Using Fenfluramine to Test the Serotonin Deficiency Theory of Depression

Using Fenfluramine to Test the Serotonin Deficiency Theory of Depression

Clinical depression is a common and disabling condition characterised by persistent low mood and loss of interest that interferes with daily functioning. Serotonin is a key brain neurotransmitter involved in mood regulation, and a leading theory proposes that depression is associated with impaired serotonin function (the serotonin deficiency hypothesis), which underpins the use of selective serotonin reuptake inhibitors (SSRIs) as first-line antidepressant treatments. However, the strength and specificity of the link between serotonin dysfunction and depressive symptoms in humans remains uncertain and requires direct evidence in living human brains.

Positron Emission Tomography (PET) allows in vivo quantification of neurotransmitter receptor systems using a radioactive tracer that binds to specific brain targets. The serotonin 2A receptor (5-HT2A) agonist tracer [11C]Cimbi-36 enables measurement of the active-state 5-HT2A receptor, which is highly expressed in cortical regions implicated in mood regulation. When combined with a pharmacological challenge that acutely increases serotonin levels, changes in [11C]Cimbi-36 binding can be used to estimate serotonin release capacity across different brain regions.

Previous work using an amphetamine challenge with [11C]Cimbi-36 has shown reduced serotonin release capacity in the frontal cortex of patients with depression compared with healthy controls, providing preliminary support for the serotonin deficiency hypothesis. However, amphetamine releases multiple neurotransmitters in addition to serotonin, limiting the ability to attribute these effects specifically to serotonergic dysfunction. Dl-fenfluramine is a more selective serotonin-releasing agent and therefore offers a targeted approach to probe serotonin release in the human brain.

This case-control observational study will compare serotonin release capacity between unmedicated adults with Major Depressive Disorder (MDD) and healthy control participants using dl-fenfluramine challenge combined with [11C]Cimbi-36 PET imaging. The primary objective is to test whether individuals with MDD show reduced fenfluramine-induced serotonin release, indexed by changes in [11C]Cimbi-36 binding, relative to healthy controls. Secondary objectives include exploring how multimodal imaging, blood biomarkers, and behavioural measures relate to serotonin release capacity and depressive symptom severity.

Following completion of imaging, participants with MDD who will start SSRI treatment as part of their usual clinical care will be followed for 8 weeks with remote assessments. The study will examine whether baseline measures of serotonin release capacity predict subsequent clinical response to SSRIs, defined primarily by change in clinician-rated depression scores over the treatment period. Together, these data aim to provide a more precise test of the serotonin deficiency hypothesis of depression and to identify potential biomarkers of SSRI treatment response in MDD.

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.

Inclusion Criteria for All Participants:

• Aged 21 years and over.
• Able to lie comfortably on their back for scanning.
• Participants must agree to use one of the contraception methods listed in Appendix 1.
• Capable of providing written informed consent and willing to comply with the requirements and restrictions listed in the consent form.
• Able to read, comprehend, and record information written in English.
• Able to access the internet through their own electronic device.

Inclusion Criteria for Participants with Major Depressive Disorder (MDD):

• Major depressive episode diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) as moderate to severe.
• Scoring above 20 on the Montgomery-Åsberg Depression Rating Scale (MADRS).
• Have never taken antidepressants, or are currently unmedicated for at least 8 weeks before signing the informed consent form.
• Not classified as treatment-resistant.
• Ongoing relationship with a general practitioner (GP) or other healthcare professional.

Inclusion Criteria for Healthy Controls:

- Healthy as determined by a responsible physician, based on a medical evaluation including medical history, physical examination, and laboratory tests.

Exclusion Criteria for All Participants:

• Presence of a general medical or psychiatric condition (excluding MDD in the relevant population), as revealed by a physical and psychiatric examination, which, in the opinion of the Principal Investigator (PI), would impair the safety of the participant or the scientific integrity of the study.
• Ongoing treatment with medication that, in the opinion of the investigator, would compromise participant safety or the scientific integrity of the study, including but not limited to compounds known to interact with the serotonin 2A (5-HT2A) receptor or to have a significant effect on the synthesis and/or release of serotonin (5-HT).
• Use of illicit compounds in the 3 months before consent, including but not limited to classic psychedelics, stimulants, 3,4-methylenedioxymethamphetamine (MDMA), and cannabis.
• Unwillingness or inability to follow the procedures outlined in the protocol.
• The participant is mentally or legally incapacitated.
• Contraindications to blood sampling and/or arterial cannulation, including but not limited to peripheral vascular disease or Raynaud's phenomenon.
• Contraindications to the administration of dl-fenfluramine include medications that have a significant effect on the synthesis and/or release of 5-HT.
• Abnormal Allen's test and/or prolonged Prothrombin Time (PT), due to the arterial cannulation required for the positron emission tomography (PET) scans.
• Participation in another research study involving ionising radiation exceeding 10 mSv within the last year.
• Contraindications to magnetic resonance imaging (MRI) scans include, but are not limited to, pacemakers, recent metallic implants, foreign bodies in the eye, or other contraindications identified by a standard pre-MRI questionnaire.
• Claustrophobia or any other condition that would render the participant incapable of undergoing MRI/PET scanning.
• Pregnancy or breastfeeding.

Exclusion Criterion for MDD Participants:

- History of suicide attempts requiring hospitalisation.

Exclusion Criteria for Healthy Controls:

• History of an Axis I psychiatric diagnosis.
• History of a neurological or general medical illness that, in the opinion of the investigator, would compromise participant safety or the scientific integrity of the study.