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so i'm gonna give a tiny bit of pharmacology because i don't know how many people here are pharmacologists

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maybe a lot maybe not a lot

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how did we study psychedelics without using humans

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when I was in a purude university

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worked with receptor targets, most of you know that, but I talk about it briefly

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and when I was doing this talk I had my talk finished

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i thought, you know there is nobody in this conference that talk about research chemicals

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and large number of people these days are getting their first exposure to a psychedelic or an enctatogen

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through a research chemical

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so what I did was I changed the whole back in my presentation to talk a little bit about research chemicals

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because lot of people don't know what they are

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sadly a large number of them was pilfered from my publications

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so i feel a little responsible for the fact they're out there

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and then I should talk a little bit about if you want to be a researcher, what should you do and that's part of the CME requirement

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so inform patients about new research, refer patients to clinical studies, develop strategies for conducting research and evaluating research

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I'm gonna touch on some of these, but obviously this talk like that would be a 2 hour talk in depth

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so I won't spend a lot of time, i do want to point out that the sort of revolution in neuroscience

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was catalysed by the discovery of LSD and then few years later the finding that serotonin was in the brain]

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and I think somebody earlier pointed that out, maybe it was Rick, that

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prior to discovery of LSD and serotonin in the brain, psychiatric disorders were thought to be due to poor parenting

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so we had mothers that were called refrigerator mothers, because they had a child with schizophrenia and they would say that's the mothers fault she didn't nurture the child properly

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so there's no connection, so whe they discovered that

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LSD had effects and serotonin was in the brain and the recognition that

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there was a similarity in the skeletal fragment of LSD and serotonin

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a number of people put together the idea well maybe behaviour and mental function is somehow related to neurochemistry

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and that seem sort of silly today because it's obvious thing but back then they thought that brain was sort of an electrical box

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nothing about neurochemistry had anything to do with behaviour

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kind of weird thinking, but that's the way it was

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and another thing to point out about psychedelics, and I used to used this definition a lot

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because you try to tell people what psychedelics were

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and Jaffe wrote a chapter in Goodman and Gilman which was a pharmacologist Bible

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and I like this definition because it says that psychedelic

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the feature that distinguishes them from other classes of drugs is their capacity reliably to induce or compel

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states of altered perception thought and feeling that are not or can not

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be experienced otherwise except in dreams or at times of religious exultation

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I often give the seminar to pharmacology departments at medical schools

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where i give this definition and I say

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do you know of any other pharmacological class that has a definition like that?

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a drug that produces religious experience? and so, very unique class

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and i'm preaching to the choir you all know that but just to mention that

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before we start

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this is a list of what I'll call recently published

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clinical studies, that have come out, and i probably dont have everything

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listed here that's been published but the point

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of the slide is, what i want you to notice is the colouring

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so these purple ones ate the 1990s

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these red ones are 2000-2010

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and then after 2010, what you see is exponential explosion

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of published studies that relate to clinical research

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on psychedelics. And i think this trend is likely to continue

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so this is really exciting trend and it really obvious when you look at these numbers

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the literature in this field is exploding, so we're having an impact

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the key brain receptors

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for psychedelic are the serotonin 2a and serotonin 2c receptors

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and for tryptamines they also activate the 1a receptor

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LSD is a little bit different

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somebody have a question?

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LSDis a little bit different because it hits lots of different receptors

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and people used to say that LSD was a promiscuous drug pharmacologicaly

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and then they switched over

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and they started sayin it had rich pharmacology

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butfrom the point of view of understanding how they work if all these drugs

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these types of compounds are considered to have a similar action in the brain

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it pretty obvious that the serotonin 2a and 2c

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receptors are probably the ones that are the most important

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things like DOM, DOB, 2C-B, the kind of things you hear about

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a lot on the streetm activate principally the serotonin

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2a receptor, they may not fully activate it but they have a high

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efficacy so they produce a good activation of the receptor

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we dont know much about the serotonin 1a receptor

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it may be important in distinguishing the effects of the tryptamines from phenethylamines

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and with lsd its uniquely potent so its doing more than just activating these 2 receptots

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and that's the quest i've been on for a long time

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and will continue work on, to try to understand why LSDhas such profound effects

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when if you look at its receptor effects at the 2a 2c or even 1a receptor

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there is no evidence that it should be as potent in humans as it actually is

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so it has unique pharmacology

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so what happens inside the brain cell when we activate these receptors?

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this is just a model of a brain synapse

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but principaly these serotonin 2a receptors are located postsynaptically

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so we have release of serotonin in a normal brain

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it comes out of these vesicles and interacts with these receptors

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so LSD or the other psychedelics will bind to these receptors and will induce a signalling in this receptor

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or within the cell

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and they can induce signalling in number of ways

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they can either fully activate the receptor

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that what serotonin does, lsd doesn't fully activate it

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its actually fairly weak agonist, so if we look at LSD on this scheme it would probably be over here

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about 30% of full activation

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so not only does LSD not have the kind of receptor affinity or stick-to-it-iveness

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we might expect from such a potent compound but it also doesn't activate the receptor fully

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so its maybe related to its mechanism of action

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and an antagonist which dont do anything, BOL as you just heard in a previous talk

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its currently thought to be either antagonist or inverse agonist, an inverse agonist

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actually drops the basal level below this

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so BOL would fit into the scheme there

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what happends when a psychedelic binds to the receptor?

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these receptors are bundles of alpha-helical protein pieces

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that are fit together in the membrane of the cell

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and when the drug interacts with the exterior part of this receptor,

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it binds inside inside an area that's in between what is called helices 3, 4, 5, 6 and 7 to some extend

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it causes the shape of this protein bundle to change

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and when the shape of that bundle changes, there are pieces of the protein that stick down into the cell

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and they also change, so as the receptor changes, the interor pieces of the protein of the receptor also change


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and when those pieces change they allow it to couple to signalling molecules within the cell

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Now years ago pharmacologists used to talk about a drug that was an agonist

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that would activate the receptor

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or not activate it. And its alwas thought that the receptor

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was sort of like a switch, you turn it on and turn it off and signal is produced.

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Now today there is a concept in pharmacology that is called functional selectivity

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which means that all drugs don't do the same thing to the receptor

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so imagine serotonin, which is the natural transmitter

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binding down inside the receptor

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it causes the shape of the receptor to change in a specific way

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and now we have an ensemble, the serotonin inside the receptor

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and that ensemble produces a change in a pieces of the receptor that are inside the cell that allow it to couple

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to some set of signalling molecules

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if we put LSD in there

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or 2C-B or you name your favourite drug

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when the receptor binds it doesn't go to the same shape

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that it had when the serotonin bound, because it's a different molecule

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this new receptor shape induces new changes

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in the interior part of the receptor and it no longer binds

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the same way to the same set of signalling molecules that serotonin bound to

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So we have a possibility of coupling to a lot of different things

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so we have what we call G proteins, those are what

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normally couple to G protein coupled receptors (GCR)

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G alpha I which generally inhibits cellular activity

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G alpha s which typically stimulates cellular activity

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theres another series of G proteins called G alpha q that I dont have shown here

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that typically also activate the cell

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it can also be phosphorylated with an enzyme

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that put phosphate groups on to serines inside these pieces of the receptor

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and that can allow them to interact with another signalling molecule

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called beta arrestin, so there are number

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of these signalling pathways that can be activated

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so when a drug binds to the receptor and activates it, it just doesn't turn it on and off, like light switch

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it turns it on and it induces certain amount of signals inside the cell

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a different drug produces a different set of signals

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and yet a third drug will produce even a different set of signals

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so we have the complexity of understanding how psychedelics work, because

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they don't just turn the receptor on and off

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they induce a whole bunch of different kinds of changes

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and these changes are related to

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the structure of the drug itself

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So how does receptor signal change consciousness?

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so this is when the hand-waving comes in

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Franz Vollenvaider will give a talk i think tomorrow

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and Robin Carhart-Harris will also talk about

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some of the newer technologies involved in looking at how the brain

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actually responds to psilocybin or to other psychoactive drugs

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the kinds of electrical changes as measured by EEG

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or glucose utilization as measure by PET

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and a number of other techniques

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so what happens?

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well basically, we don't know clearly

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but one of the things we know that the serotonin

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2a receptor, which is the esential receptor

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for producing the effects of psychedelics, is highly expresed in

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all of the structures in the brain that are involved in perception

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and counsciousness.

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So if we look at the top of the brainstem lower midbrain we have a locus coeruleus

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which sends norepinephrine projections to the whole forebrain

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we have the raphe nuclei, the dorsal rapahe is also in the same area as the brain stem

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that sends serotonin projections to the whole forebrain,

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we have the ventral tegmental area which also have serotonin 2a receptors

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we have serotonin 2a receptors both in thalamus

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but particularly in the reticular nucleus and the reticular nucleus wraps around the thalamus

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so the thalamus has the nuclei that are involved in processing all the senses

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so auditory, vision, sensing, touch

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all of that is processed through the thalamus

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and those signals are sent onto the cortex for final sort of procesing and integration

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while the reticular nucleus wraps around the thalamus as a single layer

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and sends inhibitory projections down into the thalamus

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and tells the thalamus what kind of information it should let through

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so we serotoning 2a receptors there, and we have serotonin 2a receptors located on the apical dendrites

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of these cortical cells which are the main processing units in cortex

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the cortex is where we put together our picture of the world

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its where we make our executive decisions, where we do abstract thinking

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so the cortex is critical to the funtion of higher conciousness

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it's critical to defining who we are

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it's critical to defining whether we're conscious, unconscious

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and its activity is determined by all these other areas

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so any sensory information that comes in, is filtered through

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the thalamus, that's regulated by reticular nucleus

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we have the Raphe sending signals to the cortex

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to the apical dendrites, we have a locus corelueus producing norepinephrine

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there are alpha receptors responding to norepinephine located on these pyramidal cells

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and the ventral tegmental area produces dopamine and also produces activation

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so the confluence of all these things onto these senitive processing units

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will change the way we perceive things

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changes the whole process of sensory, perception, and consciousness

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so it's not surprising that something that activate serotonin 2a receptors

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or interacts with 2a receptors will change the way we see

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and experience, because thats a fundamental receptor in the brain in all these areas

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the serotonin 2a receptors and serotonin 2 receptor, its progenitor

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is probably one of the oldest receptors

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there's known. Serotonin as a transmitter is thought to be phylogenetically

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probably the oldest of the monoamine transmitters

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we consider serotonin, dopamine and epinephrine

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because you find serotonin 2 receptors even in single cell organisms like

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paramecium, where is responsible for the movement of the cells

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so you know when nature finds something that works, like serotonin 2 receptor

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it continues to use it and exploit it in ways, and as it turns out exploited it in modulating our perception and conciousness

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one of the problems I had in Purdue university

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working in the pharmacy school in the midwest

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i had a lot of students who would come and volunteer to be a subject in my experiments

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and i would say well i can't really do that

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i can't even talk about anything i may or may not have done

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i plead the 5th amendment

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so how did we actually do our studies?

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we spend a lot of time looking at animal models

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trying to find something that would parallel the activity in animals that we saw in humans

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no animal can model what happens is humans though

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theres no animal model that can tell you i had a mystical experience

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or i remembered my childhood

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or anything like that, it's a real problem.

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turns out that in pharmacology

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the whole subject of pharmacology is the study of the action of drugs

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and if you want to understand how the system in the body works

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you apply the drug that modifies that system

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so for example we know that they are beta 1 adrenergic receptors in the heart

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so if we want to know what beta 1 receptors are doing

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we can give the organism a drug that stimulates beta 1 receptors

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and if we do we find out that beta 1 receptors control the heart rate

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give a beta 1 agonist to human their heart will start beating faster

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in a fight or flight response if you hear

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a noise at night,

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you walking down a dark street

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and you hear someone say: Hey hippie, what are you doing down here?

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what happens: your heart start beating, you start going [quick breathing]; that's epinephrine produced by adrenals

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and it stimulates beta 1 receptors in the heart

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so similarly we can argue to study consciousnesses

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we can use agents that perturbs consciousnesses, so, psychedelics

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they're the best drugs to look at consciousness and how we can perturb it

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I like to use this slide, but I don't hear the sound that goes with it

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so if you could hear this you would hear Grace Slick singing The White Rabbit, right

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anyway, the ones that mother gives you don't do anything at all, but the ones Sasha gives you would probably have some effect

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we use the techinque called drug discrimination, the two-lever drug discrimination

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and it's actually a pretty good model for what these drugs do

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we have an operant chamber that has two levers in the front

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and we can train the rats to press one lever or another

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so if we give a rat a drug, LSD in this case, what we can do is turn on the right lever

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and this is a trough that delivers 50mg sucrose pellets, so it's rat candy

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so we put the rat in the box, we turn this level on, and he'll explore the box, and learn that if he presses this lever he gets a food pellet

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if he presses the left lever, nothing happens, we turn if off

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the next day we put the rat in, we turn on the left lever, and we give him nothing, placebo

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and he presses the right lever, nothing happens, he presses the left lever he gets food pellets

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the next day we give him LSD, we put him back in, we turn on the right lever, we give him food pellets if he presses the right lever

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it takes 2-3 months to train rats to do this, but once you do, the rat will recognise very reliably the effects of a drug like LSD

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and when you train rats and they're really well trained they'll press to get a good pellet anywhere from 1500 to 2500 times in a 15 minute training session

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so they're really pressing that lever

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and the way it works is, there's something unique about this called the third state hypothesis

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if you now give the rat some other drug, like amphetamine or MDMA, and he's been trained with LSD, he won't respond on the LSD lever, he'll respond on the non-drug lever

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so they uniquely recognise the drug they were trained with

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so if we give this rat a drug that we've made that we think might have activity like LSD, and we've trained them on the right lever

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he presses the right lever, he's telling us "I think you gave me LSD"

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and if he doesn't press it, he presses the left lever, "I don't think you gave it to me"

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so we always tested our compounds after we started using this in about 1984

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we've tested our compounds with drug discrimination, and it actually is a fairly good assay

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this is just a correlation that I put together some years ago where we look at the relative potency compared with LSD in rats

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and relative potency compared to LSD in humans

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and the numbers aren't exact but you see here the ETH-LAD compound we made which is more potent than LSD in humans

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it's also more potent in rats

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we have a relative potency of about 185 which is exaggerated compared to its actualy potency which is around 140 times LSD

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LSD is 100, DOB 2.37, DOI 9.26, psilocin 2.61, MDMA 2.4, mescaline .06

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so there's a good general correlation

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this assay works well, but will sometimes give you false positive, so it will sometimes tell you something is active when in humans it may not be active

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but it was a mainstay of all the biological work in animals that we did and it served us well

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now I'm gonna talk about a compound that we worked with some time ago that some of you may of heard of called 25I-NBOMe

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that's an extremely potent compound, there have actually been a couple of deaths from overdose

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this stuff the dose of it is about 500 micrograms, 200-500 micrograms

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people have gotten hold of a pure powder, they insufflate it up their nose and die

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and I'm just going to tell you how we did a little bit of work with this before it actually came out as a so-called research chemical

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a German fellow called Rolf Heim (?) had published that these compounds with an N-benzyl were very potent

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and we were interested into whether they would actually have psychedelic-type effects

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so here's a compound called 2,5-dimethoxy phenethylamine, it's basically inactive in humans but we used it as a model for early studies

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this is the affinity, which is how tightly it sticks to the receptor, so 300nM is not a particularly potent compound

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if you put an N-methyl on this compound, you see it gets even worse, you see it gets up to 1900

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it's even less effective in binding

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add an N-propyl, which is even larger, there's about comparable decreased affinity

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so if you put something onto the nitrogen of these phenethylamines, generally it ruins their activity

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if you take DOB and you put a methyl on it, almost kills the activity

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2C-B, you put a methyl on it, almost kills the activity

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by contrast, when you put an N-benzyl on, the affinity goes from 380 to 68 nM

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and if you put a methoxy over here, it goes to 2.8nM

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so this is really curious; you put stuff onto the nitrogen, the activity goes down, way down

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but if you put on a benzyl or a methoxy-benzyl, it goes up

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and this compound with an iodo is the 25-I-NBOME, and the affinity in this case 0.04 nM, so it's a super potent compound

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so we did a lot of work trying to model the receptor, and our earliest work with an activated model of rhodopsin

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which we converted into a serotonin 2a receptor

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and this is something in March of 2013, the crystal structure of the human serotonin 2b receptor was published

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this shows it with ergotamine bound, which is the way it was crystallised

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and we've now converted that into a homology model, this is now the serotonin 2a receptor

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and this is LSD bound

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so we now have a very good receptor model to go in and look at what these things actually do

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so early on, after doing some virtual computer docking with this N-benzyl compound, it appeared to us that the N-benzyl, which is right here

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this thing that's in spheres is the N-benzyl compound

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and these things that are labelled here are the residues that are in the active site, the binding site of the serotonin 2a receptor

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and we docked it in virtually, we saw that this N-benzyl groups appeared to be interacting with phenylalanine 339

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as a kind of edge-to-face interaction

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so we thought, let's mutate all these residues and see if that assumption if that assumption or that hypothesis is true

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so we mutated that phenylalanine to leucine

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so phenylalanine is like a benzene ring, it's aromatic

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leucine is just a series of aliphatic carbon atoms, you lose the aromatic character which is necessary for these rings to stack if it binds in the receptor

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but it still keeps some hydrophobicity

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so when we looked at the mutated receptor, where that phenylalanine was mutated, now we put those compounds in, we saw the affinity drop from 380 to 4200

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so about a tenfold drop in affinity, about a fivefold drop in affinity and in this case a sixfold drop in affinity

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so that didn't tell us anything, but when we looked at the N-benzyl, you see when we did the same mutation we got a 40-fold drop in affinity, and a 500-fold loss in affinity with the methoxy-phenyl

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so this was strong was strong evidence that that residue, phenylalanine 339, was interacting with the N-benzyl group

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now with our new model now, we're trying to dock these compounds in and get a better idea of how exactly they're fitting into the receptor

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one of the other things we did though was to try to discover what shape that molecule might have when it bound to the serotonin 2a receptor

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so we made a whole series of compounds, so here's a compound 2C-B, its affinity is about 6nM in a series of experiments, and here's the N-methoxy-benzyl-2C-B, about .2nM

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so you can see the increase in potency, 6 down to .2

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so we use this is a model because the bromine atom was easier to get on than the iodine, so we made most of our compounds with bromine

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and I had a fairly large group of students so I assigned each student one of these molecules

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I said let's make all the possible combinations of 2C-B with the N-benzyl group attached

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so in each one of these although it's not immediately obvious, they all have the equivalent of an N-benzyl

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so here's the 2C-B nitrogen, and here's the N-bencyl

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here's the 2C-B nitrogen with the N-benzyl

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here's the 2C-B nitrogen and the N-benzyl is over here

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here's the N-benzyl down here

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this one is a little harder to see, 2C-B is here, here's the N-benzyl

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2C-B is here with the N-benzyl

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and then this one substituted, this is this so they're on the same side of the ring, and this is trans on the opposite side of the ring

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and the affinities for this compound is 2.6nM

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we actually crysallised this into its stereoisomers, this is a pure stereoisomer, and this is the one that's most active

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so though we don't strike the .2nM affinity, if you look at these, 70, 2000, 160, 170, 300, 800, 800, 2.8

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we're pretty sure that that represents the shape that these N-benzyl compounds mainatin when they bind to the receptor

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so we're doing docking studies now to see how that fits into the receptor

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we get a better understanding of how this super potent compound can activate the receptor

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so what I did when I changed my talk was to cut out the back end and talk about research chemicals

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and I don't think anybody here at this conference is talking about research chemicals

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so I thought that this we should really spend some time on because a lot of people are taking these

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they don't know what they are , they don't know where they came from

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and a lot of young people their first experience with a psychedelic-type compound is one of these research chemicals

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and there are several kinds of research chemicals that we could talk about

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the psychedelics, the entheogens, the psychostimulants that are like amphetamine, or entactogens or empathogens

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depending on what you prefer

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synthetic cannabinoids, and I'll briefly talk about those

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and ketamine analogues, there are one or two of those out there, I'm not going to spend time on those

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but there is a methoxy-substituted ketamine analogue out there that people seem to be using quite a bit

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also I'd point out that this paper just came out, I just got it for review a week ago, showing that lifetime use of psychedelics in the US population in 2010 is over 30 million people

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and that includes LSD, psilocybin, mescaline and a lot of other things were not classified

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so there's a large number of people that are using these

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so psychedlics, or hallucinogens, we know they're characterised by changes in perception, thought, feeling, distortion of space

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feelings of portentiousness, something important is about to happen

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high doses can elicit anxiety, or psychotic behaviour

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generally they have low toxicity and non-addictive

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now the peculiar thing about the 25I-NBOMe compounds is they have killed people

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there are no overdose deaths from LSD, mescaline, psilocybin

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some of these newer so-called research chemicals do have toxic properties that are not fully appreciated

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the psychostimulants are related to amphetamine, they produce elation and euphoria, give you energy,

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but their chronic use or high doses can produce depression and fatigue, dependence and continued can produce acute psychosis, amphetamine psychosis

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and most psychostimulants have cardiovascular effects

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they release norepinephrine into the circulation and that causes blood vessel constriction, and that's a major toxic problem of some of the research chemicals

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and entactogens also produce elation and euphoria, depending on the dose and the pharmacology

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large doses or chronic use can produce depression and fatigue

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and high doses can also produce psychotic behaviour

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people who repeatedly take large doses over short periods of time most these also have cardiovascular side effects because they release norepinephrine

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they cause vasoconstriction

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these are different from psychostimulants, the pure amphetamine, and in the beginning when MDMA was brought out and there was a lot of publicity

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there were drug abuse experts who said well, MDMA is just another methamphetamine

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we all know today that MDMA is not just another methamphetamine

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we did a number of experiments in the early days to prove that MDMA was not just another amphetamine or methamphetamine

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we also proved it wasn't another psychedelic amphetamine, that it had a specific category

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and many of you know that I created this name entactogens so set them off as a specific class of drugs

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so they wouldn't be lumped into the hallucinogenic amphetamine class

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they are dfferent; I collaborated with a fellow in Germany named Wilfred Dimpfel who planted electrodes in the brains of rats

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give them different drugs and then we'd characterise the power spectra

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so frontal cortex, hippocampus, striatum and reticular formation

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and then separated these into six frequency bins, and could do pattern recognition

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now clearly here's amphetamine, LSD and MDA, and although there's some similarities, each one is unique

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and so it's possible to show that electically in the rat brain they also were distinct and that ... psychostimulants were different from entactogens and also psychedelics

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so most of the research chemicals target receptors or reuptake sites for monoamines

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we talk about the phenethylamine type, typically they're interacting with targets for serotonin, dopamine or norepinephrine

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and depends on the substituents on the aromatic ring what kind of pharmacology you will actually get

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so we had the G-protein coupled recptor and this would include the serotonin 2a receptor showed you earlier

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these seven transmembrane helices bound together, they couple to intracellular signalling

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dopamine, serotonin, norepinephrine, cannabinoid receptors that are all members of this GPCR family

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we have things that interact with reuptake transporters

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so after transmitters are released from the nerve terminal, there are specific protein that pump the transmitter back in to conserve it, reuse it

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we have reuptake transporters for dopamine, norepinephrine, and serotonin

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and things like cocaine block these transporters so they can't transport at all

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MDMA and amphetamine actually get inside the substrates for the transporter

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once they get inside, they displace the stored dopamine, serotonin, norepinephrine

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and it spills out in the reverse direction through the transporter

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ketamine and some of the others interact with what is called an ionotropic receptor, a glutamate receptor

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and I'm not going to spend any time on those but that's a third kind of target

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so the reserch chemicals typically they hit either this or this

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the psychostimulants and entactogens they're principally interacting with the reuptake transporters

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and the psychedelics and cannabinoids they're principally interacting with a GPCR

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a G-protein coupled receptor

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in terms of preclinical characterisation, we work with a lot of these in the lab

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hallucinogens have affinity for serotonin 2a receptors, and in drug discrimination in LSD-trained rats we get generalisation

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