a Dual 5-HT Reuptake and PDE4 Inhibitor, in the Human Amygdala and its Connection to the Hypothalamus
David Terburg1,2,5, Supriya Syal2,3,5, Lisa A Rosenberger¹, Sarah Heany², Nicole Phillips², Nigel Gericke4, Dan J Stein² and Jack van Honk1,2
²Department of Psychiatry and Mental Health, University of Cape Town, Cape Town, South Africa
³Department of Psychology, University of Toronto, Toronto, ON, Canada
4HG&H Pharmaceuticals (Pty) Ltd, Bryanston, South Africa
Correspondence: Dr D Terburg, Department of Psychology, Utrecht University, Heidelberglaan 2, Utrecht 3584 CS, The Netherlands, Tel: +31 30 253 3043, Fax: +31 30 253 4511, E-mail: email@example.com
5These authors contributed equally to this work.
Received 26 February 2013; Revised 22 July 2013; Accepted 22 July 2013
Accepted article preview online 1 August 2013; Advance online publication 21 August 2013
The South African endemic plant Sceletium tortuosum has a long history of traditional use as a masticatory and medicine by San and Khoikhoi people and subsequently by European colonial farmers as a psychotropic in tincture form. Over the past decade, the plant has attracted increasing attention for its possible applications in promoting a sense of wellbeing and relieving stress in healthy individuals and for treating clinical anxiety and depression. The pharmacological actions of a standardized extract of the plant (Zembrin) have been reported to be dual PDE4 inhibition and 5-HT reuptake inhibition, a combination that has been argued to offer potential therapeutic advantages. Here we tested the acute effects of Zembrin administration in a pharmaco-fMRI study focused on anxiety-related activity in the amygdala and its connected neurocircuitry. In a double-blind, placebo-controlled, cross-over design, 16 healthy participants were scanned during performance in a perceptual-load and an emotion-matching task. Amygdala reactivity to fearful faces under low perceptual load conditions was attenuated after a single 25 mg dose of Zembrin. Follow-up connectivity analysis on the emotion-matching task showed that amygdala–hypothalamus coupling was also reduced. These results demonstrate, for the first time, the attenuating effects of S. tortuosum on the threat circuitry of the human brain and provide supporting evidence that the dual 5-HT reuptake inhibition and PDE4 inhibition of this extract might have anxiolytic potential by attenuating subcortical threat responsivity.
The South African endemic plant Sceletium tortuosum (L.) N.E. Br. (synonym Mesembryanthemum tortuosum L.), of the succulent family Mesembryathemaceae, has a long history of traditional use by San and Khoikhoi people as a masticatory and medicine (Smith et al, 1996) and later by colonial farmers as a psychotropic in tincture form (Pappe, 1868). Over the past 15 years, the plant has attracted increasing attention for its hypothesized applications in promoting a sense of wellbeing and relieving stress in healthy individuals and for treating anxiety and depression in clinically anxious and depressed patients (Gericke and Viljoen, 2008). A recent in vivo study in rats demonstrated a positive effect of an extract of S. tortuosum on restraint-induced anxiety (Smith, 2011), and a small series of case reports described preliminary evidence for antidepressant and anxiolytic activity in patients suffering from major depression who were treated with tablets of milled S. tortuosum raw material (Gericke, 2001).
Materials and Methods
The active ingredient of Zembrin is a standardized and characterized aqueous ethanolic (purified water 30% V/V and ethanol 70% V/V, spray-dried onto a maltodextrin carrier) extract of the above ground material of a cultivated traditionally used selection of the South African plant S. tortuosum. Figure 1 shows the chemical structures of the four active alkaloids (mesembrenone, mesembrenol, mesembranol, and mesembrine), which were quantified by high pressure liquid chromatography (HPLC) analysis against validated analytical reference standards (Harvey et al, 2011). See Supplementary Information for a detailed description.
Sixteen (eight female) right-handed, healthy undergraduate students (aged 18–21 years, mean=19.2 years) from the University of Cape Town participated in this study. All participants were right-handed, medication-free, had no history of neurological disease or psychopathology (screened using Mini-International Neuropsychiatric Interviews (Sheehan et al, 1998)), had normal or corrected-to-normal vision, and received transportation and remuneration for research time (research credit or ZAR200).
The PLT was adapted from Bishop et al (2007). In each trial, participants saw a string of six letters superimposed on a task-irrelevant unfamiliar face for 200 ms. Participants were required to decide whether the letter string contained an ‘X’ or ‘N’. The letter string comprised six Xs or Ns in the low-load condition, and a single target letter and five non-target letters (HKMWZ) arranged in a semi-random order in the high-load condition. Target letters appeared in each position equally often. Participants responded by a single key press using button boxes held in each hand. ‘N’ and ‘X’ responses were counterbalanced for hand across participants. The faces were of eight different individuals with fearful and neutral expressions (Ekman and Friesen, 1976). Data were acquired for a total of 12 blocks in the high-load and 12 blocks in the low-load condition wherein each block consisted of 4 trials. Load stayed constant across all trials within a block, but emotion varied on a trial-by-trial basis such that each block contained two fearful and two neutral faces presented in a random order. Within blocks, the interstimulus interval was randomly jittered with a mean of 4.5 s and a minimum of 3 s.
The EMT was adapted from Hariri et al (2002). Four blocks of an emotion-matching task were interleaved with five blocks of a sensorimotor-control task involving shape matching. During each 5-s trial in the emotion condition, participants saw a target face on top and two probe faces at the bottom. Participants’ task was to match the emotion of the face on top with one of the other faces using a left or a right key press (corresponding to the position of the face). During the sensorimotor-control shape-matching trials, participants saw oval shapes in a configuration analogous to the faces and were instructed to match the orientation of the oval (vertical or horizontal ellipses) on top to the ovals on the bottom. The emotional faces expressed anger or fear (Tottenham et al, 2009), and luminosity of the oval shapes was matched to that of the faces. Each block consisted of six trials, and blocks were interspersed by a 2-s presentation of instructions for the next block (ie ‘match emotion’ or ‘match shape’).
Participants were scanned in a 3-T Allegra Siemens head scanner (VA25 platform, Siemens Medical Systems GmBH, Erlangen, Germany), using a four-channel receiver. A high-resolution T1-weighted anatomical volume (magnetization-prepared rapid gradient echo) was collected in between the two functional runs using the following parameters: TR/TE=2.53 s/6.6 ms, FA=7°, FOV=256 × 256 × 171, and voxel-size=1.0 × 1.0 × 1.3 mm. Functional volumes (echo-planar imaging) were obtained with 36 interleaved-ascending axial slices, EPI factor=64, TR/TE=2 s/27 ms, flip angle=70°, FOV=224 × 224 × 145 mm, and voxel-size=3.5 mm isotropic. Before the start of each task, five volumes were acquired and discarded to allow for stabilization of the MR signal, and 255 (PLT) and 150 (EMT) functional volumes were acquired for data-analysis. Task-stimuli were back projected onto a screen visible via an angled mirror attached to the helmet coil. Each task was explained to the participants in detail 15 min before the scan, and they completed practice rounds in the scanner before the start of each task.
All MR images were processed and analyzed using SPM8 (www.fil.ion.ucl.ac.uk/spm; Wellcome Department of Imaging Neuroscience, London, UK). Functional volumes were realigned to the first image in the series. The anatomical volume was coregistered to the mean realigned image and transformed to standard space (Montreal Neurological Institute; MNI) using the unified model as implemented in SPM8. As the PLT is an event-related design, these images were slice-time corrected with the 19th slice as reference. Functional volumes were transformed to MNI space using the parameters from the normalization of the anatomical volume, resampled to 2 mm isotropic, and smoothed with an 8-mm full-width half-maximum Gaussian smoothing kernel.
One participant performed below chance level (47% correct), which was >3 SDs below average (85%, SD=4), and was therefore excluded from analysis. A 2 × 2 × 2 repeated-measures ANOVA on the error rates with DRUG, LOAD, and EMOTION as within-subject factors revealed a significant effect of LOAD (F(1,14)=194.2, P<0.00001) but with no other main effects or interactions (all Ps>0.27). Reaction times revealed the same pattern: a significant effect of LOAD (F(1,14)=57.4, P<0.00001) but with no other main effects or interactions (all Ps>0.28; see Figure 2a).
|Hemisphere||Cluster size||Cluster P-value||Peak T-value||Coordinate|
|Perceptual load task:|
|High>low load (see also Figure 2b)|
|Placebo>Zembrin (low load: fear>neutral) (see also Figure 3b)|
|Emotion matching task:|
|Emotion>shape matching (see also Figure 4a)|
|Functional connectivity with amygdala: placebo>Zembrin|
|Main effect (see also Figure 4b)|
Abbreviations: ACC, anterior cingulate cortex; OFC, orbitofrontal cortex; PFC, prefrontal cortex; SMC, supplementary motor cortex.
All effects are whole-brain FWE-corrected with an extend threshold of 20 voxels unless stated otherwise. Coordinates refer to MNI space.
a FWE-corrected for ROI volume.
b FWE-corrected cluster-threshold with uncorrected cluster-defining threshold P<0.001.
The participant excluded from the PLT also scored near to chance level (56% correct) on the EMT, thus was also excluded here. No differences were found in performance for CONDITION or DRUG (emotion matching: 92% correct, SD=9, shape matching: 93%, SD=13).
In this pharmaco-fMRI study, we tested the effects of a single administration of Zembrin, a S. tortuosum extract that can be characterized as a dual PDE4 and 5-HT reuptake inhibitor (Harvey et al, 2011). Using two independent fMRI designs, we show that Zembrin compared with placebo administration reduces anxiety-related (Bishop et al, 2007) amygdala reactivity and attenuates amygdala–hypothalamus coupling. These results not only have importance as the first evidence on the dampening effects of Zembrin on the brain’s threat system but support further work on the clinical applicability of dual PDE4 and 5-HT reuptake inhibitors for the treatment of anxiety disorders and depression.
Funding and Disclosure
This study was made possible by funds from H.L. Hall and Sons, Ltd, South Africa and the Netherlands Organization of Scientific Research (Brain and Cognition Grant 056-24-010). NG is the Director, Medical and Scientific Affairs, of HG&H Pharmaceuticals (Pty) Ltd, the company that has developed the extract of Sceletium tortuosum (Zembrin). DJS has received research grants and/or consultancy honoraria from Abbott, Astrazeneca, Eli-Lilly, GlaxoSmithKline, Jazz Pharmaceuticals, Johnson & Johnson, Lundbeck, Orion, Pfizer, Pharmacia, Roche, Servier, Solvay, Sumitomo, Takeda, Tikvah, and Wyeth. The other authors declare no conflict of interest.
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