No. 046 November 5, 2026 10 min read

Far Below the Documented IC50

Paracetamol was synthesised in 1893 and nobody can say how it relieves pain. What is known precisely is how it destroys a liver — and that for arthritis and back pain it is barely distinguishable from placebo.

Joseph von Mering synthesised paracetamol from p-nitrophenol in 1893. One hundred and thirty-three years later, nobody can tell you how it relieves pain.

I knew that in the way you know a piece of trivia — the drug with the unknown mechanism, a nice thing to mention. What I had not appreciated until I went through the primary literature this week is the shape of what is known. The pathway by which paracetamol destroys a liver is characterised down to the enzyme, the depleted cofactor and the antidote’s loading dose. The evidence that it barely outperforms placebo for arthritis and back pain is rated high certainty by the people whose job is rating certainty. The mechanism by which it does the thing a billion people take it for is the least settled part of the file.

That ordering is backwards from how I assume pharmacology works, and the newest result in the field — a paper from last June that the coverage described as ending the mystery — turns out to be interesting for a reason the headlines missed.

1893

first synthesised

mechanism still open

5–57 nmol/L

AM404 measured in human CSF

17 of 26 patients, 1 g IV

3.2 of 100

paracetamol's edge over placebo in osteoarthritis pain

clinical threshold is 9

39–42%

of US acute liver failure

acetaminophen, per later series

How the question got left open

Paracetamol reached people sideways. Phenacetin was marketed in 1887 as an antipyretic, fell out of favour over haemolytic anaemia and methaemoglobin, and in the 1940s Brodie and Axelrod showed that paracetamol was a major metabolite of it — so phenacetin’s effects were reassigned to the molecule it broke down into. Free availability followed in the 1950s, and aspirin’s retreat over gastrointestinal bleeding and Reye’s syndrome handed paracetamol the over-the-counter market from the 1960s on. At no point in that sequence did anyone need a mechanism.

The first serious proposal came from Flower and Vane in 1972: central cyclooxygenase inhibition. It has never closed, for a reason that is easy to state. Paracetamol does not reduce tissue inflammation, and it leaves platelets alone, which is not how a COX inhibitor behaves. Its potency in standard in vitro COX assays is weak. The ingenious repair — that paracetamol works as a reducing co-substrate at the peroxidase site, so it is potent where peroxide tone is low, as in brain, and feeble where it is high, as in inflamed tissue — is thirty years old and, as Sabah Ayoub notes in his 2021 review, has not been tested in vivo. A second repair, a COX-1 splice variant christened COX-3, came out of canine tissue; the human protein has no role in prostaglandin physiology.

The account that replaced it is more elaborate and has better genetics behind it. Paracetamol is deacetylated to p-aminophenol, which crosses the blood–brain barrier; there, fatty acid amide hydrolase conjugates it with arachidonic acid to make AM404. Injected into the brain, AM404 is analgesic. Knock out FAAH and paracetamol stops working — supraspinally, not peripherally. Knock out TRPV1, or CB1, or the Cav3.2 calcium channel, and the effect goes away too. A TRPV1 variant has been associated with paracetamol’s antinociceptive effect in healthy volunteers. Christophe Mallet and colleagues laid the whole scheme out in a 2023 review: TRPV1 in the periaqueductal grey, through mGlu5 and CB1 and on into bulbospinal serotonergic pathways.

What that review also says, carefully, is that two studies found paracetamol did not change brain endocannabinoid levels at all, that the serotonergic receptor involved has never been identified, and that the mechanism is “complex and not yet fully understood.” And it concedes the thing that bothered me most: almost all of it is rodent work.

The number that didn’t fit

There is one human measurement, and it is the crux.

In 2017 Chhaya Sharma’s group gave a single gram of intravenous paracetamol to patients having urological surgery under spinal anaesthesia and sampled their cerebrospinal fluid at lumbar puncture. AM404 was detectable in 17 of 26 evaluable samples; fourteen cleared the 5 nmol/L threshold the authors set for reliable quantitation, mostly between 5.1 and 57.4 nmol/L, with one anomalous reading at 166.8. The paper’s own abstract gives the range as 5–40, which does not match its results section — the kind of slip that makes me quote the table rather than the summary.

The authors’ verdict on their own finding is the sentence that matters: the concentrations are far below the previously documented IC50 for this metabolite. AM404 activates TRPV1 at micromolar concentrations. They measured tens of nanomolar, a thousandfold short. They offer the obvious defence — AM404 forms in FAAH-rich brain tissue, so CSF may understate what the relevant neurons see — and leave it there. Ayoub puts the same objection more bluntly: AM404’s active concentrations are well above anything detected in human CSF, and the conversion rate is very low.

So the leading mechanism had a quantitative hole in it, acknowledged by the people who found it.

What last June actually did

In June, Yaki Maatuf, Yoav Kushnir and colleagues at the Hebrew University published a different answer in PNAS. Primary sensory neurons, they report, make AM404 themselves from 4-aminophenol — no brain required. And there AM404 binds the local anaesthetic site on voltage-gated sodium channels and blocks them: NaV1.7 and NaV1.8, the two subtypes that carry nociceptive firing.

The potencies are the point. Nociceptor firing was halved at about 4.6 nanomolar in trigeminal neurons and 8.1 in dorsal root ganglion neurons. Human NaV1.8 came in at 55 nanomolar at a holding potential of −80 mV, human NaV1.7 at 21.9. Other sodium channel isoforms were much less affected at a full micromolar. Paracetamol itself did nothing to the channels; 4-aminophenol and NAPQI only moved them at millimolar, and NAPQI increased NaV1.8 current.

Set those numbers against the Sharma measurement and they overlap. Five to fifty-seven nanomolar is useless for activating TRPV1 and squarely in range for blocking NaV1.7 and NaV1.8. The new mechanism’s chief virtue is that it is potent at concentrations a human being has actually been shown to reach.

AM404 concentrations, nanomolar · log scale

Nociceptor firing, IC50, rat trigeminal

4.6 nM

Human NaV1.7, IC50 at −80 mV

21.9 nM

Human NaV1.8, IC50 at −80 mV

55 nM

Measured in human CSF after 1 g IV

5–57, one outlier at 167

TRPV1 activation

micromolar; no figure given

In human peripheral nerve or skin

never measured

And now the catch, which the chart makes plain. The compartment where AM404 has been measured in a person is cerebrospinal fluid — the compartment that matters for the theory being displaced. The new theory needs AM404 inside peripheral nociceptors, in skin and nerve, and nobody has measured it there in a human. The paper’s own trigeminal AM404 figures come from incubating rat neurons with 10 to 100 micromolar 4-aminophenol in a dish.

The objection that sank the old site was a concentration. It was answered by moving to a site where nobody has measured the concentration.

The rest of the caveats are the authors’ own, and they are unusually forthcoming about them. The neurons are from three-to-five-day-old rats; the human channels are expressed in cell lines; the in vivo work used intraplantar AM404 at 10 millimolar, a dose chosen to mimic a local anaesthetic rather than to mimic paracetamol. Behavioural groups ran about six animals. AM404 was applied from outside the cell though in vivo it is made inside, so the kinetics may not transfer. Cardiac NaV1.5 is also inhibited. No neuropathic pain model was tested. Capsazepine failed to block the peripheral effect, which they read as ruling out TRPV1 there — fair, and also the kind of negative result that is only as good as the blocker.

None of which stopped the press release cycle. One outlet ran it as the discovery that ends a longstanding mystery; others as proof that the drug stops pain at the nerves before it reaches the brain. What the paper supports is narrower and more useful: a candidate target whose potency, for the first time, sits in the range of a measured human concentration — in the wrong tissue.

The best-evidenced fact about it

Here is the part that reorganised my sense of the drug.

The Cochrane review of paracetamol versus placebo for knee and hip osteoarthritis pooled ten trials and 3,541 participants. Pain improved by 26 points out of 100 on paracetamol and 23 on placebo: a difference of 3.2 points, 95% confidence interval 1.0 to 5.4. Function differed by 2.9 points. The review’s own minimal clinically important difference is 9% for pain and 10% for function. The evidence was rated high quality, and the authors wrote that guidelines recommending paracetamol as first-line treatment should be reconsidered.

For acute low back pain the answer is flatter still. The PACE trial randomised 1,652 adults across 235 primary care practices in Sydney to regular paracetamol, as-needed paracetamol, or placebo. Median time to recovery: 17 days, 17 days, 16 days. No effect on pain, disability, function, sleep or quality of life. The Cochrane review built on it found no difference at one, two, four or twelve weeks, again at high certainty, and no eligible trials at all for chronic back pain.

So for two of the conditions it is most prescribed for, the drug’s effect is small, measured precisely, and below the threshold anyone would notice. That is the most solid quantitative claim in this whole literature, and it is a claim about absence.

Meanwhile the harm pathway is textbook. Five to ten per cent of a dose goes through cytochrome P450, mainly CYP2E1, to NAPQI; glucuronidation and sulfation saturate in overdose and push more down that route; NAPQI consumes glutathione and then attacks hepatocytes. N-acetylcysteine replenishes glutathione, works best inside eight to ten hours, and dropped mortality from as much as 5% to 0.7%. The review I took those figures from attributes about 26,000 US hospitalisations and more than 450 deaths a year to acetaminophen, with later case series putting it behind 39 to 42% of American acute liver failure and making it the commonest cause in Britain. We know the poisoning the way we know a well-mapped reaction.

When the mechanism becomes load-bearing

Mechanism was never what licensed this drug, and for most of its life nothing required one. What changes that is a question the trials were not built to answer.

Prenatal exposure is the current instance. With no mechanism to reason from, the argument is conducted entirely in epidemiology, and the strongest piece of it is a 2024 Swedish cohort of 2,480,797 children born between 1995 and 2019, of whom 7.49% were recorded as exposed. At the population level the hazard ratios were modestly above one — autism 1.05, ADHD 1.07, intellectual disability 1.05. Within sibling pairs, where much of the family background cancels, they were 0.98, 0.98 and 1.01, with confidence intervals straddling one. The authors read the population signal as familial confounding.

That is a well-designed study and it is still observational. The records carry no dose, no duration, no timing, and miss most over-the-counter use; a sibling design trades one set of assumptions for another rather than escaping them. I am not going to pretend one cohort closes a question like this. But it illustrates the asymmetry: the people arguing have a 2.5-million-child registry and no account of what the molecule does to a developing nervous system, because there is no account of what it does to an adult one.

What I take from the week is a small correction to how I rank kinds of knowledge. I had been treating mechanism as the foundation and clinical evidence as the superstructure. For paracetamol it is the reverse — the trials are the firm part, the toxicology is firmer still, and the mechanism is a hundred and thirty years of plausible stories, one of which has just been relocated from the brain to the skin on the strength of a concentration match in the wrong fluid.