This in vitro (cells) study (1995) on ibogaine revealed the mechanisms of action to be at the mu, delta, kappa, opiate, serotonin 2 and 3, muscarinic 1 and 2 receptors, and de dopamine, norepinephrine, and serotonin reuptake sites. Ibogaine also interacted with NMDA associated and sodium ion channels.
The indole alkaloid ibogaine (NIH 10567, Endabuse) is currently being examined for its potential utility in the treatment of cocaine and opioid addiction. However, a clearly defined molecular mechanism of action for ibogaine's putative anti-addictive properties has not been delineated. Radioligand binding assays targeting over 50 distinct neurotransmitter receptors, ion channels, and select second messenger systems were employed to establish a broad in vitro pharmacological profile for ibogaine. These studies revealed that ibogaine interacted with a wide variety of receptors at concentrations of 1-100 µM. These included the mu, delta, kappa, opiate, 5HT2, 5HT3, and muscarinic1 and 2 receptors, and the dopamine, norepinephrine, and serotonin uptake sites. In addition, ibogaine interacted with N-methyl-D-aspartic acid (NMDA) associated ion and sodium ion channels as determined by the inhibition of [3H]MK-801 and [3H]bactrachotoxin A 20-α-benzoate binding (BTX-B), respectively. This broad spectrum of activity may in part be responsible for ibogaine's putative anti-addictive activity.
Indolealkylamines include centrally active compounds that can produce stimulant and anxiogenic effects. One such derivative, ibogaine, has been reported to reduce behaviours related to opiate, stimulant and alcohol dependence in animal and some human observations. Previous animal studies found that ibogaine decreased morphine self-administration, attenuated morphine- and cocaine-induced dopamine turnover, reduced morphine-induced motor activity, and antagonised cocaine-induced locomotion. These findings pointed to several neurotransmitter systems — bioaminergic (dopamine/serotonin), peptidergic (opioid), and amino acidergic (GABA) — as candidate molecular targets, but no single, clearly defined mechanism has been established that would explain ibogaine’s putative anti-addictive effects. This study set out to characterise ibogaine’s in vitro pharmacological profile across a broad panel of targets. Using over 50 radioligand binding assays, the investigators aimed to identify receptor, transporter, and ion‑channel interactions and to assess potency where activity was detected. The goal was to determine whether ibogaine acts selectively at one target or interacts with multiple sites at concentrations plausibly achieved in brain, thereby informing hypotheses about its mechanisms in addiction models and guiding subsequent medicinal chemistry efforts.
Papers in Blossom that reference this study
Ona, G., Reverte, I., Rossi, G. N. et al. · Journal of Psychopharmacology (2023)
Mash, D. C. · Pharmacological Research (2023)
Rodríguez-Cano, B. J., Kohek, M., Ona, G. et al. · Drug and Alcohol Review (2022)
González, J., Cavelli, M., Castro-Zaballa, S. et al. · ACS Pharmacology and Translational Science (2021)
Radioligand competition binding assays were used to survey ibogaine’s interactions with more than 50 neurotransmitter receptors, transporters, ion channels and selected second‑messenger sites. Assays were performed in 250 µl volumes, typically containing receptor preparation, radioligand and cold ligand or test compound; ibogaine and other compounds were solubilised in DMSO and diluted to a final DMSO concentration of 0.4% in the assay. Reactions were terminated by rapid filtration onto glass fibre filters, washed with cold buffer, and radioactivity determined by liquid scintillation or gamma spectrometry. Nonspecific binding was defined in the presence of saturating concentrations of cold ligand. Initial screens were run in duplicate; activity greater than 30% inhibition at a 10 µM screening concentration prompted follow‑up concentration–response studies. Full concentration–response curves were performed in triplicate on three separate days using different tissue preparations; 14‑point reference curves, total and nonspecific binding tubes, and positive control tubes accompanied each assay run. Where feasible, concentration–response curves were verified independently by another laboratory. Apparent Ki values were calculated using the Cheng–Prusoff relationship and KD values established at NovaScreen. Data fitting used an IC50 programme proprietary to NovaScreen or a non‑linear curve‑fitting package from Lundon Associates. Specific assay conditions (tissue sources, buffers, temperatures and incubation times) were referenced in tables in the original text but are not fully reproduced in the extracted material.
Sweetnam and colleagues found that ibogaine displayed inhibitory activity at multiple targets in the low micromolar range (approximately 1–100 µM), with no high‑affinity (nanomolar) interactions detected. The compound displaced the dopamine uptake site ligand [3H]WIN‑35,248 (a putative cocaine receptor) with an apparent IC50 of 3.5 ± 0.6 µM. At related monoamine transporters ibogaine was less potent: serotonin uptake IC50 ≈ 49 ± 3.2 µM and norepinephrine uptake IC50 ≈ 15 ± 4.4 µM. Several serotonergic receptors were affected. Ibogaine inhibited 5‑HT2 receptor binding ([3H]ketanserin; reported Kd ≈ 4.8 ± 1.4 µM) and 5‑HT3 receptor binding ([3H]GR‑65630; IC50 ≈ 3.9 ± 1.1 µM). No inhibition at the 5‑HT1 receptor was observed up to 1 mM. Dopaminergic post‑synaptic receptor subtypes D1, D2, D3 and D4 showed no meaningful inhibition, although a partial (~30%) reduction in [3H]clozapine binding was seen at 100 µM. At opioid sites, ibogaine displaced the κ‑selective ligand [3H]U‑69593 with an apparent IC50 of 16 ± 2.1 µM and had weaker activity at the μ subtype (reported IC50 ≈ 26 µM). Binding at the site labelled by [3H]DTG (historically referred to as a sigma/opiate site) was inhibited with an apparent IC50 of 38 ± 4.0 µM. Ion‑channel and related findings included inhibition of [3H]batrachotoxin B (BTX‑B) binding to the voltage‑gated sodium channel (IC50 ≈ 9 ± 3.0 µM). For NMDA‑associated sites ibogaine inhibited [3H]MK‑801 binding with an IC50 of 5.6 ± 0.8 µM and [3H]TCP binding with an IC50 of 50.5 ± 11 µM, indicating interaction with the NMDA ion channel region rather than the classical competitive glutamate sites. No activity was detected at the glycine (strychnine‑sensitive) receptor, GABAA receptor sites (including benzodiazepine and chloride channel sites), GABAB receptor, GABA uptake site, peripheral benzodiazepine site, or the classical excitatory amino‑acid (AMPA, kainate, NMDA competitive) binding sites. The compound also displaced muscarinic ligands consistent with weak M1 (IC50 ≈ 7.6 ± 0.7 µM) and M2 (IC50 ≈ 5.9 ± 1.4 µM) activity, and inhibited α1‑adrenergic ([3H]prazosin) binding with IC50 ≈ 7.2 ± 3 µM. Many other receptor classes and second‑messenger binding sites tested showed no inhibitory activity in these assays. Overall, the detected interactions clustered in the micromolar concentration range and were reproducible across multiple assay runs.
Sweetnam and colleagues interpret these results as evidence that ibogaine is a relatively non‑selective CNS agent that interacts with multiple receptor, transporter and ion‑channel targets at low micromolar concentrations. The investigators identified 14 distinct sites with measurable inhibition, spanning opioid subtypes, monoamine uptake sites, serotonergic receptors, muscarinic and α1 adrenergic receptors, sodium channels and the NMDA ion channel region. Because no nanomolar‑affinity targets were found, the authors emphasise that a multiplicity of modest‑affinity interactions could underlie ibogaine’s complex behavioural effects. Pharmacokinetic considerations are invoked to argue for potential in vivo relevance: preliminary data cited by the authors indicate that a 10 mg/kg subcutaneous dose produced brain tissue levels on the order of a few micromolar, suggesting that the in vitro micromolar activities documented here could be achieved in vivo. The discussion therefore considers how different interactions might contribute to reported anti‑addictive, locomotor and tremorogenic effects. For example, inhibition of the dopamine uptake site and modulation of 5‑HT3 receptors are proposed as indirect routes to alter dopaminergic synaptic function relevant to cocaine and opioid behaviours, whereas sodium‑channel blockade is offered as a plausible mechanism for ibogaine’s tremorogenic effects and for altering dopamine release/uptake dynamics. The authors also note potential safety concerns and unresolved questions. Interaction with the NMDA ion channel is highlighted because non‑competitive NMDA antagonists have been linked to psychotropic and neurodegenerative outcomes; the paper cites reports of cerebellar Purkinje cell degeneration after ibogaine and suggests further neurotoxicity studies are warranted. Discrepancies between the present binding profile and some prior studies are acknowledged and attributed primarily to methodological differences such as choice of receptor preparation or radioligand. Finally, the investigators propose alternative explanations for ibogaine’s in vivo actions: the observed effects might arise from (a) coordinated actions across multiple modest‑affinity targets, (b) an active, more selective metabolite not assessed here, or (c) an unidentified mechanism beyond the capabilities of the applied in vitro assays. They recommend medicinal‑chemistry efforts to generate more potent and selective ibogaine analogues and suggest using the present receptor profile as a template to guide such work.
Indolealkylamines are one branch of a large number of centrally active compounds that produce stimulatory and anxiogenic effects in animals. One indolealkyl-amine derivative, ibogaine, has been shown to elicit both actions in man. It has been suggested that ibogaine may have therapeutic potential in the treatment of opiate (heroin) addiction, stimulant (cocaine) abuse, and ethanol dependence by disrupting some aspect of physiological or psychological addiction. Recent studies have attempted to substantiate these claims by using in vivo animal models, and results appear to support ibogaine's use in substance abuse therapy. Briefly, ibogaine administration decreased morphine self-administration, blocked morphine-and cocaine-induced dopamine turnover, reduced morphine-induced motor activity, and antagonized cocaine-induced locomotor stimulation. From these aforementioned studies three neurotransmitter systems, e.g., the bioaminergic (dopamine/serotonin), peptidergic (opioid), amino acidergic (GABA), appear to be potential molecular targets responsible for ibogaine's therapeutic action. By employing over 50 different radioligand binding assays we have been able to establish a broad receptor selectivity and potency profile for ibogaine, thus extending prior observations. Our results revealed that ibogaine is a nonselective agent interacting with a number of receptor systems. These include the serotonergic, dopaminergic, muscarinic, opiate, and the amino acid-ergic systems. Many of the interactions demonstrated were in the 1 to 100-gM range and no receptors examined have demonstrated nanomolar attfnity for ibogaine. The potential in vivo significance of micromolar ibogaine interactions, as revealed in preliminary pharmacokinetic studies) and the broad range of micromolar CNS activities revealed in this study suggest that any one or combination of these activities may be involved in ibogaine's putative therapeutic action. Therefore a clear delineation of ibogaine's therapeutic mechanisms of action must be determined before the efficacy of such a compound in the treatment of addiction can be accurately evaluated.
Ibogaine HCL was purchased from Sigma Chemicals (St Louis, Mo). All [3H] or [125I] radioligands used were commercially available from NEN DuPont (Boston) or Amersham (Illinois). Unlabeled ligands and peptides were purchased from a variety of commercial sources.
Competition assays were performed in 250-gl volumes containing 200 gl receptor preparation, 25 ~tl radioligand, and 25 gl cold lig- and D4 assays human receptor clones were used.• and (non-specific binding determinant) or ibogaine. All compounds were solubilized in neat DMSO diluted to a final concentration of 0.4% in the assay. Assays were terminated by rapid filtration over Whatman glass fiber filters (GFC and GFB) followed by washing with 12 ml cold assay buffer. Radioactivity was determined by either liquid scintillation or gamma spectrometry. Nonspecific binding was defined as the radioactivity remaining in the presence of a saturating concentration of cold ligand. For specific assay conditions (i.e., tissue preparation, buffers, incubation times and temperatures, filter treatments) in assays in which ibogaine was run in concentration-response format the reader is referred to references noted in Table. Initial inhibitory binding determinations were performed in duplicate. Activity of >30% at a concentration of 10 gM was verified using a freshly prepared ibogaine solution. Total and nonspecific binding tubes, positive controls tubes, and 14-point reference curves were run with each and every assay. Concentration-response studies were performed using triplicate tubes and run on 3 different days using different tissue preparations. Where possible concentrationresponse curves were verified by an independent laboratory. Results were analyzed by an ICs~ program proprietary to NovaScreen or by a non-linear curve-fitting program from Lundon Associates. Apparent K~ values reported in Tablewere determined by the Cheng-Prusoff equation using KD values established at NovaScreen, a drug discovery division of Oceanix Biosciences Corporation.
Ibogaine displaced [3H]WIN-35,248 binding to the dopamine uptake site (putative cocaine receptor) with an apparent ICs0 of 3.5+0.6 btM (Fig.). At functionally related sites, including the serotonin and norepinephrine reuptake sites, ibogaine was at least an order of magnitude less potent (ICs0 s of 49 + 3.2 and 15 _+ 4.4 mM, respectively). Ibogaine inhibited binding at 5HT2 ([3H]ketanserin, K~= 4.8 + 1.4 gM) and 5HT3 ([3H]GR-65630, ICs0 = 3.9_+1.1 btM) receptors (Fig.). Ibogaine did not inhibit binding at the 5HTI receptor at concentrations 371 as high as 1 mM. Ibogaine did not interact at other bioaminergic receptor systems examined, including dopaminergic (D~, D2, 03 and D4) and histaminergic (H~). Ibogaine partially inhibited [3H]clozapine (a putative D4 ligand) binding at a concentration of 100 mM by approximately 30% (data not shown). A recent study bysuggested that ibogaine selectively inhibited ligand binding to the kappa opioid receptor. Utilizing opiate subtype selective receptor binding assays, we confirmed the ability of ibogaine to displace fl~e kappa selective ligand [3H]U-69593 with an apparent ICs0 of 16.+2.1 mM (Fig.). At the mu subtype ibogaine was less potent with IC50 s of approximately 26 + t.7. Ibogaine displaced [3H]DTG, apparent ICs0 of 38 + 4.0 gM, from a binding site originally referred to as the sigma opioid site (Fig.). A number of ion channels were examined and we confirmed that ibogaine displaces [3H]BTX-B binding to the voltage-gated sodium channel (ICs0=9 + _ 3.0 gM) (Fig.). We were unable to detect activity at other related and non-related ion channels. It was suggested that ibogaine may interact or modulate Z 100 J 80.
Fig.A lbogaine inhibits binding at various neurotransmitter reuptake sites. inhibitory amino acid-ergic receptors, in particular the GABAA receptor complex. We employed several radioligand binding assays, each of which targets a distinct binding site on the GABAA receptor complex. No inhibitory activity was apparent at GABA, benzodiazepine or chloride channel sites. In addition, there was no evidence for interaction at related sites, i.e., glycine (strychnine-sensitive) receptor, GABA uptake site, GABAB receptor, or the peripheral benzodiazepine binding site (Table). No inhibitory activity was detected at the classic competitive excitatory amino acid receptor binding sites, i.e., RS-c~-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA), kainate or NMDA, although ibogaine bound to the NMDA ion channel associated site, e.g., noncompetitive NMDA binding site, as demonstrated by its ability to inhibit [3H]MK-801 (ICs0 = 5.6_+0.8 gM) and [3H]TCP (ICs0 = 50.5 + 11 gM) binding (Fig.). No inhibitory activity at the glycine (strychnine-insensitive) NMDA modulatory site was detected. Many drugs acting on the central nervous system have pharmacological side effect profiles which include acting as weak muscarinic agents. Ibogaine appears to be both a weak M1 and M2 agent as determined by its ability to displace [3H]pirenzepine binding from bovine striatal membranes (IC50 = 7.6 + 0.7 gM) and [3H]AF- DX 384 from rat heart membranes (IC50=5.9_+ 1.4 gM) (Fig.). Ibogaine also displaced [3H]prazosin binding from alpha1 adrenergic receptors in the rat forebrain, ICs0 = 7.2 + 3 mM (Fig.). Ibogaine was inactive in a number of other receptor binding assays, including those for brain/gut peptides, prostaglandins, and second messenger binding sites (Table).
The present study suggests that the use of ibogaine in the treatment of cocaine or opiate abuse possesses a degree of uncertainty resulting fi'om an ill-defined mechanism of action. We have identified 14 distinct receptors through which ibogaine may elicit its central nervous system effects. These included opioid receptor subtypes, catecholamine uptake sites, several serotonin receptor subtypes, and two distinct ion channels. At each of these receptors ibogaine is an inhibitory agent with low micromolar (1-100 gM) activity. In vitro micromolar activities are often described as ancillary Using this assay we were unable to determine alpha~ subtype selectivity and may initially be overlooked when attempting to define a drug's in vivo mechanism of action. However, preliminary pharmacokinetic studies revealed that administration of 10 mg/kg ibogaine subcutaneously results in levels of approximately 74 nrn/g wet weight brain tissue 4 gM concentration, suggesting that micromolar activity may be important with this compound. The potential in vivo significance of micromolar ibogaine interactions was an important factor in determining the initial screening concentration used (10 gM) and percent binding inhibition (approximately 30%) which would initiate follow-up IC50 studies. Using this particular screening strategy we were able to eliminate increases in experimental variation often associated with the use of higher concentration, i.e., 100 gM). Opiate and cocaine abuse studies, as measured by paradigms such as self-administration, often center around the ability of compounds to influence dopaminergic synaptic function. Based on this, it might be surmised that ibogaine's proposed therapeutic utility could result from interaction with receptors that directly comprise these synapses or those receptors "upstream" or "downstream" which can modulate their function. [bogaine does not inhibit binding at D~,Da,D3, or D4 receptor subtypes. Thus reports of ibogaine's ability to alter cocaine-induced dopamine metabolism in limbic and striatal brain regions does not appear to be the result of direct effects on dopaminergic neurotransmission. The ability of ibogaine to partially inhibit [3H]clozapine binding to rat brain membranes but not binding at the D4 receptor is most likely a reflection of clozapine's non-dopaminergic binding activities. Similarly, the reported inhibition of [3H]haloperidol binding may also be the result of nondopaminergic interactions. Ibogaine, as previously reported, inhibits the binding of the cocaine analogto the dopamine uptake site. Functionally, ibogaine increases extracellutar levels of dopamine in various regions of the brain). However, this increase is not thought to be through the blockade of dopamine uptake. Recently a D2 receptormediated mechanism has been suggested to modulate dopamine uptake, but ibogaine's inability to interact with this subtype eliminates this as a direct mechanism of action). Ibogaine's ability to inhibit binding at the serotonin and norepinephrine uptake sites may be problematic, as indiscriminate inhibition of bioamine uptake may actually exacerbate cocaine-induced seizures and lethality. A strong correlation has been drawn between the hallucinogenic properties of many drugs, including the indolealkylamines, and their interaction with the 5HT receptors). In the present study we have shown that ibogaine interacts at the 5HT2 receptor, confirming Repke and colleagues' findings that ibo-gaine and close structural analogs inhibitketanserin binding in the low gM range. While ibogaine's interaction, either agonistic or antagonistic, at the 5HT2 receptor may be important in the hallucinogenic potential that has been described for ibogaine, recent studies suggest that direct antagonism of the 5HT2 receptors does not appear to reduce cocaine selfadministration). Ibogaine's potential 5HT3 activity, as defined by its ability to inhibit [3H]GR65630 binding to N1E-115 neuroblastoma cell membranes, may have significance in the treatment of cocaine abuse. 5HT3 receptor-mediated dopamine release might be one of the mechanisms by which ibogaine indirectly modulates dopaminergic synaptic function and thus the physiology of cocaine substance abuse. While the 5HT3 antagonist zaclopride has been shown to partially block morphine-induced mesolimbic dopamine function and cocaine-induced locomotion, another antagonist, ondansetron, does not appear to modulate the discriminative or reinforcing stimulus of cocaine. In our study ibogaine displaced binding to kappa and mu opioid receptors in the low gM range. Interestingly it has been suggested that the opiate buprenorphine's kappa/mu crosstalk plays an important role in its ability to modify opioid and cocaine addiction. If this is so, the inability ofdetect inhibitory activity at the mu receptor would be inconsistant with such a mechanism. However, as previously suggested, this descrepancy, as with the others noted in Table, may simply be the result of procedural differences in the assays employed in each study. The attenuation of morphine self-adminisration following acute administration of ibogaine has been reported to be a rapid onset phenomenon, possibly the result of ibogaine's ability to induce whole body tremors/shakes. The ability of other indole alkaloids structurally related to ibogaine, i.e. tabernanthine, to induce tremoregenic activity through a GABAA supramolecular complex-mediated pathway suggested this as a possible molecular site of such action. However, radioligand binding assays which targeted different binding sites associated with the GABAA complex confirmed previous studies which could find no evidence to support such a mechanism of action). However, ibogaine's ability to inhibit [3H]BTX-B binding to the sodium channel binding site 2, thus altering membrane conductance and potential could result in the reported tremoregenic acitivty. In addition, such an interaction could result in the modulation of both dopamine release and uptake resulting in the increased levels of extracellular dopamine that have been reported following ibogaine administration. The ability of ibogaine to inhibit both [3H]MK-801 and [3H]TCP binding to the ion channel associated with), as well as strychnine-insensitive NMDA receptor linked glycine partial agonists) have all been shown to attenuate or reverse the development of tolerance, dependence or hyperalgesia to repeated administration of various opiates in rodents. These results have been interpreted to suggest that competitive or non-competitive blockade of NMDA receptor function could provide a new treatment for opioidtolerant or dependent patients. However, if the psychotropic and neurodegenerative potential of noncompetitive NMDA antagonists are the result of an interaction with the ion channel binding site, then it may be speculated that ibogaine's interaction at this site could also result in untoward in vivo effects. For example, ibogaine has been reported to induce purkinje cell degeneration in cerebellar tissues. Given the current results, such degeneration may occur through an interaction at the NMDA receptor. Clearly, further studies on the potential neurotoxic properties of ibogaine are warranted. As mentioned, we report a different receptor binding profile from those reported in several previous studies. These apparent differences are difficult to reconcile, but in most cases significant differences in assay protocol may be the cause. The most notable differences, i.e., choice of receptor preparation or radiotigand, are summarized in Table. In conclusion, the present data suggest that ibogaine interacts with receptor types commonly associated with opiate and cocaine abuse, e.g. dopamine uptake and 5HT2, as well as those receptor types only now being linked to addiction and tolerance. Interestingly, ibogaine's receptor profile is similar to that which has been ascribed to cocaine. These findings support a concept in which the physiological and psychological problems associated with the many aspects of substance abuse and chronic drug treatment appear to be the result of numerous but distinct molecular mechanisms performing in concert. Alternatively, these data may suggest that an unidentified and more selective and potent metabolite of ibogaine is the casual agent or that an unidentified mechanism of action is important. If the latter is so, our understanding of ibogaine's putative anti-addictive properties may be beyond the capabilities of available in vitro methodologies. Today CNS drug development efforts attempt to focus on compounds which potently and selectively target a single receptor or enzyme. The development of more potent and selective ibogaine analogs may be essential to developing a compound for therapeutic use. This receptor profile provides researchers with a template from which to monitor the medicinal chemistry efforts required to attain this goal.
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