Binding data for popular arylcyclohexamines.

[Transform]

In doing the research for the classification of these drugs, the UK ACMD has apparently commissioned research into the binding profiles. It's a shame that this ban will blanket a huge range of compounds but at least something positive has come from it. The full report can be found here (p14) and is incredibly interesting as a review of what's known about the arylcyclohexamines.

Spoiler: Screened Receptors

Compounds were initially screened in quadruplicate primary assays at a fixed concentration of 10μM. Those which yielded inhibition of tracer binding of more than 50% at this concentration were subjected to Secondary assays in which test substance concentrations ranging from 10pM to 10μM were tested in triplicate to yield concentration effect curves from which pKi and Ki values were calculated.
For experimental details refer to the PDSP web site http://pdsp.med.unc.edu/ and click on "Binding Assay" or "Functional Assay" on the menu bar. All compounds were screened against the targets listed in Table 1 below.

Table 2: Results of Binding assays – pK_i , (K_i) nM
Open boxes with – indicate that compounds failed the Primary Screen criterion of >50% inhibition at 10μM. All the compounds failed the Primary Screen criterion in the other screening targets listed in Table 1.

Compound	NMDA	SERT	NET	Sigma1	Sigma2
Ketamine	6.18 (659)	-	-	-	-
Phencyclidine	7.23 (59)	5.65 (2234)	-	-	6.82 (136)
MXE	6.59 (259)	6.32 (481)	-	-	-
4-MeO-PCP	6.39 (404)	6.07 (844)	6.1 (713)	6.5 (296)	7.93 (143)
3-MeO-PCP	7.69 (20)	6.7 (216)	-	7.4 (42)	-

Conclusions from binding data
The results obtained in receptor screening confirm that the novel analogues share the profile of ketamine and PCP as ligands for the glutamate NMDA receptor. The binding data revealed methoxetamine to have an affinity for the NMDA receptor comparable to or higher than the parent compound ketamine. The methoxy analogues of PCP also had appreciable affinities for the NMDA receptor, and 3-MeO-PCP in particular proved particularly active, with a Ki of 20 nM placing it among the most potent known NMDA antagonists (cf dizocilpine (MK-801) Ki = 4.8nM).

Methoxetamine and the phencyclidine analogues also had appreciable affinity for the serotonin transporter (SERT) (Table 2). The affinity of methoxetamine for SERT was similar to its affinity for the NMDA receptor, suggesting that inhibition of SERT and a resulting increase in the release of serotonin in the brain may contribute to its psychopharmacological profile and the additional features seen in acute methoxetamine toxicity.

Spoiler: Original data (.jpg)

 

[Dr. Drugs]

On the previous page, the authors include a table of all the receptors that they tested for binding affinity.

According to this study, all of the compounds tested showed no affinity for any receptors other than NMDA, SERT, NET, and Sigma1/2. I find it surprising that they weren't able to observe any binding affinity for ketamine at the mu-opioid receptor (MOR), given that previous studies have associated the site with some of ketamine's activity.

Here are some references:

PMID 2823161: Properties of the interaction between ketamine and opiate binding sites in vivo and in vitro.

A dose-dependent (80-120 mg/kg i.p.) inhibition of the binding of [3H]naloxone was observed in both brain and spinal cord. All regions of the brain (except the cerebellum) were affected, but the reduction was significant in the cortex, hippocampus, thalamus and striatum. Thus, a component of ketamine-induced analgesia could be related to a functional interaction with opiate receptors.

PMID 11726430: The involvement of the mu-opioid receptor in ketamine-induced respiratory depression and antinociception.
In this study, transgenic mice with the MOR gene knocked out showed a lower response to ketamine than wild-type.

We assessed the involvement of the mu-opioid receptor in S(+) ketamine-induced respiratory depression and antinociception by performing dose-response curves in exon 2 mu-opioid receptor knockout mice (MOR(-/-)) and their wild-type littermates (WT). The ventilatory response to increases in inspired CO(2) was measured with whole body plethysmography. Two antinociceptive assays were used: the tail-immersion test and the hotplate test. S(+) ketamine (0, 10, 100, and 200 mg/kg intraperitoneally) caused a dose-dependent respiratory depression in both genotypes, with greater depression observed in WT relative to MOR(-/-) mice. At 200 mg/kg, S(+) ketamine reduced the slope of the hypercapnic ventilatory response by 93% +/- 15% and 49% +/- 6% in WT and MOR(-/-) mice, respectively (P < 0.001). In both genotypes, S(+) ketamine produced a dose-dependent increase in latencies in the hotplate test, with latencies in MOR(-/-) mice smaller compared with those in WT animals (P < 0.05).

 

[endotropic]

I'm so glad to see this data! There's been enough speculation on these compounds, it's nice to finally have some hard data!

According to this study, all of the compounds tested showed no affinity for any receptors other than NMDA, SERT, NET, and Sigma1/2. I find it surprising that they weren't able to observe any binding affinity for ketamine at the mu-opioid receptor (MOR), given that previous studies have associated the site with some of ketamine's activity.

Here are some references:

PMID 2823161: Properties of the interaction between ketamine and opiate binding sites in vivo and in vitro.


PMID 11726430: The involvement of the mu-opioid receptor in ketamine-induced respiratory depression and antinociception.
In this study, transgenic mice with the MOR gene knocked out showed a lower response to ketamine than wild-type.

They used a pretty whopping dose of ketamine in both of those studies, and it's possible that brain concentrations were over the 10uM cutoff used in the binding study. It also wouldn't be surprising if ketamine were causing an endorphin release, and indirectly activating mu receptors, in which case you wouldn't expect any affinity.

 

[pofacedhoe]

what about the dopaminergic effects or am i missing something?

 

[MyDoorsAreOpen]

what about the dopaminergic effects or am i missing something?

Yeah I was wondering about this too. I'd always heard phencyclidine and ketamine both have affinity for DA receptors.

I don't buy ketamine having much activity at mu opioid receptors, precisely because it does not depress breathing.

 

[sekio]

they do, just not at micromolar concentrations (millimolar maybe)
see also: mu, 5ht2a, kappa opioid (which kertamine binds to, weakly, millimolar concentrations)

 

[TheAzo]

Yeah I was wondering about this too. I'd always heard phencyclidine and ketamine both have affinity for DA receptors.

Possibly they're acting as DARIs or DA releasers? Release/reuptake inhibition was not assessed in that study.

they do, just not at micromolar concentrations (millimolar maybe)
see also: mu, 5ht2a, kappa opioid (which kertamine binds to, weakly, millimolar concentrations)

With normal doses, ketamine and PCP do not approach millimolar concentrations in the brain, so it's unlikely that this is relevant to the effects produced.

 

[sekio]

Possibly they're acting as DARIs or DA releasers? Release/reuptake inhibition was not assessed in that study.

Actually it was, they assayed all 3 transporter protiens and apparently there is little binding at DAT.

 

[nuke]

Actually it was, they assayed all 3 transporter protiens and apparently there is little binding at DAT.

That's pretty interesting, I would have figured there was some DAT activity.

Strangely, they wish to schedule methoxetamine but in the report they state that neither deaths nor chronic toxicity has occurred. The only adverse effects noted clinically appear to be persistent ataxia/neurological problems of 4-5 days duration and moderate-high tachycardia.

International data
3.14 The ACMD are not aware of any confirmed deaths solely related to methoxetamine reported in Europe or elsewhere in the world.
3.15 There has been one published case of potential acute methoxetamine toxicity from Switzerland and one from the USA. These cases are summarised below.
3.16 The US case was an individual who presented to the Emergency Department after self-reported methoxetamine use in a dissociative state. In addition he had mild stimulant features (HR 105 bpm, BP 140/95 mm Hg) and bilateral nystagmus (abnormal eye movements). Analysis of a drug specimen suggested that the drug used was methoxetamine, however it is not possible to determine definitively whether
8
toxicity was due to methoxetamine as there was no toxicological screening of biological samples to confirm actual use of methoxetamine (Ward J 2011).
3.17 The case from Switzerland was an individual who reported injection of methoxetamine. On arrival in the Emergency Department, he was agitated, had ataxia, and was disoriented. He also had stimulant features (HR 134 bpm, BP 168/77 mm Hg) and a mild pyrexia (37.6°C). However, this individual had also injected MDMA (3,4-methylenedioxymethamphetamine) 2 days previously. Analysis of serum samples taken 5 hours after injection of methoxetamine showed both methoxetamine and MDMA and therefore it is not possible to determine the relative role of these drugs in the presentation (Hofer KE 2012).
3.18 In addition there are anecdotal reports of acute methoxetamine toxicity presentations to hospitals in other countries in Europe including in Belgium.

It's kind of a curiosity as to what is causing the tachycardia, as they don't report of any NET/DAT/adrenoreceptor activity

3.7 In summary, methoxetamine appears to be associated with some features that are similar to ketamine (hallucinations, drowsiness, and dissociative effects). Ketamine can cause mild hypertension, however the hypertension reported in methoxetamine toxicity is greater than what would generally be expected with ketamine toxicity. In
7
addition to more significant hypertension, methoxetamine appears to be associated with additional toxicity compared to ketamine including agitation, other stimulant effects such as tachycardia (a fast heart rate) and cerebellar features. Cerebellar features such as ataxia (unsteadiness on the feet), are rarely seen with other recreational drugs and are not seen with acute ketamine toxicity.

...what? Ataxia is a normal finding for both acute ethanol and ketamine intoxication.

 

[Dondante]

The SERT affinity of methoxetamine is interesting though I'm not sure it'd be relevant at normal doses. Regarding dopaminergic effects of arylcyclohexylamines, I think there may be more to the story...

"While the clinical anti-dopaminergic actions of antipsychotic drugs are compatible with the hyperdopamine hypothesis of psychosis and schizophrenia, the psychoses caused by glutamate antagonists such as phencyclidine or ketamine suggest a hypo-glutamate component in psychosis. However, phencyclidine also lowers plasma prolactin and elicits rotation, suggesting a direct or indirect dopaminemimetic action of phencyclidine. Although phencyclidine and ketamine are not selective for glutamate NMDA receptors, the precise affinities of these drugs for dopamine D2 receptors need to be clarified in order to determine their dopaminergic and non-dopaminergic components of action. More specifically, although phencyclidine had a dissociation constant of 37 000 nM at the D2 receptor in rat striatal homogenate, phencyclidine had a dissociation constant of 1.3 nM for the functional high-affinity site of the cloned D2 receptor, or the D2High receptor.

This apparent discrepancy may be resolved by the recent finding that dopamine itself has a dissociation constant of 3000 nM when competing vs [3H]raclopride at striatal D2 receptors, but has a dissociation constant of 1.5 nM at the D2High receptor when the link between dopamine D1 and D2 receptors is blocked by the D1 antagonist SCH23390. … [3H]Domperidone is a selective ligand for D2 that readily detects D2High receptors [and doesn't require the addition of SCH23390]."

Seeman P, Ko F, Tallerico T. Dopamine receptor contribution to the action of PCP, LSD and ketamine psychotomimetics. Mol Psychiatry. 2005 Sep;10(9):877-83.

 

[ungelesene_bettlek]

I am also surprised by the action on the SERT and the missing action as opioid receptor agonist, sigma agonist and especially DAT. to me it seemed always completely clear that the highly stimulating and euphoric properties of MXE were due to dopamine reuptake inhibition.

have I understood it correctly that there was just affinity of MXE for the SERT confirmed, and that it is still unclear what kind of action (reuptake inhibition, release, reuptake enhancement) it causes there?

am I right in assuming the MK-801 binding site is where all dissociatives act at (at least all of the arylcyclohexylamine family)? isn't it usually called "PCP binding site"? any ideas why they prefer MK-801 as model compound?

 

[JackiesBabyy]

I thought NMDA antagonism itself caused an increase in dopamine somehow, though. I never thought dissociatives were DRIs.

 

[ungelesene_bettlek]

I thought NMDA antagonism itself caused an increase in dopamine somehow, though. I never thought dissociatives were DRIs.

I just checked william white's DXM FAQ; he writes about a PCP2 binding site which is likely responsible for dopamine reuptake inhibition (whereas the PCP1 binding site is the one on the NMDA receptor). somewhere else in the FAQ he writes about the PCP2 binding site also being the target of cocaine and methylphenidate.

see:
http://www.erowid.org/chemicals/dxm/faq/dxm_physiological.shtml#toc.9.2.2
http://www.erowid.org/chemicals/dxm/faq/dxm_neuropharm.shtml#toc.10.4
and the references therein.

 

[Limpet_Chicken]

Regarding the tachycardia nuke, that particular patient had also taken MDMA.

Could be due to interaction between the SERT affinity of methoxetamine and the well known serotonergic effects of MDMA?

 

[ebola?]

he writes about a PCP2 binding site which is likely responsible for dopamine reuptake inhibition

This doesn't make sense. Activity at another receptor cannot "block reuptake" downstream.

ebola

 

[ungelesene_bettlek]

This doesn't make sense. Activity at another receptor cannot "block reuptake" downstream.

I'm not entirely sure if I have understood it correctly, but is it possible that the PCP2 binding site is located at the dopamine transporter?

compare the following paper; it is also about the "cocain binding site" at the dopamine transporter:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2692229/

 

[King Kong]

I am maybe stupid... But I think we can't guess the pharmacologic activities of those compounds only with biding affinities. For example, we can’t know from SERT affinity if the compounds are releasers or inhibitors of the reuptake of serotonin (or maybe something else).
Also, those compounds could have active metabolites!

Those values are nice to do some hypothesis and thinking, but nothing more.

 

[amanitadine]

Interesting stuff, good find! Hopefully this will dispel the oft repeated conjecture that methoxetamine has significant effects on the MOR. I've been arguing against such since the beginning, albeit from subjective effects moreso than SAR.

 

[Dondante]

"While the clinical anti-dopaminergic actions of antipsychotic drugs are compatible with the hyperdopamine hypothesis of psychosis and schizophrenia, the psychoses caused by glutamate antagonists such as phencyclidine or ketamine suggest a hypo-glutamate component in psychosis. However, phencyclidine also lowers plasma prolactin and elicits rotation, suggesting a direct or indirect dopaminemimetic action of phencyclidine. Although phencyclidine and ketamine are not selective for glutamate NMDA receptors, the precise affinities of these drugs for dopamine D2 receptors need to be clarified in order to determine their dopaminergic and non-dopaminergic components of action. More specifically, although phencyclidine had a dissociation constant of 37 000 nM at the D2 receptor in rat striatal homogenate, phencyclidine had a dissociation constant of 1.3 nM for the functional high-affinity site of the cloned D2 receptor, or the D2High receptor.

This apparent discrepancy may be resolved by the recent finding that dopamine itself has a dissociation constant of 3000 nM when competing vs [3H]raclopride at striatal D2 receptors, but has a dissociation constant of 1.5 nM at the D2High receptor when the link between dopamine D1 and D2 receptors is blocked by the D1 antagonist SCH23390. … [3H]Domperidone is a selective ligand for D2 that readily detects D2High receptors [and doesn't require the addition of SCH23390]."

Seeman P, Ko F, Tallerico T. Dopamine receptor contribution to the action of PCP, LSD and ketamine psychotomimetics. Mol Psychiatry. 2005 Sep;10(9):877-83.

Scratch that. According to the Director of the NIMH Psychoactive Drug Screening Program, the above data is "completely fallacious." Apparently, nobody outside of Seeman's lab has been able to reproduce the results (and the guy has 20 publications on the D2High affinity state)! Maybe there's no direct dopamine effect for arylcyclohexamines after all...

 

[skillet]

Really? Where did Roth say that?