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

maybe a lot maybe not a lot

how did we study psychedelics without using humans

when I was in a purude university

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

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

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

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

through a research chemical

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

because lot of people don't know what they are

sadly a large number of them was pilfered from my publications

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

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

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

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

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

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

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

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

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

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

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

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

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

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

nothing about neurochemistry had anything to do with behaviour

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

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

because you try to tell people what psychedelics were

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

and I like this definition because it says that psychedelic

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

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

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

I often give the seminar to pharmacology departments at medical schools

where i give this definition and I say

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

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

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

before we start

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

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

listed here that's been published but the point

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

so these purple ones ate the 1990s

these red ones are 2000-2010

and then after 2010, what you see is exponential explosion

of published studies that relate to clinical research

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

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

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

the key brain receptors

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

and for tryptamines they also activate the 1a receptor

LSD is a little bit different

somebody have a question?

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

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

and then they switched over

and they started sayin it had rich pharmacology

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

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

it pretty obvious that the serotonin 2a and 2c

receptors are probably the ones that are the most important

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

a lot on the streetm activate principally the serotonin

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

efficacy so they produce a good activation of the receptor

we dont know much about the serotonin 1a receptor

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

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

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

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

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

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

so it has unique pharmacology

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

this is just a model of a brain synapse

but principaly these serotonin 2a receptors are located postsynaptically

so we have release of serotonin in a normal brain

it comes out of these vesicles and interacts with these receptors

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

or within the cell

and they can induce signalling in number of ways

they can either fully activate the receptor

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

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

about 30% of full activation

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

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

so its maybe related to its mechanism of action

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

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

actually drops the basal level below this

so BOL would fit into the scheme there

what happends when a psychedelic binds to the receptor?

these receptors are bundles of alpha-helical protein pieces

that are fit together in the membrane of the cell

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

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

it causes the shape of this protein bundle to change

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

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

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

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

that would activate the receptor

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

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

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

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

so imagine serotonin, which is the natural transmitter

binding down inside the receptor

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

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

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

to some set of signalling molecules

if we put LSD in there

or 2C-B or you name your favourite drug

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

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

this new receptor shape induces new changes

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

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

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

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

normally couple to G protein coupled receptors (GCR)

G alpha I which generally inhibits cellular activity

G alpha s which typically stimulates cellular activity

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

that typically also activate the cell

it can also be phosphorylated with an enzyme

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

and that can allow them to interact with another signalling molecule

called beta arrestin, so there are number

of these signalling pathways that can be activated

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

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

a different drug produces a different set of signals

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

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

they don't just turn the receptor on and off

they induce a whole bunch of different kinds of changes

and these changes are related to

the structure of the drug itself

So how does receptor signal change consciousness?

so this is when the hand-waving comes in

Franz Vollenvaider will give a talk i think tomorrow

and Robin Carhart-Harris will also talk about

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

actually responds to psilocybin or to other psychoactive drugs

the kinds of electrical changes as measured by EEG

or glucose utilization as measure by PET

and a number of other techniques

so what happens?

well basically, we don't know clearly

but one of the things we know that the serotonin

2a receptor, which is the esential receptor

for producing the effects of psychedelics, is highly expresed in

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

and counsciousness.

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

which sends norepinephrine projections to the whole forebrain

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

that sends serotonin projections to the whole forebrain,

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

we have serotonin 2a receptors both in thalamus

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

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

so auditory, vision, sensing, touch

all of that is processed through the thalamus

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

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

and sends inhibitory projections down into the thalamus

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

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

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

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

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

so the cortex is critical to the funtion of higher conciousness

it's critical to defining who we are

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

and its activity is determined by all these other areas

so any sensory information that comes in, is filtered through

the thalamus, that's regulated by reticular nucleus

we have the Raphe sending signals to the cortex

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

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

and the ventral tegmental area produces dopamine and also produces activation

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

will change the way we perceive things

changes the whole process of sensory, perception, and consciousness

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

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

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

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

is probably one of the oldest receptors

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

probably the oldest of the monoamine transmitters

we consider serotonin, dopamine and epinephrine

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

paramecium, where is responsible for the movement of the cells

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

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

one of the problems I had in Purdue university

working in the pharmacy school in the midwest

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

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

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

i plead the 5th amendment

so how did we actually do our studies?

we spend a lot of time looking at animal models

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

no animal can model what happens is humans though

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

or i remembered my childhood

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

turns out that in pharmacology

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

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

you apply the drug that modifies that system

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

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

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

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

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

in a fight or flight response if you hear

a noise at night,

you walking down a dark street

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

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

and it stimulates beta 1 receptors in the heart

so similarly we can argue to study consciousnesses

we can use agents that perturbs consciousnesses, so, psychedelics

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

so they're really pressing that lever

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

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

so they uniquely recognise the drug they were trained with

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

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

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

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

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

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

and relative potency compared to LSD in humans

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

it's also more potent in rats

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

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

so there's a good general correlation

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

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

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

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

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

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

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

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

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

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

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

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

it's even less effective in binding

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

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

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

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

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

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

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

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

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

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

which we converted into a serotonin 2a receptor

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

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

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

and this is LSD bound

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

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

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

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

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

as a kind of edge-to-face interaction

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

so we mutated that phenylalanine to leucine

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

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

but it still keeps some hydrophobicity

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

here's the N-benzyl down here

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

2C-B is here with the N-benzyl

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

and the affinities for this compound is 2.6nM

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

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

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

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

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

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

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

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

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

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

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

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

depending on what you prefer

synthetic cannabinoids, and I'll briefly talk about those

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

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

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

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

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

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

feelings of portentiousness, something important is about to happen

high doses can elicit anxiety, or psychotic behaviour

generally they have low toxicity and non-addictive

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

there are no overdose deaths from LSD, mescaline, psilocybin

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

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

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

and most psychostimulants have cardiovascular effects

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

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

large doses or chronic use can produce depression and fatigue

and high doses can also produce psychotic behaviour

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

they cause vasoconstriction

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

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

we all know today that MDMA is not just another methamphetamine

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

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

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

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

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

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

so frontal cortex, hippocampus, striatum and reticular formation

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

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

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

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

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

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

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

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

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

we have things that interact with reuptake transporters

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

we have reuptake transporters for dopamine, norepinephrine, and serotonin

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

MDMA and amphetamine actually get inside the substrates for the transporter

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

and it spills out in the reverse direction through the transporter

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

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

so the reserch chemicals typically they hit either this or this

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

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

a G-protein coupled receptor

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

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