Deciphering Ibogaine’s Matrix Pharmacology: Multiple Transporter Modulation at Serotonin Synapses

Abstract

Ibogaine is the main psychoactive alkaloid produced by the iboga tree (Tabernanthe iboga) that has a unique therapeutic potential across multiple indications, including opioid dependence, substance use disorders, depression, anxiety, posttraumatic stress disorder (PTSD), and traumatic brain injury (TBI). We systematically examined the effects of ibogaine, its main metabolite noribogaine, and a series of iboga analogs at monoamine neurotransmitter transporters, some of which have been linked to the therapeutic effects of these substances. We report that ibogaine and noribogaine inhibit the transport function of the vesicular monoamine transporter 2 (VMAT2) with sub-micromolar potency in cell-based fluorometry assays and at individual synaptic vesicle clusters in mouse brain as demonstrated via two-photon microscopy. Examining the uptake and release of radiolabeled serotonin in isolated brain synaptic vesicles, we confirmed that ibogaine and noribogaine act as inhibitors of VMAT2 and showed that noribogaine induces partial serotonin release from synaptic vesicles. Noribogaine does not compete with binding of dihydrotetrabenazine, an established VMAT2 inhibitor, indicating that noribogaine engages the VMAT2 transporter at a distinct binding site or conformational state. The iboga compounds also inhibit the plasma membrane monoamine transporters (MATs), prominently including the serotonin transporter (SERT), and a novel iboga target, the organic cation transporter 2 (OCT2). SERT transport inhibition was demonstrated in serotonin axons and somata in mouse brain slices and in rat brain synaptosomes, where ibogaine and its analogs did not act as substrate-type serotonin releasers. Noribogaine, oxa-noribogaine, and several analogs displayed dual inhibition of VMAT2 and SERT with comparable potency, a relatively uncommon activity in the pharmacopeia of monoamine transporter inhibitors, and were hence labeled as “Synaptic Reuptake Inhibitors” (“SynRIs”). The SynRI profile provides an explanatory model for previously reported neurochemical effects of ibogaine in rodents. Together, the updated profile of the monoamine transporter modulation offers insight into the grand complexity of the iboga pharmacology, which we termed “matrix pharmacology”. The matrix pharmacology hypothesis is outlined and used to conceptualize why ibogaine and noribogaine do not induce catalepsy, as demonstrated in our study, in contrast to other VMAT2 inhibitors.

Graphical Abstract

Introduction

Ibogaine is the main psychoactive principle of the iboga plant (Tabernanthe iboga), indigenous to West Central Africa.¹ The iboga root has been used for centuries to induce visionary states and transpersonal spiritual experiences during ceremonies and has served as a centerpiece for indigenous spiritual practices.² In addition to traditional use, ibogaine and iboga have been reported to interrupt dependence on opioids and other reinforcing drugs, including alcohol and cocaine.^3,4 Through the work of several generations of pioneers, pursued mostly outside the mainstream medical system, ibogaine appears to provide a new paradigm for treating substance use disorders (SUDs) – albeit a controversial one due to its adverse cardiac effects.^5,6 This paradigm consists of one or a few treatment sessions with ibogaine, which have acute and long-lasting effects as reported in numerous observational surveys and case studies.^3,7 The acute effects include dream-like experiences (oneiric effects) that often provide psychological insights and reduction in physical drug dependence (i.e., large effects in reducing acute withdrawal symptoms). Long-term effects include attenuation of drug craving and improvement in mood and anxiety symptoms. It may thus be that ibogaine provides a treatment that addresses multiple core aspects of SUDs.^8,9 The clinical observations have been supported by numerous preclinical studies that report therapeutic-like effects of ibogaine in rodent models of SUDs and depression.^10,11

However, administration of ibogaine or iboga materials can lead to rare but severe cardiac adverse effects and sudden death which have presented substantial obstacles to the development of ibogaine as an FDA-approved medication.¹² Nevertheless, individuals desperate for relief from their suffering have sought ibogaine treatment in clinics abroad, a trend that has provided pilot clinical observations.¹³

In addition to the reported anti-addiction effects of ibogaine, its putative therapeutic scope encompasses other psychiatric and neurological conditions, often co-morbid with SUDs, including PTSD and mild traumatic brain injury (mTBI), according to surveys of combat veterans with blast exposures.^14,15 Although the clinical data supporting therapeutic efficacy of ibogaine are almost exclusively based on open label observational studies, the reported effect sizes are large across neurological and mental health assessment scales (for example, > 90% response rates and 80% remission after a single treatment).¹⁵ Collectively, the current evidence indicates a trans-diagnostic therapeutic potential of ibogaine rooted in neurorestorative effects across biological scales including molecular, physiological, psychological, and social-spiritual domains.

The emerging therapeutic profile of ibogaine leads to a fundamental question of how such a range of neurorestorative effects may be induced and mediated on a molecular level. Ibogaine is metabolized in vivo to form noribogaine, in both humans and rodents, where the two compounds likely act in tandem due to overlapping pharmacokinetic profiles.^11,16–18 The reported pharmacological targets and associated effects provide a window on the complexity of signaling pathways that ibogaine and noribogaine engage. The combined action of the two compounds modulates several classes of molecular targets and pathways, including ion channels, neurotransmitter transporters, G protein coupled receptors (GPCRs) and their signaling, and kinase signaling pathways via several modes of action such as ion channel blockade,¹⁹ monoamine transporter inhibition via an atypical mechanism,²⁰ monoamine transporter pharmaco-chaperoning effect,^21,22 and kinase signaling pathway potentiation.²³ Interestingly, these wide-ranging effects typically involve low potency drug interactions in the micromolar range (Figure 1).^24–27

Figure 1. Proposed global model of ibogaine’s pharmacology, downstream molecular signaling, neuro-restorative processes, and clinical therapeutic and adverse effects.

(A) Ibogaine is metabolized to noribogaine and both compounds contribute to the complex pharmacology. (B) Examples of known primary molecular targets; the present study focuses on monoamine transporters including the vesicular monoamine transporter 2 (VMAT2) and organic cation transporter 2 (OCT2), disclosed as novel targets in this study. Ibogaine and noribogaine act as weak modulators of several targets and signaling pathways via multiple modes of action, such as blockers of ion channels, atypical inhibitors of monoamine transporters, pharmaco-chaperones of monoamine transporters (SERT and DAT), and potentiators of downstream signaling pathways – a functional profile termed “matrix pharmacology”. Functional potency or affinity at targets shown in grey is lower in comparison to those shown in black. (C) The downstream effects include, for example, expression changes of neurotrophic factors such as glial cell derived neurotrophic factor (GDNF) and brain derived neurotrophic factor (BDNF), and potentiation of receptor tyrosine kinase signaling pathways. Oxytocin (OT) signaling has been implicated in mediating reopening of the critical development-like learning states. (D) The indicated pathways mediate the neuroplasticity and neurorestorative processes, to which oneiric and psychedelic states may contribute. (E) These processes collectively likely mediate the profound therapeutic effects across biological scales.

Here, we introduce the term “matrix pharmacology” to describe this mechanistic model, defined as modulation of multiple molecular targets and signaling pathways via several modes of action with weak or modest-potency molecular interactions. As the collective data suggests that iboga compounds act throughout the signalome’s information-processing matrix, we wish to differentiate this mechanistic model (matrix pharmacology) from “selective polypharmacology”, which is characterized by potent agonism or antagonism at several targets, such as G protein coupled receptors (GPCRs), as exemplified by LSD.^28,29 A molecular logic of iboga matrix pharmacology is emerging in which iboga compounds do not directly modulate GPCRs (with some exceptions) but rather 1) modulate neurotransmitter dynamics via direct inhibition and modulation of the monoamine transporters, 2) engage ion channels as channel blockers and or antagonists, and 3) potentiate downstream signaling pathways of GPCRs and receptor tyrosine kinases (RTKs).

As downstream effectors activated by the matrix pharmacology, iboga compounds modulate gene expression and protein levels of neurotrophic factors in multiple brain regions with relevance to SUDs and other mental health disorders.^30,31 Increased myelination markers were reported in morphine-dependent mice after a single ibogaine administration, indicating the possibility of white matter restoration.³² It was suggested that ibogaine increases oxytocin gene expression,³³ while another report demonstrated induction of a form of metaplasticity in the nucleus accumbens (NAc) that sensitizes local synaptic connections to oxytocin, which correlated with reopening a critical learning period for social reward in adult mice (Figure 1).³⁴ These results together with the compounds’ behavioral effects indicate induction of both specific (e.g., reversal of drug dependence phenotype) and broad (e.g., induction of metaplasticity) restorative programs and processes.

In this report, we focus on mapping the actions of iboga compounds at monoamine transporters, some of which have previously been implicated in ibogaine’s anti-addiction and oneiric effects.²⁰ The plasma membrane monoamine transporters (MATs), including the dopamine transporter (DAT), norepinephrine transporter (NET), and serotonin transporter (SERT), are essential regulators of monoamine neurotransmission that directly modulate the magnitude and duration of the synaptic activity by rapid removal and recycling of the monoamine neurotransmitters from the extracellular space. They constitute a branch of a solute carriers 6 family (SLC6; also known as neurotransmitter sodium symporters, or NSS) that mediate the directional transport of endogenous monoamine transmitters by coupling their translocation to the transmembrane sodium ion gradient.^35,36 DAT and NET are located in the corresponding catecholamine neurons, whereas SERT is expressed in 5-HT neurons, blood-brain barrier (BBB) cells, enterochromaffin cells, and blood platelets.^37,38 MATs are prominent targets of psychoactive substances and medications that inhibit one or more monoamine transporters, including cocaine, methylphenidate, tricyclic antidepressants, and selective serotonin reuptake inhibitors.^39–41 They are also targets for monoamine releasers, such as amphetamine and MDMA, that act as MAT substrates and induce efflux of monoamine transmitters from the cytoplasm via these transporters, often referred to as “reverse transport”.⁴²

The functional dynamic range of MAT transporters, described as high-affinity, low-capacity transporters (uptake 1) is complemented by the low-affinity, high-capacity transporters (uptake 2) comprised of transporters from several different SLC families including organic cation transporters 1–3 (OCT1–3, SLC22A1–3), plasma membrane monoamine transporter (PMAT, SLC29A4), and additional more recently identified transporters.⁴³ These transporters generally show as much as two orders of magnitude lower apparent affinity for biogenic monoamines but greater maximal transport velocity than MATs, and are equilibrative transporters driven by the concentration gradient of the substrates. OCT1–3 and PMAT have distinct but often overlapping expression patterns in the CNS on neurons (presynaptic and postsynaptic) and glial cells (Figure 2).⁴³

Figure 2. Ibogaine and noribogaine exert dual inhibition of vesicular and plasma membrane transporters.

(A) Graphic representation of ibogaine’s (left panel) and noribogaine’s (right panel) inhibitory profile at the vesicular monoamine transporter 2 (VMAT2), uptake 1 and uptake 2 monoamine transporters. Ibogaine inhibits hVMAT2 with submicromolar potency (IC_{50, hVMAT2} = 390 nM) and modest selectivity of around one log unit over hSERT (IC_{50, hSERT} = 2980 nM) and hNET (IC_{50, hNET} = 3670 nM), while having a weak effect at hOCT2 (IC_{50, hOCT2} = 9310 nM). Noribogaine inhibits hVMAT2 (IC_{50, hVMAT2} = 570 nM) and hSERT (IC_{50, hSERT} = 280 nM) with comparable potency. This dual activity is separated by more than one log unit from its effect at hDAT (IC_{50, hDAT} = 6760 nM) and hOCT2 (IC_{50, hOCT2} = 6180 nM). (B) A schematic showing a 5-HT axon (blue line), 5-HT release sites (blue dots) and 5-HT synapses (lighter and larger blue clouds) on a pyramidal neuron’s soma and dendrite (left graphic). Right, A cartoon of 5-HT synapse and location of VMAT2 on synaptic vesicles, SERT on presynaptic boutons, and uptake 2 transporters OCT2 and PMAT on presynaptic and postsynaptic cells (localization on glial cells is omitted for clarity). Noribogaine exerts a dual inhibitory effect on VMAT2 and SERT, suggesting complex, non-linear effects on 5-HT and monoamine neurotransmission.

MATs work in tandem with the vesicular monoamine transporters, VMAT1 (SLC18A1) and VMAT2 (SLC18A2), which load synaptic vesicles or large dense core vesicles with monoamine neurotransmitters as an essential step prior to activity-dependent release via exocytosis (Figure 2B). VMAT1 is expressed in neuroendocrine cells, while VMAT2 is largely found in neurons in the CNS. Both transporters concentrate neurotransmitters in the vesicle lumen, driven by the proton gradient between the cytoplasm and vesicular lumen, via an amine-proton antiport mechanism.^44,45 The cytosolic monoamines that are not packaged into synaptic vesicles are oxidatively deaminated by the monoamine oxidases. Reserpine and tetrabenazine (TBZ) are classical VMAT2 inhibitors. Reserpine (a dual VMAT1 and 2 inhibitor) was previously used to treat hypertension and psychosis, while TBZ and its deuterated analog, which are selective VMAT2 inhibitors, are currently prescribed to treat choreic movements associated with the Huntington’s disease.⁴⁶ The functional interaction between SERT and VMAT2 was demonstrated by pharmacological VMAT2 inhibition or genetic VMAT2 deletion in 5-HT neurons, which resulted in a decrease of SERT-mediated synaptic 5-HT uptake, affecting both the rate (3-fold decrease of V_max) and the amount of synaptic 5-HT (50% reduction). Also, VMAT2 deletion or inhibition resulted in a measurable increase in potency of the SERT substrate MDMA as measured by the reverse transport of 5-HT from 5-HT neurons.⁴⁷

VMAT2 and the plasma membrane uptake 1 and uptake 2 transporters jointly shape the spatial and temporal landscape of monoamine neurotransmission in the CNS and its perturbation by pharmaceutical agents. Biogenic monoamines act as modulatory neurotransmitters via their respective receptors by augmenting or attenuating the excitatory and inhibitory synapses and serve as critical neuroplasticity mediators.⁴⁸

Guided by the framework of matrix pharmacology and the substantial corpus of neurochemical studies with ibogaine and noribogaine in rodents, we hypothesized that there are one or more important molecular targets of iboga alkaloids in the monoamine transporter families, which have not been examined in detail previously (the “iboga X project”). The effects of ibogaine or noribogaine on monoamine neurotransmitter dynamics in vivo are complex; for example, brain tissue analysis in mice and rats showed, in terms of total tissue concentrations, a decrease in dopamine and increase in the dopamine metabolite, DOPAC (dihydroxyphenylacetic acid), which is typically a tell-tale sign of VMAT2 inhibition.²⁰ Weak binding affinities of ibogaine and noribogaine at VMAT2 in human brain striatal homogenates (ibogaine, IC₅₀ = 14.6 μM; and noribogaine, IC₅₀ = 29.5 μM) have been reported, supporting the hypothesis that VMAT2 may be directly inhibited by the iboga compounds.⁴⁹ Ibogaine and noribogaine were also reported to acutely raise extracellular 5-HT levels in the nucleus accumbens (NAc), while the total tissue content analysis revealed minimal or no 5-HT decrease and a more pronounced decrease of the 5-HT metabolite, 5-HIAA (5-hydroxyindolylacetic acid), consistent with SERT inhibition.²⁰ These measurements indicate a complex monoamine modulation scenario and provides a rationale to systematically examine monoamine transporters, including VMAT2 and the uptake 1 and uptake 2 plasma membrane monoamine transporters.

Our previous work revealed that even small structural perturbations of the iboga system can have large effects on in vitro and in vivo pharmacology, as demonstrated by the oxa-iboga class of compounds (benzofuran-based iboga analogs).³¹ We therefore examined a focused SAR space of the ibogamine core in terms of the monoamine transporter activity.

Results

Ibogaine and noribogaine inhibit the function of multiple monoamine transporters.

To examine the effect of ibogaine and noribogaine on human VMAT2 (hVMAT2), we used HEK293 (human embryonic kidney 293) cells stably transfected with hVMAT2 (hVMAT2-HEK cells) and a VMAT2 fluorescent substrate, FFN206 (Figure S2).⁵⁰ For uptake 1 plasma membrane monoamine transporters, we used APP+ [4-(4-dimethylamino)phenyl-1-methylpyridinium] as a validated fluorescent substrate and the corresponding stably transfected HEK cells (hSERT-HEK, hDAT-HEK, and hNET-HEK). These cell-based assays provide an optical readout for the fluorescent substrate uptake (fluorescence units) mediated by the selected transporter.⁵¹ The extent of inhibition induced by the tested compounds is quantified by the difference in fluorescence signal between the vehicle and the iboga compound treatments (Figures S1, S4, and S5). Similar protocols were developed for the uptake 2 transporters, OCT1–3 and PMAT, using APP+ (OCT1 and PMAT) and ASP+ (4-[4-(dimethylamino)styryl]-N-methylpyridium; OCT2–3) as fluorescent substrates (Figures S6–S9). Established transporter inhibitors, including indatraline (DAT; Figure S4), reboxetine (NET; Figure S5), imipramine (SERT; Figures 3 and S1), tetrabenazine (VMAT2; Figures 3 and S2), and decynium-22 (OCT1–3 and PMAT; Figures S6–S9) were used for each transporter as positive controls.⁵²

Figure 3. Iboga compounds define a new class of pharmacological agents, termed “synaptic reuptake inhibitors” or “SynRIs”.

(A) Structure-activity relationship (SAR) examination of two positions of ibogamine and oxa-ibogamine (indicated on top, the 5-position in blue, the 1-position in yellow and purple) with respect to VMAT2 and SERT transport. We introduce a new numbering system in line with the numbering of simple tryptamines and indoles. The table provides IC₅₀ values for transport inhibition obtained by fluorometry based on inhibition of uptake of fluorescent substrates, APP+ for hSERT and FFN206 for hVMAT2, n = 4 biological replicates, mean ± SEM. Brick red shading highlights SynRI compounds with balanced inhibitory profiles; i.e., with IC₅₀ values for SERT and VMAT2 that fall within a three-fold range (one-half log). (B) Dose-response curves for control inhibitor (for hSERT: imipramine and for hVMAT2: tetrabenazine) and selected compounds in hSERT-HEK cells (left panel) and hVMAT2-HEK cells (right panel). The y-axis shows normalized inhibition of fluorescent substrate uptake, 100 × [(drug - nonspecific)/(total uptake - nonspecific)] ± SEM, derived from four separate experiments (n = 4). (C) Graphical representation of the dual inhibitory activity at SERT and VMAT2 exhibited by noribogaine, N-methyl-ibogaine, N-methyl-noribogaine, and oxa-noribogaine. Minor modifications of the iboga structure yield a range of activities in the SERT and VMAT2 continuum including potent and selective SERT inhibitors and VMAT2 inhibitors, as well as balanced SynRI compounds.

We found that both ibogaine and noribogaine inhibit hVMAT2 transport with comparable potency (IC₅₀, _{ibogaine, hVMAT2} = 390 nM, IC₅₀, _{noribogaine, hVMAT2} = 570 nM, Figure 2A, Figure 3, and Figure S2). The values indicate more potent inhibition in functional assays than those previously reported for hVMAT2 binding in a radioligand displacement assay in post-mortem human brain homogenates (IC₅₀, _{ibogaine, striatum} = 14.6 μM and IC₅₀, _{noribogaine, striatum} = 29.5 μM).⁴⁹ Ibogaine shows approximately a one-log higher affinity for hVMAT2 over hSERT (IC_{50, hSERT} = 2980 nM) and NET (IC_{50, hNET} = 3670 nM) (Figures 3, S1, and S5). Noribogaine inhibits hVMAT2 (IC_{50, hVMAT2} = 570 nM) and SERT (IC_{50, hSERT} = 280 nM) with comparable potency, which represents an unusual pharmacological profile (Figures 2–3, and S1–S2). VMAT2 and SERT dual inhibition is over 10-fold more potent than the effect of noribogaine at DAT (IC_{50, DAT} = 6760 nM) (Figure S4). In terms of uptake 2 transporters, we found that ibogaine (IC_{50, OCT2} = 9310 nM) and noribogaine (IC_{50, OCT2} = 6180 nM) inhibit transport by hOCT2 with weak but measurable potency, while showing no inhibition below 10 μM at the other uptake 2 transporters examined (Figures 2A and S6–S9).

Synaptic reuptake inhibitors (SynRIs) are defined by dual inhibition of the vesicular monoamine transporter 2 and a plasma membrane monoamine transporter with comparable inhibitory potencies.

The inhibition of both VMAT2 and a plasma membrane monoamine transporter is relatively uncommon in the pharmacopeia. There is a precedent for VMAT2 inhibitors that also block DAT transport with comparable potencies, as exemplified by deoxygenated lobeline analogs.⁵³ S-amphetamine has comparable potency as a [³H] 5-HT releaser via SERT in rat synaptosomes (EC₅₀ = 1.8 μM) and uptake blocker at VMAT2 (IC₅₀ = 3.3 μM) in crude vesicular rat brain fractions; whereas its releasing potency was reported to be much greater at NET and DAT in synaptosomes (EC_{50, NET} = 7.1 nM and EC_{50, DAT} = 24.8 nM, respectively).^42,54

Nevertheless, while there are reports suggesting cross-activity between the MATs and VMAT2 for some classical compounds,^55,56 the potent VMAT2 inhibitors such as reserpine or TBZ are selective for VMAT2 versus plasma membrane monoamine transporters (> 1000-fold potency differences for reserpine and > 70-fold for TBZ, Figure S10),⁵⁷ whereas potent SERT inhibitors such as imipramine and fluoxetine are selective for SERT over VMAT2 (typically > 100-fold, Figure S10).^55,58–60 Thus, VMAT2 inhibitors do not block MATs in brain tissue or in vivo at concentration exposures that lead to vesicular depletion.^57,61 We therefore propose a new class of pharmacological agents - termed “synaptic reuptake inhibitors” or “SynRIs” – as agents exerting dual inhibition of VMAT2 and SERT (or the other plasma membrane monoamine transporters including either DAT or NET) with comparable inhibitory potencies (within a half log in IC₅₀ values, Figures 3, S1–S2, and S4–S5). The potency cutoff range was chosen based on a common usage of semi-quantitative terms in the field of pharmacology to describe the degree of selectivity of an agent based on in vitro assays. 100-fold or greater differences in binding affinity or functional potencies are typically considered as “high selectivity”, a 10-fold difference as “modest to good selectivity”, and ~ 3-fold or ½-log as “low selectivity”, where < 3-fold difference typically borders on the experimental error and assay variability. The selected potency range for the SynRI definition is rooted in practical pharmacology as we aim to assure that the dual MAT/VMAT2 activity is likely operative in tissue preparations and in vivo with a comparable degree of engagement of both targets.

The dual transporter inhibition profile suggests complex effects at 5-HT synapses and release sites, as well as other monoamine synapses, as noribogaine directly modulates the synaptic vesicular, cytoplasmic, and extracellular pools of 5-HT (Figure 2B). Some of the in vivo neurochemical measurements reported previously are consistent with this pharmacological profile (see Introduction and Discussion). Ibogaine is outside the SynRI range (seven-fold more potent at VMAT2), but it is a pro-drug to noribogaine which is a prototype SynRI compound.

Synthesis of iboga alkaloids and their analogs from natural voacangine.

We next examined the effect of substitution in the 1-position and 5-position (indole substituents) on the balance between the VMAT2 and SERT inhibition (we propose a new, more logical iboga system numbering as shown in Figure 3A). These two structural positions are readily modifiable via late-stage functionalization of naturally derived iboga alkaloids (Scheme 1), with the exception of (±)-oxa-noribogaine and (−)-fluoro-ibogamine analogs, which required a total synthesis as reported previously.^23,31,62,63,⁶⁴ We do not use Tabernanthe iboga materials for analog synthesis for eco-cultural reasons (to avoid overharvesting this resource) and instead use natural (−)-voacangine obtained by extraction from the root bark of Voacanga Africana (Scheme 1).^31,65 Voacangine was converted to desmethyl voacangine, using ethanethiol and boron tribromide, and its subsequent hydrolysis and decarboxylation yielded noribogaine. We used noribogaine as the starting point to generate a number of readily accessible derivatives, by taking advantage of reactive sites on the indole core.

Scheme 1. Synthesis of iboga analogs via late-stage functionalization of natural voacangine.

Noribogaine, accessible semi-synthetically from voacangine isolated from root bark of Voacanga Africana, serves as a versatile intermediate for preparation of analogs with amplified and targeted transporter inhibitory profiles. Detailed synthetic procedures are provided in supplementary information.

The phenol moiety can be derivatized selectively under mild basic conditions to obtain alkoxy analogs (with alkyl halide, metal carbonate) or the reactive triflate intermediate [by bis(trifluoromethanesulfonyl)aniline, tertiary amine base]. 5-Triflate-ibogamine can be further transformed by palladium-mediated hydrogenation into ibogamine,⁶⁶ a component of the total iboga alkaloid content of Tabernanthe iboga. The nitrile group is a prominent feature of the selective SERT inhibitor citalopram, and as such we hypothesized that this group could have a dramatic effect on ibogamine’s SERT inhibition. Aryl halides and pseudohalides, like triflates can be transformed into nitriles by a widely used palladium-mediated reaction using a suitable nitrile source (zinc cyanide), carried at elevated temperatures (80 – 160 °C). However, we found that a prolonged reaction time and high excess of reagents were required to achieve a desirable level of conversion at a lower temperature range required to limit degradation.

Indole nitrogen and phenol oxygen of noribogaine are alkylated in good yields when treated with alkyl halide in dimethylsulfoxide in the presence of potassium hydroxide. Milder alkylation conditions (metal carbonate in hot acetonitrile) can be used to alkylate more labile analogs, that would not tolerate the harsher KOH/DMSO conditions. For analogs where restoring the free phenol group was desirable, the alkoxy group was cleaved using ethanethiol and boron tribromide.

Oxa-noribogaine was prepared and characterized as a racemate by a total synthesis as reported previously,³¹ while (−)-5- and (−)-6-fluoro-ibogamine analogs were prepared from a chromatographically resolved isoquinuclidine intermediate to match the natural iboga stereochemistry (see the Supporting Information for detailed synthetic schemes and reaction conditions).

Structure-activity relationship (SAR) of iboga alkaloids at hVMAT2 and hSERT.

Removal of the methyl group in the 5-position transforms VMAT2-prefering activity of ibogaine to a balanced inhibitory profile of noribogaine, by increasing the SERT potency 10-fold (Figures 2–3 and S1–S2). Excision of the entire 5-methoxy group of ibogaine, rendering another natural iboga alkaloid, ibogamine, inverts the ratio of the two activities by shifting the potency about 10-fold at each target, giving a SERT-preferring pharmacological profile (IC_{50, hSERT} = 330 nM, IC_{50, hVMAT2} = 3010 nM; Figures 2–3 and S1–S2). In contrast, extending the steric bulk of the methoxy to ethoxy group in the same position, accomplished by synthesizing 5-ethoxyibogamine, affords a potent VMAT2 inhibitor with more than 100-fold selectivity over SERT (IC_{50, hVMAT2} = 80 nM, IC_{50, hSERT} = 9030 nM, Figures 3A–C and S1–S2). In further contrast, placing a nitrile group in the 5-position gives 5-cyano-ibogamine, which is a potent SERT inhibitor with high selectivity over VMAT2 (IC_{50, hSERT} = 26 nM, IC_{50, hVMAT2} = 3340 nM, Figures 3A–B and S1–S2). Thus, the 5-position has a strong effect on both transporters and the observed relative inhibitory potencies. The hydroxy or alkoxy group is important for the observed sub-micromolar inhibitory potency at hVMAT2, as the corresponding 5-fluoroibogamine analogs and ibogamines (H in the 5-position) are weak hVMAT2 inhibitors (Figure 3A and Figure S2). With respect to hSERT activity, the nitrile and fluorine 5-substituents give potent, low nanomolar hSERT inhibitors (Figure 3A and Figure S1).

N-alkylation of the indole nitrogen (1-position) can also have robust SAR effects. For example, N-methylation of noribogaine resulted in increasing the potency two- to three-fold at both targets, giving a SynRI ligand, N-methyl-noribogaine (IC_{50, hVMAT2} = 59 nM, IC_{50, hSERT} = 170 nM, Figures 3A–C and S1–S2). Conversely, N-ethyl-noribogaine is a potent hVMAT2 compound with good selectivity over hSERT (IC_{50, hVMAT2} = 70 nM, IC_50, h_SERT = 1860 nM, Figures 3A–C and S1–S2). N-methylation of the potent hSERT inhibitor 5-cyano-ibogamine further increases the inhibitory potency at hSERT, yielding a compound with low nanomolar potency at hSERT (IC_{50, hSERT} = 5 nM, IC_{50, hVMAT2} = 440 nM; Figures 3A–B and S1–S2). Replacing the nitrogen with oxygen, rendering the oxa-iboga system, as exemplified by oxa-noribogaine (IC_{50, hSERT} = 900 nM, IC_{50, hVMAT2} = 670 nM; Figures 3A–C and S1–S2) results in a nearly perfect balance between hSERT and hVMAT2 due to a modest decrease of the hSERT potency versus noribogaine. In the oxa-iboga class we have focused on detailed characterization of oxa-noribogaine, not oxa-ibogaine (O-methylated analog), to eliminate the confounds of CYP-mediated demethylation in in vivo experiments.³¹

We found that hOCT2 has its own SAR trends. Removal of the 5-methoxy group or N-methylation of ibogaine, yielding ibogamine (IC_{50, hOCT2} = 2.0 μM) and N-methyl-ibogaine (IC_{50, hOCT2} = 2.3 μM), respectively, gives the most potent compounds of the series at hOCT2 in the range of low micromolar inhibitory potencies (Figure S6).

The combined SAR at these two positions enabled us to generate a series of iboga compounds with a broad range of activities, including potent hVMAT2 or hSERT inhibitors, balanced SynRI compounds (Figure 3C), and compounds with varying relative potencies at these two targets. The rest of the monoamine transporters were also examined for each of the featured compounds (Figures S1–S2 and S4–S9). The data above was obtained by fluorometry of the transfected cells using a plate reader. We confirmed these results with epifluorescence and confocal fluorescence microscopy, which showed that the fluorescence signals originate from intracellular localization of the fluorescent substrates (FFN206, APP+, and SERTlight), which is dependent on the function of relevant transporters (Figure S3).

Iboga alkaloids inhibit serotonin (5-HT) uptake via VMAT2 in brain synaptic vesicles.

To complement the optical assays in cell cultures, we examined the effect of iboga compounds on the uptake and release of [³H]5-HT via endogenous rat VMAT2 (rVMAT2) in synaptic vesicles. The synaptic vesicular fractions were prepared from rat brain and incubated with [³H]5-HT in the presence of the test compounds, adapting an assay previously established for [³H]DA uptake.⁵⁴ The results are in a good agreement with those obtained in the optical assays, showing similar potency trends, despite the methodological differences between the optical and radioactive assays (Table 1). For example, ibogaine showed sub-micromolar inhibitory potency (IC_{50, rVMAT2} = 560 nM versus IC_{50, hVMAT2} = 390 nM); the most potent analog, N-ethyl-noribogaine, mid-nanomolar activity (IC_{50, rVMAT2} = 54 nM versus IC_{50, hVMAT2} = 70 nM); whereas noribogaine relatively weaker, low micromolar inhibitor potency (IC_{50, rVMAT2} = 1311 nM versus IC_{50, hVMAT2} = 570 nM). The potency trends at VMAT2 (rat or human) did not show correlation with lipophilicity of the ibogaine compounds, which was estimated by determining log D values (Figure S17). With the exception of N-ethyl-noribogaine, the most potent compound of the series, the iboga analogs showed weaker functional potency (≤ approximately 3-fold) at the native rVMAT2 in synaptic vesicles compared to the overexpressed hVMAT2 in cell-based assays. This trend in potency across compounds may reflect a species difference and or a distinct membrane milieu of VMAT2 molecules in the native system with low VMAT2 expression levels versus the over-expressed transporter in transfected cells.

Table 1. Effect of iboga alkaloids on SERT and VMAT2-mediated uptake and release.

(A) Iboga analogs inhibit SERT-mediated [³H]5-HT uptake in rat-derived brain synaptosomes, and VMAT2-mediated [³H]5-HT uptake in rat-derived brain vesicular fraction (n = 3). No [³H]5-HT release was observed via SERT and only partial release for noribogaine and several of its analogs via VMAT2 (n = 3). *Two criteria were used to differentiate active/inactive releasers (EC₅₀ < 10 μM and E_max > 30%). (B) Comparison of inhibitory activity determined for iboga analogs in cell-based fluorescent uptake assay (n = 4) with values observed in rat brain synaptosomes/vesicular fraction presented on panel A.

A) Compound	[3H]5-HT Uptake Inhibition IC50 ± SD (nM) Values		[3H]5-HT Release EC50 ± SD (nM) Values (% Emax)*
	SERT	VMAT2	via SERT	via VMAT2
ibogaine	3037.0 ± 291.0	559.6 ± 68.3	inactive (<30)	inactive (<30)
noribogaine	326.1 ± 47.6	1311.0 ± 160.0		6480 ± 1,787 (50)
ibogamine	554.1 ± 49.0	>10000		inactive (<30)
N-methyl-noribogaine	13.6 ± 1.2	239.4 ± 27.2		219 ± 36 (32)
N-ethyl-noribogaine	80.1 ± 7.2	54.3 ± 6.4		711 ± 254 (31)
5-cyano-ibogamine	65.2 ± 5.4	9616.0 ± 1400.0		inactive (<30)
N-methyl-5-cyano-ibogamine	9.8 ± 0.8	1324.0 ± 145.0		inactive (<30)
cocaine	295.7 ± 29.5	n.d.	n.d.	n.d.
amphetamine	n.d.	n.d.	3336.0 ± 445.0 (91.0)	2671.0 ± 617.0 (98)
serotonin (5-HT)	n.d.	n.d.	n.d.	585.3 ± 129.0 (113)
tetrabenazine (TBZ)	n.d.	n.d.	n.d.	inactive (<30)
reserpine	n.d.	186 ± 26.8	n.d.	inactive (<30)
B) Compound	Uptake Inhibition at SERT IC50 Values (nM)		Uptake Inhibition at VMAT2 IC50 Values (nM)
	[3H]5-HT	APP+	[3H]5-HT	FFN206
ibogaine	3037	2980	560	390
noribogaine	326	280	1,311	570
ibogamine	554	330	>10000	3010
N-methyl-noribogaine	14	59	239	170
N-ethyl-noribogaine	80	1860	54	70
5-cyano-ibogamine	65	26	9616	3340
N-methyl-5-cyano-ibogamine	10	5	1324	440
cocaine	296	n.d.	n.d.	n.d.
reserpine	n.d.	>10000	186	52

We also examined the ability of iboga compounds to release [³H]5-HT from synaptic vesicles by first loading them with [³H]5-HT, followed by incubation with the test compounds and measuring the released radioactivity. Majority of iboga analogs showed modest releasing potency and low to partial efficacy near or below the detection sensitivity of the assay (<30% compared to 5-HT control set as E_max = 100%), with noribogaine (EC₅₀ 6.48 uM, E_max = 50%, Table 1 and Figure S13) acting as the most efficacious releaser from the selected derivatives investigated. However, the estimated [³H]5-HT release potencies (EC_50’s) were markedly weaker (~10-fold) compared to the uptake inhibitory potencies (IC_50’s). As true VMAT2 substrates such as 5-HT typically show comparable IC₅₀ and EC₅₀ values in purified synaptic vesicles, the low-potency, low-partial-efficacy releasing effects of iboga compounds likely represent non-specific effects associated with high compound concentrations (for example ibogaine, ibogamine, 5-CN-, and N-Me-5-CN-ibogamine exceed E_max of 30% at concentrations > 10 μM).⁵⁴ A notable exception may be N-methyl-noribogaine whose estimated release potency (EC₅₀ ~ 220 nM) is comparable to its inhibitory effect (IC₅₀ ~ 240 nM).

Finally, we tested the interaction of noribogaine with dihydrotetrabenazine (DTBZ) at rVMAT2 in a binding competition assay using rat brain synaptic vesicles incubated with [³H]DTBZ.⁶⁷ As expected, TBZ competed with [³H]DTBZ with high potency (IC_{50, rVMAT2} = 14.5 nM, > 90% displacement), consistent with the literature value (IC_{50, rVMAT2} = 16 nM).⁵⁴ In stark contrast, noribogaine showed essentially no effect on [³H]DTBZ binding (IC_{50, rVMAT2} > 10 μM, < 20% displacement, Figure S19), indicating that noribogaine engages a distinct transporter binding site or a conformational state from the occluded binding state of TBZ identified in cell preparations.⁶⁸

Iboga alkaloids inhibit uptake but do not induce release of serotonin (5-HT) in brain synaptosomes.

We next examined the effect of iboga compounds on native monoamine transporters in rat brain tissue preparations. For this purpose, synaptosomes enable measurements of both uptake and release of radiolabeled neurotransmitters including tritiated dopamine ([³H]DA), norepinephrine ([³H]NE), and serotonin ([³H]5-HT).⁴² Synaptosomes are prepared by fractionation of brain tissue. Caudate tissue was used for DAT assays whereas whole brain minus caudate and cerebellum was used for NET and SERT assays. The synaptosome fraction preserves much of the molecular machinery and physiological functions of presynaptic boutons, such as respiration, membrane potential, depolarization-induced exocytosis, neurotransmitter uptake, and pharmacologically induced monoamine efflux.⁶⁹

We examined a set of iboga compounds on [³H]5-HT uptake in synaptosomes, in the presence of DAT and NET inhibitors, effectively examining the function of native rat SERT (rSERT, Table 1).⁷⁰ Noribogaine was about 10-fold more potent than ibogaine in this assay, consistent with the literature and the inhibitory potency values determined in the optical assays with hSERT-transfected cells as detailed above (ibogaine: IC_{50, rSERT} = 3037 nM versus IC_{50, hSERT} = 2980 nM; noribogaine: IC_{50, rSERT} = 326 nM versus IC_{50, hSERT} = 280 nM, Table 1 and Figure S12). Both sets of values are similar to those reported previously in rat synaptosomes (ibogaine: IC_{50, rSERT} = 3150 nM, noribogaine: IC_{50, rSERT} = 330 nM).¹⁷ Close similarities of the obtained values are noteworthy considering the differences between these two assays. For the other iboga analogs, the values between the synaptosome and fluorescence assays also matched well, except for N-ethyl-noribogaine where the inhibition of [³H]5-HT uptake in synaptosomes was more than 20-fold more potent than the fluorescent SERT substrate inhibition in transfected cells (IC_{50, rSERT} = 80 nM versus IC_{50, hSERT} = 1860 nM, Table 1B and Figure S12). The reasons for the large difference in potency for this specific compound are unclear; however, we note that N-ethyl-noribogaine differs from the rest of tested compounds by its high potency of VMAT2 inhibition. The greater relative potency of N-ethyl-noribogaine in synaptosomes versus transfected cells was also found at NET (Figure S12). This leads to a speculation that the increased potency at MATs may be due to a functional coupling between the VMAT2 and MAT transporters in the native system, but not in the singly transfected cell lines, rather than species differences (rat versus human).

Generally, transporter substrates differ from non-transported inhibitors by stimulating the efflux of pre-accumulated substrates (“trans-acceleration” in the terminology of W. Stein).⁷¹ Synaptosomes allow compounds to be assayed for promoting the release of preloaded [³H]5-HT, which would be indicative of acting as SERT substrates. A previous study showed that ibogaine did not release [³H]5-HT from synaptosomes, but noribogaine was not tested.⁷² Considering the ability of ibogaine and noribogaine to stabilize conformations of SERT distinct from those induced by SSRIs, and their SERT pharmacochaperone activity,^20–22 we re-examined this question with a wider set of iboga analogs (Table 1 and Figure S12). No [³H]5-HT release was observed for ibogaine, noribogaine, or ibogamine; nor for any of the synthetic iboga analogs tested. This contrasts with the robust [³H]5-HT release by amphetamine, a drug shown to be a SERT substrate by numerous lines of evidence (positive control, Table 1 and Figure S12).⁴²

The ratio of binding to uptake inhibitory constants at SERT is low (~ 1) which also supports the model in which ibogaine and noribogaine are non-transported SERT inhibitors. In contrast, transporter substrates typically exhibit large ratios (10s-100s) due to markedly weaker potency in ligand binding displacement assays relative to the potency obtained in uptake inhibition assays.¹⁷

Imaging SERT by iboga alkaloids with subcellular resolution in mouse brain.

We next set out to examine SERT inhibition by the iboga compounds in acutely sectioned living brain slices in several brain regions that contain either serotonergic axons or cell bodies and dendrites. We used the recently developed molecular probe, SERTlight, which is a selective fluorescent SERT substrate that labels 5-HT neurons in mouse brain with high morphological fidelity in a SERT-dependent manner (Figure 4A).⁵¹ This imaging probe enables selective visualization of SERT function in the context of 5-HT neuronal morphology by incubating brain slices with SERTlight (10 μM, 30 min incubation), or injecting SERTlight into the cerebral cortex in living animals, and acquiring images inside tissue via two-photon fluorescence microscopy.

Figure 4. Inhibition of SERT in axons, cell bodies, and dendrites of 5-HT neurons in mouse brain slices visualized by SERTlight and two-photon imaging.

(A) Left panel: A schematic graphic of the concept of the imaging method. A 5-HT axonal bouton and release site is shown featuring SERT in the plasma membrane, which transports 5-HT and SERTlight (as the fluorescent substrate) to yield fluorescently labeled 5-HT neurons. Inhibition of SERT with noribogaine or other iboga analogs diminishes the labeling of 5-HT neurons. Examples of the other transporters examined in this study (OCT2, OCT3 and PMAT) are also shown. Right panel: A schematic representation of a sagittal slice of mouse brain (Allen Brain Atlas) showing the brain areas chosen for imaging, including dorsal raphe (DR), the seat of 5-HT cell bodies and dendrites; substantia nigra pars reticulata (SNpr) and dorsal striatum (DS), the regions with relatively dense 5-HT axonal innervation; and somatosensory cortex layers 2/3 (SSp2/3), a location with relatively sparse 5-HT axons.

(B) Left, image in SNpr (Scale bar: 20 μm) showing axonal strings and the location of the inset image shown right (Scale bar: 7 μm). In-between the microscopy images, mouse brain atlas image highlighting the SNpr region (Bregma: 1.8 mm, Allen Brain Institute). For the inset images to the right, SERTlight (10 μM for 30 minutes) accumulates in 5-HT axons in WT mice. Co-incubation of acute mouse brain slices with the SERT inhibitor citalopram (2 μM for 30 minutes) and SERTlight (10 μM for 30 minutes) inhibited SERTlight uptake (positive control). Co-incubation of acute mouse brain slices with ibogaine (2 μM for 30 minutes) and SERTlight (10 μM for 30 minutes) did not alter probe uptake into 5-HT axons, but co-incubation with noribogaine (2 μM for 30 minutes) or 5-cyano-ibogamine (2 μM for 30 minutes) ablated SERTlight probe uptake.

(C) Left, image in DS (Scale bar: 20 μm) showing axonal strings and the location of the inset image shown to the right (Scale bar: 7 μm). In between images, a mouse brain atlas representation highlighting the dorsal striatum (DS) in red (Bregma: −3.3 mm, Allen Brain Institute). For the inset images to the right, the same treatment conditions as in (B) above.

(D) Two-photon images in DS showing the dose-response of co-incubating SERTlight (10 μM for 30 minutes) with 5-cyano-ibogamine. Marked labeling inhibition is observed at 0.2 μM concentration of 5-cyano-ibogamine. Quantification of the images was not feasible due to the heterogeneity of labeling patterns.

(E) Left, mouse brain atlas image highlighting the barrel cortex (SSp2/3) region containing sparse 5-HT axonal projections (Bregma: −1.0 mm, Allen Institute). Images to right (scale bar: 7 μm), the same treatment conditions as in B above.

(F) Vertical organization of panels, top: mouse brain atlas image highlighting the small DR region (Bregma: −4.5 mm, Allen Brain Institute). The image below shows SERTlight staining of 5-HT neuronal cell bodies, the larger objects with darker nuclei, and punctate staining of dendrites. From top to bottom, co-incubation of SERTlight with citalopram (positive control) and increasing concentrations of noribogaine (10, 30, 50, and 100 μM) shows weak inhibitory potency of noribogaine, but not citalopram, in this brain region. For all panels, representative images from n = 3 mice, three slices per mouse per region.

Serotonergic cell bodies and dendrites were imaged in the dorsal raphe (DR), the locus of most 5-HT neuronal cell bodies that project throughout the forebrain (Figure 4A, F). The 5-HT axonal projections appearing as punctate strings were imaged in the substantia nigra pars reticulata (SNpr) and dorsal striatum (DS), regions with a relatively high density of 5-HT axons, and somatosensory cortex (SS, or barrel cortex), which has more sparse 5-HT axonal innervation (Figure 4A–E). In these regions, citalopram (2 μM, pre- and co-incubation with SERTlight) used as a positive control abolished SERTlight labeling in 5-HT axons, consistent with in situ axonal SERT inhibition. In contrast, ibogaine (2 μM) had no effect on SERTlight labeling using the same concentration and incubation protocol as for citalopram, consistent with its relatively weak, micromolar inhibitory effect determined in the fluorometric cell assays and synaptosomes (Figure 4B–C, E). In contrast, noribogaine (2 μM) completely blocked the SERTlight signal in the axons, in line with its sub-micromolar SERT inhibition potency (Figure 4B–C, E, F). Similarly, the potent SERT inhibitor, 5-cyano-ibogamine (2 μM), fully inhibited SERTlight label; and a qualitative dose-response study indicated a marked suppression of SERTlight fluorescent labeling at 200 nM concentration of this iboga analog (Figure 4D). These results indicate that noribogaine, and its more potent analog, 5-cyano-ibogamine, can inhibit axonal SERT function in native brain tissue in a concentration range that matches the brain exposures of free drug at behaviorally active doses (greater than 2 μM, see the pharmacokinetic studies below).

In the DR, the somatodendritic labeling pattern of SERTlight was also completely inhibited by citalopram (2 μM); however, noribogaine showed no effect at either 2 μM or 10 μM concentrations, with the latter concentration being five-fold higher than a concentration effecting complete SERTlight labeling suppression in 5-HT axons (Figure 4F). A dose-response study demonstrated that even 30 μM concentration of noribogaine had no apparent effect on the somatodendritic SERT function. A clear inhibition was only achieved at 100 μM concentration of noribogaine (Figure 4F). Thus, in semi-quantitative terms, there is greater than a 50-fold difference in the inhibitory potency of noribogaine between the axonal and somato-dendritic brain regions. A possible explanation, that the cell bodies and dendrites of 5-HT neurons express other transporter(s) that take up SERTlight but are noribogaine-insensitive, was discarded by showing that: 1) there was no SERTlight somatodendritic labeling in SERT knock-out (SERT-KO) mice, 2) SERTlight was not a substrate for DAT, NET, or any of the uptake 2 transporters.⁵¹ Alternatively, our results could indicate that noribogaine interaction may be regulated by a different functional status of the SERT molecules in cell bodies versus axons, rendering noribogaine an “axon-selective” or “brain-region-selective” SERT inhibitor.

Imaging VMAT2 inhibition by iboga alkaloids at individual synaptic boutons in mouse brain slices.

We next examined the effect of ibogaine, noribogaine, and 5-cyano-ibogamine on mouse VMAT2 (mVMAT2) function in acute mouse brain slices using the fluorescent VMAT2 substrate, FFN200. We previously demonstrated that FFN200 labels synaptic vesicle clusters in a mVMAT2-dependent manner in the monoaminergic axons in the striatum (Figure 5A).⁷³ FFN200 affords a highly dense punctate labeling pattern which is attenuated by VMAT2 inhibitors, such as DTBZ (2 μM), (Figure 5B). In addition to a qualitative assessment, the punctate pattern was quantified (see Methods for details) and confirmed strong inhibitory effects by ibogaine and noribogaine (each at 2 μM), consistent with their sub-micromolar potency at VMAT2. To further probe the inhibition potency, we examined the dose-response effect of noribogaine on FFN200 uptake in acute mouse slices (Figure 5D). The inhibition constant (IC₅₀ = 160 nM) is comparable to the values obtained in fluorometric assays of hVMAT2 in transfected cells (IC_{50, hVMAT2} = 570 nM, Figure 3) and [³H]5-HT uptake by rVMAT2 in rat brain synaptic vesicles (IC_{50, rVMAT2} = 1311 nM, Table 1), providing confirmation of the VMAT2 inhibitory range within acute brain tissue. In comparison, the weaker VMAT2 inhibitor, 5-cyano-ibogamine (2 μM), showed no inhibitory effects on FFN200 uptake in brain slices at the equivalent concentration (despite its greater lipophilicity compared to noribogaine and ibogaine, Figure S17). These outcomes support the mechanistic model in which ibogaine and noribogaine directly inhibit VMAT2, an intracellular molecular target expressed on monoamine synaptic vesicles, and thus are expected to limit the synaptic vesicular neurotransmitter pools by drug exposures reached by administering biologically active doses (> 2 μM, after 10 mg/kg, see pharmacokinetics results below).

Figure 5. Inhibition of VMAT2 by iboga alkaloids visualized with resolution of individual synaptic vesicle clusters of monoamine axons in the brain.

(A) A graphic representation of the imaging method visualizing VMAT2 transport in the brain. A monoaminergic axonal bouton and release site in DA, NE, or 5-HT axons where intracellular neurotransmitter or FFN200 (a fluorescent substrate) are transported into the synaptic vesicles. The puncta in the microscopy images represent individual axonal vesicle clusters.

(B) Left panel, mouse brain atlas image highlighting the dorsal striatum (DS) region containing high density of DA, but also 5-HT and NE neuronal axonal projections (Bregma: −3.3 mm, Allen Brain Institute). Top middle panel (vehicle, DMSO), incubation of acute mouse brain slices with FFN200 (10 μM for 30 minutes) leads to probe accumulation in VMAT2-expressing vesicle clusters in WT mice giving a dense punctate pattern imaged by two-photon microscopy. Top right panel (DTBZ), co-incubation of acute mouse brain slices with FFN200 (10 μM for 30 minutes) and the VMAT2 inhibitor DTBZ (2 μM for 30 minutes) inhibited the labeling pattern (positive control). Bottom right and middle panels: co-incubation of FFN200 (10 μM for 30 minutes) with ibogaine (2 μM for 30 minutes) or noribogaine (2 μM for 30 minutes) completely inhibited FFN200 uptake. Bottom left panel: coincubation with 5-cyano-ibogamine (2 μM for 30 minutes) under the same conditions as above had no effect on labeling pattern of FFN200. Ex./em. for FFN200: 740 nm exc, 460 nm em/50 nm (FWHM). Scale bar for images: 20 μm. Representative images from n = 3 mice, three slices per mouse.

(C) Quantification of the number of puncta per image under different conditions. Y-axis: % of control number per unit area.

(D) Quantification of the number of puncta per image under increasing noribogaine concentration, IC₅₀ value was calculated from the constructed dose-response curve.

The results obtained with SERTlight and FFN200 support a model in which noribogaine modulates both SERT and VMAT2 in the 5-HT presynaptic boutons, as well as VMAT2 in the DA and NE presynaptic boutons and release sites at relevant brain exposures (Figure 2B).

Ibogaine and noribogaine do not induce catalepsy despite their VMAT2 inhibitory activity.

VMAT2 inhibitors at high doses induce catalepsy in animals, characterized by an immobile position and lack of volitional motor behavior, a consequence of dopamine depletion in the striatum and basal ganglia. In humans, therapeutic doses of TBZ, an established selective VMAT2 inhibitor, lead to Parkinsonism as an adverse effect in 15–24% of subjects treated for various forms of dyskinesias.^74,75 We used a simple bar test to determine catalepsy in mice where the animals are situated in the instrument in a rearing position with the front paws placed on an elevated bar (Figure 6A). Under vehicle control conditions, animals come off the bar immediately (< 2 to 4 seconds), whereas the cataleptic state is determined by a time threshold (30 – 60 s) of remaining with both paws on the bar.^76,77 In this manner, catalepsy in mice can be readily differentiated from sedation, which is a non-specific readout of suppression of spontaneous locomotor behavior as measured by placing animals in a novel arena (open field, OF, test, (Figure 6A)).⁷⁸

Figure 6. Ibogaine and noribogaine do not show behavioral signs of monoamine depletion.

(A) Left panel: tetrabenazine time profile at TBZ (30 mg/kg, s.c.). Catalepsy was tested at indicated time points after TBZ injection (n = 5). Left middle panel: TBZ dose-response in the catalepsy test was tested at 60 min post TBZ injection (s.c., n = 5 – 9). Right middle panel: diminished locomotor activity after TBZ (s.c.) injection was observed in an open-field test (n = 4 – 8), measured as ambulatory distance in 10 min bins. Right panel: total locomotory distance traveled over 60 min after TBZ administration. (B) Noribogaine administration does not result in catalepsy in male (n = 5), nor female mice (n = 4), but produce sedation-like behavior resulting in diminished ambulatory behavior (n = 4 – 8), presented as total distance traveled over 60 min. (C) High doses of N-ethyl-noribogaine induces catalepsy (n = 4 – 9) and suppresses locomotor activity in an open field (n = 4 – 8, male mice). (D) Noribogaine administration rapidly produces high, freely available brain concentration levels. N-ethyl-noribogaine is no less brain penetrant, but significantly less available compared to noribogaine due to its high nonspecific brain tissue homogenate binding. Pharmacokinetic data and derived parameters are available in the supplementary information file (Figure S11). Compounds were administered by s.c. injection. Data were analyzed using one- and two-way ANOVA followed by Dunnett’s post hoc test. All values represented as mean ± SEM, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Using TBZ (s.c. administration) as a positive control, we observed both sedation in the open field test and catalepsy in the bar test in a dose-dependent manner (Figure 6A). In contrast, noribogaine did not induce catalepsy even at high doses (30, 50 and 80 mg/kg, s.c.), whereas the locomotion was strongly suppressed already at 30 mg/kg in both male and female mice (Figure 6B). Similarly, ibogaine did not induce catalepsy while suppressing locomotion, but the assessment of catalepsy at higher doses (30 mg/kg and higher) was prevented by strong tremors, a recognized effect of ibogaine and several other natural iboga alkaloids (Figures S13–S15).⁷⁹ After administration of high doses of noribogaine or other iboga compounds, we did not observe a loss of righting reflex (LORR), indicating that the heavy sedation and catalepsy states were not confounded by anesthesia (Figure S14).^80–82

The potent VMAT2 inhibitor, N-ethyl-noribogaine, also showed a lack of catalepsy up to 30 mg/kg, while this dose completely suppressed locomotion. However, at a still higher dose of 50 mg/kg, catalepsy was induced at the 60-minute mark, while trending toward cataleptic states at the 30- and 90-minutes post drug administration (Figure 6C). The difference in cataleptic effects between noribogaine and N-ethyl-noribogaine is not due to a relatively lower brain penetration by noribogaine, as both compounds produce comparable total brain concentrations (Figure S11). However, due to significantly higher nonspecific protein retention of N-ethyl-noribogaine (both in plasma and brain tissue), noribogaine at the same dose shows a nearly three-fold greater estimated free drug exposure in the brain, in terms of both maximum concentration (C_max) and total drug exposure (AUC, area under the curve, Figure S11) (given 50 mg/kg of each compound, s.c.: C_max, noribogaine = 31 μM versus C_max, _{N-Et-noribogaine} = 11 μM (Figure 6D)). Thus, the greater VMAT2 inhibitory potency of N-ethyl-noribogaine (IC_{50, hVMAT2} = 70 nM) versus noribogaine IC_{50, hVMAT2} = 570 nM), as determined in vitro, likely underlies the observed differences in catalepsy in vivo. The observation that N-ethyl-noribogaine, a potent VMAT2 inhibitor with high brain bioavailability, requires high doses and brain concentrations to induce catalepsy suggests that the iboga matrix pharmacology, the complex pharmacological background of iboga alkaloids, exerts a protective effect against monoamine tissue depletion and catalepsy.

To our knowledge, catalepsy had not been previously reported following ibogaine or noribogaine in rodents or humans, neither the application of specific neurological tests to confirm cataleptic state. In humans, high doses of ibogaine (> 10 mg/kg, p.o.) can induce ataxia,¹⁸ while very high doses of ibogaine (> 20 mg/kg) can lead to anesthesia-like states.⁸

Discussion

In this report, we examine the pharmacological profile of iboga alkaloids at monoamine neurotransmitter transporters, with a focus on the parent compound, ibogaine, its main metabolite, noribogaine, and a small series of iboga analogs. We find that ibogaine and noribogaine exhibit a complex monoamine modulatory profile by inhibiting three or more transporters within a pharmacologically relevant concentration range. Although the relative exposure of these two major circulating compounds varies widely between individuals, typically at doses lower than 8 mg/kg of ibogaine (p.o.), noribogaine is the dominant compound in plasma, while at doses greater than 9 mg/kg, ibogaine becomes the major circulating compound for at least 12 hours post ibogaine administration. In a recent study conducted with opioid-dependent human subjects, ibogaine administration (10 mg/kg, p.o.) led to a median plasma concentration of ~ 4.8 μM of ibogaine.¹⁸ When plasma protein binding correction is applied (65% bound / 35% available),¹² the resulting plasma free concentration of ibogaine is ~ 1.7 μM. In rodents, noribogaine and ibogaine display > 4-fold greater concentration of free drug in the brain over plasma (rats¹¹ and mice, Figure S11). As such, the estimated free ibogaine in human brain is expected to be in the range of ~ 2 μM (assuming conservatively a 1:1 ratio of brain/plasma) to 8 μM (assuming a similar ratio of brain/plasma as in rodents), well within the relevant concentration range with respect to VMAT2 modulation. The PK profile in rodents shows similar trends where both species are present with ibogaine dominant in the early phase, followed by noribogaine becoming the major species in the longer exposure times.^11,16–18

Ibogaine has been assumed to mediate the oneiric and visionary states of consciousness, since noribogaine induces mild subjective effects of when administered alone (e.g., light sensitivity, brighter light and colors, no hallucinations). However, the reported doses of noribogaine in human studies were limited due to the cardiac QT interval prolongation (top dose of 180 mg, p.o., ~ 2.6 mg/kg), constraining inferences about whether psychedelic effects could appear at higher exposures.⁸³ Therefore, the comparative psychoactive effects of ibogaine and noribogaine are largely based on correlations between PK and subjective effect profiles, where the oneiric phase overlaps with the peak ibogaine plasma concentrations.¹⁸ We speculate that the complex modulation of 5-HT (e.g., transient increases in extracellular 5-HT) and dopamine (e.g., persistent decreases in extracellular and tissue dopamine) dynamics likely contributes to the shape of the subjective experiences induced by ibogaine and the other iboga alkaloids.

We demonstrated that ibogaine and noribogaine inhibit the transport function of the vesicular transporter VMAT2 with sub-micromolar potency, using a fluorescent VMAT2 substrate (FFN206) in cell-based assays. We also demonstrated in acute mouse brain slices that ibogaine and noribogaine inhibit VMAT2 transport in synaptic vesicle clusters of intact monoamine axons using two-photon microscopy imaging. Finally, we confirmed the inhibitory effects on VMAT2-mediated transport of [³H]5-HT in isolated brain synaptic vesicles. Thus, we provide evidence for VMAT2 transport inhibition by iboga alkaloids and the potency range of this effect (sub-micromolar to low micromolar, Figure 5) in three experimental systems. Ibogaine (IC_{50, hVMAT2} = 390 nM) and noribogaine (IC_{50, hVMAT2} = 570 nM) have similar inhibitory potencies at VMAT2. In the isolated synaptic vesicle fractions prepared from rat brain, we found that iboga compounds are not efficient releasers of preloaded [³H]5-HT via rVMAT2; noribogaine shows negligible efficacy of release at concentrations that half-saturate rVMAT2. This data suggests that iboga alkaloids largely act as VMAT2 non-substrate inhibitors or low efficacy “partial VMAT2 substrate inhibitors”. The lack of correlation between lipophilicity (estimated by log D) and VMAT2 inhibitory potency (IC₅₀) does not support the lipophilic weak base effect as the main mechanism of VMAT2 transport inhibition. For example, ibogaine and noribogaine possess markedly different lipophilicity (difference of 1.5 log D units, Figure S17) while exerting comparable potencies on the uptake by VMAT2; or ibogamine versus noribogaine where the latter is less lipophilic (by 1.75 log D units) and more potent at VMAT2. However, a weak base effect might contribute to the greater potency of more lipophilic N-ethyl-noribogaine and 5-ethoxy-ibogamine, in a similar manner as proposed for amphetamine, which can function at least at high concentrations as a lipophilic base and alkalize synaptic vesicles.⁸⁴ In the synaptic vesicles we also found that noribogaine does not compete with [³H]DTBZ indicating a distinct binding site or conformational state of the transporter bound to noribogaine in comparison to TBZ. These findings warrant future kinetic and structural studies to determine in detail the mode of inhibition of VMAT2 by iboga compounds.

We also examined the effect of iboga compounds on two types of monoamine plasma membrane transporters, the uptake 1 and uptake 2 transporters. In line with previous results reported in the literature,²⁰ we show that noribogaine is about 10-fold more potent than ibogaine at inhibiting hSERT as determined using the SERT fluorescent substrate APP+ in cell-based assays. Noribogaine has a comparable inhibitory potency at hSERT (IC_{50, hSERT} = 280 nM) and hVMAT2 (IC_{50, hSERT} = 570 nM, Figures 3 and S1), exhibiting an uncommon pharmacological profile of dual inhibition of these two critical transporters at 5-HT synapses and release sites. We proposed the term “synaptic reuptake inhibitors” or “SynRIs” to highlight this interesting mechanistic feature and contrast it to the well-known VMAT2 inhibitors and SSRIs which have very different potencies at these two transporters (Figures 3 and S10). The inhibition of OCT2, identified here as a novel molecular target of iboga alkaloids, may be pharmacologically relevant in vivo, considering the estimated free brain concentrations of noribogaine based on the pharmacokinetic studies reported here (C_max ~ 6.6 μM after 10 mg/kg, and 31 μM after 50 mg/kg, s.c., Figures 6 and S11), and previous reports in humans (C_max ~ low micromolar after 10 mg/kg of oral ibogaine in opioid-dependent individuals).¹⁸ OCT2 certainly needs to be entered into the iboga matrix of known targets, particularly for SAR studies with new analogs.⁸⁵

The presented SAR study showed that the relative potencies at SERT and VMAT2 can be tuned over a broad range, identifying analogs with a balanced SynRI profile (e.g., noribogaine, oxa-noribogaine), dominant VMAT2 (N-ethyl-noribogaine), and dominant SERT (5-cyano-ibogamine) (Figures 3 and S1–S2). We also determined that none of the iboga compounds - with varying SERT inhibitory potency - induce release of [³H]5-HT in synaptosomes, confirming that they act as non-transported inhibitors at SERT, consistent with a previous report on ibogaine (Figure S12).⁷² The structural dimensions of the iboga compounds, in which the aliphatic amino group of the tryptamine unit is embedded in the bulky isoquinuclidine ring system, are probably too large for them to serve as SERT substrates, in contrast to the smaller known SERT substrates and 5-HT releasers such as amphetamine analogs. However, a recent report claimed a partial [³H] 5-HT release (E_max ~ 30%) induced by noribogaine in SERT-transfected cells.⁶⁴ With regard to in vivo neurochemistry, although there is a single report claiming a 5-HT release induced by ibogaine in nucleus accumbens,⁸⁶ other studies described results consistent with a SERT non-substrate inhibitory profile for ibogaine and noribogaine.^17,20 The contrast between MDMA’s and ibogaine’s/noribogaine’s activities as 5-HT releasers is consistent with the downstream signaling divergence between these substances where β-arrestin2 knock-out blocked the learning plasticity induced by MDMA (5-HT releaser) but not by ibogaine (SERT blocker/chaperone).^34,87

We further probed the effect of iboga alkaloids on transport by SERT in acute mouse brain tissue with subcellular spatial resolution enabled by the state-of-the-art imaging probe, SERTlight, and two-photon microscopy.⁵¹ While noribogaine (2 μM) completely inhibited the axonal SERT in three different brain regions, as expected, it was far less potent at inhibiting SERT in 5-HT neuronal cell bodies and dendritic compartments of the dorsal raphe nucleus (> 50-fold difference; Figure 4). While this finding was surprising, there is a precedent for different binding affinities of noribogaine at VMAT2 in distinct human brain regions (IC₅₀, _{binding, striatum} = 29.5 μM versus IC₅₀, _{binding, cortex} = 5 μM).⁴⁹ It has been previously demonstrated that ibogaine is an atypical SERT inhibitor (non-competitive inhibition) that binds and stabilizes SERT in inward-open conformational states, in contrast to cocaine-based blockers or SSRIs that bias the SERT conformational dynamics toward outward-facing conformations.^88,89 It has been proposed that the pharmaco-chaperone effect of iboga compounds at SERT (along with DAT and likely NET) is related to the stabilization of inward-open conformational states sampled by the transporter in its folding and trafficking pathway.⁹⁰ Inspired by ibogaine’s mode of action, more potent SERT pharmacochaperones were generated and conformation-selective SERT ligands were developed via computational methods.^22,91 On this background of rich iboga SERT pharmacology, we speculate that noribogaine “detects” different SERT conformational or functional states in the brain, which may be related to post-translational modifications and or complexation dynamics with ancillary proteins, transporter-associated chaperones, or cytoskeleton proteins, as part of the local membrane composition and cellular functional states.^92,93

Considering the findings of this study and prior literature, a complex picture of monoamine neurotransmission modulation by ibogaine and noribogaine emerges (Figure 2). The iboga compounds inhibit monoamine transporters known to play critical roles in monoamine neurotransmission, including the synaptic vesicle transporter VMAT2, plasma membrane monoamine transporters of uptake 1 family (most notably SERT), and the uptake 2 transporter OCT2, expressed on neuronal and glial cells. The dual or multiple transporter site modulation predicts complex, non-linear effects on monoamine neurotransmission, especially when considering functional couplings between SERT and VMAT2 and the different neurotransmitter pools (i.e. vesicular, cytoplasmic, and extracellular)⁴⁷ and the complex mutual interactions between 5-HT and catecholamines.^94–96

The updated monoamine transporter profile of the iboga compounds provides a new explanatory model to address questions previously posed by the extensive body of neurochemical literature: namely, why do iboga compounds have such a profound effect on dopamine metabolism (a strong decrease of dopamine and an increase in its metabolite DOPAC in total tissue content in most brain regions), while the effect on 5-HT metabolism is modest (small or no decrease in 5-HT and a modest decrease of metabolite 5-HIAA).²⁰ The direct inhibition of VMAT2 has an important role in the explanatory hypothesis; while ibogaine and noribogaine act as VMAT2 transport inhibitors in vitro and in vivo, they poorly inhibit DAT in vivo (in vitro inhibition potency at DAT is more than one log unit weaker compared to VMAT2; Figure S4). Thus, the effect of the iboga compounds on dopamine metabolism and neurotransmission is dominated by the VMAT2 inhibitory effect and largely the absence of DAT inhibition. This model is also consistent with the effect of ibogaine and noribogaine on extracellular dopamine: generally, a decrease in striatum, and no effect or a decrease in NAc, as well as attenuation of cocaine-induced dopamine increase in NAc and striatum.^20,97 The lack of DAT inhibition by iboga compounds in vivo, and VMAT2-mediated depletion of vesicular dopamine pools explains the behavioral effects in which ibogaine decreases the ambulatory stimulation by cocaine, an effect dependent on the vesicular release of dopamine via exocytosis.²⁰ Thus, ibogaine and noribogaine as VMAT2 inhibitors limit loading of dopamine into synaptic vesicles, resulting in increased cytoplasmic catabolism of dopamine and the neurochemical signature of VMAT2 inhibitors (decreased tissue dopamine and increased dopamine metabolites, Figure 5).

In 5-HT axons, noribogaine acts as a SynRI, a dual inhibitor of SERT and VMAT2. In this manner, the uptake of 5-HT released via activity-dependent exocytosis is limited by SERT inhibition (and potentially OCT2 inhibition), which protects 5-HT from extensive metabolism within 5-HT neurons.²⁰ VMAT2 inhibition also modulates rates of 5-HT uptake via SERT,⁴⁷ and likely contributes to the temporal profile and firing pattern of 5-HT transmission, leading to transient increases of extracellular 5-HT in the NAc and striatum, following ibogaine or noribogaine administration (Figure 5).⁶¹ In synaptic microcircuits, 5-HT modulates excitatory and inhibitory synapses via both volume neurotransmission and direct modulation of triad synaptic connections.^98,99 The dynamics of extracellular 5-HT concentrations and the downstream synaptic sequelae may be further modulated by other transmitter systems, such as cholinergic and opioidergic axonal inputs (e.g., at hub synapses), where iboga compounds can exert direct effects including the inhibition of nicotinic acetylcholine receptor^25,100 or potentiation of ongoing MOR signaling.¹⁰¹

We propose that the complexity of iboga’s modulation of monoamine neurotransmission provides clues and insights about the global picture of the iboga matrix pharmacology. The present results do not suggest indiscriminate inhibition or activation of many random targets by iboga substances, but rather modulation of a combination of molecular targets that are functionally coupled in critical and highly tuned processes, such as monoamine neurotransmission. Furthermore, the effects at certain individual targets are atypical revealing higher order molecular functions. The prime exhibit is SERT, a pivotal element of serotonergic transmission, in which several modes of action of the iboga compounds have been reported: including the acute inhibition of SERT via distinct conformational states, pharmaco-chaperone effect (the latter effects may modulate the long-term function of SERT), and now the axon selective inhibitory effects of noribogaine (which may lead to targeted 5-HT circuit modulation). Hence, we extrapolate the existing evidence and speculate that the iboga matrix pharmacology may have a deep sophisticated logic based on unique molecular effects at specific targets and complex combinations of molecular targets and interventions.

We also propose that the matrix pharmacology concept is essential for the interpretation and re-interpretation of the experimental results in the iboga structural space. For example, selective single-target perturbations ought not to be considered in isolation, but in context of the iboga matrix pharmacology.

In the present study, the conceptual framework of matrix pharmacology may be useful for explaining why ibogaine or noribogaine do not induce catalepsy even at high doses, in contrast to the standard VMAT2 inhibitors. To this end, we examined the effect of varying VMAT2 potency within the iboga system on mouse behavior. We confirmed that catalepsy can be readily induced in mice by TBZ, a potent VMAT2 inhibitor, as determined by using a bar test (Figure 6). Unlike the TBZ positive control, noribogaine did not show any catalepsy even at very high doses (50 and 80 mg/kg, s.c.) in which the 50 mg/kg dose resulted in > 20 μM estimated maximum concentration of free drug in the brain (Figure 6D); a level that is 10-times greater than the concentration that completely inhibited substrate transport by VMAT2 in striatal brain slices (2 μM, Figure 5). Similarly, N-ethyl-noribogaine, a compound with the highest VMAT2 potency among iboga analogs tested (IC_{50, hVMAT2} = 70 nM in vitro) did not induce catalepsy at 10 and 30 mg/kg doses, but only by the top administered dose of 50 mg/kg (Figure 6C). Pharmacokinetic experiments showed that the non-cataleptic 30 mg/kg dose yielded an estimated maximum brain concentration of 7,000 nM of free drug – approximately 100-fold higher than its IC_{50, hVMAT2}. We therefore propose that the background iboga matrix pharmacology mitigates or integrates the consequences of the VMAT2 inhibition effect, dopamine depletion and catalepsy.¹⁰²

How could one begin to describe the lack of catalepsy after iboga alkaloids? Simple explanations based on known mechanisms that mitigate effects of VMAT2 inhibitors, such as monoamine oxidases inhibition, DAT blockade, or direct dopamine receptor agonism, do not apply in this case because ibogaine lacks these activities (e.g., ibogaine showed no activity at MAO A, Figure S16).¹⁰⁴ We propose that iboga compounds modulate targets other than VMAT2 to facilitate: 1) moderation of dopamine depletion, thereby limiting the extent of decrease of extracellular dopamine concentrations, and/or 2) potentiation of signaling at relevant dopamine receptors in the basal ganglia. It is not clear at this time which specific targets and mechanisms accomplish these putative actions, but we propose that the effects are driven by a complex mechanism that involves crosstalk and synergism of multiple molecular targets, locally within synaptic microcircuits (axonal dopamine release) and or distally (control of dopamine cell firing) across brain regions, conceptualized as the matrix pharmacology. Modulation of dopamine axonal release may be accomplished either directly (e.g., by nicotinic receptors on dopamine axons) or indirectly via modulating firing patterns of the cholinergic interneurons which in turn control striatal dopamine release.¹⁰³

Brain-wide circuit effects involving 5-HT-dopamine crosstalk could also be at play,⁹⁶ which may include control of dopamine neuron firing patterns. For example, VMAT2 inhibition blocks the uptake of dopamine and other monoamine neurotransmitters into synaptic vesicles, which in turn leads to a sequence of events including decreased exocytotic release of dopamine and increased dopamine cell firing (and elevated dopamine biosynthesis).⁶¹ As a specific example, it is plausible that the matrix pharmacology limits (locally or distally) the VMAT2-inhibition-induced increase in dopaminergic cell firing and thus attenuates dopamine depletion.

This indirect circuit model is also useful for explaining the opposite effect of ibogaine; namely, attenuation of a transient dopamine increase induced by cocaine (DAT inhibitor) or opioids (indirect circuit mechanism via MOR agonism).²⁰ The iboga compounds attenuate extracellular dopamine elevations, and the accompanying increases in rodent locomotion, not by direct DAT modulation and competition with cocaine, but by indirect mechanisms that may include inhibition of VMAT2, and subsequent decreases in vesicular dopamine available for exocytotic release, and/or moderation of dopaminergic neuron firing rates. Hence, evidence points to a complex matrix mechanism that restrains both neurochemical extremes – high and low dopamine levels – and the behavioral consequences as indicated by attenuating the effects of both DAT and VMAT2 inhibition. Importantly, the specific physiological mechanisms noted here such as firing rates of dopamine neurons are testable experimentally and will be a subject of future studies.

Our discussion here focuses on the acute (drug-on) neurochemical and behavioral effects, which likely shape the long-term sequels of iboga compounds.³⁰ While much work lies ahead on the path toward elucidation of the full complexity and sophistication of iboga matrix pharmacology, the observed mitigation of behavioral consequences of VMAT2 inhibition is an important example.

Conclusions

The present study offers two major contributions. First, we expand the map of monoamine transporters as pharmacological targets of the iboga alkaloids. For this task, we used a combination of in vitro and ex vivo experimental methods, including the state-of-the-art optical imaging of transporter function in cultured cells and brain tissue, to provide the first direct evidence for VMAT2 and OCT2 inhibition. The optical methods were complemented by uptake and release of radiolabeled serotonin in brain synaptosomes and synaptic vesicle preparations. Second, we propose the concept of iboga matrix pharmacology to advance the understanding of molecular mechanisms, interpretation of results including the therapeutic and psycho-spiritual effects, and development of novel iboga analogs. Our results support a proposed theory that ibogaine and iboga alkaloids are molecular entities operating via a novel mechanism that likely underlies their remarkable therapeutic and healing effects. The present study adds a few new cornerstones to the molecular puzzle of the iboga pharmacology and puts forth a conceptual framework for the mechanistic complexity it presents.

Supplementary Material

The Supporting Information is available free of charge at the following link.

Experimental details include in vitro functional assays, rat brain synaptosome radioligand uptake and release, and behavioral catalepsy and locomotor activity evaluation protocols, data analysis, and results, synthetic schemes and protocols, and NMR spectra.