A new study published in PNAS has found that AM404, a metabolite of paracetamol (acetaminophen), directly blocks pain by acting on peripheral sensory neurons.
Researchers reported that AM404 inhibits two key voltage-gated sodium channels—NaV1.7 and NaV1.8—which are essential for generating pain signals.
Paracetamol, or acetaminophen, is widely used for mild to moderate pain, but its mechanism of action has remained unclear. While past research focused on effects in the central nervous system, this study examined AM404’s action in the peripheral nervous system.
The researchers showed that peripheral sensory neurons, including those in the trigeminal and dorsal root ganglia, produce AM404 from paracetamol’s precursor, 4-aminophenol. When exposed to AM404, these neurons failed to generate action potentials, the electrical signals that transmit pain.
Patch-clamp experiments demonstrated that AM404 suppressed NaV1.7 and NaV1.8 channel activity at nanomolar concentrations. The inhibition was strong, concentration-dependent, and occurred within minutes. This effect was also observed in engineered cells expressing the human forms of these channels.
In vivo tests confirmed the compound’s analgesic effect. Rats injected with AM404 in the hind paw exhibited significantly higher thresholds for both thermal and mechanical pain. The effect was local and lasted about an hour. No change was observed in the untreated paw.
AM404 also reduced inflammatory pain. In formalin and complete Freund’s adjuvant (CFA) models, AM404 lowered pain responses when injected directly into the affected area. These results were consistent across both sexes.
The study confirmed the specificity of AM404’s action. Paracetamol and its other metabolites, including 4-aminophenol and NAPQI, did not block the sodium channels or reduce pain responses. Only AM404 had this effect.
The mechanism of inhibition resembled that of local anesthetics such as bupivacaine. AM404 bound to the same site on the sodium channels and blocked them in a use- and state-dependent manner. A point mutation at the local anesthetic binding site eliminated AM404’s effect, confirming this mode of action.
These findings indicate that AM404, formed from paracetamol in peripheral neurons, directly inhibits nociceptive sodium channels. This supports a peripheral mechanism for paracetamol-induced analgesia and suggests potential for developing targeted pain therapies using AM404 or similar compounds.
The authors declared no competing interests.
Source: Proc Natl Acad Sci U S A