fever reliever in the U.S., how it relieves pain is still a mystery.

“There are hypotheses, but we still don’t know precisely how it works,” Dvorakova said. “Up until now we thought that elevated endocannabinoids in our body meant less pain, but our study shows that in the case of 2-AG, it might be the opposite. Actually, reduced levels of 2-AG leads to decreased pain.”

In high doses, acetaminophen can be toxic to the liver. Acetaminophen toxicity causes around 500 deaths per year in the U.S. and is the second leading cause of liver transplantation around the world. Due to its wide availability and its combination in other products, more than 60 million Americans consume acetaminophen weekly, making it crucial to understand how it targets pain receptors.

"If you don’t know what the target is, you can’t design an alternative,” Straiker said. “Our research suggests that this enzyme might be the target, in which case you can start developing drugs that target that specific enzyme but without that toxicity.”

There was skepticism in the research community about a new mechanism of action for acetaminophen, Straiker said. When 50 years of research showed that activating CB1 receptors produced pain relief in the body, scientists settled on that concept.

“It can be hard to break through that dogma,” Straiker said.

Going forward, the research team is looking into evaluating other common pain relievers, like ibuprofen and aspirin, to determine whether they have similar mechanisms of action.

Reference: Dvorakova M, Bosquez-Berger T, Billingsley J, et al. Acetaminophen inhibits diacylglycerol lipase synthesis of 2-arachidonoyl glycerol: Implications for nociception. CR Med. 2025. doi: 10.1016/j.xcrm.2025.102139

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