A Single Administration of the Atypical Psychedelic Ibogaine or Its Metabolite Noribogaine Induces an Antidepressant-Like Effect in Rats

Article abstract of DOI:10.1021/acschemneuro.0c00152

Anecdotal reports and open-label case studies in humans indicated that the psychedelic alkaloid ibogaine exerts profound antiaddictive effects. Ample preclinical evidence demonstrated the efficacy of ibogaine, and its main metabolite, noribogaine, in substance-use-disorder rodent models. In contrast to addiction research, depression-relevant effects of ibogaine or noribogaine in rodents have not been previously examined. We have recently reported that the acute ibogaine administration induced a long-term increase of brain-derived neurotrophic factor mRNA levels in the rat prefrontal cortex, which led us to hypothesize that ibogaine may elicit antidepressant-like effects in rats. Accordingly, we characterized behavioral effects (dose- and time-dependence) induced by the acute ibogaine and noribogaine administration in rats using the forced swim test (FST, 20 and 40 mg/kg i.p., single injection for each dose). We also examined the correlation between plasma and brain concentrations of ibogaine and noribogaine and the elicited behavioral response. We found that ibogaine and noribogaine induced a dose- and time-dependent antidepressant-like effect without significant changes of animal locomotor activity. Noribogaine's FST effect was short-lived (30 min) and correlated with high brain concentrations (estimated >8 μM of free drug), while the ibogaine's antidepressant-like effect was significant at 3 h. At this time point, both ibogaine and noribogaine were present in rat brain at concentrations that cannot produce the same behavioral outcome on their own (ibogaine ～0.5 μM, noribogaine ～2.5 μM). Our data suggests a polypharmacological mechanism underpinning the antidepressant-like effects of ibogaine and noribogaine.

Full text of DOI:10.1021/acschemneuro.0c00152

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Article
A Single Administration of the Atypical Psychedelic Ibogaine or its
Metabolite Noribogaine Induces an Antidepressant-like Effect in Rats
Paola Rodríguez, Jessika Urbanavicius, Jose Pedro Prieto, Sara Fabius, Ana
Laura Reyes, Vaclav Havel, Dalibor Sames, Cecilia Scorza, and Ignacio Carrera
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.0c00152 • Publication Date (Web): 24 Apr 2020
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A Single Administration of the Atypical Psychedelic Ibogaine or its Metabolite
Noribogaine Induces an Antidepressant-like Effect in Rats
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Paola Rodríguez1,2, Jessika Urbanavicius2, José Pedro Prieto2, Sara Fabius2, Ana
Laura Reyes3, Vaclav Havel4, Dalibor Sames4*, Cecilia Scorza2*, Ignacio Carrera1*
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1Laboratorio de Síntesis Orgánica, Departamento de Química Orgánica, Facultad de
Química - Universidad de la República, Montevideo, Uruguay
2Departamento de Neurofarmacología Experimental, Instituto de Investigaciones
Biológicas Clemente Estable, Montevideo, Uruguay
3Centro Uruguayo de Imagenología Molecular, Montevideo, Uruguay
4Department of Chemistry, Columbia University, New York, USA
*Corresponding authors: Dalibor Sames, Cecilia Scorza and Ignacio Carrera
Keywords: psychedelic drugs, depression, preclinical test, serotonin, SERT
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Abstract
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Anecdotal reports and open label case studies in humans indicated that the
psychedelic alkaloid ibogaine exerts profound anti-addictive effects. Ample preclinical
evidence demonstrated the efficacy of ibogaine, and its main metabolite noribogaine, in
substance use disorder rodent models. In contrast to addiction research, depression-
relevant effects of ibogaine or noribogaine in rodents have not been previously
examined. We have recently reported that the acute ibogaine administration induced a
long-term increase of brain-derived neurotrophic factor mRNA levels in the rat
prefrontal cortex, which led us to hypothesize that ibogaine may elicit antidepressant-
like effects in rats. Accordingly, we characterized behavioral effects (dose and time-
dependence) induced by the acute ibogaine and noribogaine (20 and 40 mg/kg, i.p.)
administration in rats using the forced swim test (FST). We also examined the
correlation between plasma and brain concentrations of ibogaine and noribogaine and
the elicited behavioral response. We found that ibogaine and noribogaine induced a
dose- and time-dependent antidepressant-like effect without significant changes of
animal locomotor activity. Noribogaine’s FST effect was short lived (30 minutes) and
correlated with high brain concentrations (estimated > 8 µM of free drug), while the
ibogaine’s antidepressant-like effect was significant at 3 hours. At this time point, both
ibogaine and noribogaine were present in rat brain at concentrations that cannot
produce the same behavioral outcome on their own (ibogaine ~ 0.5 µM, noribogaine ~
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2.4 µM). Our data suggests a polypharmacological mechanism underpinning the
antidepressant-like effects of ibogaine and noribogaine.
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INTRODUCTION
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Ibogaine is the main indole alkaloid isolated from the root bark of the African
shrub Tabernanthe iboga (Figure 1).1-3 It is considered an atypical psychedelic drug
capable of inducing waking dream-like states (oneirogenic effects) and vivid memory
recall and replay.4, 5 Anecdotal reports and open label case studies with volunteers
seeking detoxification from heroin and cocaine indicated ibogaine’s ability to interrupt
the drug dependence phenotype via rapid and lasting relief of drug withdrawal
symptoms and cravings.6-8 Two recent open label observational clinical studies with
subjects diagnosed with opioid dependence confirmed the earlier reports by showing a
significant reduction of withdrawal symptoms (3 days post-treatment) and an
improvement of quality of life (up to 12 months) after a single ibogaine therapeutic
session.9, 10 In addition, a sustained antidepressant effect (evaluated at 1 and 12 months
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post-treatment) was recently reported,7, 10 consistent with other observations of ibogaine’s
,5, 11, 12
attenuation of depressive symptoms in humans.
The addiction-interrupting and
antidepressant properties of ibogaine are of high interest considering the renaissance of
psychedelic research and re-emerging use of psychedelic compounds as experimental
therapeutics in neuropsychiatric disorders.13-15
Extensive preclinical work recapitulated the clinical effects of ibogaine in
rodent models of substance use disorders (SUDs), including attenuation of self-
administration of opioids, cocaine, nicotine, and alcohol, as well as a reduction of
opioid withdrawal symptoms in opioid-dependent animals.16, 17 In addition, it has been
shown that noribogaine, ibogaine’s major metabolite (Figure 1), exhibits a similar
potency and efficacy profile in rodent models of SUDs, leading to a mechanistic model
where noribogaine contributes to the observed anti-SUD effects.18
We recently reported in rats that ibogaine induces a dose-dependent
upregulation of glial cell-derived neurotrophic factor (GDNF) in specific brain regions of
the rat brain related to the mesocorticolimbic and nigral dopaminergic circuits,19 an
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effect which was previously proposed as a molecular mechanism underlying the
attenuation of drug self-administration and drug craving by ibogaine.20, 21 In addition, we
also found a large upregulation of brain-derived neurotrophic factor (BDNF) expression
in the prefrontal cortex after a single ibogaine administration, suggesting that ibogaine
may exert an antidepressant-like effect, since an increased BDNF signaling plays a
critical role in the antidepressant-like efficacy of selective serotonin uptake inhibitors
(SSRIs) in rodents.22-24 Also, we previously showed that ibogaine administration in rats
promoted an increase in wakefulness time and a long-lasting rapid eyes movement
(REM) sleep suppression, a profile which is also shared with SSRIs.25 The increase in
wakefulness time in rodents has been also reported for rapidly acting antidepressants
such as ketamine and mGlu2/3 receptor antagonists.26 Since it is well known that
ibogaine and noribogaine inhibit the plasma membrane serotonin transporter (SERT)
(noribogaine displaying approximately ten-fold greater inhibitory potency at SERT, IC50
~ 50-300 nM than ibogaine),27-29 we therefore postulated that ibogaine administration in
rats would exhibit antidepressant-like effects. In this manner, we hypothesized that the
above noted physiological effects and the BDNF upregulation would be, at least in part,
downstream consequences of the serotonin neurotransmission enhancement28, 30, 31
generated by SERT inhibition. However, ibogaine can also block N-methyl-D-aspartate
receptors (NMDA-R),32 and NMDA-R non-competitive antagonists such as ketamine
and dizocilpine were also shown to exhibit antidepressant-like effect in rodents.33-36
Moreover, noribogaine was recently found to promote neuritogenesis in rat cortical
primary cultures, which could also contribute to a potential antidepressant-like effect in
rats.37
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In contrast to the large body of literature on SUD models, to our knowledge
the preclinical examination of antidepressant-like effects of ibogaine has not been
reported. Considering the clinical observations and the mechanistic considerations
mentioned above, we set out to examine the effects of ibogaine and noribogaine in the
forced swim test (FST) in rats, a widely used preclinical assay, that measures coping
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strategy to an acute inescapable stress, with well-established responsiveness to a
broad range of clinically efficacious antidepressants.38-40 In this manner, the present
study was designed to characterize the behavioral effect (dose and time-dependence)
induced by acute administration of ibogaine and noribogaine in rats using the FST.
Additionally, the plasma and brain concentration of ibogaine and noribogaine were
measured to associate the behavioral outcomes to the pharmacokinetic profiles of both
drugs.
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Figure 1. The psychoactive alkaloid ibogaine, its source, metabolism, and summary of
clinical and preclinical effects. Illustration of different parts of Tabernanthe iboga. Ibogaine is
isolated from the root bark. It is converted in vivo by the CYP2D6 isoform to its main active
metabolite, noribogaine. In contrast to extensive examination of ibogaine in preclinical SUD
models, antidepressant-like effects in rodents have not been investigated despite the clinical
signals observed in anecdotal and open label studies.
RESULTS AND DISCUSSION
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Ibogaine elicits dose-dependent and time-dependent reduction in the
immobility time in the forced swim test. The FST has been extensively used as a
behavioral screening test for antidepressant drugs with different mechanisms of
action.41, 42 When rats are subjected to the standard FST, two swimming sessions are
carried out 24 hours apart. The first one called “pre-test” has a duration of 15 minutes
and is aimed to stress and sensitize the animals to develop immobility in the second
session, the actual “test”, that lasts only 5 minutes (where the time animals spend
immobile, swimming or climbing is measured).38, 43 Antidepressant drugs are typically
administered 2-3x before the test swimming session (e.g. 24, 5 and 1 hour before the
test session) and induce significant reduction in the time animals spend immobile in
comparison to the vehicle control group (i.e., rats pursue more active escape
strategy).38, 40 We decided to examine the effect of acute administration of ibogaine in
the FST, since most of the previous pre-clinical studies using SUDs models were
carried out with a single intra-peritoneal (i.p.) administration of 20 mg/kg (I20) or 40
mg/kg (I40).17 Based on previous behavioral and pharmacokinetic (PK) studies using
i.p. administration,28, 44-46 and our own previous work,19, 25 we selected two time points
for the FST as 3 and 24 hours after ibogaine administration. At 3 hours, ibogaine and
noribogaine are expected to have pharmacologically and behaviorally relevant
concentrations in the rat brain, while the transient behavioral effects observed
immediately after the i.p. administration of I40 (i.e. hypoactivity, tremor and
piloerection), which interfere with both the passive (immobility) and active (swimming
and climbing) behaviors, are absent at this time point. In contrast, at 24 hours ibogaine
would be no longer present in the brain and noribogaine would be at pharmacologically
irrelevant concentrations, allowing for examination of potential long-lasting effects
produced by both substances.
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Figure 2A shows the effect of both doses of ibogaine in the FST evaluated 3 h
after the acute administration. For immobility time, one-way ANOVA revealed a
significant effect for treatment [F(2,20) = 4.11, P < 0.03]. The Tukey post hoc test
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showed that only I40 was able to induce a significant decrease in the immobility time
compared to the control group (P < 0.05; Fig. 2A). This can be interpreted as an
antidepressant-like effect induced by I40, and not due to nonspecific alterations on the
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animal motor activity since One-way ANOVA showed no significant changes [F(2,15)
=
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2.58, P = 0.11] in the first 5 min of the animals motor activity evaluated 3 h after the
drug i.p. administration in the open field test (OFT, Fig. 2A,d). Remarkably, whereas a
wide range of clinically efficacious antidepressants typically require two or more
administrations to induce an antidepressant-like behavior in the rat FST, a single acute
I40 administration elicited a statistically significant effect. In contrast, one-way ANOVA
did not reveal a significant effect for the treatment in the swimming [F(2,20) = 2.73, P =
0.08] or climbing time [F(2,20) = 0.65, P = 0.53]. Thus, neither active behavior was
significantly altered by ibogaine administration, however there was an upward trend in
the swimming readout. Figure 2B shows the effect of ibogaine in the FST evaluated 24
h after its i.p. administration. One-way ANOVA did not reveal a significant effect for the
treatment; neither for immobility [F(2,17) = 0.85, P = 0.44], swimming [F(2,17) = 1.31, P =
0.29] nor climbing time [F(2,17) = 0.53, P = 0.59], although the swimming time showed a
qualitative trend toward a dose-dependent increase.
In order to correlate the behavioral results and the in vivo concentrations of
ibogaine and noribogaine, we carried out PK studies in brain (Figure 2C) and plasma
(See Supporting Information, SI) for both drugs after I40 using the same route of
administration, evaluation time, and rat strain.
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Figure 2. Ibogaine i.p. acute administration elicits dose-dependent and time-dependent
antidepressant-like effects in rats. A) Effect of ibogaine (i.p. 20 and 40 mg/kg) evaluated in
the FST (a-c) 3 h after the acute administration. The locomotor activity was recorded in a
separate group of animals in the Open Field Test (OFT, d). The inset graph shows the distance
traveled by the animals in the first 5 min, in accordance with the test session period of the FST.
Only I40 was able to induce a significant decrease in the immobility time compared to the control
group (P < 0.05) without any significant change in locomotor activity. B) Effect of Ibogaine (i.p.
20 and 40 mg/kg) evaluated in the FST 24 h after the administration. No significant
antidepressant-like effect was found for either dose. C) Pharmacokinetic profile showing
ibogaine and noribogaine total concentrations in the rat brain after I40 i.p. administration
(concentrations of ibogaine and noribogaine at FST relevant time points are highlighted). Error
bars represent mean ± SEM. One-way ANOVA followed by Tukey's multiple comparisons test.
* = P < 0.05 as compared to vehicle control. N = 7 - 8
Our results confirmed earlier PK reports in rats with regard to ibogaine´s fast
metabolism to noribogaine (which becomes the major species in plasma and brain
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relative to ibogaine after ~ 45 min) and a high brain/plasma concentration ratio for both
drugs (ibogaine ~7.7 and noribogaine ~7.9). Nevertheless, there were some
differences between our data and that reported by Baumman et al28: namely, our
results showed ibogaine´s Cmax being greater than the noribogaine’s value, both in
plasma (1.5 fold) and in brain (2.2 fold) (for PK details see Supporting Information),
while the reversed scenario was found by the noted report. A possible explanation for
this discrepancy can be related to differences between whole blood (Baumann) and
plasma (our study) drug content and or a different strain of rats used.47
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Correlation of the PK values and the behavioral outcome suggests that the
response observed in the FST depends on the simultaneous presence of both ibogaine
and noribogaine in the brain. At 3 hours where a significant reduction in the immobility
was found, both drugs are circulating and evident in the brain tissue, where the mean
total concentration of 21 µM was found for noribogaine and 9.9 µM for ibogaine (Figure
2C). In contrast, at 24 hours, where no effect was observed in the FST, ibogaine was
not detectable while a residual amount of noribogaine (a mean value of 0.5 µM, ~2% of
its Cmax) was found in the brain. These results led to the next question of which
compound is responsible of the reduction of the immobility time at 3 hours after the I40
treatment.
Norbogaine induces a dose-dependent antidepressant-like effect in rats
but with a different time profile than ibogaine. In order to study the contribution of
noribogaine to the behavioral effect induced by ibogaine, we examined the acute effect
of two doses of noribogaine, 20 mg/kg (N20) and 40 mg/kg (N40) in the FST. Since no
previous complete pharmacokinetic profiles were found in the literature for noribogaine
administered i.p. in rats, concentrations in plasma and brain were determined up to 24
hours for N40 (see Figure 3C) in order to select the time points for the FST study. The
pharmacokinetic data obtained is consistent with previous results in several species
showing high bioavailability and high brain penetration of noribogaine after direct
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noribogaine administration (for key parameters, see Supporting Information).18, 27, 45, 46,
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Guided by the PK results, we carried out the FST at 0.5 h after noribogaine i.p.
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administration, where the maximal brain concentration of noribogaine is achieved.
Additionally, to compare the results to those obtained after 3 h of i.p. ibogaine
administration, another experimental group was assayed at this time point after
noribogaine administration.
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Results showed a significant and robust effect for the noribogaine treatment
on the time rats spent immobile after 0.5 hours of the i.p. administration [F(2,18) = 7.24,
P < 0.01] (Figure 3A). Tukey post-hoc analysis showed a significant decrease in the
immobility time following N40 relative to vehicle-pretreated animals (P < 0.01) and the
lower dose N20 treatment (P < 0.05). In contrast, one-way ANOVA did not reveal
significant changes for swimming [F(2,18) = 2.44, P = 0.11] or climbing behaviors [F(2,18)
= 1.85, P = 0.18]. To evaluate potential behavioral confounds, the motor activity of
animals was examined (Figure 3A). One-way ANOVA indicated that no significant
changes in the animal locomotor performance were seen after N40 treatment [F(2,12)
=
0.60, P = 0.56]. However, at 3 hours one-way ANOVA did not show significant changes
[F(2,14) = 0.77, P = 0.48] in the immobility time of noribogaine-treated animals (Figure
3B). Further, neither swimming [F(2,14) = 0.65, P = 0.53] nor climbing behavior [F(2,14)
=
0.48, P = 0.62] were altered significantly by noribogaine (Figure 3B), although there
was a qualitative upward trend in the swimming time.
The PK results showed that at 0.5 hours a mean total concentration of 144
µM of the drug was reached in the brain. Since N20 was not effective at significantly
altering the behavioral patterns of the animals, assuming a linear concentration
dependence of the pharmacokinetic profile, total concentration of ~ 72 µM of
noribogaine (half of 144 µM found for the Cmax after N40 dose) was not sufficient to
produce a statistically significant effect, although a clear trend was observed (Figure
3A).
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Figure 3. Noribogaine i.p. acute administration elicits dose-dependent and time-
dependent antidepressant-like effects in rats. A) Effect of noribogaine (20 and 40 mg/kg)
evaluated in the FST 0.5 h after the acute administration. The locomotor activity was recorded
in a separate group of animals in the Open Field Test (OFT, d). The inset graph shows the
distance traveled by the animals in the first 5 min, in accordance with the test session period of
the FST. N40 was able to induce a significant decrease in the immobility time relative to vehicle-
pretreated animals (P < 0.01) and N20 (P < 0.05), without any significant change in the distance
traveled by the animals in the OFT. B) Effect of noribogaine (i.p. 20 and 40 mg/kg) evaluated in
the FST 3 h after the acute administration. No antidepressant-like effect was found for either
dose. C) Pharmacokinetics profile showing noribogaine concentrations in rat brain and plasma
after N40 i.p. administration (concentrations of noribogaine at FST relevant time points are
highlighted). Error bars represent mean ± SEM. One-way ANOVA followed by Tukey's multiple
comparisons test. ** = P < 0.01 as compared to vehicle control and + = P < 0.05 as compared to
noribogaine. N = 5 - 9
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Similarly, the mean total brain concentration of 31 µM of noribogaine
determined at 3 hours is not enough to exert an antidepressant-like effect in the FST.
Also, the brain exposure to noribogaine over the period of 3 hours (AUC) after direct
noribogaine administration is much greater than the corresponding exposure after
ibogaine administration (and even the combined exposure to ibogaine and
noribogaine). Consequently, it is evident that noribogaine at exposures and
concentrations found in the rat brain 3 hours after i.p. administration of ibogaine (I40, 21
µM, compare concentration values at 3 hours in Figure 2C and 3C) cannot alone drive
the antidepressant-like effect of ibogaine. Therefore, the next question was whether
ibogaine itself may actuate the antidepressant-like effect at the brain concentration at
or above that found 3 hours post drug administration (9.9 µM, I40 treatment). In order to
investigate this possible scenario, we examined the capacity of ibogaine to evoke an
antidepressant-like effect in the FST under conditions where metabolism to noribogaine
is minimized.
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Ibogaine itself does not drive the antidepressant-like effect found in the
FST after ibogaine i.p. administration. In this experiment, we administered ibogaine
through the intravenous route (i.v.) and evaluated the behavioral response in the FST.
As reported previously,28 ibogaine i.v. administration greatly reduced its metabolism to
noribogaine because of liver first-pass avoidance, producing maximal ibogaine
concentration values one-minute post-injection, with only trace amounts of noribogaine
detected in blood. An ibogaine dose of 10 mg/kg i.v. used in that study, produced
significant transient behavioral impairments in rats, such as tremors, forepaw tapping,
abnormal postures, body sway and staggering-type locomotion that lasted for 30
minutes after administration. In order to avoid these behavioral impairments that could
interfere with the swimming or climbing performance, we tested the effect in the FST 1
minute after the i.v. administration of 1 mg/kg (I1) and 5 mg/kg (I5) of ibogaine (Figure
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4A). In addition, concentration profiles for ibogaine and noribogaine in plasma and
brain were determined after the higher dose (Figure 4B).
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For immobility time, one-way ANOVA did not reveal a significant effect for the
treatment [F(2,20) = 0.73, P = 0.49]. In addition, neither swimming [F(2,20) = 1.81, P =
0.18] nor climbing behavior [F(2,20) = 2.80, P = 0.08] were altered significantly by either
ibogaine i.v. doses. Nevertheless, a trend for reduction in the immobility time can be
observed for the I5 treatment. While it is likely that ibogaine would show a reduction in
the immobility time at higher concentrations, it is not feasible to examine higher doses
due to the above-mentioned behavioral side effects that are not compatible with animal
performance in the FST. Examining brain PK results we confirmed that total
noribogaine concentrations at 1 minute after injection were minimal (~0.07 µM) and
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ibogaine total concentration was determined to be 23 µM. Therefore, this brain level of
ibogaine on its own was not able to produce a robust antidepressant-like effect in the
FST. Comparing this result with the concentration of ibogaine detected 3 hours after
i.p. I40 administration (~ 10 µM), it seems that ibogaine itself cannot be responsible for
reduction in the immobility time found in the FST during this experimental condition.
There is a caveat to this interpretation related to the limited brain exposure to ibogaine
after 1 minute, compared to the much greater exposure after 3 hours.
Figure 4. Ibogaine i.v. acute administration does not elicit an antidepressant-like effect in
rats. A) Effect of ibogaine (i.v. 1 and 5 mg/kg) evaluated in the FST 1 minute after the acute
administration. No antidepressant-like effect was found at either dose. B) Pharmacokinetics
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profile showing ibogaine and noribogaine total concentrations in brain after I5 i.v. administration.
Error bars represent mean ± SEM. One-way ANOVA followed by Tukey's multiple comparisons
test. Non significance was found. N = 7 - 9
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Interpretation: Both ibogaine and noribogaine are required for expression of
antidepressant-like effect in the FST after ibogaine i.p. administration. Figure 5 A-
B summarizes the relationship between brain PK and behavioral data obtained in the
FST described previously. As can be seen, 23 µM of ibogaine found in the rat brains at
1 minute after i.v. I5 administration, or 31 µM of noribogaine found after 3 h of i.p. N40
administration, did not produce a statistically significant antidepressant-like response in
the FST. In contrast, when both drugs are present in the brain together at even lower
individual concentrations (9.9 µM ibogaine, 21 µM noribogaine) after 3 h i.p. I40
administration, a significant reduction in the immobility time was observed, suggesting
a putative additive or synergistic effect produced by both drugs after I40 administration.
Alternatively, these results could be rationalized by formation of an additional unknown
metabolite of ibogaine (that would be responsible for the antidepressant-like effect),
which would be formed after the i.p. but not intravenous administration.
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Figure 5. Correlation between pharmacokinetic data of ibogaine and noribogaine and the
behavioral outcomes induced by both drugs in the FST at different times. Total
concentrations of ibogaine and noribogaine found in rat brain after treatment A) without effect
and B) with antidepressant-like effect. C) Binding of ibogaine and noribogaine to plasma
proteins and brain tissue. D) Estimated free brain concentrations of ibogaine and noribogaine
found in rat brain that correlate with an antidepressant-like effect. Error bars represent mean ±
SEM.
Regarding a possible neurochemical mechanism that explains the observed
antidepressant-like effect, both drugs have been shown to inhibit SERT, noribogaine
being approximately ten-times more potent than ibogaine (IC50 for noribogaine ~ 50-
300 nM).27-29 We determined the plasma protein binding (in rat and human plasma) and
rat brain tissue binding for both ibogaine and noribogaine, which allows for estimation
of free plasma and free brain concentrations (Figure 5C and 5D). We estimate that at 3
hours post I40 free brain concentration of ibogaine is ~ 0.5 µM and noribogaine 2.4 µM,
which is within a range relevant for SERT modulation. Thus, both drugs could inhibit 5-
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HT reuptake and produce an enhancement in serotoninergic transmission. In vivo
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microdialysis studies in rats treated with ibogaine and noribogaine (1 and 10 mg/kg,
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i.v.),28,
showed that both drugs can induce significant increments in the
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extracellular 5-HT content in the nucleus accumbens (NAcc) within the same range
(two to threefold compared to the control group, noribogaine being more potent),
comparable to effects induced by SSRIs. Similar results were obtained evaluating the
effect of ibogaine on 5-HT tone using in vivo microvoltammetric studies.49 However,
additional neurochemical studies should be done to investigate the per se effect of
ibogaine or noribogaine and their combination at the same doses assayed here, on 5-
HT extracellular levels in other brain regions relevant to depression and mood
disorders (e.g., prefrontal cortex and hippocampus).
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Finally, it is well documented that antidepressant drugs that selectively inhibit
noradrenaline uptake reduce immobility and increase climbing behavior without
affecting swimming, while antidepressants that inhibit serotonin uptake decrease
immobility but increase swimming behaviour.42 Neither of active behaviors were
significantly modified by ibogaine and noribogaine treatments used in this study,
although there were trends toward increasing the swimming time compared to the
control groups. This may reflect the abovementioned enhancement in the
serotoninergic transmission owing to SERT inhibition and 5-HT re-uptake attenuation.
However, SERT modulation does not provide a comprehensive explanatory model as
the estimated free brain concentration of > 8 µM is required for noribogaine to elicit
significant FST effects when used alone: N20 was not significant, which is estimated to
give ~ 72 µM of noribogaine total tissue concentration (½ of 144 µM measured at 30
minutes), which is approximately equivalent to ~ 8 µM of free noribogaine brain
concentration). This estimated concentration range is > 20 greater than the in vitro
SERT inhibitory potency of noribogaine, the more potent SERT inhibitor of the two
compounds. The two-drug effect suggests additional target(s) at play. To further probe
this hypothesis, we carried out an additional experiment to test whether fluoxetine (a
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classical SSRI) was able to induce an antidepressant-like effect in the FST after a
single dose of 40 mg/kg.
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Fluoxetine does not induce an antidepressant-like effect after a single dose
treatment. To compare the efficacy of the iboga alkaloids used in this study to classical
SSRIs, we examined the effect elicited by fluoxetine, the prototypical SSRI, following
the same injection schedule and dose as those for noribogaine (Figure 6). In contrast
to noribogaine, fluoxetine (40 mg/kg) did not modify the immobility (t10 = 0.62, P =
0.55), swimming (t10 = 0.32, P = 0.75) or climbing time (t10 = 0.28, P = 0.78),
suggesting that noribogaine was more effective than fluoxetine at inducing an
antidepressant-like effect in this model. In order to obtain a significant reduction in the
immobility time, three doses of fluoxetine were required in our experimental setting
(See supporting information, consistent with previous reports).38-40 Fluoxetine is a
potent SERT blocker (low nM range)50 and produces significant increases in
extracellular 5-HT as established by in vivo microdialysis studies.51 Our results suggest
substantial mechanistical differences between the iboga alkaloids and classical SSRIs.
It is known that ibogaine and noribogaine display differences in their interaction with
SERT in comparison to SSRIs and other common antidepressant drugs. While typical
SSRIs inhibit SERT in a competitive manner to 5-HT, generally stabilizing an outward-
facing conformation,52 ibogaine and noribogaine act non-competitively at the
transporter but display competitive binding towards SSRIs and stabilize an inward-
facing conformation.29, 53-55 Moreover, ibogaine and noribogaine have been classified
as pharmacochaperones, being able to rescue folding-deficient SERT mutants, an
effect not exhibited by SSRIs.56 The functional and biological consequences for these
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different interaction modes need further investigation. Alternatively,
a
polypharmacological mode of action for the iboga alkaloids with respect to
antidepressant-like behavior represents a rational mechanistic model (e.g. NMDA-R
non-competitive antagonism, which has been described for ibogaine, can contribute to
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its antidepressant-like effects). Although ibogaine and noribogaine trigger long-term
plasticity effects, such as the expression upregulation of GDNF and BDNF shown in
our recent report,19 the FST readouts in the present study did not indicate significant
delayed behavioral effects. The potential long-term behavioral effects will be examined
in future studies.
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Efforts toward elucidating the neurochemical mechanisms behind the
antidepressant-like effects (found in this study) and anti-SUD effects are ongoing in our
laboratories, along with our medicinal chemistry program aimed at mapping the SAR of
iboga-inspired analogs.57
Figure 6. Fluoxetine 40 mg/kg i.p. acute administration does not elicit an antidepressant-
like effect in rats. Acute effect of F40 administration in the FST 30 minutes after the i.p.
administration. No antidepressant effect was found. Error bars represent mean ± SEM.
Unpaired Student t-test. Non significance was found. N = 6
CONCLUSIONS
In the present study we demonstrated for the first time that an i.p.
administration of ibogaine and noribogaine in rats produce an acute antidepressant-like
effect in a dose- and time-dependent manner. Using a single dose of 40 mg/kg, the
effect was observed after 3 hours of administration of ibogaine, while noribogaine had
a shorter duration (present 30 minutes after administration but lost at 3 hours). We
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demonstrated that the antidepressant-like response produced after ibogaine i.p.
administration correlates with the presence of both ibogaine and noribogaine in rat
brain at concentrations that cannot produce the same behavioral outcome on their own,
indicating a potential additive/synergistic effect of both drugs or the existence of an
unknown active metabolite. Moreover, the effect found using a single dose of ibogaine
or noribogaine could not be reproduced by an equivalent dose of the classic SSRI
fluoxetine.
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The translational utility of FST has been challenged owing to numerous
failures in clinical studies of compounds that showed positive results in FST and other
preclinical models of psychiatric disorders. However, drug development is a complex
process and the notion that the preclinical animal models are not predictive of clinical
outcomes may be an inaccurate generalization, according to recent surveys of the
available data.58 The pharmacological validity or reverse translational validity of FST is
excellent as many different classes of clinically efficacious antidepressants are active in
this test. FST serves well for the purposes of this study: to gain mechanistic insights
about ibogaine’s effects by reverse engineering the clinical signal to animal models. In
this context, our results indicate a potentially different neurochemical mechanism for
the iboga alkaloids as compared to classical SSRIs, and constitute the first step toward
identifying putative differences between classical antidepressant drugs (e.g., fluoxetine)
and ibogaine or noribogaine as a potential new class of antidepressant drugs.
Additional behavioral tests will be carried out to characterize the antidepressive-like
effect of ibogaine/noribogaine on other aspects of animal behavior that bear face
validity to human disease such as motivational and anhedonic readouts (e.g. chronic
unpredictable stress paradigm, sucrose preference test, learned helplessness test
among others).
Finally, our findings provide preclinical confirmation of the recently reported
antidepressant effects of ibogaine in opioid-dependent subjects. It is therefore
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conceivable that the antidepressant efficacy may contribute to the anti-addictive
property of ibogaine and noribogaine.
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METHODS
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Animals
Forced Swim Test and Open Field Test were carried out in the Instituto de
Investigaciones Biológicas Clemente Estable (IIBCE, Uruguay) using adult male Wistar
rats weighing 290-320 bred in the animal facility of the School of Medicine (Universidad
de la República - Uruguay). All animals were acclimated in IIBCE facility for 5 days
after they arrived. Animals were housed in groups of 4-5 in plastic cages (50 cm × 37.5
cm × 21 cm) and kept under controlled conditions (temperature 22 ± 2°C, 12-h day-
night cycle, lights on at 7:00 am) with food and water available ad libitum. All
procedures were carried out in accordance to the Bioethics Committee Guidelines of
the IIBCE and following the “Guide for use and care of Animals in the Laboratory” of
the National Institute of Health, (NIH Publications N° 8023, revised 1978) and current
ethical regulations under animal experimentation law N° 18.611 (Montevideo Uruguay).
Pharmacokinetic studies were conducted at Sai Life Sciences Limited, Pune,
India, with approval of Institutional Animal Ethics Committee (IAEC) in accordance with
requirement of The Committee for the Purpose of Control and Supervision of
Experiments on Animals (CPCSEA), India. Healthy male Wistar rats (8-12 weeks old)
weighing between 280 to 300 g were procured from Global, India. Three rats were
housed in each cage. Temperature and humidity were maintained at 22 ± 3 ºC and 30-
70 %, respectively and illumination was controlled to give a sequence of 12 h light and
12 h dark cycle. All the animals were provided laboratory rodent diet (Envigo Research
private Ltd, Hyderabad). Reverse osmosis water treated with ultraviolet light was
provided ad libitum.
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In all the experiments involving animals, adequate measures were taken into
account to minimize pain, discomfort or stress, and all efforts were made to use the
minimal number of animals necessary to obtain reliable scientific data.
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Drugs
Ibogaine·HCl
The ibogaine used in this study was chemically synthesized in the Laboratorio
de Síntesis Orgánica, Facultad de Química – Universidad de la República, using
voacangine as starting material, which was extracted from the root bark of Voacanga
africana using a modification of a previously described procedure.59 Briefly, 100 g of
grounded root bark of Voacanga africana was extracted with a 1% aqueous solution of
HCl (6 x 500 mL). The combined aqueous extracts were basified by adding
concentrated NH4OH until pH 10 - 11. A brown precipitate was separated by
centrifugation and dried at 60°C for 24 h. This solid was taken in acetone and filtered to
discard root impurities. The solvent was evaporated in vacuo to afford a total alkaloid
extract of 3.5-4.0 g. Column chromatography (SiO2, Hex:EtOAc:NH4OH, 90:10:0.01)
allowed to obtain 1 g of pure voacangine which was analyzed by 1H and 13C NMR (See
supporting information). Voacangine was decarboxylated as follows. To a solution of
voacangine in EtOH (0.45 M) in a double necked round bottomed flask, KOH in pellets
(10 equivalents) was added. The solution was heated to reflux until consumption of the
starting material was evident by thin layer chromatography (TLC) analysis. EtOH was
removed under reduced pressure, and the residue was dissolved at 0 °C in a round
bottomed flask using a 6% (v/v) aqueous solution of HCl (enough quantity to adjust pH
to 1). The system was then heated to reflux for five minutes. Once the starting material
consumption was evident by TLC analysis, the solution was carefully basified using
50% NaOH (pH 10 - 11). Precipitation of ibogaine as a white solid was observed. Ethyl
acetate was added, and the resulting biphasic system was transferred into a separation
funnel. The aqueous phase was extracted three times with EtOAc. The combined
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organic layers were dried over Na2SO4, and the solvent was removed in vacuo.
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Ibogaine free base was obtained with an 86% yield and was analyzed by H and 13C
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NMR (see supporting information). Crystallization from EtOH afforded a crystalline solid
which was converted to the corresponding hydrochloride by treatment with diethyl ether
saturated with HCl(g). Purity of ibogaine·HCl was determined by GC-MS analysis as
98.3 % (see supporting information). Dissolution of ibogaine-HCl to prepare the
samples for i.p. injection was carried out using warm saline/ethanol (9:1) vehicle that
was previously degassed by nitrogen bubbling.
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Noribogaine·HCl
Noribogaine·HCl used for the FST studies was prepared by demethylation of ibogaine
in the Laboratorio de Síntesis Orgánica, Facultad de Química, Universidad de la
República. Briefly, under Nitrogen atmosphere, aluminium (3.0 equivalents, previously
washed with hexanes and dried overnight at 70 °C) was suspended in MeCN (enough
volume for a 0.3 M suspension), and iodine (3.0 equivalents) was added. The
suspension was heated under reflux until brownish color from iodine disappeared. The
resulting suspension was cooled to reach room temperature where a solution of 0.2 M
of ibogaine in MeCN (0.3 equivalents) was added. The suspension was heated under
reflux for at least 3 hours, until analysis by TLC (hexanes: ethyl acetate, 1:1 + 1%
NH4OH) revealed complete consumption of ibogaine. After cooling to room
temperature, the suspension was added to aqueous HCl 5 % to destroy excess of
aluminium species. The resulting aqueous phase was basified by adding NaHCO3, and
further extracted exhaustively with ethyl acetate. Combined organic layers were dried
using Na2SO4, and solvent distilled under vacuo to obtain a crude material, which was
purified using column chromatography (SiO2, hexanes: ethyl acetate, 1:1 + 1%
NH4OH) to obtain noribogaine as a pure white solid with 76 % yield. The free base was
converted to the corresponding hydrochloride by treatment with diethyl ether saturated
with HCl (g). Purity of noribogaine·HCl was determined by GC-MS analysis as 95.2 %
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(see supporting information). Dissolution of noribogaine·HCl to prepare the samples for
i.p. injection was carried out using warm saline/ethanol (95:5) vehicle that was
previously degassed by nitrogen bubbling.
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Noribogaine·HCl used for the PK studies was prepared at Columbia University by
Vaclav Havel.
A solution of voacangine (0.15 M) in CH2Cl2 (anhydrous) was cooled in ice bath (0 °C)
and treated with EtSH (4.5 equiv.) and BBr3 (1M in CH2Cl2, 1.5 equiv., added dropwise
over 5 min). After all reagents were added, reaction mixture was allowed to warm to
room temperature. Progress of reaction (from small aliquots of reaction mixture
quenched with saturated NaHCO3 solution) was monitored by TLC (hexanes: ethyl
acetate, 1:2), until no more starting material was observed (usually 1-2 h). Reaction
was quenched with a small amount of CH3OH (until oily precipitates completely
dissolved) and poured to a saturated NaHCO3 solution. The mixture was extracted 3x
with CH2Cl2 (until no more extraction was observed by TLC), combined extracts were
dried over Na2SO4 and concentrated under vacuo. Desmethyl-voacangine (typical
yield >95%) is obtained as an off-white foamy solid. Material is of sufficient purity to
use for the next step without further purification, if necessary, it can be purified by
column chromatography (SiO2, hexanes: ethyl acetate, gradient of 20 to 30%).
Desmethyl-voacangine and either KOH or LiOH·H2O (5 equiv.) were combined in a
mixture of EtOH and H2O (2:1, 0.3 M). Reaction mixture was further heated to 80 °C
under argon atmosphere in a closed reaction vessel (a dark brown solution is formed).
After TLC indicated full conversion of starting material (hexanes: ethyl acetate, 1:2, <24
h) reaction mixture was cooled to room temperature and concentrated under vacuo.
Residue was dissolved in a small amount of H2O (~15 - 20 mL for 1g of starting
material) and the solution was added to a hot 2M HCl (~30 mL for 1g of starting
material, pH must be ~1 after the solutions are combined) and the mixture was further
refluxed for 5 min under argon atmosphere. After cooling to RT, the mixture was
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partially neutralized with 50% NaOH solution (to pH 5 - 6) and final adjustment was
done using excess solid NaHCO3 (careful addition). Mixture was extracted repeatedly
with CH2Cl2 (until no more material was extracting as evidenced by TLC, if needed
CH2Cl2:iPrOH 9:1 mixture can be used to increase the speed of extraction). Combined
organic extracts were dried over Na2SO4, concentrated under vacuo and the dark
brown residue was pre-purified by a short column chromatography (SiO2, hexanes:
ethyl acetate + 2% Et3N, 1:2 to 1:1). Excess Et3N was removed from isolated
noribogaine by repeated evaporation from MeOH/H2O mixture. The still slightly colored
noribogaine free base was dissolved in CH3OH (~10 - 15 mL for 1g of free base) and
the solution was acidified using aq. HCl (36%) to pH (~2, estimated by a pH paper).
Upon cooling to RT noribogaine·HCl started to crystallize from the solution. The
suspension was further cooled in freezer (-20 °C), the solid was collected by filtration
and washed repeatedly with cold CH3OH to obtain an off-white (grey to light pink)
crystalline noribogaine·HCl (average yield of 60 - 65%) and a less pure methanolic
fraction. The crystalline noribogaine·HCl contains ~0.8 equiv. of CH3OH bound in its
crystal structure which is not removed even after extensive drying under vacuum.
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Behavioral tests
Forced Swim Test (FST)
FST is one of the most commonly used assays for the study of depressive-
like behavior in rodents, since it examines certain aspects of despair and coping
behavior in rodents43. Its sensitivity to a broad range of antidepressant drugs makes it a
suitable screening test38. The procedure used for the FST was performed as previously
described.38, 42, 60 The apparatus consisted of a transparent cylindrical tank (50 cm
height and 20 cm diameter) filled with water (24 - 25 °C) to a depth of 34 cm (in order
to allow rats to swim or float without touching the bottom of the tank with their paws).
Two experimental swimming sessions within an interval of 24 h were carried out as
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previously described.60 Briefly, in the first pretest session, the animals were placed in
the tank for 15 min, immediately dried and returned to their home cage. During the test
session, the total time spent immobile (making only the movements necessary to
remain afloat), swimming (horizontal movements throughout the cylinder) and climbing
(vigorous and upward-directed movements of the forepaws along the cylinder wall)
were scored by a single, trained observer in real time during 5 min.42, 61 Sessions were
recorded for later analysis or confirmation when necessary.
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These experimental groups were independently used for the behavioral studies:
1) animals received an intraperitoneal (i.p.) injection of ibogaine at 20 mg/kg, 40 mg/kg,
or vehicle (ethanol 5 % and saline; control group) and then exposed to the FST test
session 24 h and 180 minutes after the administration; 2) animals received an i.p.
injection of noribogaine at 20 or 40 mg/kg i.p. or vehicle (control group), and tested 30
or 180 min after the administration; 3) animals received an intravenously (i.v.) injection
of ibogaine at 1 and 5 mg/kg, or vehicle (control group) and exposed to the FST test
session immediately after the administration; 4) animals received an i.p. injection
Fluoxetine at 40 mg/kg (equivalent dose of noribogaine) only once or vehicle (saline).
Open Field Test (OFT)
This behavioral test was carried out in an independent group of animals to
discard the possibility that the behavioral changes observed in the FST were affected
by alterations in the locomotor activity. The OFT apparatus consisted of a square arena
(45 cm wide × 45 cm long × 40 cm high) with transparent plastic walls indirectly
illuminated (35 luxes) to avoid reflection and shadows. Locomotor activity was recorded
automatically by a camera located above the field connected to a computer equipped
with the Ethovision XT 12.0 software (Noldus, The Netherlands). We measured the
horizontal locomotor activity defined as the total distance moved in meters (m). After
recording the animal behavior, the OF was cleaned with alcohol 30 % before placing
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the following rat .53 In all the experiments rats were naive to the OF and were used only
once. All the experiments were performed between 9:00 and 14:00 h.
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Pharmacokinetic Experiments
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These experiments were conducted at Sai Life Sciences Limited. Plasma and
brain pharmacokinetic distribution of ibogaine and its metabolite noribogaine were
determined in three study groups of male Wistar rats following a single intravenous (5
mg/kg) and intraperitoneal (40 mg/kg) administration of ibogaine and intraperitoneal (40
mg/kg) administration of noribogaine. Solutions of nor/ibogaine were administered in
10% ethanol and 90% normal saline via intraperitoneal/intravenous route. The dosing
volume administered was 5 mL/kg for intravenous and 10 mL/kg for intraperitoneal
route. Blood samples (approximately 120 µL) were collected under light isoflurane
anesthesia from retro orbital plexus from a set of three rats at specified time intervals.
Samples were collected into labeled micro-tubes, containing K2EDTA solution (10 μL of
20% K2EDTA of solution per mL of blood) as an anticoagulant. Plasma was
immediately harvested from the blood by centrifugation at 4000 rpm for 10 min at 4 ± 2
ºC and stored below -70 ± 10 ºC until bioanalysis. Immediately after blood collection,
animals were euthanized with excess CO2 and their brains were isolated at each time
point. Brains were rinsed three times in ice cold PBS (5 - 10 seconds rinse, using ~ 5 -
10 mL fresh PBS in disposable petri dish for each rinse) and dried on blotting paper.
Brain samples were homogenized using ice-cold phosphate buffer saline (pH 7.4) and
homogenates were stored below -70 ± 10 ºC until analysis. Total homogenate volume
was three times the tissue weight. See Supporting Information for detailed results.
Statistical Analysis
One-way analysis of variance (ANOVA) followed by post hoc Tukey’s multiple
comparisons tests and Unpaired Student t-test were conducted to assess the main
effects of treatments in the FST and OFT. Calculations were carried out using
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GraphPad Prism 7 software. All data are presented as mean ± SEM, and significance
was determined at P < 0.05.
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ASSOCIATED CONTENT
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Supporting information contains analytical and NMR spectra of the drugs administered
to the animals, and details from the pharmacokinetic study and the FST. This
information is available free of charge via the Internet at http://pubs.acs.org/.
AUTHOR INFORMATION
Corresponding authors
Dr. Ignacio Carrera. Departamento de Química Orgánica, Facultad de Química –
Universidad de la República, Av. General Flores 2124, C.P. 11800, Phone:
+59829247881 Fax: +59829241906, E-mail: icarrera@fq.edu.uy
Dra. Cecilia Scorza. Departamento de Neurofarmacología Experimental, Instituto de
Investigaciones Biológicas Clemente Estable, Av. Italia 3318, C.P. 11600, Phone:
+59824871616, E-mail: cscorza@iibce.edu.uy
Dr. Dalibor Sames. Department of Chemistry, Columbia University, 3000 Broadway
MC3101, New York, NY 10027 USA, E-mail: sames@chem.columbia.edu
Author Contributions
Experiments were designed by P.R., D.S., C.S. and I.C. Experiments were performed
by P.R., J.U., J.P.P., S.F. A.L.R. and C.S. Data analyses were performed by P.R. C.S.
V.H. D.S. and I.C. Manuscript was written by P.R., V.H., D.S., C.S. and I.C.
Funding Sources
This research was supported by Agencia Nacional de Investigación e Innovación (ANII-
Uruguay, Fondo María Viñas FMV_1_2014_1_103488), Comisión Sectorial de
Investigación Científica (Universidad de la República, CSIC-Grupos I+D 1063),
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Programa de Desarrollo de las Ciencias Básicas (PEDEClBA - Uruguay), and
Columbia University, New York, NY, USA. P.R. acknowledges ANII for a MSc and PhD
scholarships and V.H. acknowledges the Experientia Foundation Postdoctoral
9
Fellowship.
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ACKNOWLEDGEMENTS
We thank Kevin Wulff for the original drawing of Tabernanthe iboga in Figure 1. We
also thank Dr. Madalee Gassaway Wulff for facilitating the commission of this drawing.
Dr. Andrew Kruegel, Dra. Mariana Pazos and Q.F. Bruno Gonzalez are acknowledged
for valuable discussions.
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