The Endocannabinoid System’s Contribution to Placebo Analgesia

Background

Pain perception is not solely determined by nociceptive input; it is well established in the literature that pain is strongly modulated by cognitive and contextual factors, such as expectations and beliefs¹. One example of this modulation is placebo analgesia, a phenomenon in which the administration of an inert treatment reduces perceived pain through factors such as expectations of an analgesic effect and learning mechanisms.

Most studies on this topic have shown that the endogenous opioid system plays a central role in placebo analgesia. For instance, studies have demonstrated that administration of naloxone, an opioid receptor antagonist, reduces placebo effects². Neuroimaging studies have also reported activation of µ-opioid receptors during placebo responses³.

However, opioids do not fully account for the phenomenon. In several studies, naloxone does not fully eliminate placebo analgesia, suggesting the involvement of additional neurobiological systems⁴. Evidence indicates that administration of rimonabant, a CB1 antagonist, can reduce certain non-opioid placebo responses⁵. Furthermore, preclinical studies suggest a functional interaction involving the opioid and endocannabinoid systems in pain modulation, although this interaction remains poorly understood in humans^6-9.

Novel contribution of the preprint

This preprint aims to better understand the neurobiological basis of placebo analgesia and the sources of interindividual variability in this response. To date, most research primarily focuses on the role of the opioid system, while the contribution of the endocannabinoid system (and especially its interaction with the opioid system in humans) remains largely unexplored.

The main objective of this study is therefore to examine whether circulating endocannabinoids contribute to individual differences in placebo analgesia, and, more importantly, whether their effect depends on the level of endogenous opioid system activation, measured here by peripheral β-endorphin levels.

In other words, this article does not simply aim to show that multiple systems contribute to placebo analgesia but rather proposes a more specific framework: placebo analgesia may arise from a dynamic interaction between different neuromodulatory systems, rather than from a single biological mechanism.

By simultaneously assessing circulating endocannabinoids and opioids levels, this study seeks to address an important gap in the literature and to propose a more integrated model of the biological mechanisms underlying placebo analgesia, with potential implications for biomarker-guided personalized medicine and endogenous pain modulation.

Key Findings

The authors induced placebo analgesia using a validated paradigm. Two creams were applied to the participants’ dominant arm. The first was presented as an inert control cream, while the second was presented as a potent analgesic, when in reality both creams were petroleum jelly.

FAAH substrates contribute to placebo analgesia.

The authors claim the endocannabinoid system constitutes a non-opioid biological mechanism essential to placebo analgesia. They postulate that the mobilization of FAAH’s substrates contributes to individual differences in placebo response.

They measured changes in circulating anandamide (AEA), PEA, and OEA in blood samples collected before and after the experimental paradigm. Since the three substances are strongly correlated, they used principal component analysis (PCA) to create a composite score. They then used mixed linear models to link these changes to pain assessment.

The individuals with a greater increase in FAAH substrates experienced significantly stronger placebo analgesia (reduced pain). It is important to note that it was the combined increase in these molecules, not any taken in isolation, that predicted the pain relief.

Beta-endorphins moderate the relationship between FAAH substrates and analgesia

The authors claim the endocannabinoid and opioid systems don’t function independently. They interact in a state-dependent way. The authors hypothesized that beta-endorphin levels modulate the contribution of the endocannabinoid system to analgesia.

They measured beta-endorphins levels by ELISA testing. They tested a statistical model that included three factors: condition (placebo/control), FAAH substrate score, and variations in beta-endorphins. They analyzed this relation with levels of beta-endorphin that were low, moderate and high.

The study showed a significant interaction (p=0.044) when the rise in beta-endorphins is low: endocannabinoids strongly predict the decrease in pain. However, when beta-endorphins are high, endocannabinoids don’t predict pain relief. This suggests opioids could saturate modulating pathways.

Sex, cannabis history and FAAH genotype have no influence on the placebo effect or on circulating endocannabinoid levels

The authors wanted to verify if covariables, such as biological or behavioural factors known to influence the endocannabinoid system, could modify the placebo response. They looked at sex, cannabis usage history and FAAH C385A genotype.

They included these variables as cofactors in their statistical analyses (ANOVA and mixed models). For the genotype, they did a genetic analysis by PCR to separate the carriers of allele A and non-carriers. For cannabis, they compared past and current users to non-users.

None of these factors had a significant effect on the placebo effect or on circulating endocannabinoid levels. Although a trend toward greater placebo effects in women was observed, it didn’t reach statistical significance (p=0.099). FAAH genotype and cannabis use did not modulate the response (p=0.381 and 0.610 respectively).

Why we highlight this preprint

Pain management remains a major clinical challenge in medicine. With widespread opioid dependence, few therapeutic alternatives have emerged for pain management. In this study, the authors elegantly highlight the functional complementarity of the endogenous opioid and endocannabinoid systems in placebo analgesia. This finding not only supports the idea that optimizing clinical analgesia requires simultaneously targeting multiple neuromodulatory pathways but also underscores the importance of accounting for the distinct activation states of these pathways between individuals.

We were initially drawn to this article because we have a substantial interest in pharmacology and pain management. We were initially drawn to this article because, while none of us studies pain directly, our group’s interests in clinical pharmacology, neuroimmunology, and developmental neuroscience all intersect with the questions it raises. From a clinical pharmacology standpoint, the inter-individual variability in placebo response mirrors challenges we encounter in predicting drug efficacy, making the prospect of peripheral biomarkers as predictive tools feel both intuitive and compelling. From a neuroimmunology perspective, the idea that circulating neuromodulators might serve as windows into central processes is a familiar framework, yet seeing it applied to something as elusive as placebo analgesia was a genuine point of excitement for us. Perhaps what struck us most, however, was that despite coming from quite different research backgrounds, we all found ourselves drawn to the same core question this paper raises — which speaks to its breadth.

What captivated us was not pain per se, but the broader idea that subjective experience and expectation can be traced back to identifiable neuromodulatory systems — and that this knowledge could one day translate into more personalized care. Beyond its theoretical implications, this study also appealed to us through its methodological approach. By exploring placebo analgesia using peripheral blood biomarkers, the authors indeed present a strategy likely to deepen our understanding of the molecular mechanisms underlying this phenomenon. Once the relationship between peripheral levels of neuromodulators and the central mechanisms of placebo analgesia is validated, such serum indicators will open a promising avenue for accessible, low-cost personalized medicine, as well as effective stratification of patients with pain.

Questions for the authors

Question 1: It is well established that hormonal fluctuations across the menstrual cycle modulate circulating AEA levels¹⁰. To what extent might the menstrual cycle contribute to the inter-individual variations observed in the placebo response within the female cohort?

Question 2: The endocannabinoid CB1 receptor is broadly expressed in the central nervous system, but also in the peripheral nervous system¹¹. Could placebo analgesia engage local mechanisms in the peripheral nervous system, in addition to the well-documented central pathways?

Question 3: Chronic cannabis use leads to functional neuroadaptation of the CB1 receptor that persists for several weeks after cessation, without significantly influencing serum concentrations of circulating FAAH substrates^12-14. By including both chronic and occasional cannabis users in the cohort without distinction or stratification, could this source of variability nuance the contribution of these substrates to the placebo analgesia demonstrated in this study?

References

1. Benedetti F. Placebo effects: Understanding the other side of medical care. 3rd ed. Oxford: Oxford University Press; 2021.
2. Eippert F, Bingel U, Schoell ED, Yacubian J, Klinger R, Lorenz J, et al. Activation of the opioidergic descending pain control system underlies placebo analgesia. Neuron. 2009;63(4):533-43.
3. Wager TD, Scott DJ, Zubieta JK. Placebo effects on human mu-opioid activity during pain. Proc Natl Acad Sci U S A. 2007;104(26):11056-61.
4. Pecina M, Zubieta JK. Molecular mechanisms of placebo responses in humans. Mol Psychiatry. 2015;20(4):416-23.
5. Benedetti F, Amanzio M, Rosato R, Blanchard C. Nonopioid placebo analgesia is mediated by CB1 cannabinoid receptors. Nat Med. 2011;17(10):1228-30.
6. Cichewicz DL. Synergistic interactions between cannabinoid and opioid analgesics. Life Sci. 2004;74(11):1317-24.
7. Maguire DR, Yang W, France CP. Interactions between mu-opioid receptor agonists and cannabinoid receptor agonists in rhesus monkeys: antinociception, drug discrimination, and drug self-administration. J Pharmacol Exp Ther. 2013;345(3):354-62.
8. Welch SP. Interaction of the cannabinoid and opioid systems in the modulation of nociception. Int Rev Psychiatry. 2009;21(2):143-51.
9. Rodriguez-Munoz M, Onetti Y, Cortes-Montero E, Garzon J, Sanchez-Blazquez P. Cannabidiol enhances morphine antinociception, diminishes NMDA-mediated seizures and reduces stroke damage via the sigma 1 receptor. Mol Brain. 2018;11(1):51.
10. Cui N, Wang L, Wang W, Zhang J, Xu Y, Jiang L, Hao G. The correlation of anandamide with gonadotrophin and sex steroid hormones during the menstrual cycle. Iran J Basic Med Sci. 2017;20(11):1268-1274.
11. Finn DP, Haroutounian S, Hohmann AG, Krane E, Soliman N, Rice ASC. Cannabinoids, the endocannabinoid system, and pain: a review of preclinical studies. Pain. 2021;162(Suppl 1):S5-S25. doi:10.1097/j.pain.0000000000002268.
12. Hirvonen J, Goodwin RS, Li CT, Terry GE, Zoghbi SS, Morse C, et al. Reversible and regionally selective downregulation of brain cannabinoid CB1 receptors in chronic daily cannabis smokers. Mol Psychiatry. 2012;17(6):642-9.
13. Fatemi SA, Abssy SS, Bourke SL, Murray KB, Kyeremaa-Adjei C, Honigman L, et al. The contribution of baseline circulating endocannabinoids to individual differences in human pain sensitivity: a quantitative sensory testing study. bioRxiv [Preprint]. 2025 [cited 2026 Mar 22]. Available from: https://doi.org/10.1101/2025.08.22.671762.
14. Ceccarini J, Kuepper R, Kemels D, van Os J, Henquet C, Van Laere K. [18F]MK-9470 PET measurement of cannabinoid CB1 receptor availability in chronic cannabis users. Addict Biol. 2015 Mar;20(2):357-67. doi: 10.1111/adb.12116. Epub 2013 Dec 27. PMID: 24373053.

Tags: beta-endorphin, endocannabinoid system, faah substrates, human experimental study, opioid system, pain modulation, placebo analgesia

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