Effect of Iboga Alkaloids on µ-Opioid Receptor-Coupled G Protein Activation

Objective The iboga alkaloids are a class of small molecules defined structurally on the basis of a common ibogamine skeleton, some of which modify opioid withdrawal and drug self-administration in humans and preclinical models. These compounds may represent an innovative approach to neurobiological investigation and development of addiction pharmacotherapy. In particular, the use of the prototypic iboga alkaloid ibogaine for opioid detoxification in humans raises the question of whether its effect is mediated by an opioid agonist action, or if it represents alternative and possibly novel mechanism of action. The aim of this study was to independently replicate and extend evidence regarding the activation of μ-opioid receptor (MOR)-related G proteins by iboga alkaloids. Methods Ibogaine, its major metabolite noribogaine, and 18-methoxycoronaridine (18-MC), a synthetic congener, were evaluated by agonist-stimulated guanosine-5´-O-(γ-thio)-triphosphate ([35S]GTPγS) binding in cells overexpressing the recombinant MOR, in rat thalamic membranes, and autoradiography in rat brain slices. Results And Significance In rat thalamic membranes ibogaine, noribogaine and 18-MC were MOR antagonists with functional Ke values ranging from 3 uM (ibogaine) to 13 uM (noribogaine and 18MC). Noribogaine and 18-MC did not stimulate [35S]GTPγS binding in Chinese hamster ovary cells expressing human or rat MORs, and had only limited partial agonist effects in human embryonic kidney cells expressing mouse MORs. Ibogaine did not did not stimulate [35S]GTPγS binding in any MOR expressing cells. Noribogaine did not stimulate [35S]GTPγS binding in brain slices using autoradiography. An MOR agonist action does not appear to account for the effect of these iboga alkaloids on opioid withdrawal. Taken together with existing evidence that their mechanism of action also differs from that of other non-opioids with clinical effects on opioid tolerance and withdrawal, these findings suggest a novel mechanism of action, and further justify the search for alternative targets of iboga alkaloids.


Ibogaine source A:
Ibogaine HCl obtained from Slater & Frith Ltd (Wroxham Norwich, UK). Samples were analyzed on a Varian Saturn 2100T gas chromatograph-ion trap mass spectrometer with a Varian 3900 GC and CP-8400 autosampler. The mass spectrometer was operated in positive chemical ionization mode using methanol as the chemical ionization reagent gas. One microliter of sample was injected onto a Phenomenex Zebron ZB-5 (5% phenyl-95% dimethyl-polysiloxane) GC column (30m x 0.25 mm ID x 0.25 um film) with the following chromatographic conditions: injection port temp 270 °C; Initial GC temp 50 °C; Initial Time 2 min; ramp rate 30°C/min; final temp 310 °C; final time 15 min.

Pages 1-3:
Overall purity of this free base sample is 95%. In this sample the first GC peak on the left is for ibogamine, the smaller between ibogamine and ibogaine may be tabernanthine (a comparison standard was not available), the largest peak is ibogaine and the third peak is an unknown and corresponds to ibogaine minus 2H.

Ibogaine source B:
Ibogaine HCl source B was obtained from National Institute on Drug Abuse Research Resources Drug Supply Program.

Pages 1-3:
Research Triangle Institute (RTI) data sheet with HPLC-MS. Overall purity of this sample is 97.2 ± 0.06% by reversed phase HPLC-MS (total area analysis).

Noribogaine Source C:
Noribogaine HCl was prepared by conversion from ibogaine source A by Kuehne lab with preparative HPLC purification at the Bornmann lab. 400 mg of ibogaine HCl (source A above) was demethylated with boron tribromide by the Archer procedure [1] at the Kuehne lab to provide 220 mg of crude product which had a reverse phase HPLC-MS composition (retention times/MW+1) of 65% noribogaine (5.6 min/297), 29% ibogaine (10.9 min/311), and 6% ibogamine (11.4 min/281).
Preparative HPLC was carried out at the Bornmann lab on a Varian Prepstar SD-1 semi-preparative system equipped (UV detection at 250 nm) using a Prep Microsorb-MWC18 column (250 X 41.4 mm; 6µ; 60 Å) with the following solvent system A= 5mM ammonium formate in water and B=acetonitrile and a gradient of 20%B to 44%B over 65 minutes with a flow rate of 20 ml/min.140 mg of the crude material above was dissolved in 10 ml methanol and filtered. The filtrate was divided into ten 1 ml aliquots for subsequent injection. Once completed the fractions corresponding to the correct mass were collected and freeze dried. Analytic HPLC-MS at the Bornmann lab was performed on an Agilent Accurate-Mass 6200 TOF LC/MS system equipped with an Agilent LC1200 HPLC using a Varian Microsorb-MW C18 column (250 X 4.6 mm; 5 µ) with the following solvent gradient system: A= H 2 O /0.1% TFA and B=acetonitrile/0.1% TFA.
10%B to 95%B over 30 min with a flow rate of 1ml/min Nuclear magnetic resonance (NMR)spectra were recorded at the Bornmann lab on an IBM-Bruker Advance 500 (500 MHz for 1 H NMR and 125.76 MHz for 13 C NMR), spectrometers. The chemical structures of ibogamine, ibogaine and noribogaine were confirmed by 1 H and 13 C NMR via correlation spectroscopy (COSY) and heteronuclear correlation (HETCOR) analysis.

18-MC:
18-MC was obtained from Obiter Research LLC, Champaign, IL, USA. is 16 higher than the mass of the compound. This is not an impurity in the sample, but is generated in the MS/HPLC system and appears to correspond to an extra oxygen on the molecular ion.

Comment regarding the possible effect of a laboratory contaminant:
Overall, it appears unlikely that a laboratory impurity in the iboga alkaloids utilized in this study could account for the finding that these compounds are not MOR agonists. Ibogaine, noribogaine and 18-MC, which produced similar effects on tenth that of naltrexone [2] Therefore, the contaminant, when present at 0.1%, would need to have potency close to that of naloxone; at lower levels of contamination, potencies higher than that of naloxone would be needed for the presently observed antagonist activity.
Furthermore, the above assumes that the iboga alkaloids are neutral with regard to a functional effect at the MOR, i.e., are not agonists (or antagonists). This would not negate the essential finding of the study, that these compounds, including noribogaine are not agonists. If the iboga alkaloids were really MOR agonists with the potency previously reported elsewhere [3], the hypothetical contaminant would have to overcome the putative agonist effect to truly artifact the main finding. We have calculated (assuming an EC 50 of 324 nM for agonist action as previously reported [3]) that this would require the presence of an antagonist (at 0.1%) that is at least 30 times more potent than naloxone. As noted above, it would also need to be present in 18-MC as well despite the very different synthetic process. All together, it appears unlikely that a laboratory impurity could account for the finding that ibogaine, noribogaine and 18-MC are not MOR agonists.