5-Hydroxytryptamine (5-HT), a monoamine neurotransmitter, first recognised in 1948, 2 has a wide range of functions in the central nervous system (CNS), including modulation of attention, cognition, behaviour, memory, and thermoregulation, as well as in the peripheral nervous system (PNS), where it regulates, for example, gastrointestinal (GI) motility, uterine contraction, vasoconstriction, and bronchoconstriction. 3 The first report of a clinical picture consistent with what nowadays is termed SS was in 1960, 4 which described the coadministration of L-tryptophan (the substrate of the rate-limiting enzyme, tryptophan hydroxylase [TPH], in the biochemical synthesis of serotonin) with a monoamine oxidase inhibitor (MAOI) inducing delirium. The first use of the term SS came 20 years later, describing the characteristic features in rats of tremor, rigidity, hypertonicity, hind-limb abduction, Straub tail, lateral head shaking, hyperactivity to auditory stimuli, myoclonus, generalised seizures, and various autonomic responses such as salivation, penile erection, and ejaculation. 5 Two years later, the first contemporaneous case in humans was reported by Insel et al, 6 followed by numerous case reports and reviews. 7 - 11

Epidemics of ‘convulsive ergotism’ were widespread and well described east of the Rhine river from 1085 to 1927. 1 Given that ergot alkaloids, a known contaminant of human cultivated grains, are now recognised to induce serotonin syndrome (SS), this suggests that the syndrome may have been a public health problem long before its more recent ‘discovery’ as a complication of modern pharmacology. 1

The true incidence of SS, however, is unknown, as is the number of cases that are mild, moderate, or severe. There are a number of reasons for this: it is a relatively uncommon condition that cannot be easily picked up in randomised clinical control trials, 20 and the condition is under-recognised and under-reported by physicians (85% of general physicians [GPs] were not familiar with the condition in one survey, 21 and mild cases are often dismissed or self-limiting). 20

Serotonin syndrome is observed across the full range of age groups, from neonates all the way through to the elderly, with an increasing incidence likely to represent the increasing use of serotonergic drugs in clinical practice. 7 , 12 , 13 The percentage of adults taking antidepressants in the United States nearly doubled between 1999 and 2010, increasing from 6% to 10.4%. 14 In 2016, the Toxic Exposure Surveillance System, which receives case descriptions from emergency departments, inpatient settings, and office-based practices, reported 54 410 incidences of exposures to selective serotonin reuptake inhibitors (SSRIs; 43% of which were single exposures), with 102 deaths (the ninth most common cause of fatality in drug overdoses in the United States in this period). This represented an 18% increase in cases between 2002 and 2016 and an 8% increase in the number of deaths. 15 - 17 Large case series suggest that moderate SS occurs in approximately 15% of poisonings with SSRIs. 18 A recent study found that close to half of U.S. Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS) SS reports involved a single drug (depending on the diagnostic criteria used), which is, perhaps, a little surprising, given the common perception of a multidrug aetiology. 19

Pathophysiology and Molecular Mechanisms

The serotonin signalling pathway The cells of the raphe nuclei located in the midline of brainstem (from the midbrain to the medulla) are the source of neuronal 5-HT in the CNS.22 The 9 raphe nuclei (B1-B9) are centred in the reticular formation, and axons from the serotonergic neurons in this region form a neurotransmitter system that functionally affects most parts of the CNS. The rostral group of nuclei project into multiple cortical and subcortical structures contributing to the regulation of wakefulness, attention, affective behaviour (depression and anxiety), sexual behaviour, appetite, thermoregulation, and migraine.22 The caudal group project into the spinal regions and are involved in motor tone as well as nociception.22 In the periphery 5-HT, produced in the enterochromaffin (EC) cells of the GI tract, is involved in the regulation of GI motility as well a number of roles in vascular biology.22 These include control of blood pressure through vasoconstriction or vasodilation depending on the expressed receptor type in the vessel wall, as well as control of haemostasis and platelet function. Serotonin is secreted during platelet activation, playing an important role in their aggregation and concomitant vasoconstriction of the surrounding blood vessels during haemostasis. Platelets lack enzymes to synthesise serotonin,23 and are therefore reliant on taking up serotonin from the plasma via the serotonin transporter. Selective serotonin reuptake inhibitors can inhibit the uptake of serotonin by platelets leading to decreased aggregation responses and thereby increasing bleeding time.24 For this reason, SSRIs should be used in caution in those patients on anticoagulants or at higher risk of bleeding. Intriguingly, there is emerging evidence that SSRI treatment may lower myocardial infarction risk though their inhibition of platelet aggregation.25,26 The occurrence of disseminated intravascular coagulation in severe SS27 may be due to an exaggerated platelet aggregation response driven by excess peripheral serotonin. The initial and rate-limiting step in the biosynthesis of 5-HT, the decarboxylation and hydroxylation of the essential amino acid tryptophan, is catalysed by TPH.28 There are 2 isoforms of this enzyme, TPH1 and TPH2, which are highly homologous but differ in their kinetic properties and their tissue distribution.29,30 Tryptophan hydroxylase 1, the source of most peripheral 5-HT, is predominantly expressed in the EC cells of the gut and other peripheral tissues, such as the pancreas, lung, and adipose tissue, though is also expressed in the pineal gland where 5-HT serves as the precursor molecule for melatonin biosynthesis.29,31,32 Tryptophan hydroxylase 2, the source of the central neurotransmitter pool of 5-HT, is predominantly expressed in the raphe projection neurons of the brainstem,29,33 though is also found in the myenteric neurons of the gut.32,34 Work with knockout animals suggests that TPH1 expression in the pineal gland does not significantly contribute to the central 5-HT pool,35 and similarly, TPH2 expression in the myenteric plexus does not significantly contribute to the peripheral 5-HT pool.36 Once synthesised from tryptophan, 5-HT is then stored in presynaptic vesicles until it is required for neurotransmission and released into the synaptic cleft. Serotonergic signalling is a tightly regulated process characterised by a combination of feedback loops, multiple reuptake mechanisms, and metabolising enzymes (Figure 1). Depolarisation of the presynaptic axon leads to the release of serotonin into the synaptic cleft, which leads to multiple downstream effects; activation of presynaptic serotonin autoreceptors inhibits exocytosis of further vesicles, while binding to postsynaptic 5-HT receptors effects neurotransmission. Serotonergic signalling is terminated primarily by the uptake of 5-HT from the synaptic cleft back into the presynaptic neuron. This is accomplished predominantly by the serotonin reuptake transporter protein (SERT) on the presynaptic membrane, though there is evidence that the plasma membrane monoamine transporter (PMAT) may also contribute.37 5-Hydroxytryptamine is then metabolised into 5-hydroxyindoleacetic acid (5-HIAA) primarily by monoamine oxidase A (MAO-A). Download Open in new tab Download in PowerPoint There are at least 7 families of 5-HT receptors (5-HT 1 to 5-HT 7 ), some of which have multiple subtypes, resulting in a total of 14 structurally and pharmacologically distinct receptors.38 The 5-HT 1 receptor family consist of 5 subtypes: 5-HT 1A , 5-HT 1B , 5-HT 1D , 5-HT 1e , and 5-HT 1F receptors are G-protein-coupled receptors (GPCRs) that signal via the inhibition of adenylate cyclase or G-protein-sensitive K+ channels (note that the 5-HT 1e receptor is still just recognised as a gene product, since no function has been attributed to this receptor in native tissue). While a number of clinically used drugs affect these receptors, the 5-HT 1A receptor provides a pharmacological target for the anxiolytic agent, buspirone, which lacks potential for dependency. However, 5-HT 1B/1D (and possibly 5-HT 1F ) receptors are targeted by the antimigraine ‘triptans’, examples of which include sumatriptan and zolmitriptan (which are also used for cluster headaches). The 5-HT 2 receptor family consists of 3 members: 5-HT 2A , 5-HT 2B , and 5-HT 2C receptors that are excitatory GPCRs signalling via Gq to activate, for example, phospholipase C to generate Inositol 1,4,5-triphosphate (IP3) and diacyl glycerol. Many antipsychotic drugs at relevant clinical concentrations interact with 5-HT 2 receptors, with antagonism of the 5-HT 2A receptor likely to contribute to therapeutic effects (eg, risperidone). More recently (2012), the 5-HT 2C receptor has been targeted selectively with the agonist, lorcaserin, to treat obesity by reducing food intake (although not yet approved in Europe). The 5-HT 3 receptor is a ligand-gated ion channel of the cys-cys loop class that conducts Na+/K+/Ca2+; this excitatory receptor is expressed predominantly by neurons and is responsible for initiating emesis following aggressive anticancer therapy (that releases copious quantities of 5-HT from the EC GI tract) such that antagonists such as ondansetron offer effective antiemetic treatment. The 5-HT 3 receptor also mediates prokinetic activity of the GI tract – as occurs in irritable bowel syndrome (IBS) or carcinoid patients; for both patient cohorts, 5-HT 3 receptor antagonists are beneficial to reduce diarrhoea, although currently, they are only indicated for IBS (and only in some territories like the United States and Japan). The 5-HT 4 receptor is a GPCR that promotes adenylate cyclase activity via Gs. The receptor increases gastric motility by increasing the activity of, for example, cholinergic neurons in the GI tract and is activated by some drugs used to reverse constipation (eg, prucalopride). The 5-HT 5 receptors are GPCRs and the family has 2 members: 5-HT 5A and 5-HT 5b receptors. The latter receptor is not functional in humans due to the insertion of a stop codon into the gene that results in a truncated protein. Little is known about the 5-HT 5A receptor, and it is not actively targeted for therapeutic benefit. The 5-HT 6 and 5-HT 7 receptors are both GPCRs that couple via Gs to promote cAMP synthesis via adenylate cyclase. There has been much interest in these as potential clinical targets, largely due to the known interaction of a wide range of antipsychotic and antidepressant drugs that antagonise these receptors. However, to date, no clinically used drug selectively targets either receptor. Further structural and functional diversity occurs in 5-HT receptors through allelic polymorphisms, splice variants, and the formation of receptor heterodimers.34,39

Pharmacology of serotonin and cellular toxicity Serotonin syndrome can result from agonism (either from increased concentrations of 5-HT or medications that act directly as receptor agonists), and/or antagonism, of varying combinations of the 5-HT receptor subtypes. Stimulation of the postsynaptic 5-HT 1A and 5-HT 2A receptors has been implicated in SS, but no single receptor is likely to be solely responsible. The classical view, mainly supported by evidence from animal studies (with few studies in humans), is that the life-threatening effects of SS, in particular, severe hypertonicity and hyperthermia, are primarily mediated by the activation of 5-HT 2A receptors at higher serotonin concentrations.18 The 5-HT 1A receptors, which have a higher affinity for 5-HT and are therefore likely to be nearly fully occupied at much lower extracellular 5-HT concentrations, possibly contribute to some of the milder symptoms including anxiety and hyperactivity.40 This may provide a pharmacological explanation for why severe SS is usually only seen with particular combinations of drug classes, in particular MAOIs (that increase 5-HT concentration in the synapse) and SSRIs (that modulate 5-HT receptor signalling at the postsynaptic membrane in addition to inhibiting serotonin reuptake via the SERT).18,41,42 Recently, a bioinformatics approach has demonstrated an association between second-generation antipsychotics (SGAs) and SS.43 Counter to the classical view that 5-HT 2A agonism is a significant contributor to severe SS, the authors have suggested a potential role for 5-HT 2A antagonism in concert with 5-HT 1A agonism as a common mechanism that may explain the SGA-SS association. It is this effect on the serotonergic system that distinguishes the SGAs (‘atypical’ antipsychotics) from the older ‘typical’ antipsychotics (butyrophenones, diphenylbutylpiperidines, and phenothiazines) that predominantly antagonise D2 dopaminergic receptors and are not associated with SS. They postulate that in the presence of SERT inhibition, 5-HT levels are increased throughout serotonergic synapses, and 5-HT 2A antagonism then shunts elevated levels of 5-HT to the co-localised 5-HT 1A receptor.44,45 This selective activation of the 5-HT 2A receptor not only appears to increase its sensitivity to 5-HT46-48 but may also enhance the effects of 5-HT 1A agonists.49,50 In addition, there is evidence that 5-HT 2A receptor antagonists may elevate 5-HT neurotransmission via a disinhibitory feedback loop, through inhibition of local gamma-aminobutyric acid (GABA) interneurons in the dorsal raphe nucleus.51 This association (and potential mechanistic explanation) is reinforced by a recent meta-analysis of SS cases, in which 3% (10/299) of cases were associated with SGAs as the decisive triggering factor. Eight of these cases occurred in combination with antidepressants and 2 in the context of an SGA swap. Interestingly, in this study, 45.4% of the SS cases had rigidity/hypertonicity, and 14.0% of cases had rhabdomyolysis and symptoms also commonly associated with neuroleptic malignant syndrome (NMS).20 Whether 5-HT 2A receptor antagonism in concert with 5-HT 1A agonism plays a role in all cases of SS or is the exception in the case of SGAs remains to be determined. An additional question is whether other neurotransmitters play a role, particularly in the development of severe SS. A full review of the large body of evidence in this area is beyond the scope of this review, though it is important to emphasise that active research is ongoing, and many questions remain to be definitively answered. In animal models, it has been shown that 5-HT can cause the release of noradrenaline from the anterior hypothalamus, a neurotransmitter known to cause CNS hyperexcitability, which may correlate with the clinical outcome.52-54 Gamma-aminobutyric acid, N-methyl-D-aspartate (NMDA), and dopamine may also play a role through their recruitment, in addition to direct serotonin stimulation, in severe SS.52,55 A recent study using a systematic bioinformatics approach to analyse off-target effects of the drugs most commonly involved in SS supports the hypothesis that multiple neurotransmitter pathways may be recruited in addition to direct serotonin stimulation in severe SS.19 As well as highlighting the importance of SERT inhibition (46/71 serotonergic drugs interacted with this protein), there was a higher than expected association of SS cases in the triple receptor drug cohort (drugs that interact at SERT, norepinephrine transporter [NET], and muscarinic receptors).19 Off target receptor activity may help to explain the differences in the severity of the syndrome as well as the spectrum of clinical features. These findings raise the possibility that when the offending drug (or combination of drug classes) has (have) both antidopaminergic and serotonergic activity,20 the resulting toxidrome may share features of both SS and NMS.

Drugs commonly associated with SS A wide range of drug types and combinations have been implicated in SS, with the final common pathway thought to involve a net increase in serotonergic neurotransmission. The main drug classes classically implicated in SS can be divided into serotonin precursors, inhibitors of serotonin reuptake from the synaptic cleft, inhibitors of serotonin metabolism, direct serotonin receptor agonists, and drugs that sensitise serotonin receptors (Table 1). As discussed in the previous paragraph, there is evidence that serotonin antagonism (in particular 5-HT2A antagonism) may also play a role, based on the observed association between SGAs and SS.43 Table 1. Drugs associated with serotonin syndrome. View larger version Pharmacokinetic drug interactions are also implicated through the inhibition of the cytochrome P450 pathway, a pathway that SSRIs themselves inhibit (in particular CYP2D6 and CYP3A4.56,57 At least 25 serotonergic drugs are metabolised by the cytochrome P450 pathway,19 and a recent study has shown that 50% of the top 20 drugs associated with SS have known pharmacokinetic interactions in which coadministration of cytochrome P-450 inhibitors may elevate drug concentrations to toxic levels. These drugs collectively were shown to participate in > 70% of all the reported SS reports in their study.19 Examples include the concomitant use of SSRIs and tramadol,12,58 SSRIs and ciprofloxacin,59 as well as of citalopram and fluconazole.60 Although SS has been described after overdose of a single drug,61 and occasionally from increasing therapeutic doses in susceptible individuals, this only usually results in mild to moderate SS.4,13,62 Severe SS usually only occurs with the concomitant administration of 2 or more serotonergic drugs (even at therapeutic doses) (Table 2), with the combination of serotonergic drugs with MAOIs being especially dangerous, causing serious adverse outcome including death.8,41,42 Table 2. Reported drug combinations causing moderate to severe serotonin syndrome.7,8,11 View larger version Taken together, these observations are consistent with the hypothesis that SS is a dose-dependent spectrum of adverse effects mediated by a combination of elevation of endogenous intrasynaptic or extrasynaptic serotonins and direct or indirect activation of 5-HT receptors.63 However, the fact that different patients present with SS at differing drug dosages and/or combinations suggests that there may also be underlying genetic and pharmacodynamic factors that modulate susceptibility.11 Genetic factors could include polymorphisms in the CYP450 pathway,64 the SERT gene,65 as well as the 5-HT 2 receptor.18,13,66