Using Fenfluramine to Test the Serotonin Deficiency Theory of Depression

Major depressive disorder (MDD) is a leading cause of disability worldwide and is associated with substantial personal, social, and economic burden. Despite the availability of antidepressant medications, fewer than half of patients achieve an adequate response to first-line treatments, and many fail to reach full remission, highlighting the need for a clearer understanding of the underlying neurobiology to guide more effective and personalised interventions. The most established biological theory of depression is the serotonin deficiency hypothesis, which proposes that in a proportion of patients, depression arises from impaired brain serotonin neurotransmission, particularly within cortical and limbic regions involved in mood regulation. While the efficacy of selective serotonin reuptake inhibitors (SSRIs) and other serotonergic agents is consistent with this hypothesis, most supporting data have been indirect, and prior imaging and peripheral biomarker studies have not provided a definitive, mechanistic test in living humans. Recent advances in positron emission tomography (PET) have made it possible to measure endogenous serotonin release capacity in the human brain. The agonist radiotracer \[11C\]Cimbi-36 binds to the active state of the serotonin 2A receptor (5-HT2A), which is densely expressed in frontal and other cortical regions implicated in mood and affect regulation. Because agonist tracers are more sensitive than antagonist tracers to competition from endogenous neurotransmitters, increases in synaptic serotonin can be inferred from reductions in \[11C\]Cimbi-36 binding following a pharmacological challenge that releases serotonin. Using this methodology with a d-amphetamine challenge, prior work in healthy volunteers established that acute monoamine release reduces \[11C\]Cimbi-36 binding in the frontal cortex and other cortical areas. In a subsequent case-control PET study, patients undergoing a major depressive episode were found to have significantly reduced serotonin release capacity, defined as the percentage reduction in \[11C\]Cimbi-36 binding after d-amphetamine compared to placebo, providing the first direct in vivo evidence of impaired serotonin release in depression. However, d-amphetamine releases dopamine and noradrenaline in addition to serotonin, so it remains unclear whether the observed deficit in release capacity is specific to the serotonin system or reflects broader monoaminergic dysfunction. Dl-fenfluramine is a more selective serotonin-releasing agent that has previously been used as a single-dose challenge in neuroendocrine and neurobiological studies of mood and anxiety disorders. Fenfluramine increases presynaptic serotonin release and leads to downstream effects such as prolactin elevation and changes in thermoregulation, which can be blocked by 5-HT2A receptor antagonists, indicating that its functional effects depend on serotonin acting at postsynaptic 5-HT receptors rather than direct agonist activity at 5-HT2A itself. Preclinical PET and microdialysis work has shown that fenfluramine produces robust serotonin release and larger reductions in \[11C\]Cimbi-36 binding than d-amphetamine, suggesting that a fenfluramine challenge may offer a more sensitive and specific assay of cortical serotonin release capacity. The FenDep study is a case-control, observational brain imaging study designed to use dl-fenfluramine and \[11C\]Cimbi-36 PET to test the serotonin deficiency hypothesis of depression and to explore whether baseline serotonin release capacity predicts subsequent SSRI treatment response. The study will enrol approximately 26 adults with moderate to severe MDD and 20 healthy control (HC) participants. Following an initial pre-screening telephone contact, potentially eligible individuals will be screened for mental and physical health. Screening also includes completion of a battery of psychometric instruments assessing depression, anxiety, stress, rumination, anhedonia, well-being, personality traits, and connectedness. Eligible participants then attend two imaging visits, separated by approximately 14 days. The study uses a single-blind, balanced-order design in which each participant receives dl-fenfluramine (60 mg oral liquid) on one imaging day and a placebo on the other, with the order counterbalanced across participants. Dosing occurs approximately 3.5 hours before the PET scan to coincide with peak plasma levels of dl-fenfluramine. PET imaging is performed with intravenous administration of up to 300 MBq of \[11C\]Cimbi-36, followed by 90 minutes of dynamic data acquisition and arterial blood sampling via a radial arterial cannula to allow kinetic modelling of radiotracer uptake and metabolism. A venous cannula is used for tracer injection and additional venous sampling. The primary region of interest is the frontal cortex, with the cerebellum serving as a reference region for calculation of non-displaceable binding potential (BPND) using the multilinear analysis-1 (MA1) model. Serotonin release capacity (SRC) is operationalised as the relative reduction in \[11C\]Cimbi-36 BPND after dl-fenfluramine compared with placebo. Alongside PET, a comprehensive multimodal imaging, including Magnetic Resonance Imaging (MRI) and electroencephalography (EEG), and a behavioural task battery is collected during the imaging visits. Visit procedures also include repeated measurements of blood pressure and heart rate, pharmacokinetic assessment of dl-fenfluramine and prolactin, and subjective drug effect ratings. After completing both imaging visits, MDD participants return to their usual clinical care. In case participants initiate SSRI treatment as part of standard practice, the study team conducts three remote follow-up assessments by video call over an 8-week observational period. At these contacts, clinician-rated and self-report depression scales and other relevant questionnaires are administered. The primary clinical outcome is change in the Montgomery-Åsberg Depression Rating Scale (MADRS) score after 8 weeks of SSRI treatment, with response defined as a 50% or greater reduction from baseline, enabling investigation of whether baseline serotonin release capacity and related imaging/biomarker measures predict antidepressant response. The primary objective of FenDep is to compare fenfluramine-induced serotonin release capacity in cortical regions between unmedicated adults with MDD and healthy controls using \[11C\]Cimbi-36 PET. By combining a selective serotonin releaser with a validated PET methodology for measuring serotonin release, multimodal imaging, behavioural tasks, blood-based biomarkers, and prospective SSRI treatment follow-up, FenDep aims to deliver a rigorous test of the serotonin deficiency hypothesis in MDD and to identify mechanistically informed biomarkers that could support future stratified treatment approaches.