Our project is intended to be a therapeutic as research is
beginning to show that psilocybin-assisted psychotherapy
could be helpful for numerous mental health disorders. The
nature of our project has many layers as it could be considered
quite controversial! To make sure we were happy with the track
we had chosen we had to understand psilocybin and to
make sure it really will be a beneficial alternative to chemically
synthesised psilocybin we had to understand the scalability of
our project, and whether current academics had any demand
for the product. Click below to learn more!
For centuries, wherever psychedelic substances were
found around the world, religious rituals followed. The
peyote cactus in the Mexican back country (Clarke, 1968),
the Amanita muscaria associated with the Siberian tribes
of the 18th and 19th century (Miller, 2013), even the
psychedelic Mandrake plant was mentioned in the bible as
a possible way to restore fertility (Lee, 2006), to name a
few.
1000BC
In many areas of South America and Mexico various cult
practices formed around psilocybe mushrooms and their
relations (Guzmán, 2008). In pre-Hispanic times these
mushroom rituals were practiced widely and recorded in
the earliest written records about the region in the 17th
century (Feinberg, 2003). Indeed, further evidence found in
the form of stone mushroom artefacts indicates that
mushroom use dates back as early 1000BC (Pollock, 1975).
The mushrooms were called fruit or flesh of the gods and
were believed to bring the user closer to the spirits
(Feinberg, 2003). However, due to the Spanish perception
that mushrooms brought the users into the presence of the
devil, psilocybin use went into obscurity and was
completely unrecorded from the beginning of the colonial
period until the early 20th century (Feinberg, 2003).
1900s
At the beginning of the 20th century, around the 1930s,
various ‘hallucinogenic adventurers’ began travelling to
Latin America and documenting their experiences with
native hallucinogenic plants such as peyote (Feinberg,
2003). This increased interest in the area drew
entrepreneur Gordon Wasson to the Sierra Mazateca area
to study the indigenous use of mushrooms and wrote
about it for the mass audience in the 1950s (Feinberg,
2003). His writings prompted experts from many fields including botany, ethnology, and chemistry to descend
upon the region (Feinberg, 2003). Shortly after their return,
in 1957, the term Magic Mushrooms was coined by LIFE
magazine (Carhart-Harris and Goodwin, 2017). One such
expert was Albert Hofman, who discovered LSD. He
isolated and identified the active ingredient, psilocybin, in
the mushrooms in 1958 and first synthesised it in the
laboratory (Carhart-Harris and Goodwin, 2017).
1960s Mexico
In the mid-60s large numbers of hippies from North
America began to make pseudo-pilgrimages to the Sierra
Mazateca region, camping around the town of Huautla
appeared to take mushrooms indiscriminately (Feinberg,
2003). This caused a lot of tension between these visitors
and the local community as they were not respecting the
traditional ‘rules’ surrounding mushroom use - locals
viewed the plant with reverence and believed that they
should only be taken at night, and that no hedonistic
behaviours such as giving or accepting gifts should be
indulged in for three days after consuming the mushrooms
(Feinberg, 2003). The tourists were disruptive, spent as little
money in local businesses as possible, and mixed drugs
such as marijuana with the psilocybin (Feinberg, 2003). The
situation got so tense that in 1968 the municipal president
asked the government to remove the hippies from the
region, by 1976 the whole region was guarded by military
checkpoints and closed off to outsiders (Feinberg, 2003).
1960s USA
Back in the United States, and around the world, magic
mushrooms began to be used recreationally and also
began to garner scientific attention. In the 1960s the first major
conference on psychedelics was held in Europe, but within
five years the research into these substances ceased
(Carhart-Harris and Goodwin, 2017). This was largely due to
the questionable research performed by two major
institutions: the CIA and Harvard! The CIA was interested in
psilocybin and LSD in the context of either conducting or
preventing mind control, the project was called MKUltra
(Linville, 2016). Some civilians were experimented on, and
only some were told that they were going to be
given drugs beforehand (Linville, 2016). They even spiked
their own agents with the drugs to train them to resist mind-altering substances (Linville, 2016). On a similar vein, two
Harvard academics, professor Richard Alpert and
instructor Timothy Leary conducted a series of trials using
psilocybin and LSD on students from 1960 to 1962 (Hiatt,
2016). Calling them trials is quite questionable as often the
experiments would have no control group and the
experimenters would often take the drugs with their
subjects to better empathise (Hiatt, 2016). The two
academics recruited their subjects by offering their
students a choice: participate in the trials or take the final
exam (Hiatt, 2016).
1970s USA
Following an inquiry into the ethics, legality, and validity of
these experiments, Leary was dismissed from Harvard in
1963 (Carhart-Harris and Goodwin, 2017). This seemed to
signal the end of psychedelic research in the United States
as LSD and psilocybin manufacture ceased in 1965 and
psychedelics use and research began to be prohibited in
the United States in 1966 (Carhart-Harris and Goodwin,
2017). The Controlled Substances act formally made
psilocybin and LSD schedule 1 substances that were signed into
the law in 1970 (Carhart-Harris and Goodwin, 2017). This
reflects a general global shift toward making these
substances illegal as the United Nations Convention on
Psychotropic Substances in 1971 ruled that psychedelics
such as psilocybin should be controlled substances in
member countries (United Nations, 1971). Some countries
such as the United Kingdom and the Netherlands left
magic mushrooms as decriminalised substances - at least
in their fresh form - until quite recently. The UK only made
psilocybin-containing mushrooms fully illegal in 2005 (BBC,
2005), and the Netherlands only making possession of
psilocybin forbidden in 2008 (van Amsterdam et al., 2011).
1970s Australia
To bring things into a more Australian context, whilst
Australia has its own native psilocybe mushroom, no
literature can be found describing any use of this
mushroom by indigenous Australians. The native
Australian psilocybe mushrooms were, however, definitely discovered in 1969 in
Southern Queensland spawning what was described as a large “surfer mushroom cult”, which was so large and prominent that transport businesses were instituted to supply other major Australian cities with the mushroom
(Pollock, 1975). Others cite that it is more likely that tourists
from the United States and the media attention
surrounding the rise of hallucinogens in the United States
were more likely contributors to the widespread increase in
the popularity of magic mushrooms in Australia (Allen et al.,
1991). The apparent apathy of the psychedelics users
toward life and society is cited as the reason that the
Queensland governor outlawed possession of magic
mushrooms in 1971 (Pollock, 1975). Similarly, others cite the
ease of access to magic mushrooms by young people in
Australia as many schools had local crops of magic
mushrooms nearby as being the main cause for concern
and stricter regulation across Australia (Allen et al., 1991).
Present Mexico
Since being made largely illegal around the world,
psilocybin culture has dropped below the radar. Some
psilocybin tourism still exists in Mexico, but it is believed
that this tourism has distorted the local practices to better
suit tourists and as such hold less spiritual significance to
the local people (Guzmán, 2008). Authentic traditional
mushroom use only survives in more remote communities,
and in some places, the Catholic religion inherited from the
Spanish has merged with traditional mushroom worship to
create ‘Mushroom Churches’ where crucifixes are affixed
below mushroom specimens (Guzmán, 2008). The
mushroom culture in these regions has quietened down,
but has not completely died, showing how deeply the
spiritual significance afforded by magic mushrooms is held
by these communities.
Present Global
Over the last 15 years, however, renewed interest in
psilocybe mushrooms has surfaced in the scientific
community. Unlike previous less ethical studies, these
scientists are conducting ethical, safe, well-managed
safety trials. Whilst the sample sizes are small, they have
been providing promising results for the potential efficacy
of psilocybin mediated psychotherapy for the treatment of
end of life anxiety (Grob et al., 2011), OCD (Moreno et al.,
2006), and more.
References
Allen, Merlin and Jansen (1991). An Ethnomycological Review of Psychoactive
Agarics in Australia and New Zealand. Journal of Psychoactive Drugs. 23(1). pp.39-69.
BBC (2005). Magic mushrooms ban becomes law. http://news.bbc.co.uk/2/hi/
uk_news/4691899.stm
Carhart-Harris and Goodwin (2017). The Therapeutic Potential of Psychedelic
Drugs: Past, Present, and Future. Neuropsychopharmacology. 42(11). pp.
2105-2113.
Clarke (1968). Religious Aspects of Psychedelic Drugs. California Law Review.
56(1).
Feinberg (2003). Devil's Book of Culture: History, Mushrooms, and Caves in
Southern Mexico. Austin: University of Texas Press. pp. 126-191.
Grob, Danforth, Chopra, Hagerty, McKay, Halberstadt, and Greer (2011). Pilot
study of psilocybin treatment for anxiety in patients with advanced-stage cancer. Archives of General Psychiatry. 68(1). pp. 71-78.
Guzmán (2008). Hallucinogenic Mushrooms in Mexico: An Overview. Economic Botany. 62(3). pp. 404-412.
Hiatt (2016). A Trip Down Memory Lane: LSD at Harvard. The Harvard Crimson.
Lee (2003). The Solanaceae II: The mandrake (Mandragoraofcinarum); in
league with the Devil. Journal of the Royal College of Physicians of Edinburgh.36. pp. 278-285
Linville (2016). Project MKULTRA and the Search for MindControl: Clandestine
Use of LSD Within the CIA. History Capstone Research Papers. 6.
Miller (2013). Religion as a Product of Psychotropic Drug Use. The Atlantic.
Moreno, Wiegand, Taitano, and Delgado (2006). Safety, tolerability, and efcacy
of psilocybin in 9 patients with obsessive-compulsive disorder. Journal of Clinical Psychiatry. 67(11). pp. 1735-1740.
Pollock (1975). The Psilocybin Mushroom Pandemic. Journal of Psychedelic Drugs. 7(1). pp. 73-84.
United Nations (1971). Convention on Psychotropic Substances.
van Amsterdam, Opperhuizen, and van den Brink (2011). Harm potential of
magic mushroom use: a review. Regulatory Toxicology Pharmacology. 59(3). pp.423-429.
If Psilocybin treats depression, is it just another SSRI?
Whilst psilocybin acts upon SERTs
(serotonin re-uptake transporters),
similarly to antidepressants, there appears
to be little structural similarity. Key
features of SSRIs are aromatic rings and
large halogen groups, designed to block
the SERT protein from uptaking serotonin,
as seen in Figure 1.
Figure 1 : Chemical structure of fluoextine (A) and sertraline (B) SSRIs (wikipedia.org)
Hallucinogens such as LSD and Psilocybin,
on the other hand, look much more like
serotonin itself. In Figure 1 the chemical
bonds the compounds share are
highlighted using bolded lines.
The chemical structures alone highlight
how hallucinogenic drugs work differently than SSRIs, even if the idea is that we will
use them for the same purpose: treating disorders such as anxiety and depression.
Figure 2 : The chemical structures of LSD, serotonin, and psilocybin (Nichols, 2013)
(wikipedia.org)
How can they work on the same receptor but work
differently?
There are many different types of serotonin receptors, broadly it is
postulated that 5-HT1A receptors are involved with passive coping (tolerating stress)
and that 5-HT2A receptors are involved with active coping (addressing stress)
(Carhart-Harris and Nutt, 2017). 5-HT1A receptors appear to be more targeted by
conventional antidepressants while 5-HT2A receptors are more readily targeted by
psychedelics (Carhart-Harris and Nutt, 2017). Both of these receptors are expressed
widely throughout the brain, but both can be targeted to improve the treatment of
mental health disorders (Carhart-Harris and Nutt, 2017). 5HT1A and its 5-HT1AR
signalling pathway works to moderate anxiety and stress and promote patience and
coping, whilst 5HT2A and its 5-HT2AR signalling pathway promotes adaptation and
brain plasticity in times of stress (Carhart-Harris and Nutt, 2017).
Figure 3 : Heat map of serotonin receptors in healthy individuals where blue represents
reduced expression and red represents increased expression. (Beliveau et al., 2017)
Whilst these two pathways work together, their functions are actually quite opposed:
5HT1A is inhibitory whilst 5HT2A is excitatory (Carhart-Harris and Nutt, 2017). The two
receptors are also expressed in vastly different areas of the brain, as seen in Figure
3, rarely are areas that are red for 5-HT1AR red for 5-HT2AR (Beliveau et al., 2017).
Carhart-Harris and Nutt (2017) suggest a two-part treatment regime for future
depression cases whereby antidepressants are supplied to enable patients to better
tolerate their current stress, and this is supplemented by psychedelic mediated
psychotherapy to capitalise on the active coping and willingness to change that
drugs such as psilocybin provide via the 5-HT2AR pathway.
What does this mean for brain function while on psilocybin?
Within the brain there is a process known as the Default Mode Network (DMN), whilst
not much is specifically known about its function it is believed to be involved in
consciousness, rumination, and the idea of the self (Carhart-Harris et al., 2012). DMN
regions are connector hubs of the brain, allowing efficient information transfer
between different regions using the fewest number of connections (Carhart-Harris et
al., 2012). Hence, when psilocybin was found to reduce DMN activity during treatment
(Carhard-Harris et al., 2012) it was postulated that the unrestricted cognition it
permitted was the mechanism behind the beneficial effects of psilocybin. However,
later studies have found that post-treatment with psilocybin DMN activity actually
increases (Carhart-Harris et al., 2017), they liken the mechanism to a soft reset of the
brain. Psilocybin may act similarly to electroconvulsive
therapy where DMN activity is acutely reduced and then normalises or increases,
resulting in a more positive mood (Carhart-Harris et al., 2017). However, this is still a
theory and would need to be tested using a healthy control group and a placebo
treatment.
Figure 4 : Simplified visualisation of the communication pathways between diferent regions
of the brain when on (a) placebo, or (b) psilocybin. (Petri et al., 2014)
The notion of ‘unrestricted cognition’ when under the influence of psilocybin has
been explored by other academics, Petri et al. (2014) explored the differences in brain
activity and communication pathways between subjects dosed with placebo and
subjects dosed with psilocybin. They found that the stability of “mesoscopic
association cycles” - i.e. the kinds of brain waves that can be detectable with devices
such as an EEG - is reduced under psilocybin, supporting the notion of unrestrained
thought (Petri et al., 2014). However, when they explored the communication
pathways that were active in the brain under the influence of either placebo or
psilocybin they found that more functional connections emerge in the psilocybin
state (Figure 4) and that they possess their own organisational features and
functional ‘edges’ (Petri et al., 2014). This means that psilocybin does not necessarily
induce randomness in the brain, but rather new brain organisation that cannot exist
in the placebo state (Petri et al., 2014). This increased connection could also explain
how psilocybin enhances cognitive flexibility and creative thinking (Carhart-Harris
and Nutt, 2017).
Studies have also shown that psilocybin reduces threat signalling from the amygdala (lizard brain cortex) to the visual cortex which leads to reduced threat sensitivity in
the visual cortex (Kraehenmann et al., 2016) which could explain some of psilocybin’s
effectiveness in PTSD and anxiety cases when dealing with triggers. The reduced
threat signalling is thought to be related to the reduced CBF (cerebral blood flow)
found in areas such as the amygdala, the left Heschl’s gyrus, left precentral gyrus,
left planum temporale, left superior temporal gyrus, left amygdala, right
supramarginal gyrus and right parietal operculum - however, only the amygdala is
involved in fear responses, all the other regions are involved in auditory processing
or visuospatial thought (Carhart-Harris et al, 2017).
This means that patients with depression, anxiety, PTSD, and more would have their
threat sensitivity reduced while on psilocybin (Kraehenmann et al., 2016) enabling
them to be more receptive to talking about their triggers and fears. This, coupled
with the increased cognitive flexibility (Carhart-Harris and Nutt, 2017), increased
functional connections (Petri et al., 2014), increased active coping from the
stimulation of the 5HT2A receptor (Carhart-Harris and Nutt, 2017), and pseudo-
cognitive reset from decreased DMN activity (Carhart-Harris et al., 2017) would
enable individuals with these mental illnesses to address them in the supportive
environment of the complementary psychotherapy in a manner unlike that which
psychology can currently achieve. This explains a lot of the incredible preliminary
results that have been seen in clinical trials on psilocybin’s efficacy in treating various
mental health conditions (Grob et al., 2011)(Moreno et al., 2006).
References
Beliveau, Ganz, Feng, Ozenne, Højgaard, Fisher, Svarer, Greve, and Knudsen (2017).
A High-Resolution In Vivo Atlas of the Human Brain's Serotonin System. Journal of
Neuroscience. 37(1). pp. 120-128.
Carhart-Harris and Nutt (2017). Serotonin and brain function: a tale of two
receptors. Journal of Psychopharmacology. 31(9). pp. 1091-1120.
Carhart-Harris, Erritzoe, Williams, Stone, Reed, Colasanti, Tyacke, Leech, Malizia,
Murphy, Hobden, Evans, Feilding, Wise, and Nutt (2012). Neural correlates of the
psychedelic state as determined by fMRI studies with psilocybin. Proceedings of
the National Academy of Sciences of the United States of America. 109(6). pp.
2138-2143.
Grob, Danforth, Chopra, Hagerty, McKay, Halberstadt, and Greer (2011). Pilot study
of psilocybin treatment for anxiety in patients with advanced-stage cancer.
Archives of General Psychiatry. 68(1). pp. 71-78.
Kraehenmann, Schmidt, Friston, Preller, Seifritz, and Vollenweiderb (2016). The
mixed serotonin receptor agonist psilocybin reduces threat-induced modulation of
amygdala connectivity. NeuroImage: Clinical. 11. pp. 53-60.
Moreno, Wiegand, Taitano, and Delgado (2006). Safety, tolerability, and efficacy of
psilocybin in 9 patients with obsessive-compulsive disorder. Journal of Clinical
Psychiatry. 67(11). pp. 1735-1740.
Nichols (2013). Serotonin, and the Past and Future of LSD. MAPS Bulletin Special
Edition. 23(1). pp. 20-23.
Petri, Expert, Turkheimer, Carhart-Harris, Nutt, Hellyer, and Vaccarino (2014).
Homological scaffolds of brain functional networks. Journal of the Royal Society
Interface. 11(101).
Essentially, no. Not easily at least! When tested in mice the LD50 (the
dose that killed 50% of the mice tested - the clinical standard for drug
toxicity) was 293.07mg/kg (Zhuk et al., 2015) which is over 900 times
the dose that tends to be used in current clinical trials (Moreno et al.,
2006)(Hasler et al., 2003)(Studerus et al., 2011).
Figure 5: An artistic representation of the number of potential pills an individual would have to take to overdose on psilocybin.
The standard for clinical trials is
for the doses to be delivered to
the patient pre-portioned for their
weight and for the entire duration
of symptoms to occur entirely
under researcher supervision, not
for patients to be given pills to
take home (Moreno et al., 2006)(Hasler et al., 2003)(Studerus et al.,
2011). However, even if patients were to be given pills to take home
they would need to take over 900 of their prescribed tablets to
overdose.
Does psilocybin affect the body in other ways?
The base effects of psilocybin consumption include slight drowsiness,
intensification of pre-existing mood states, and changes in sensory,
time, space, and self-perception (Hasler et al., 2003). Psilocybin has no
significant effects on the electrical activity of the heart or mean
arterial pressure, but high doses (~300ug/kg) do tend to increase
systolic and diastolic blood pressure between an hour and 90mins
after dosage, but these effects do normalise as the other symptoms
subsided (Hasler et al., 2003). Hence, psilocybin is not recommended
for use by individuals with a history of cardiovascular conditions but
the Hasler et al. study (2003) did conclude that psilocybin use was
safe for most healthy individuals physiologically.
Other physical side effects that have been self-reported during
psilocybin use include lack of energy, difficulty concentrating, a ‘gone
feeling’, headaches or head pressure, lack of appetite, and heavy or
tired legs (Studerus et al., 2011).
Most of the negative side effects of magic mushrooms such as
tachycardia, nausea, and anxiety have been attributed to other
compounds present in the whole mushroom such as phenylethylamine
which is found in relatively large concentrations in a wide variety of
mushrooms (Van Amsterdam et al., 2011). This risk would hence be
eliminated with the use of pure psilocybin in treatment regimes.
What about mental illnesses? Can psilocybin give you
schizophrenia?
One of the primary fallacies with psilocybin is that it can cause
schizophrenia. However, whilst psilocybin affected individuals have
been used as a model for schizophrenia (Vollenweider et al., 1998) it is
exactly that, a model. These individuals are dosed with psilocybin to
induce the mild hallucinations and ‘ego-dissolution’ characteristic of
the early stages of schizophrenia, but these effects resolve within 6
hours or can be halted early using medication (Vollenweider et al.,
1998). However, as it is being theorised that the serotonin receptor that
psilocybin acts upon may be involved in schizophrenia, it would not be
wise for schizophrenics to take psilocybin.
Whilst subjects experience significant psychological effects during
dosing including dissolution of ego boundaries (altered perception of
the self, often resulting in euphoria), hallucinations and synaesthetic
phenomena, reduced concentration, and auditory input distortions
(Hasler et al., 2003), the main criteria for psychosis - anxious ego
dissolution, the measure of loss of control over the body and thoughts
resulting in derealisation - does not increase with increased dosage of
psilocybin (Hasler et al., 2003). Additionally, Studerus et al. (2011)
contacted past participants from 8 clinical trials with follow up surveys
on the long-term effect of psilocybin on their lives and they found that
only 12% of participants reported any negative changes in their mental
health after their clinical trial, and four out of eleven of those
individuals noted that those changes were unrelated to their psilocybin
consumption. Of the remaining seven participants, only one
participant reported that the change was severe enough to warrant
seeking further psychological help, and the reason for their desire to
seek further help was that the psilocybin dosing resulted in him being
confronted with suppressed memories, thoughts, and feelings
(Studerus et al., 2011). His negative feelings were resolved with a few
sessions with a psychotherapist (Studerus et al., 2011). This highlights
the importance of psilocybin sessions being conducted in the format
of psilocybin-assisted psychotherapy.
More broadly, several population studies have been conducted both
via web-based questionnaire and via random survey looking to find if
there is any relationship between lifetime psilocybin (or psychedelic
more generally) use and the incidence of mental illness. The
population study by Krebs et al. (2013) used data drawn from the
2001-2004 US national survey on drug use which involved 130,152
randomly selected respondents. All respondents were assessed for
various mental illnesses including panic disorder, PTSD, non-affective
psychosis, and major depressive episodes (Krebs et al., 2013). 13.4% of
respondents reported lifetime psychedelic use and no significant
associations were found between psychedelic use and any of the
assessed mental health issues (Krebs et al., 2013). In fact, psychedelic
use was associated with a lower rate of mental health issues.
This was mirrored in another population study by Johansen and Krebs
(2015) which used the following period’s national drug survey data and
involved 135,095 respondents including 19,299 psychedelics users.
Haijen et al. (2018) conducted a web-based survey of individuals with
the intention of taking a psychedelic drug, looking to assess the
various factors that influence the positivity of users experiences,
including baseline well-being. They found that respondents who
identified as long-time psychedelics users tended to have higher-
baseline well-being scores, consistent with the notion that the use of
drugs like psilocybin do not correlate with worse mental health
(Haijen et al., 2018).
What about Hallucinogen Persisting Perceptual Disorder?
Hallucinogen persisting perceptual disorder (HPPD) describes a condition where users of certain psychedelic drugs will have
spontaneous alterations in consciousness, anxiety, flashbacks to past
hallucinations, or even new hallucinations despite not having recently
consumed any hallucinogens recently (Krebs and Johansen, 2015).
HPPD is one of the most famous negative side effects of psychedelic
drug use (Bonson, 2012). Whilst symptoms that coincide with HPPD
have been observed in some psychedelic users it is considered rare
(Bonson, 2012) and more frequently associated with LSD use rather
than psilocybin use (Carhart-Harris and Nutt, 2010). In the population
studies above, however, psychedelic use was not strongly associated
with any of the having symptoms of HPPD within the past 12 months
(Krebs et al., 2013)(Johansen and Krebs, 2015), and the authors have
expressed scepticism at the notion of HPPD due to the high potential
of the listed symptoms in the general population. Additionally, in most reports of HPPD, the subject was a poly-drug user or a psychiatric
patient at the time of use (Van Amsterdam et al., 2011).
Can you get addicted to psilocybin?
Experimental data does not support the notion that psilocybin is a
physiologically addictive substance nor a drug of dependency
(Johnson et al., 2018). Studies have shown that psilocybin has weak
reinforcing potential (ability to form addictions through positive
association) and weak generalisation to substances of high abuse
potential (a low tendency for individuals already addicted to one drug
to respond similarly to psilocybin) (Johnson et al., 2018). Psilocybin has
failed to generate reliable self-administration in monkeys previously
able to self administer MDMA, and psilocybin failed to act as a
substitute for amphetamine in rats trained to distinguish
amphetamine from saline (Johnson et al., 2018). Whilst individuals who
take psilocybin can quickly develop a tolerance, this tolerance does
not equate to dependency as individuals who stop taking it do not
experience withdrawal symptoms (Van Amsterdam et al., 2011)
(Bonson, 2012).
In clinical trial follow-ups specifically, it has been
found that participants reported that their drug
consumption habits were mostly unchanged
after their study (Studerus et al., 2011). Some
individuals even reported decreased
consumption of drugs (Studerus et al., 2011). For
psilocybin specifically, most participants
reported no change to their consumption whilst
5.6% of subjects reported to consume it less
often than prior to the study and 3.3% of subjects reported that they
consume it more often than before the study they participated in
(Studerus et al., 2011). It is important to note that the subjects who
reported consuming psilocybin on their own said that they only used it
twice to three times a year (Studerus et al., 2011). This is not consistent
with an abusive pattern of consumption.
This is promising as it suggests that patients prescribed psilocybin-
assisted psychotherapy would not develop an addiction following their
therapy, nor would they experience any withdrawals once therapy
concluded.
What about bad trips? They might happen during
treatment sessions!
Bad trips definitely happen. Bad trips can be likened to a drunken rage
in an alcohol user or a manic craving in a cocaine user - terrible when
they occur, but for most people very rare (Bonson, 2012). Bad trips are
characterised as a psychedelic experience where the user experiences
severe anxiety and disconnection with reality (Bonson, 2012). Whilst
some suggest that as these bad trips are more associated with
enduring benefits than regular experiences, implying that the
therapeutic potential of bad trips might actually be greater than other
experiences, it is important to remember that these negative
experiences are absolutely not necessary for positive therapeutic
outcomes (Carbonaro et al., 2017). In fact, many researchers are
incredibly interested in understanding what causes these negative
experiences to prevent them from occurring during their patient
sessions due to the discomfort they cause (Haijen et al., 2018)
(Carbonaro et al., 2017)(Bradstreet et al., 2014).
Studies surrounding bad trips rely on self-reported data, which is
always flawed due to its reliance on participants to willingly surrender
information, tell the truth, and due to sampling bias - most of these
studies relied on recruiting from psychedelic websites or other
communities likely to have a more positive view upon drug use.
However, as we don’t really want to intentionally give people bad trips
to test influences upon drug experience, it’s the best we can do!
Generally, it’s been found
that having clear intentions, feeling well-
prepared and ready for the experience, and being
engaged by the
therapeutic process
correlated with a reduced
likelihood of having a
challenging experience
due to the individual’s willingness to confront any anxieties they would
encounter (Haijen et al., 2018).
People taking higher doses than usual
and younger individuals were found to be more likely to have a negative experience (Bradstreet et al., 2014)(Carbonaro et al., 2017).
Not having a guide or sitter, or more specifically not having a guide
who was experienced in supporting psychedelic sessions was
correlated with longer, more challenging, psychedelic experiences
(Carbonaro et al., 2017).
Taking other drugs such as
cannabis or alcohol during
the session, being physically
uncomfortable, in a
negative emotional state
beforehand, and lacking
social support were also
indicators of having longer,
more challenging
experiences (Carbonaro et al., 2017).
So why do we think that these experiences won’t happen in clinical
trials or in treatment settings? Labs have rigorous protocols of
supervision, keeping participants away from physical harm, and lack
of interactions with other substances such as alcohol and cannabis
(Carbonaro et al., 2017). Studies often involve psychological
preparation for the drug experience, most recreational experiences do
not (Carbonaro et al., 2017)(Haijen et al., 2018). All lab studies involve
the following that psychedelic users themselves described as being
conducive to good experiences: conducive emotional state, physical
comfort and safety, social support and trust of others, a sober trusted
guide experienced in psychedelic guidance (Carbonaro et al., 2017). In
clinical trials and treatment regimes doses are controlled and
moderate, using pure psilocybin to control dosage. Individuals in their
private sessions tend to use dried or fresh mushrooms of unknown
concentration and many of the individuals said that in their
challenging psychedelic experience they believed their dose to have
been large (Carbonaro et al., 2017). Finally, if negative experiences do
occur within studies or treatment sessions, any participants in the lab
studies that have negative outcomes are immediately offered therapy
whereas individuals on their own have to rely on their willingness
to seek psychological help (Carbonaro et al., 2017)(Studerus et al.,
2011).
So... it’s safe?
Johnson et al. (2018) supports the moving the scheduling of psilocybin
down from Schedule I to Schedule IV. We know that psilocybin is not
significantly correlated with decreases in mental well-being (Hasler et
al., 2003)(Studerus et al., 2011)((Krebs et al., 2013)(Johansen and Krebs,
2015)(Haijen et al., 2018). Hallucinogen persisting perceptual disorder is
rare (Bonson, 2012), more associated with LSD (Carhart-Harris and
Nutt, 2010) and polydrug users (Van Amsterdam et al, 2011), and
potentially not as much of a concern as previously thought due to the
prevalence of symptoms in non-drug users (Krebs et al., 2013)
(Johansen and Krebs, 2015). Lastly, when Van Amsterdam et al. (2011)
reviewed magic mushroom use in the Netherlands during the period
that it was legal they found no significant links between psilocybin and
crime, and significantly fewer lock-ups for psilocybe-mushroom-related
public nuisance or traffic violation related offences than for alcohol
related offences.
This does support that psilocybin can generally be considered safe for
healthy individuals to take in the supervised conditions that psilocybin-
assisted therapy will be conducted in. However, individuals with
cardiac issues and hypertension (Hasler et al., 2003) or individuals who
already experience schizophrenic symptoms as it is being postulated
that the receptor that psilocybin acts upon is also involved in that
condition (Vollenweider et al., 1998).
Reviewing this information makes our team comfortable to designate
our project as a therapeutics project and to look into the
commercialisation of our end product. It is important, however, to
always remember:
“Nothing in life is free from risk – risk is simply impossible to avoid...
Even ordinary activities – eating breakfast, watching television,
walking the dog – carry risks, however minor” (BMA, 2012)
Bradstreet, Johnson, MacLean, and Grifths (2014). Characterization of challenging
experiences (i.e., bad trips) after ingesting psilocybin. Drug and Alcohol
Dependence. 140. pp. 18-19.
British Medical Association (2012). Risk: What's Your Perspective?: A Guide for Healthcare Professionals.
Carbonaro, Bradstreet, Barrett, MacLean, Jesse, Johnson, and Grifths (2017).
Survey study of challenging experiences after ingesting psilocybin mushrooms:
Acute and enduring positive and negative consequences. Journal of
Psychopharmacology. 30(12). pp. 1268-1278.
Carhart-Harris and Nutt (2010). User perceptions of the benefits and harms of
hallucinogenic drug use: A web-based questionnaire study. Journal of Substance Use. 15(4). pp. 283-300.
Haijen, Kaelen, Roseman, Timmermann, Kettner, Russ, Nutt, Daws, Hampshire,
Lorenz, and Carhart-Harris (2018). Predicting Responses to Psychedelics: A Prospective Study. Frontiers in Pharmacology. 9.
Hasler, Grimberg, Benz, Huber, Vollenweider (2004). Acute psychological and
physiological effects of psilocybinin healthy humans: a double-blind, placebo-
controlled dose–effect study. Psychopharmacology. 172. pp. 145-156.
Johansen, and Krebs (2015). Psychedelics not linked to mental health problems or suicidal behavior: A population study. Journal of Psychopharmacology. 29(3).
Johnson, Garcia-Romeu, and Grifths (2017). Long-term follow-up of psilocybin-
facilitated smoking cessation. The American Journal of Drug and Alcohol Abuse. 43(1). pp. 55-60.
Johnson, Grifths, Hendricks, and Henningfield (2018). The abuse potential of
medical psilocybin according to the 8 factors of the Controlled Substances Act. Neuropharmacology. 142. pp. 143-166.
Krebs, and Johansen (2013). Psychedelics and Mental Health: A Population Study. Public Library of Science. 8(8).
Studerus, Kometer, Hasler, and Vollenweider (2011). Acute, subacute and long-term subjective effects of psilocybin in healthy humans:a pooled analysis of
experimental studies. Journal of Psychopharmacology. 25(11). pp. 1434–1452.
Van Amsterdam, Opperhuizen, and Van den Brink (2011). Regulatory Toxicology and Pharmacology. 59(3). pp. 423-429.
Vollenweider, Vollenweider-Scherpenhuyzen, Bäbler, Vogel, and Hell (1998).
Psilocybin induces schizophrenia-like psychosis in humans via a serotonin-2 agonist
action. Cognitive Neuroscience. 9(17). pp. 3897-3902.
Zhuk, Jasicka-Misiak, Poliwoda, Kazakova, Godovan, Halama, and Wieczorek
(2015). Research on Acute Toxicity and the Behavioral Effects of Methanolic Extract
from Psilocybin Mushrooms and Psilocin in Mice. Toxins. 7. pp. 1018-1029.
Integrated Human Practices
Throughout our project we have been keenly aware of the need to integrate the things that we've learned during this project into our final design and future research avenues. This section aims to highlight the ways we've gone about making sure we're on the right track and preparing for future research endeavours in a way that will maximise the good our project can perform and minimise any potential harm.
To make sure our project was right for the people who need it most, and to scope out future development opportunities we wanted to interview some experts. Due to the controversial nature of our project we needed to get human ethics approval to conduct these interviews. This was done to ensure experts were aware of any risk or implications inherent in putting their name to a discussion about psilocybin.
Once this was approved, we talked with experts in the therapeutics side of psilocybin, and the legality and scale-up side of our project.
We have also explored future research and scale-up possibilities that are in line with the lessons we've learned throughout our project.
Dr Prashanth Puspanathan - The Alfred Hospital, Australia.
I am a psychiatry registrar and psychedelic researcher. My primary
interest would be in psychedelic-assisted psychotherapy with a particular
niche currently in the role of psychedelics and addiction treatment.
Psychedelics research has been my soapbox since about 2013 or 2014.
Psychedelics are the whole reason I did psychiatry. I was a surgical
trainee, who switched paths and started psychiatry because once you
really discover psychedelics and, as a result, the untapped potential of
the mind, then a profession like psychiatry that really allows you to
operate on the mind and in the mind, really makes the mind so much
more interesting than simply being able to operate on the body.
Dr Margaret Ross - St Vincents Hospital, Australia
I have a doctorate of clinical psychology and I’ve been working as
a clinical psychologist for 15 years. I’ve been a senior clinical psychologist
at St Vincents for the past 8 years. My interest resides in psychedelic-assisted psychotherapy, particularly for existential distress, particularly
as a result of the fact that we’ve got a number of people who are not
responding to their current treatments, the main two treatments being
psychotherapy or medication. I remember looking at some of the
preliminary research that came out of UCLA, JH, NYU, looking at
psilocybin psychotherapy for people who had cancer. Reducing their
distress and their depression and anxiety. They found a very rapid and
dramatic reduction of symptoms, that were sustained at 26 weeks, so
that absolutely propelled me into action and I wanted to bring that
research to Australia, so here we are. I am running the first-ever
psychedelics clinical trial in Australia, looking into the use of psilocybin as
an adjunct to psychotherapy for those with end of life anxiety and
depression.
Dr Alan Davis - John Hopkins University, USA.
I am a clinical psychologist and my training is in that field. I’m also
an assistant professor of social work at Ohio State University and adjunct
assistant professor at John Hopkins University with the centre for
psychedelic and consciousness research. My research focuses on
substance use, broadly speaking, however, related to psychedelics I’m
very interested in the epidemiology of psychedelic use. Understanding
how and where and for what reasons people use psychedelics in the
natural environment. I’m also interested in the use of psychedelics as an
adjunct to therapy for people with mental health problems.
Dr Francisco Moreno - The University of Arizona
Dr Francisco Moreno is a professor of psychiatry at the University of
Arizona. He was part of a clinical trial into the safety and effectiveness
of psilocybin as an adjunct to therapy for individuals with Obsessive
Compulsive Disorder.
Theraputic Interviews
Psilocybin’s effectiveness and safety
The interviewees’ backgrounds had
exposed them to trials involving the use of
psilocybin for depression, OCD, end of life
anxiety, and the treatment of addiction. As
we’ve learned through our own literature
reviews, psilocybin is incredibly effective at
treating these conditions. The effects of a
single dose last far longer than traditional
medications and the effect is unlike any
other currently on the market. Instead of
masking symptoms, psilocybin produces
mystical experiences that can unblock rigid thought patterns by inducing insight in
users.
Alan Davis at John Hopkins revealed they would be starting trials for conditions
such as anorexia, Alzheimer’s, PTSD, and opioid use disorder. They believe that as
psilocybin has been shown to be effective against alcohol and heroin abuse, as
well as various mood disorders, it is prudent to investigate their effect on other
disorders that have almost no effective treatment methods at present. He believes
that there might be similar root causes behind all these different aetiologies and
that the transformative experience psilocybin-psychotherapy provides could help numerous more people.
In terms of safety, all the interviewees reiterated that psilocybin is incredibly safe
physiologically. None of the symptoms are worse than those experienced by
patients prescribed traditional
antidepressants. However, they all
emphasised the importance of the
supportive, safe, and supervised setting that
clinical trials provide.
Psilocybin experiences can be
challenging, and having a guide and
support person for before, after, and
during the experience is crucial to the psychological safety of the patient and
to ensuring the transformative effects of
the experience are consolidated properly.
In clinical trials, some individuals are excluded for their own safety. People with a
history of psychoses such as schizophrenia, or with first degree relatives who have
experienced that condition are encouraged not to participate in this therapy.
Psychedelic experiences generally are known to possibly trigger relapses in these
individuals or possibly even trigger psychotic episodes as they act on the
serotonergic pathways that are often dysfunctional in these individuals.
Psilocybin is also not recommended for people who have experienced a deep
trauma, as it can be challenging for these people to feel safe in the therapy
environment.
When asked to compare psilocybin-assisted
psychotherapy to traditional therapies such
as antidepressants, the major benefit of
psilocybin was, of course, dosage. Patients
with depression currently, have to take
antidepressants daily, often with undesirable
side effects and poor adherence.
Additionally, for patients with cancer such as
those Margaret Ross deals with, combining antidepressants with other medications
can cause interaction effects such as delirium! Thus, for many people, simply not
having to take a pill every single day
would be a massive improvement to
their lives.
Then, there’s the staggering
difference in effectiveness. A huge
proportion of patients prescribed
antidepressants and other psychiatric
medications do not experience any relief from their
symptoms. Not only do the effects of
psilocybin last longer, but the proportion of patients who experience
the effect in a given clinical trial is
significantly larger. According to Alan
Davis, the effect sizes that they’re
seeing in their depression study are
almost ten times greater than those
seen when antidepressants were first released to the public!
One of the things that the researchers did
point out to us, though, is that the screening
processes for these trials is extensive.
Patients will be excluded for having co-
occurring conditions, the nature of their
problems not being a good fit for the study,
and sometimes if the patient failed to develop a good rapport with the clinicians
they may be excluded. These are all important measures in a clinical trial setting,
especially when pioneering a new treatment method. Psilocybin-assisted-
psychotherapy is all about trust and a feeling of safety, so it’s important to only
include patients who will feel that trust and safety. It does mean, however, that
when this treatment is rolled out to the public we may see a slight decrease in
effectiveness as many people may have co-occurring conditions, have had their
condition for longer than those tested, or have more complex problems. Alan Davis
suggests that some individuals may need
three or four dosing sessions rather than
the current standard of one or two to feel
the full effects. However, even reducing
the proportion of people helped by this
treatment by half still leaves psilocybin-
assisted psychotherapy miles ahead of
antidepressants.
Expert Opinions on our Project
Whilst the experts largely said that they haven’t had problems accessing psilocybin
due to its production cost, thanks in large part to their university connections or
charitable partnerships, the resounding feeling was that yes, cheaper psilocybin
would play a huge part in making this treatment more accessible to patients.
Key things that were important to our experts about our theoretical final product
were: dosage accuracy and reliability, no additional side effects, and shelf stability.
Being able to buy the quantities that they need and not be concerned about shelf
life, even if the product required refrigeration, was a big drawcard for them.
We mentioned the possibility of giving universities their own DNA constructs so
they could make their psilocybin in-house to Alan Davis. He responded that whilst
this would be a possibility for larger universities like John Hopkins, this wouldn’t be
accessible to smaller institutions and eventually to clinical practices. However, he
liked the idea that institutions could distribute psilocybin to their region, in which
case the previous considerations about shelf life and stability would still apply.
Whilst the prospect of making
psilocybin in E.coli excited people, some
were worried about the perception the
public has about this bacteria. E.coli is
not generally recognised as safe, and
many believe it to be synonymous to
food poisoning - definitely a potential
branding issue! This began to foster
within us the idea of thinking about
shuttle vectors to move our construct into a Bacillus spp. or Lactobacillus spp.
Some experts suggested that our
system, or parts of it, could also be
repurposed to make other compounds
of interest such as some other
compounds in the mushroom, or short
acting tryptamines such as DMT. The
cost of the therapy sessions themselves
would be a major contributor to the total
treatment cost for individuals, especially
in places without socialised healthcare.
Whilst short-acting tryptamines might be
too short-acting to produce the
profound experiences psilocybin does,
Margaret Ross says, these are exciting
times where a lot of research is being done and these avenues are only beginning
to be explored. If DMTs or other related compounds are found to be useful, our system could be easily adapted to accommodate the production of these
compounds.
Sam Bannister - University of Sydney and Lambert Initiative
I'm a senior research fellow and a team leader in medicinal chemistry at the Lambert Initiative for cannabinoid therapeutics. My role is to guide the discovery of chemistry efforts for new cannabinoid-based medicines, mostly for the treatment of epilepsy but also for some other conditions that are of interest to the initiative. Then, as a side stream of research, I've been working for a long time on the structure-activity relationships of cannabinoid agonists, which are an emerging class of designer drugs, quite a large class. I was referred to the iGEM team’s supervisor, Nick, to help him grapple with the various regulatory forms required for this project because I’ve spent many months researching the regulations in Australia for my research.
Dr Dale McClure - University of Sydney
I’m a post-doctoral research associate at the University of Sydney in the Chemical and Biomolecular Engineering department. My background is bio-industrial biotechnology and chemical engineering, so my research is focused on the production and scale-up of biotechnological processes.
Adam Bandt - Greens MP
Adam Bandt, a former industrial lawyer, is the federal Greens spokesperson for science and representative for the Division of Melbourne.
Legalisation and Industrialisation
We talked to Adam Bandt, MP, and Sam Bannister about the legal conundrums surrounding our project. Sam Bannister also helped us to evaluate some of the production considerations for our system alongside Dale McClure.
In Australia the Therapeutic Goods Act designates whether or not substances are controlled countrywide and to what level, but further restrictions are determined by each state. The TGA lists psilocybin and psilocin as schedule 9 - controlled/prohibited substances with no recognised medicinal value. In NSW the Drugs Misuse and Trafficking Act [DMTA] of 1985 lists psilocybin and psilocin as similarly prohibited and there are laws regarding the possession, use, trafficking, manufacture, distribution, and more. Sam Bannister pointed out that the language regarding drug regulation in many countries is quite vague - the DMTA, for example, says “psilocybin and its derivatives” to guard against novel drug development, but where does that leave our system? Is a gene sequence a derivative? It appears, according to Sam’s reading, that in Australia you have to be in possession of the substance itself to be prosecuted - for example. the possession of mushroom spores does not appear to be heavily regulated. One can assume that in that case, possession of a DNA sequence or even an uninduced cell would likely not be controlled, but possession of an induced cell or cell lysate would be regulated… or so it seemed!
Sam dug up a really interesting obscure law that he felt we ought to be aware of. In the DMTA there is a section on possession of instructions for manufacture, and possession of equipment for manufacture. Sam did note that many scientists have published ‘instructions’ to produce various illicit substances using chemistry for scientific purposes, and there have been no cases that he could find where someone was actually prosecuted for that. Even so, whilst these laws are rarely enforced they could apply to our plasmid(s). Thus, we want to strongly recommend that only persons with a license to produce/research psilocybin as we do possess our plasmids. We also intend to try to keep our system somewhat restricted to persons with institutional resources.
When talking to Adam Bandt, he expressed that he felt a definite reluctance in some areas of parliament to change the regulatory framework surrounding therapeutic restricted substances like cannabinoids. However, he thinks that these people could have their minds changed as more and more promising research comes out about these substances. This mirrored what Sam Bannister had to say, he feels that there is a massive trend of decriminalisation for substances like these, with cannabis leading the charge. Adam did feel that substances of this nature would always need to be regulated appropriately, so long as the regulation did not massively restrict research. He feels that, if the appropriate steps were taken, it would be entirely possible for us to take our system all the way to the industrialisation step within the current regulatory framework, which is extremely promising.
Concerning genetically engineered products generally, Adam expressed that concerns and regulations around genetically modified products in Australia are largely related to agricultural concerns, contamination of non-GM products with GM products, and restriction of exportation possibilities to markets with tighter GM legislation. Luckily for us, our project would only really be concerned with one of those - export - which would, of course, be a bridge we would have to cross once we had managed the various hurdles of getting into the Australian market. Adam Bandt did seem quite pleased with the project generally as he wants GM-products in the medical sphere to be in the interests of the public good, and he found it admirable that we want to ensure our project is open source. Unfortunately, he wasn’t able to on the spot clarify whether or not making our project open source would get us in any hot water, but we’re cautiously optimistic.
When it came to production, we decided to take advantage of Sam Bannister’s experience as a medicinal chemist to discuss the current psilocybin production paradigm. He explained that generally, chemical manufacture has a very high environmental footprint due to the waste from solvents, especially organic and chlorinated solvents. However, he did point out that even with an E.coli based construct we would likely have to use chromatography to purify out the psilocybin from the proteins of the cell - that involves some of those same solvents. This fed a burgeoning idea we were having as a team to investigate how we would move our construct into a generally recognised as safe (GRAS) organism like bacillus.
He also brought up a point that we had briefly talked about with Margaret Ross - in Australia chemical synthesis of compounds like the cannabinoids he works on, or psilocybin in Margaret Ross’s case, has to be conducted in facilities that use ‘good manufacturing practice’ (GMP). However, there are currently no GMP manufacturers in Australia. Researchers like him and Margaret Ross need to get their compounds imported from Europe, where there are numerous GMP manufacturers. However, importing over large distances carries additional costs, difficulties, and environmental detriment. This made us feel as though our construct could help a lot in this way as it could easily be transported to different institutions and grown in facilities a lot closer to any given researcher.
With Dale, we tried to get a grasp of the realities of scaling up production of a genetically engineered organism. Whilst we had always thought that the higher the product yield, the better it would perform in an industrial setting, Dale informed us that this was not always the case. Sometimes the best performing lab strain isn’t always the most suitable for industrialisation, often they’re happy to sacrifice some yield for a more robust strain. In industrial fermentation, you can’t always get the specific temperature and pH conditions you’re able to achieve in the lab, and that can impact the yield of less robust strains greatly. He also reinforced that purification, especially for pharmaceuticals, is often lengthy and complex, which greatly adds to the cost.
When we asked him if using a different organism than E.coli, such as Bacillus subtilis, would complicate the industrialisation process, he said that any well-characterised organism is usually amenable to industrial growth processes. Most organisms that aren’t filamentous, fastidious, or pathogenic are usually going to work industrially - but he reminded us that working with microbes is never easy!
From these discussions we felt more confident about where we stand in terms of the current regulations in our country, especially regarding our theoretical ability to scale. We also felt more encouraged with some of our future avenues, including notions of moving our system into a GRAS organism like Bacillus subtilis.
Armed with all our data, literature, and interviews, we are now able to put considerable thought into where our project next. Here's a selection of the things we want to do with our project in the future to showcase all the things we've learned.
Next we believed it important to follow up on our current LC/MS data from our colonies containing both pUS387 constructs. The LC/MS results from our resting pUS387 cell assays which showed weak signals for baeocystin, one of the products of PsiM, and no signals for psilocybin. Through late sequencing and restriction digestion of our pUS387 constructs, we learned that we had a potentially truncated PsiM protein present in our system. Going forward we want to explore two possible avenues that might explain the presence of baeocystin in our samples:
The truncated PsiM is non-functional and an endogenous methyltransferase in E.coli may be responsible for the norbaeocystin to baeocystin conversion.
The truncated PsiM protein still retains some methyl-transferase activity and is able to perform the norbaeocystin to baeocystin conversion.
Alongside these investigations we would also seek to reconstruct our pUS387 constructs using Golden Gate cloning once more to make sure the full psiM gene is inserted properly.
Quantifying yield
The LC/MS we performed was largely qualitative. It told us which metabolites we had and which we didn’t. One of the key hurdles to optimising the flux of PsiM and ensuring that if we move our system into Bacillus we output only pure psilocybin and none of the other metabolites would be this qualitative measurement.
In order to make LC/MS quantitative, standard curves of all metabolites at different concentrations would need to be made up to compare to our samples (Sargent, 2013). This would be a major research hurdle as getting ahold of pure norbaeocystin, baeocystin, and psilocybin is both expensive and highly regulated. There are also no producers of these substances in Australia. For this project to continue, it would need to be performed on a longer timescale to allow for the time it would take to get the substances from a manufacturer, likely in Europe… and of course, it would need a much bigger budget!
Once the yield is quantified at a lab-scale, we’d be ready to move onto the industrialisation side of things.
Additionally, we’d have the capacity to examine the stability of our product in refrigerated and unrefrigerated conditions, both as purified product and within Bacillus subtilis cells. Our experts expressed that whilst refrigeration wouldn’t be a complete turn off for our product, they would want to be assured that our psilocybin would have a decent shelf life. These experiments would be crucial to meet their demands before moving our product to market.
Scalability
Scaling the production of any therapeutic made by a synthetic organism remains a significant obstacle when attempting to produce a commercially viable product. In speaking with Dale McClure we learned that it isn’t uncommon for your strain to perform poorly in fermentation-based scale-up practices despite performing well in a laboratory setting. Lab strains that generate the most amount of product may not be the most robust strain for an industrial setting. Conversely, the most robust strains may not be the highest performers when it comes to product yield. A balance between these two factors makes for a more desirable strain that can be successfully scaled for production of your therapeutic. For this reason, it is common to use mutagenesis methods to improve the performance of your organism and screen hundreds to thousands of clones in a lab setting before taking the highest performing ones though to scale up exercises. With Dale’s help, we set about exploring how well our system might perform in industrial fermentation through a small scale fermentation exercise. In this exercise we monitored the growth of our empty final chassis organism (DH5-alpha E.coli cells) at 37C in our supplemented TB medium with which we planned to grow our Magi.coli.
Figure 2: Dale's fermentor that we borrowed for our scale experiment.
Our goal was to track and compare the growth rates and cell densities achieved a fermentation system against a lab-grown culture (see results). Through this practice, we learned the value of optimizing our system for the fermentation-based scale-up and recognized a need to optimize our Psi proteins to better express under these conditions.
Through our engagement with Dale we also gained an understanding over how costly the purification of therapeutics can be from recombinant chassis E.coli. The separation of potentially harmful endotoxins from the therapeutic of interest is vital to the recovery of a product suitable for medical application. There is a continuing need for endotoxin-free systems to increase biosafety and cut down on the costs associated with this purification step (Sanchez-Garcia et al., 2016). In recognizing this need we developed a future plan to investigate the expression of Psi enzymes in Bacillus Subtillus.
Moving the system into Bacillus
When talking to experts, we were surprised by the general reactions when we told them that we were making our system with an E.coli chassis… there’s definitely a marketing issue there! People were concerned about pathogenicity, public perception, and the complications that come with purification. This prompted our very earliest thoughts of moving the system into a GRAS (generally recognised as safe) organism. Then, the more we looked at it, the more it made sense!
Moving the production of psilocybin into Bacillus subtilis could further reduce the cost of psilocybin production. Bacillus subtilis is a GRAS organism (Sewalt et al., 2016), meaning that we might be able to avoid having to purify psilocybin from the bacteria. Instead, patients could be given a capsule containing the Bacillus with the psilocybin, like a probiotic. Alternately, Bacillus subtilis might be able to pump out the drug into the culture media, which could just be syphoned off and the process of cell lysis could be avoided. In order to test either of these possibilities, the Psi gene would need to be transferred into Bacillus subtilis, ideally in an E. coli and Bacillus shuttle vector. This vector would need an E. coli origin of replication, and an antibiotic resistance marker, and a screenable marker to tell if the genes have been inserted. Additionally, it would be useful for the plasmid to ultimately integrate into the Bacillus genome, to avoid the use of antibiotics in the culture media used to grow the cells. We have found a vector that fulfils these characteristics and has been used by a previous USYD iGEM team: pUS258.
Figure 3: Our proposed shuttle vector to move our system into Bacillus, Sydney Australia 2017's PUS258 vector.
Another consideration to test this system is which B. subtilis strain to use. Many B. subtilis strains have endogenous proteases, which could cause problems since our system relies on four enzymes. Therefore, a strain with the proteases knocked out should be chosen. One such strain is Bacillus subtilis KO7 (Zeigler, 2016).
Further Research
Fermentation trial of Magi.coli
The scaling of an organism producing a currently illegal therapeutic, such as psilocybin, presented barriers in setting up our experiment with Dale in his lab. In compliance with our NSW order for the manufacture and possession of psilocybin, we did not pursue testing any of our recombinant organisms outside of our lab. Going forward we hope to generate a more accurate indication of cell growth by testing our Magi. coli organism under induction conditions in the industrial fermenter.
Optimizing Psi proteins for fermentation conditions
From the results of our scaled growth exercise and our interview with Dale we realised it was important to work towards a more robust Magi.coli system, adapted to express Psi protein at 37C. By doing so we could hopefully generate higher yields of psilocybin and take advantage the level of growth evidence in our preliminary experiments with DH5-alpha cells. Since we had such success using error-prone PCR to generate a more fluorescent VVD protein we believe that we can leverage this technique to hopefully generate clones that express functional Psi genes at higher temperatures.
Alternative Applications
Numerous migraine medications are indole alkaloids which have a tryptamine moiety (Baumann et al., 2011). We believe that our PsiD part could be used to reduce the costs associated with producing this drug by being integrated into the chemical synthesis process.
Additionally, experts such as Alan Davis believe that an avenue of future research could involve short-acting tryptamines such as DMT (also known as N,N-Dimethyltryptamine). Short-acting tryptamines, if found to induce similar introspective experiences to psilocybin, could significantly reduce the psychotherapy costs required for individuals to access psychedelic-assisted-psychotherapy due to their shorter periods of activity. In plants, DMT is produced from tryptophan, via tryptamine, and then methylated twice to form N,N-Dimethyltryptamine (Rosengarten and Friedhoff, 1976). We believe that PsiD could be used to perform part of this synthesis process. Additionally, if future research shows that PsiM can act upon tryptamine, our PsiM part could be used to complete this process.
In Conclusion
Whilst our project started with psilocybin, it certainly does not end there. We believe that our project can be more broadly applied to reduce costs and democratise numerous aspects of current and future medicine.
References
Baumann, Baxendale, Ley, and Nikbin (2011). An overview of the key routes to the best selling 5-membered ring heterocyclic pharmaceuticals. Beilstein Journal of Organic Chemistry. 7. Pp. 442-495.
Rosengarten and Friedhoff (1976). A review of recent studies of the biosynthesis and excretion of hallucinogens formed by methylation of neurotransmitters or related substances. Schizophrenia Bulletin. 2(1). Pp. 90-105.
Sanchez-Garcia, L., Martín, L., Mangues, R., Ferrer-Miralles, N., Vázquez, E., & Villaverde, A. (2016). Recombinant pharmaceuticals from microbial cells: a 2015 update. Microbial cell factories, 15, 33. doi:10.1186/s12934-016-0437-3
Sargent (2013). Guide to achieving reliable quantitative LC-MS measurements. Royal Society of Chemistry Analytical Methods Committee. 1st ed.
Sewalt, V., Shanahan, D., Gregg, L., La Marta, J. and Carrillo, R. (2016). The Generally Recognized as Safe (GRAS) Process for Industrial Microbial Enzymes. Industrial Biotechnology, 12(5), pp.295-302.
Zeigler, D. (2016). BGSC - Home. [online] Bgsc.org. Available at: http://bgsc.org/ [Accessed 18 Oct. 2019].
