BEST OF

NEUROSCIENCE
 Drug Discovery News
Best of Neuroscience
Contents
On good behavior
Holding out hope
Intriguing approaches to
Parkinson’s disease
Beyond ‘reefer’madness
Next-gen neuroscience
The ‘Tao’ of tau?
Targeting α-synuclein and the
pathogenesis of Parkinson’s disease
Image-ine that
Bringing precision medicine to
the evaluation and treatment
of mental illness
Foreword
Life-sciences research and pharma R&D
is fraught with daunting challenges, from
difficulties in fighting viruses like HIV and
SARS to finding “a cure” for cancer when
tumors vary so much across the oncology
spectrum. But one of the standout areas for
3 challenges seems to be neuroscience. The
complexity of the human brain, problems
9 in getting past the blood-brain barrier,
teasing out environmental vs. neurochemical
effects on mental health and understanding
15 cognition are just a few of the areas that
continue to vex researchers.
18
And while progress has been made
23 in many other disease areas despite
challenges—turning AIDS into a chronic
26 illness rather than a sudden death sentence
and beating back cancer more and more
each year—Alzheimer’s disease, multiple
28 sclerosis, schizophrenia and so many other
neurological conditions continue to pose
30 huge challenges for diagnosis and treatment.
So, DDN presents this collection of articles
and commentaries from recent years of the
magazine on progress that is being made
34 in neuroscience. We hope that what lies
between the covers here will expand your
own understanding and also let you know
that while the road may be rough, we are
getting someplace. We just don’t know how
long it will take to get there.
FROM 2020
SPECIAL REPORT ON NEUROSCIENCE
ON GOOD BEHAVIOR
To translate to humans, neurobehavioral models must first translate to animals
BY RANDALL C WILLIS, DDN FEATURES EDITOR
A lab-coated intern excitedly corrals her lab mates to share
her great discovery. She carefully places a housefly in the
middle of a cleared space and asks everyone to observe.
Suddenly, she claps two pans together, making a loud noise, and
the housefly takes off.
No one is particularly impressed.
She then places a second housefly in the same spot, but carefully
immobilizes its wings.
Again, the pans clash, but this time, the fly stays put.
“See,” the intern proclaims. “When you disable a fly’s wings,
it goes deaf.”
An old joke and unfair comparison, perhaps, but neurobehav-
ioral and neuropsychiatric research present challenges rarely
experienced in most other scientific efforts—and sometimes it
feels a lot like the scenario above.
Unlike many areas of research where the molecular and medi-
cal align discretely, here that alignment can be significantly hazier,
criss-crossed with a multitude of confounding and disguising factors.
“For many of these complex behavioral assays ... the ultimate goal
is to identify disease-relevant endpoints that are robust, reliable, and
reproducible, and that can be employed to evaluate potential novel
therapeutic agents,” wrote Jill Silverman of the UC Davis MIND
Institute and Jacob Ellegood of Toronto’s Mouse Imaging Centre in
a 2018 review. “The impact of a competing or confounding behavior
on the behavioral endpoints ... cannot be understated.”
They stressed that mutations can cause physical impairments
that limit a subject’s abilities to perform a task, much as in the
housefly example above.
“Motor defects in [autism spectrum disorder] models, including
hypo- and hyper-locomotion, can also have consequences on the
behavioral outcome of interest by competing or preventing the
subject from engaging in the tasks of core symptomology testing,”
the researchers noted. “Just as it is important to understand the
Drug Discovery News
limitations of a behavioral task itself, it is important to investigate,
acknowledge, and report the limitations of the rodent model being
tested so as not to be short-sighted in the interpretations and
applications of the data.”
Elucidating the scale of this complexity and designing exper-
iments and models to mitigate at least some of those challenges
is a significant focus of neurobehavioral researchers who seek to
improve translation of findings not just to humans, but also to the
same animals in a more native state.
Teasing out complexity
“My background and training is in Parkinson’s disease, but I stud-
ied signaling and inflammation components as an environmental
modifier of neurodegenerative disease,” says Taconic Bioscienc-
es field application specialist Terina Martinez. “So, I was keenly
aware of the fact that we were talking of diseases that have a
complex etiology.”
That applies not only to neurodegenerative disorders, she con-
tinues, but also to neuropsychiatric diseases where genetics is
clearly not the only factor involved. Rather, she points to a vari-
ety of environmental and social cues that, alongside genetics and
other pathologies, converge in a manifestation of disease.
“Behavior is one of those modalities that is both loved and
loathed,” Martinez offers.
“In a complex disease that has maybe six or seven different
molecular pathways that converge on a pathologic mechanism,
behavior is one of the few modalities to integrate multiple path-
ways,” she suggests. “So, it is very important as a measure.”
She is quick to add, however, that this ability to integrate mul-
tiple pathways to a pathology is also why behavioral studies and
models are so challenging. Rather than presenting in discrete
terms, where it is simple to reproduce findings from one experi-
ment to another, these analyses offer significant variability.
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 DDN Best of Neuroscience
“The challenge is in acknowledging [behavior’s] importance,
knowing what the limitations are, and being thoughtful about
interpretation so you don’t over-interpret,” Martinez states. “And,
on the front end, designing studies so you can try to optimize the
behavioral observations in a way that’s interpretable.”
And that complexity is, in some ways, confounded by the nature
of animal models and testing.
Experimental models are, by necessity, a gross simplification of
what is happening in a human patient, as well as what is happen-
ing with test animals under natural circumstances, suggests Lucas
Noldus, founder and CEO of Noldus Information Technology and
recently appointed professor at Radboud University in Njimegen,
the Netherlands.
“In the laboratory, we try to eliminate as many uncontrollable
variables and reduce the experiment to a very simple set of stimuli
and outcome measures, which we then record and from which
we tease out the results of the effects of treatment,” he continues.
This has resulted in a vast collection of test paradigms that
address single aspects of behavior or functional domains of the
brain. These could be tests of locomotion, where the animal goes
or how long it stays there, how it interacts with cage-mates, or
even its diurnal rhythms.
“And all these different aspects of behavior were traditionally
tested in separate devices, apparatuses and tests,” Noldus notes.
This isn’t to say that these tests have had questionable value.
Instead, Noldus suggests they have been quite helpful in estab-
lishing specific relationships—say, between an administered com-
pound and a behavioral outcome. He is quick to note, however,
that translational or ecological validity has long been a weakness.
“The behavior of an animal in a barren cage, devoid of any stim-
ulation, with the animal being observed for 10 minutes, during
which you record whether the animal moves to the center of the
cage, is hardly a valid representation of what happens to a human
patient suffering from an anxiety disorder during his daily pattern
of life, at home, at work, in the open space in the street,” Noldus
offers as an example. “The environment in which we humans
operate and perform is so much more complex than the simplistic
representation in the test arena for an animal that the translational
value is inherently weak.”
Martinez concurs.
“That translatability is very key,” she says. “Looking at some of
these complex neuropsychiatric questions, how do you ask your
mouse if it’s depressed?”
In DDN’s April 2019 Special Report on Neuroscience, Aptinyx’s
president and CEO Norbert Reidel and Brain and Cognition Dis-
covery Foundation’s executive director Roger McIntyre offered
much the same observation while discussing efforts to develop
new treatments for depressive disorders.
“It is hard to measure depression in a rodent,” acknowledges
Riedel. “There are ways in which we look at this, but I would say
that it is of limited value in predicting a human disease course in
a depressive disorder.”
“There is no question that the animal models we have are not
just imperfect, but are serving as a major limitation to progress
in the field,” McIntyre adds.
Drug Discovery News
“I have done that forced swim test [on rodents],” offers Riedel.
“Why would you stop swimming? Well, maybe the rat doesn’t
know how to swim. Maybe it’s weak. Maybe it did well the day
before. But to say that it is because they’re depressed is a stretch.”
“Certain human symptoms like hallucinations, delusion, guilt;
obviously, those aren’t going to replicate in an animal,” Martinez
presses. “But other things have the potential to be replicated.”
She points to examples like executive function, motivation,
working memory and emotion, which she argues are more rea-
sonable to ask an animal to model.
Pushing reproducibility
Further complicating the translational validity of neurobehavioral
models and experiments, says Noldus, is the manner in which the
experiments have been conducted.
“Because there were no ways in the past to automate the mea-
surements,” he explains, “all the measurements were performed
by human beings, who would handle the animals, transport the
animals, administer the compound, do the observations.”
Despite the use of common protocols, the natural variability in
how the human researchers handled the animals, performed the
manipulations, and even how they dressed and smelled was an
inherent confounding factor for these studies.
“We sometimes forget that mice, rats, all other animals have
much more heightened senses of smell,” Martinez echoes. “They
use other senses to engage with their environments and register
stress and respond to their environment.”
“We can literally turn on or off certain behavior outcomes in
mice depending on whether or not the person running the study
is male or female,” she offers. “The animal will smell testosterone
on a male operator, and it will automatically enhance their fight-
or-flight response.”
And because these hidden variables influence experimental
outcomes, Noldus adds, animal behavior studies have a reputation
of being very difficult to reproduce.
Highlighting this challenge was a 2018 report from Anne
Andrews and colleagues at UCLA, who looked at questions of
reproducibility and validation in the rodent anxiety test novel-
ty-suppressed feeding (NSF).
NSF monitors hyponeophagia, a state where the normal ten-
dency to avoid new environments competes with the need to find
food. Thus, the longer the latency period to the first bite of food,
the greater the state of anxiety.
Following previously reported NSF test parameters on a
strain of mice genetically engineered to lack SERT expression,
the researchers found they were unable to reproduce the earlier
results. It was only through a systematic modification of different
test parameters that they were able to achieve the same outcomes.
From this, the researchers posited two conclusions.
Firstly, they wrote, “had we assumed the NSF test was ‘work-
ing’ without first validating the test in our hands, the results
of experiments investigating novel phenotypes could have been
wrongly interpreted.”
Secondly, they opined, “it is less critical to reproduce exact con-
ditions reported by others, though these are a logical starting point.
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 DDN Best of Neuroscience
In contrast, it is more important to determine experimental con-
ditions in individual laboratories that produce expected results,
acknowledging that precise conditions can vary across laboratories.”
The researchers went one step further, however, reporting on
challenges of reproducing behavioral studies even within the same
lab following their move from Penn State to UCLA.
Having re-established their SERT-modified mice from cryopre-
served embryos, the researchers repeated a different anxiety-related
behavioral test, the elevated plus maze, only to realize that yet again,
they could not reproduce results from other labs or their own.
As the scientists reported, they were using the same strain of
mice, the same maze, similar lighting conditions and even the
same experimenter.
A PROPER HOME: One of the most important aspect of building
better animal models for neurobehavioral research might be providing
an environment that not only feels more natural to the animal but
also prevents them from being aware of researchers’ presence.
“Even within the same research group, behavior phenotypes
can shift, and differences may go unnoticed without ongoing test
validation,” the authors noted. “Behavior changes can be due to
differences in laboratory or animal care personnel, changes in
environmental/housing conditions, or genetic or epigenetic drift.
“Thus, even laboratory-specific conditions benefit from peri-
odic revalidation.”
Martinez offered her takeaways from this study.
“I found it absolutely fascinating that when they tried to control
for all variables, every single one possible, that this didn’t actually
contribute to success, because it didn’t magically solve the prob-
lem,” she opines. “As with so many things, there’s a balance to it.
One of the thoughts that resonated with me is that researchers
really need to pay close attention to that initial validation step.”
“But then there also needs to be really close attention to report-
ing in a more accurate and detailed fashion the parameters of the
behavior testing that was done in the environment,” she adds.
This is something that is becoming increasingly important at
Taconic, for example.
“We’re understanding that even within the ecosystem of, say,
one company or in Taconic, from one animal room to another,
the environmental conditions may be different, the number of
Drug Discovery News
animals in a cage, the people taking care of the animals,” Martinez
explains. “Even just the food, water and microbiome.”
Fortunately, this expanding recognition is being met with action.
Noldus points to the Innovative Medicines Initiative (IMI) as
one international effort to recognize and address challenges like
these. One project under the IMI umbrella is European Quality
in Preclinical Data, or EQIPD.
An assembly of 29 pharmaceutical companies, universities and
technology developers, EQIPD is investigating the variables that
influence the quality of preclinical data, seeking to improve not
only the quality of this data, but also the processes involved in
generating it.
In January, along with the American Society for Pharmacology
and Experimental Therapeutics, three members of the EQIPD
consortium published new instructions to authors outlining new
methods to display and report experimental data with an eye to
greater transparency, less risk of bias and ultimately, greater sci-
entific rigor (see sidebar article on Page 8 titled “Full disclosure”).
“It is trying to identify the sources of variability and secondly, to
define protocols and standards by which the variation is reduced
by adhering to more rigorous, robust designs of experiments,”
summarizes Noldus.
Without such guidelines, he adds, nobody can ever reproduce
your study and reuse your results.
Martinez also sees opportunities for publishers to step up and
help facilitate both model validation and reproducibility.
“It’s hard to get validation data published and into the public
domain,” she argues.
Journals prefer to publish really tight, hypothesis-driven stories,
she complains, and validation or confirmatory studies simply do
not fit that cookie-cutter approach.
For this reason, she highlights different formats that can cater
to this unique need, offering the video journal JoVE as an example.
“This is perfect for behavior,” she enthuses. “If there are certain
aspects of an animal behavior study that really are operator-de-
pendent or have a very acute environmental component, you can
capture that in a video format.”
Integral to making progress in this area, Martinez presses, is an
awareness of being really accurate and detailed at a more enhanced
level than has been the norm for the past several decades.
And, as suggested earlier, part of enhancing reproducibility will
also come in minimizing some of the sources of variability in the
first place through experimental design and execution.
Hands-off
Platform companies, including Noldus IT, are stepping up to
improve the ways in which behavioral neuroscience is studied.
As Noldus describes the approach: Rather than bring the animal
to the experiment, we are bringing the experiment to the animal.
The goal, he continues, is to move away from artificial arenas
and test apparatus, environments completely unlike how an ani-
mal lives normally or how the downstream patient experiences
a neurological or psychiatric disorder, and move toward more
natural or near-natural settings using automated measurements
and multiple testing modalities.
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 DDN Best of Neuroscience
By examining multiple outcomes or even a single outcome but
from multiple perspectives, there is an opportunity to discover
much more meaningful behavioral data and insights.
The home cage environment—Noldus’ PhenoTyper is a good
example—is enriched with food, drink, shelter, bedding, whatever
the animals need to behave as they might normally. And using
recording techniques such as video, audio or other sensors,
researchers can unobtrusively observe the animals for days or weeks.
Likewise, technologies for stimuli or challenges can be brought
to the home cage, where changes in behavior can be monitored
while the animals interact in a social setting.
“That has been a big shortcoming of previous generations of tech-
nology—the study of social behavior wasn’t really possible,” Noldus
says. “And many of the psychiatric disorders that we are dealing
with in our current society in the West have a social component.”
He offers examples of conditions like schizophrenia, which
leads to social isolation, and autism or anxiety disorders.
“Many of them must be addressed taking the social aspect into
account, because we humans are social beings, and our perfor-
mance is often determined in a group setting at work or in a
family,” he stresses.
A good example of this is efforts at high-throughput automated
olfactory phenotyping reported by Janine Reinert and colleagues
at Heidelberg University late last year. The researchers used group-
housed RFID-tagged mice to monitor investigator-free training
and response of mice to rewarded and unrewarded odors.
The mouse cohort was housed in a variant of a behavioral testing
platform known as the AutonoMouse.
“Such a design can house two cohorts of mice (i.e., genetically
modified mice and their littermate controls) for simultaneous
behavior testing using a single olfactometer, thereby reducing
potential sources of variation,” the authors described. “As animals
are able to freely access the testing area whenever they are moti-
vated to obtain a reward, this setup produces a large number of
trials performed by highly incentivized animals.”
The researchers noted that once a cohort had been trained
on one odor pair, the number of trials to learn a second odor
pair was typically shorter. And although they noted a correlation
between smaller cohorts and higher success rates, the difference
from larger cohorts was small and they could not rule out con-
founding factors.
Interestingly, where cohort size seemed to have the most sig-
nificant impact was in the circadian rhythm of activity. In smaller
cohorts, the mice tended to perform their daily trials during the
dark phase, with peak activity in the fourth hour of the night
phase. In larger cohorts, the activity was distributed throughout
the day with no peak in activity.
Such a difference in activity between day and night phases is
something that has until recently been either largely unappreci-
ated or ignored.
“Mice and rats are naturally nocturnal, so any behavior test that
is run on them in the human day with the lights on, effectively
yanks them from their otherwise peaceful night of sleep and makes
them perform under sleep deprivation,” notes Martinez. “It seems
reasonable to infer that this can be a major factor for behaviors
Drug Discovery News
or disease models that include stress, motivation, etc., as either
dependent mechanisms or confounds.”
Reinert and colleagues also reported high reproducibility both
for animals within a given cohort and across multiple experiments.
“In addition to simultaneously phenotyping large cohorts
of mice or testing a large variety of arbitrary odor mixtures,
the setup could also be a useful tool to prepare mice for more
complex experiments like in-vivo imaging or electrophysiological
recordings,” the authors suggested. “As these experiments
themselves can be very time-consuming, the setup could be used
to, for example, generate a continuous supply of pre-trained
animals without the need for potentially time- and labor-intensive
manual training.”
They also noted that the modular and non-proprietary nature
of the equipment meant it could be further modified for addi-
tional stimuli, such as visual or tactile cues, or for more complex
olfactory tests.
In 2018, Patrick Nolan and colleagues at MRC Hartwell
Institute and Actual Analytics reviewed efforts to assess mouse
behavior through the light/dark cycle using home-cage analysis
(HCA) platforms.
“Robust changes in social interactions over the dark and light
phase can be observed in the mouse home cage using the HCA
system, where cumulative time spent in close proximity (<75 mm)
to other individual cage mates can be recorded over time,” the
authors wrote.
SHINY NEW TOOLS: The automation of behavioral testing using
advanced sensor technologies, digital imaging and video tracking—
along with AI-powered data analysis—is allowing researchers to
provide robust, quantitative phenotyping of rodents, including
genetically modified animals, that is on par with molecular techniques.
But beyond the anticipated night activity and day nesting
observations, they continued, the HCA system allows for social
and behavioral monitoring of animals of mixed genotype, both in
snapshots and longitudinally.
“True home-cage phenotyping over long periods has the poten-
tial to greatly enhance the study of a wide range of neurobiological
diseases by enabling the accurate measurement of progressive
behavioural changes in the same animals over weeks and months,”
the team noted, as exemplified by a particular experimental obser-
vation of three lab animals.
“While the three animals in the cage show similar activity during
the light phase, one of the mutants shows sustained hyperactivity
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 DDN Best of Neuroscience
during the dark phase up until dawn,” the researchers described.
“Without continuous monitoring over the light/dark phase, it would
not have been possible to observe this phenotype, and its potential
impact on the welfare of the animals would have gone unnoticed.”
They also described the automated analysis of unprovoked cage-
bar climbing activity as a subtle measure of motor function.
“We used this automated system to analyse climbing activity in
detail over three consecutive days in a mouse line with progressive
motor deficits with wildtype littermate controls at eight and 13
weeks of age,” the authors wrote. “Preliminary data indicates that
a specific time-dependent decrease in climbing activity, detected
using the automated system, is a strong indicator of disease onset
in this line.”
Although many of these tests offer discrete measurements—e.g.,
swim time, frequency of events—observing a behavior or making
a correlation with disease is still viewed by many as qualitative and
lacking in the rigor of the more quantitative molecular techniques.
Room for all
Noldus acknowledges this has long been a challenge, but he sug-
gests that efforts like those described above are helping to change
that perception.
EXPERIMENTAL AND ANIMAL WELL-BEING: Using the PhenoTyper
automated home-cage, Jean-Luc and Jean-Claude do Rego of the
University of Rouen limited the number of users while reducing the
number of test animals and the stress upon them.
The automation of behavioral testing using advanced sensor
technologies, digital imaging and video tracking, and AI-powered
data analysis is allowing researchers to provide robust, quantita-
tive phenotyping of rodents, including genetically modified ani-
mals, that is on par with molecular techniques.
Martinez goes even further.
“The other thing that I think is really important is taking a page
from the playbook of machine learning and big data, and really
acknowledging that a lot of care and attention needs to be put into
what the input is,” she remarks. “And then, if you have 40 differ-
ent things that you’re observing, which six provide a signature or
provide less sensitivity to noise and environmental influence?”
Drug Discovery News
“It’s really an interesting way to take what is probably the oldest
play in the scientist playbook, which is observing animals in their
environment, and then integrating these really next-gen technol-
ogies to get to the high-level functioning of what would otherwise
be a very simple observation,” she enthuses.
That said, neither Noldus nor Martinez see phenotyping and
genotyping as competitive efforts, but instead look to the synergies
between the two approaches.
Martinez posits that molecular biomarkers may in fact be one
way to ensure translatability from animal model to human patient.
Once you have a behavior or series of behaviors that show a
connection to human disease, there is an opportunity to supple-
ment or enhance that connection by searching for companion
biomarkers that correlate with the behavior(s).
“Whether you do imaging or biochemistry or some other
non-invasive type of means, you can build a case of correlation
and more confident translatability of that behavior modality from
animals to humans,” she notes.
Silverman and Ellegood expanded on this idea in their review.
“In conjunction to behaviorally relevant outcome measures,
the search for biomarkers of [neurodegenerative disorders] has
grown and heavily relied upon visualizing the brain in an effort
to understand the neurodevelopmental differences in preclinical
genetic models and to determine if those neuroanatomical alter-
ations can be reversed or corrected,” they wrote.
They highlighted efforts to examine models both at the cellular
level, using platforms such as histology, two-photon microscopy
and electron microscopy, as well as at the mesoscopic level with
CT, PET and MRI.
Silverman and Ellegood then offered further thoughts on the
applications of MRI.
“The non-invasive nature of MRI also means that it can be
performed repeatedly to track disease progression and loss
of skills and/or symptom onset (or regression by reversals of
brain phenotypes), extremely beneficial to neurodevelopmental
research,” they suggested.
“In collaboration with prominent behavioral scientists, we have
spearheaded an effort to correlate neuroanatomical differences
with behavioral metrics, which allows for powerful inferences
and biochemical hypotheses to be pursued for any given study,”
the authors continued. “In fact, showing direct relationships and
links amongst behavior and any of our numerous MRI readouts
(e.g., regional volume, DTI, cortical thickness) can be used as
biological markers, outcome measures, and may define targets
for genetic or pharmacologic intervention.”
For Noldus—the founder and the company—the value of such
collaborations cannot be underestimated.
“The molecular biologists tell us what the genome looks like
and where exactly the mutations have occurred,” Noldus explains.
“The behavioral scientists and the physiological monitoring
experts provide digital readouts of the outcomes of these muta-
tions at the phenotypic level.”
“We work with the genetics companies that make tools for the
genetic analysis,” he offers. “Our behavioral data sets are merged
with the genetics data sets to find the correlations between, say,
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 DDN Best of Neuroscience
specific changes in the genome and changes in the behavioral
readouts.”
As an example, he offers his company’s efforts with Sylics, which
has developed software tools to integrate these otherwise dispa-
rate data sets.
Sylics’ AHCODA platform, for example, rapidly processes,
quality checks and analyzes data automatically. The company
has also developed a variety of tests to monitor spontaneous
behavior, as well as prespecified behaviors and even disease-
specific behavioral parameters.
At Neuroscience 2019 in Chicago, Sylics CEO Maarten Loos and
collaborators presented their efforts to develop better models of
Alzheimer’s disease by seeding tau protein into the brains of different
mouse strains. The goal was to facilitate testing of antibody-based
therapies for protection against tau spreading and cognitive decline.
Although they were able to see differences in tau pathology
between transgenic htau mice and C57BL/6J wildtype mice, a
battery of behavioral tests found no signs of cognitive impairment
in either model six months after seeding.
Thus, such experiments are critical in determining the limita-
tions of current models of neurological disease.
“Behavioral science is becoming more quantitative, reproducible,
standardized,” says Noldus. “So, I hope that these critical voices will
be silenced in the near future as we produce more robust results.”
Last year, Noldus and colleagues at organizations such as Sylics,
Pfizer, Roche, University of Groningen and others demonstrated
the changing reproducibility landscape in a multi-center study of
a genetic rat model for autism spectrum disorder.
The study, led by Groningen’s Martien Kas, involved replicating
previous observations of autistic-like hyperactive and repetitive
behavior phenotype in a Shank2 knockout rat model of synaptic
dysfunction. Beyond simply reproducing previous results in a sin-
gle lab, however, the collaboration sought to reproduce the results
across three study sites, as well as examine the response to phar-
macological intervention with mGluR1 antagonist JNJ16259685.
As the researchers explained, the study design was adapted from
earlier work but with additional focus on preventing bias in the
design, collection and analysis of data, and with this analysis per-
formed using automated scoring.
They noted “that rigorous alignment of experimental protocols
between three research centers resulted in comparable experimental
findings across sites for both genotype and treatment effects.”
Phenotypic differences between the Shank2 knockout and
wildtype rats were observed across all three sites, including
consistently heightened motor activity and stereotypic circling
behavior in the knockouts.
“Likewise,” the authors reported, “a consistent and dose-depen-
dent attenuation of motor activity and circling behavior in both
[knockout] and [wildtype] rats by JNJ16259685 was found across
the three study sites.”
“These results show that reproducibility in preclinical studies
can be obtained and emphasizes the need for high-quality and
rigorous methodologies in scientific research,” they concluded.
“Considering the observed external validity, the present study also
Drug Discovery News
suggests mGluR1 as potential target for the treatment of autism
spectrum disorders.”
For Noldus, the study highlights the value of diligence to
agreed-upon protocols and standardization of methods, tools
and procedures.
“It is not as simple yet as ordering a DNA sequencer from
PerkinElmer,” he says, “putting it on your benchtop in Los Angeles,
and putting exactly the same device on the benchtop in New York
and getting exactly the same outcome from the same samples.”
“We want it to be like that,” he adds, “but we’re heading in
that direction.”
And that progress is vital, he presses, as the genome alone has
yet to give us the outcomes we sought.
“Eventually, we’re talking about disorders and diseases that
manifest themselves as behavioral problems in reality,” Noldus
concludes. “You’re not suffering from the knock-out in your gene;
you’re suffering from anxiety in daily life. And that’s what even-
tually needs to be cured.”
FULL DISCLOSURE
I
n continuing efforts to address the so-called reproducibility
crisis in experimental biology, Martin Michel of Johannes
Gutenberg University, T.J. Murphy of Emory University and
Harvey Motulsky of GraphPad Software developed revisions to
the Instructions to Authors for the American Society for Pharma-
cology and Experimental Therapeutics (ASPET) journals.
A subset of the ASPET recommendations included:
• Detail how data were analyzed, including normalization,
transforming, subtracting, baselines, etc.
• Identify if all or part of the study tested a hypothesis with
prespecified design or was exploratory
• Explain whether sample size or experiment number were
predetermined or adapted after results were obtained
• Explain whether statistical analysis was predetermined
or adapted after results were obtained
• Explain whether outliers were removed, the criteria for
removal and if this was predetermined
• Use P values sparingly, but instead focus on con-
fidence intervals
• Avoid or clearly define the meaning of “significant”
in reporting
• Present graphs with as much granularity as reasonable,
e.g., scatter plots rather than bar graphs
“We believe that these revised guidelines will lead to a less
biased and more transparent reporting of research findings,” the
authors concluded.
(Adapted from Michel et al. JPET. 2020;372:136-147.)
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FROM Best of Neuroscience
SPECIAL REPORT ON NEUROSCIENCE
HOLDING OUT HOPE
Trying to reframe the depression conversation
BY RANDALL C WILLIS, DDN FEATURES EDITOR
L ook around as you read this article.
Assuming you’re not hidden in a corner somewhere, 7
percent or more of the people you can see have had a major
depressive episode within the past year, according to National
Institute of Mental Health stats. And more than a third of those
people received no treatment for their condition.
An even larger percentage, if not everyone, has had to deal
with a loved one or friend who has experienced depression in
the past, or does now.
And according to the Centers for Disease Control and Prevention,
at least 80 percent of adults experiencing depression will report
some difficulty with their work, home or social lives because of the
depressive symptoms.
“When you have depression, you also have comorbidity of
depression,” says Aptinyx President and CEO Norbert Riedel.
“You have sleep disturbance. You have cognitive dysfunction.”
And even when a patient’s mood symptoms have been stabilized,
a significant proportion of people still struggle with these
comorbidities, finding it difficult to get back to work or take care
of the kids.
Aggravating this situation further, according to Roger McIntyre,
executive director of the Brain and Cognition Discovery Foun-
dation, is that the longer someone has depression or the more
episodes they have, the less reliable treatments become.
“Something is happening at the neurobiological level that’s
changing the biology of the brain, making it less cooperative
with treatments,” he presses. “That is why it is so important for
us to have timely early accurate diagnosis and get people on the
right treatments.”
Unfortunately, clinicians have not had a lot of options from
which to choose.
“When you look back in history, you had tricyclic antidepressants in
the 1950s and then the SSRIs [selective serotonin reuptake inhibitors]
Drug Discovery News
in the 1980s,” explains Stephen Peroutka, vice president of global
product development and neuroscience therapeutic lead for PPD.
McIntyre recounts cases he has seen where a patient is put on
an antidepressant treatment that is only partially effective, but
they are left on that same medication for two, three, four years.
As he terms it, the patient’s depression simply smolders along.
“Can you imagine going on a cancer drug that just has a partial
effect on your tumor?” he asks. “Your oncologist says ‘just stay
on it and in three or four years, we’ll see what happens.’ No one
thinks like that outside of psychiatry.”
Alternatively, Riedel offers, the clinician intervenes when the
patient experiences side effects or poor response, but to question-
able effect.
“They put you on another one and then a third one, but they
are all the same class of molecules,” he complains. “I’m not sure
what we are gaining by experimenting with different molecules
of the same class.”
This unfortunate scenario may soon change, however, as the
understanding of the pathophysiology of depression expands and
new insights are resulting in a growing pipeline of novel class
candidates, some of which recently received blessing from the FDA.
Expanding repertoire
“The landscape really has changed and has evolved into a much
more comprehensive, much more coherent and powerful inflam-
matory model,” says McIntyre.
The notion that there is an alteration in your serotonin levels
has been with us for a long time, he continues, suggesting that
most FDA-approved agents for depression are monoamine-based.
“It turns out, however, that a good 30 to 40, maybe 45 percent
of people with depression taking Prozac-type drugs do not get
better,” he claims. “So therefore, one can only conclude that there
must be some other target that we can look at.”
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McIntyre points to the array of preclinical and clinical work
showing that disturbances in brain neurochemistry extend well
beyond monoamine, impacting other areas such as glutamate
pathways, inflammation and neurosteroids.
“This hangs together because glutamate, among other things,
plays a key role in brain plasticity, known to be abnormal in
depression,” he explains. “Moreover, inflammation plays multi-
ple roles involved in plasticity, regulating other neurochemistries
like dopamine and serotonin, and having its own direct effect on
brain systems relevant to the symptoms that we see in depression.”
As proof, he points to drugs being developed to target glu-
tamate and target inflammation, many of which, he suggests,
look very promising.
EXITING THE TUNNEL VIEW: A wide array of preclinical and clinical
work shows that disturbances in brain neurochemistry extend well
beyond monoamine, impacting other areas such as glutamate
pathways, inflammation and neurosteroids. This wider view is
expanding and refining the definition of, research into and treatment
options for depression.
One such candidate is rapastinel, which was licensed to Aller-
gan as a treatment for depression from what is now Aptinyx. The
compound is a partial agonist of the NMDA receptor, which is
also the target of ketamine and its derivatives.
“That receptor is very well known to be associated with mem-
ory, learning, chronic pain, neurodegeneration,” explains Riedel.
“When that mechanistic approach was applied to MDD [major
depressive disorder] with rapastinel, we saw very striking recovery
in patients in a Phase 2a study, as well as a Phase 2b study. This
became the foundation on which Allergan acquired rapastinel.”
These other impacts are why Aptinyx continues to explore the
NMDA receptor as a therapeutic target for chronic pain, PTSD
and cognitive impairment in Parkinson’s disease.
Riedel suggests that this allows the company to take a more
holistic approach to the various comorbidities that overlap
between the many disorders.
“They are always multifactorial diseases,” he says. “None of
them are just one thing only.”
The early success of rapastinel hit a bit of a bump in March,
however, with an announcement from Allergan, bringing into
Drug Discovery News
question rapastinel’s development as adjunctive to antidepressant
therapy for MDD.
In three acute studies, rapastinel did not differentiate itself from
placebo on primary and key secondary endpoints.
“We are deeply disappointed with these results, and they are a
vivid reminder that drug development is extremely challenging,
especially in mental health,” said Allergan’s chief R&D officer,
David Nicholson, in an announcement. “We will evaluate the
impact of these data on the ongoing monotherapy MDD program
and [Phase 2] suicidality in MDD study. We expect to make a
decision on these programs during the course of 2019.”
March was a good month for Sage Therapeutics and Janssen,
however, as both companies saw FDA approval of their lead products
in depression.
In a Phase 3 program, Janssen’s nasal esketamine formulation
(Spravato) was found to be superior to placebo when given on top
of an oral antidepressant, providing significant improvement in
symptoms of treatment-resistant depression. And in long-term
studies, esketamine patients who achieved remission were 51
percent less likely to suffer a relapse than placebo patients.
Because esketamine is a derivative of illicit street drug ket-
amine, precautions are being taken with dosing such that patients
will not take the drug home, but rather self-administer under
clinical supervision.
Like rapastinel, esketamine targets the NMDA receptor, but
rather than modulate activity, it completely blocks receptor func-
tion. This may help facilitate the rapid efficacy for which the drug
has become known, but as Aptinyx’ Riedel explains, it may also
bring a lot of safety concerns.
“In oncology,” Riedel continues, “when you have [a] molecule
go out of control, like a tyrosine kinase, for example, the goal has
to be to shut that molecule down, to block it completely.”
In the brain, however, the NMDA receptor is a very important
player in normal brain physiology and is involved in a variety of neu-
rological conditions, such as pain, PTSD and cognitive impairment.
“The idea of using a sledgehammer to shut that receptor off, in
my view, is highly unphysiological,” Riedel offers.
“The right approach is to ask has that receptor gone off-track
a little and can you put it back on track by modulating it like a
dimmer switch, as opposed to an on-off switch,” he presses. “That,
we have shown repeatedly and across multiple indications, gives
you a much better efficacy and a remarkably better safety profile.”
Within two weeks of the esketamine announcement, the FDA
then announced approval of brexanolone (Zulresso) from Sage
Therapeutics for the treatment of postpartum depression, the
first and only such treatment.
A derivative of the naturally occurring progesterone metabolite
allopregnanolone, brexanolone is an allosteric modulator of both
synaptic and extrasynaptic GABAA receptors. In its three clinical
trials, the drug achieved significant mean reductions in Hamilton
Rating Scale for Depression (HAMD) total scores from baseline
vs. placebo. Symptom reduction was seen as early as 24 hours,
and could be maintained for up to 30 days.
“The potential to rapidly reduce symptoms in this critical disorder
is an exciting milestone in women’s mental health,” offered clinical
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trial lead Samantha Meltzer Brody, director of perinatal psychiatry
at UNC’s Center for Women’s Mood Disorders. “[Postpartum
depression] is recognized to have significant and long-term impact
on women and their families, but with Zulresso, we may finally have
the opportunity to change that.”
As McIntyre suggested above, however, there are numerous
other areas being explored to understand depression pathophys-
iology and treatment, including inflammatory pathways.
Early this year, for example, Lisa Kalynchuk and colleagues at
University of Victoria and University of Saskatchewan reviewed
the evidence for antidepressant mechanisms of TNF-α antagonists.
“Treatment with proinflammatory cytokines, including IL-1, IL-6
or TNF-α, or lipopolysaccharide (LPS), induces sickness behavior
and corresponding depression-like behavior on the forced swim
test,” they wrote. “Mice that lack certain cytokines or cytokine
receptors do not display stress-induced depression-like behavior,
which suggests that lower levels of cytokines confer a protective
effect on the development of depression-like behavior.”
The authors then highlighted evidence from several studies of
patients being treated for autoimmune disorders with different
TNF-α inhibitors.
“Patients with rheumatoid arthritis and plaque psoriasis taking
prescribed etanercept, which is a TNF-α antagonist, reported
significant reductions in depressive symptoms,” they noted.
“Similarly, patients with Crohn’s disease receiving infusions
of infliximab experienced significant reductions in depressive
symptoms, and this decrease was associated with corresponding
reductions in proinflammatory cytokines. Finally, psoriasis
patients with comorbid psychiatric conditions report improvement
in mood and overall well-being when taking infliximab.”
Because etanercept does not cross the blood-brain barrier, the
authors could not identify the mechanisms by which the anti-TNFs
addressed depression, although they speculated that in binding
peripheral TNF-α, it may promote activation of microglia, which
results in decreased central secretion of TNF-α.
Jumping off findings like these, McIntyre initiated his own
studies directly linking anti-TNFs and depression.
“I have a study that’s in-press in JAMA, where we gave patients
infliximab as a treatment of their depression,” he recounts. “We
found that people who’ve had depression, who told us that they
had trauma in their history, had a significantly greater improve-
ment on the infliximab than they did with placebo.”
Perhaps not surprisingly, even the microbiome is being explored
as a possible entry point to understanding depression and response
to treatment.
In January, for example, Tonji Medical Hospital’s Chun Yang
and colleagues examined the role of gut microbiota on the anti-
depressant effects of ketamine in an LPS-induced mouse model of
depression. They noted that 30 bacterial species were significantly
altered in prevalence between mice treated with LPS alone versus
those treated with LPS plus ketamine.
In particular, the presence of two bacteria—the phylum
Actinobacteria and the class Coriobacteriia—correlated with immobility
time in a forced swim test, a test of stress meant to parallel depression
Drug Discovery News
in mice. To the authors, this suggested these microbes had potential
as biomarkers of ketamine effectiveness.
Working in humans rather than mice, McIntyre looked for
bacterial signatures or enterotypes in the feces of people with
depression that differed from those found in healthy controls.
And indeed, he found significant differences, particularly in the
microbial diversity.
Thus, he says, “we wonder whether for some people, the altered
inflammatory burden that we observe in depression—which, by
the way, is observed in 35 to 50 percent of patients—may have
some type of contribution from the gut biome.”
Despite these extended avenues of exploration, however, many
challenges remain in the understanding of depressive pathophysi-
ology and the ability to test new therapeutic candidates in a mean-
ingful way. Part of that challenge is a lack of quantifiable markers
of disease status and the largely subjective endpoints commonly
used to diagnose patients and monitor response to treatment.
Begging for biomarkers
According to PPD’s Peroutka, subjective endpoints are problem-
atic, pointing to pain as the simplest example.
“We ask patients, zero to 10, what’s your pain like?” he explains.
“And what is a five to me versus you versus another person—we
don’t know that they’re the same; there’s no way to check that.”
With depression, that uncertainty is amplified dramatically. 
“Primary endpoint for HAMD is depressed mood: zero to four
scale, how are we, no depression to severe depression,” he says,
adding that is just the first of 17 questions.
BRAIN BIOMARKERS: Efforts have been made to identify biomarkers
for neurological conditions like depression, but these efforts have met
with limited success because researchers have managed to make only
modest correlations between a particular biomarker and the disease.
Feelings of guilt, zero to four, he recounts. Suicidal thoughts,
zero to four. Insomnia, early at night…the questions and scales
keep coming.
He quickly notes that he is not arguing the tests are spurious—
HAMD and its ilk have been validated.
“But along all those different scales, you can see the challenge
is that it’s just too subjective,” he says.
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And even if you could convince yourself that one patient’s score
in one category is every other patient’s score in the same category,
there is the broader question of the continuum of scores.
“Is a 15 depression score different than a 25?” Peroutka asks. “The
answer is yes. But in clinical trials, we lump all of them together.”
To get more meaningful data in a clinical trial, he suggests, it
might be preferential to narrow the range of subjects as a statistical
way of homogenizing the patient population.
A molecular biomarker or panel might clarify some of this
uncertainty, but finding one has proven elusive.
“I don’t think we have a really good handle on the underlying
molecular pathology of defined depressive disorder,” explains Rie-
del. “It is a very complex disease and varies as to which pathology
matters to which patient or another—similar, I think, to the exam-
ple in oncology where not every tumor is the same.”
Efforts have been made to identify biomarkers, he continues,
and these efforts have met with limited success, in that researchers
have managed to make only modest correlations between a
particular biomarker and the disease. He questions, however,
whether those correlations have been consistent enough to offer
any predictive value.
“There is a prevailing view that if we ever have such a thing,”
McIntyre adds, “it’s going to be exceptionally complicated because
of the complexities of the biology.”
“Part of the challenge we have is that because we don’t have
a blood test or chest X-ray or CT scan to make the diagnosis, we
rely on symptom ratings,” McIntyre presses. “We rely on clinician
impressions, and they are far from perfect.
“They’re not useless—that’s what we base our whole careers
on—but they’re not perfect. And that introduces, as you might
expect, variability.”
That variance makes it very difficult to achieve any consensus,
he adds, because clinicians and researchers can’t even agree on
what the phenotype is.
“In cancer, they agree that there’s the tumor,” he offers as an
example. “In heart disease, they agree you’ve had a heart attack.
This is something we agree on.
“But the challenge is that we just can’t agree on what the lesion
looks like in depression, which makes the search for a biomarker
to make the diagnosis not believable at this point in time.”
Peroutka sees a strong parallel between today’s analysis of
depression and that of multiple sclerosis 25 years ago.
“MS was defined as two separate neurological lesions or deficits
in space and time,” he explains. “So, if I was to ask you has your
left or right foot fallen asleep in the last year, and you’d probably
say yes. I’d then ask did your opposite hand or fingers ever fall
asleep in the past year, and you’d probably say yes. Technically,
you could be diagnosed 25 years ago with MS.”
Now, with MRI-based brain scans, he says, it really doesn’t matter
how the patient answers questions or even why the patient has
come in for the scan.
“If you’re scanned for a neck injury, and you have plaques in
your head, then you have MS,” he adds.  subjects, although they noted no significant differences between
The desire for and potential of recognized biomarkers has led to
several labs approaching the problem from a multitude of directions.
Drug Discovery News
Earlier this year, for example, Peng Xie and colleagues at
Chongqing Medical University described their efforts to identify
metabolite signatures in the urine of patients experiencing
depressive episodes in bipolar disorder (BD).
“A clinical review recommended that the antidepressants
should be stopped in period of mania, and the antidepressants
should be used with a mood stabilizer in period of depression,”
the authors explained. “Given these facts, there is an urgent need
to develop objective diagnostic methods for BD patients during
different episodes.”
In considering their approach, the researchers suggested that
no single methodology would give the wide coverage or mean-
ingful results they would need, so they opted for a dual-platform
approach using GC-MS and NMR spectrometry to profile urine
from young and middle-aged subjects.
Comparing samples from healthy controls and affected patients,
the scientists identified 13 differentially regulated metabolites,
most of which were involved in carbohydrate and energy metabo-
lism. Of these, they defined a biomarker panel of five metabolites
that they suggested could be helpful in developing an objective
diagnostic method.
“A previous study reported that the brain energy metabolism
in BD patients was altered,” they stated, finding their results con-
sistent with others. “Our previous studies have also identified
some differential urinary metabolites that were involved in energy
metabolism in depressed patients. Meanwhile, we also observed
the perturbed energy metabolism in the cerebellum of chronic
mild stress-treated depressed rats.
“Based on these results, we deduced that the pathogenesis of BD
might be associated with the disturbance of energy homeostasis
that was caused by the dysfunctional [hypothalamic-pituitary-
adrenal] axis.” (For more about metabolism and mental regulation
and dysregulation, see the sidebar article on Page 14 titled
“Metabolism and mood.”)
In a similar effort also reported this year, Daihui Peng and col-
leagues at Shanghai Jiao Tong University & Deakin University used
UPLC-3Q-MS to examine blood samples from healthy controls and
subjects with MDD or bipolar disorder. Specifically, they wanted
to identify any correlations between metabolites in the kynurenine
pathway (KP) and depression presence and severity, as determined
with the HAMD-24 survey.
Part of the attraction for KP in particular is a growing under-
standing of its possible role in a number of neurological disorders,
including depression.
“Myint and Kim’s widely accepted ‘KP metabolic imbalance
hypothesis’ proposes that metabolic imbalance is the major mech-
anism of KP-induced depression,” the authors noted. “In line
with this, variations in metabolic factors in the KP and relevant
enzymes have been found in MDD, bipolar affective disorder and
several neurodegenerative diseases.”
The researchers identified several metabolites that were dif-
ferentially expressed between healthy controls and depressed
subjects with MDD or bipolar disorder.
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As well, there was an inverse correlation between the severity
of depression and metabolite levels; for example, lower levels
of tryptophan and kynurerine were associated with more severe
symptoms. And although not perfect, there was a strong indication
that many of these levels could be used for clinical diagnosis.
“The associations between HAMD-24 scores and metabolic
factors in MDD suggest variations in [tryptophan] exert a critical
role in disease remission,” the authors concluded. “Most impor-
tantly, [kynurenic acid] is a potential biomarker discriminating
MDD and healthy controls, offering a novel diagnostic method
for clinicians.”
THE PROBLEM OF ANIMALS: Unlike molecular biomarkers, which may
at times be found as readily in model organisms as they are in humans
(or have analogues), subjective diagnostic methods are difficult with
animal models, as you will not find a rat, mouse or non-human primate
that can directly answer questions about their mood or severity of
mood dysfunction.
Glen Baker and colleagues at the University of Alberta, mean-
while, recently reviewed efforts to understand the roles of D-ser-
ine in both schizophrenia and depression. Beyond the compound’s
potential as an NMDA receptor co-agonist, the researchers sug-
gested it has also shown potential as a predictive biomarker for
antidepressant response to ketamine.
“Several interesting pharmacodynamic and pharmacokinetic
interactions between D-serine and ketamine have been report-
ed, and, interestingly, evidence suggests that low plasma levels
of D-serine may predict positive antidepressant response to ket-
amine,” the authors summarized. “Several animal model and
clinical studies also indicate that D-serine may be effective in
reducing cognitive deficits, but that further study is necessary
before considering it an effective cognitive enhancer for routine
use in humans.”
Whether looking at metabolite panels, cytokine levels or protein
markers, however, there is still a significant gap between exper-
imental correlations and validated, broadly accepted biomarker.
In fact, Riedel is not completely sure that the FDA is ready to imme-
diately accept an objective biomarker that correlates with disease.
“Even if we had [a good biomarker] and we could show that it
can be changed with a drug, the FDA would say that’s not good
Drug Discovery News
enough, I still need to have patient-reported or physician-reported
subjective methods of verification,” he suggests. “The biomarker
may be supportive, but it will not become the endpoint upon
which you decide the drug gets regulatory approval. We are a long
distance from that.”
And until biomarkers are accepted as objective endpoints,
studies of depression will continue to struggle with another major
obstacle: the placebo effect.
Because diagnostic panels like HAMD rely on patient-report-
ed feedback—Do I feel better? Do I feel less depressed?—many
clinical studies can suffer from a very strong placebo response,
says Riedel.
In part, according to Peroutka, it is the nature of the disorder.
It’s the natural history of the disorder that people get better on
their own, he says. Depression is episodic, by definition in the case
of bipolar disorder, and so even without therapeutic intervention,
a percentage of patients in a clinical trial may feel better.
This sets an artificially high bar over which any study treatment
must show an effect.
Riedel also offers the example of functional unblinding of the
study, where patients and clinicians know whether the patient
is receiving the study drug vs. placebo because of the associated
side effects.
Thus, a positive response may have little to do with drug efficacy
but rather the awareness of treatment and the belief that it will
have a positive impact.
“We probably leave a lot of good compounds behind because
they really can’t overcome the placebo effect even if they are per-
fectly active drugs,” Riedel says.
“There may have to be different thinking about how we design
and execute trials,” he continues. “Maybe we measure the activity of
a drug against the baseline that is known in each of these patients.”
Long before any candidate reaches clinical trials, though, the
search for effective and safe antidepressants is also challenged by
the models on which these drug leads are initially tested.
Middling models
Another issue of using subjective, query-based diagnostics criteria
such as the HAMD is that unlike molecular biomarkers, which may
also be found in model organisms or have analogues, it is difficult
to find a rat, mouse or non-human primate that can directly answer
questions about how depressed they are feeling.
“In mood disorders like depression, it is hard to measure
depression in a rodent,” acknowledges Riedel. “There are ways in
which we look at this, but I would say that it is of limited value in
predicting a human disease course in a depressive disorder.”
“There is no question that the animal models we have are not
just imperfect, but are serving as a major limitation to progress
in the field,” McIntyre adds.
In place of the HAMD, researchers are forced to use stress- and
anxiety-inducing tests on lab animals, such as the forced swim test
mentioned earlier, or the tail suspension test. Another commonly
used test is a measure of anhedonia—losing the ability to derive
pleasure from something that usually produces pleasure. In the case
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of rodents, this is often measured in the preference for sweetened
over unsweetened water.
But how well does the rodent response reflect the human condition?
“I have done that forced swim test [on rodents],” offers Riedel.
“Why would you stop swimming? Well, maybe the rat doesn’t know
how to swim. Maybe it’s weak. Maybe it did well the day before.”
“But to say that it is because they’re depressed is a stretch,” he adds.
In a recent review, Karolinska Institutet’s Johan Söderlund and
Maria Lindskog chose to see these tests in a different light.
“When we compare animal models with clinical conditions,
we need to use less well-established clinical descriptions,” they
opined. “These conditions are often associated with an increased
risk of developing major depression, but are not major depression
per se.”
“It becomes clear that the animal models are not diagnostic
models, but rather models of risk and vulnerability factors of
depression,” they continued. “Importantly, this is not a drawback
of the animal models, but again illustrates that the current diag-
nostic system is neither compatible with neuroscience nor is it
sufficient to describe the underlying mechanisms of depression.”
And just as the array of approaches to depression pathophysi-
ology has expanded in human profiling, the pair noted that this
is also the case in animal model development, suggesting that
we are moving well beyond simply behavioral assays and into
more molecular modalities such as gene knockouts, optogenetic
stimulation and neuroinflammatory models.
McIntyre also sees hope in efforts to model depressive disorders
using stem cell approaches.
“If you can take a stem cell from someone who has been affected
by depression and induce it into a neuron, what you’ve effectively
done is you’ve put into your dish a neuron which presumably is
identical to the neuron in the brain,” he says, acknowledging that
this is yet a work in progress. “But you can imagine that this is a
better methodology than the animal models.”
Indeed, companies like StemonX and Stem Cell Technologies,
as well as organizations like the Wyss Institute, have put a lot of
effort into the development of brain cell organoids and spheroids,
as well as brains-on-a-chip and blood-brain barrier mimics to offer
researchers everything from precision medicine approaches to
specific patients or simply deeper molecular understandings of
cell communication and drug pharmacology.
But if we thought it was difficult to ask a mouse how it felt, we
can only imagine the effort to do so with a microwell of cells, no
matter how well-organized.
For Söderlund and Lindskog, animal models and human
experience can only improve by taking a feedback approach.
“Clinical experience is necessary to develop fruitful animal
models, but the animal models are also needed to delineate the
complex patterns of different exposures and vulnerability factors
characterizing the clinical situation, indicating the potential
mutual benefit between research and clinic,” they concluded.
As the repertoire of possible treatments slowly expands, perhaps
a similar feedback loop needs to tighten between patients and
clinicians, clinicians and researchers, and patients and communities.
Drug Discovery News
METABOLISM AND MOOD
A
s recent efforts to study the gut-brain axis with
microbiomics or correlations between metabo-
lic syndrome and the onset of neurodegenerative
diseases like Alzheimer’s would indicate, there is a slowly
growing awareness of links between metabolism and mood
disorders like depression.
As the University of Toronto and University Health Network’s
Roger McIntyre explains, although insulin is prevalent in the
brain, it is not being utilized for glucose homeostasis as it is
elsewhere in the body.
“The evidence indicates that insulin is a brain peptide that
serves a tropic role,” explains McIntyre, who is also executive
director of the Brain and Cognition Discovery Foundation. “It’s
responsible for neurodifferentiation, neuroplasticity and also
reducing apoptosis.”
And just as too much or too little insulin is problematic in
the periphery, so too does the imbalance create problems
in the brain.
According to McIntyre, upward of 30 to 40 percent of
people with depression—including those without diabe-
tes—exhibit impaired insulin-glucose homeostasis.
Thus, he continues, “because people with diabetes and
depression have hyperinsulinemia for a long time, the
amount of insulin getting into your brain over time begins to
diminish. The brain adapts, it decreases the insulin uptake.”
“And the problem with diminishing insulin uptake in
response to peripheral hyperinsulinemia is that now you’re
losing those key roles that insulin performs in your brain:
neurodifferentiation, neuroplasticity, as well as inhibiting
apoptosis,” he says, bringing the discussion full circle.
It’s a double hit, he remarks.
“In the short-term, you get this amyloid deposition. And
in the long-term, you lose the tropic support.”
Given this paradigm, McIntyre and colleagues have exa-
mined the impact of the GLP-1 receptor agonist liraglutide
on non-diabetic subjects experiencing depression. In a pilot
study, they found that a four-week regimen was not only
safe and well tolerated, but also subjects showed signifi-
cant improvements in cognitive function.
Similar effects have been seen with other diabetes the-
rapies, including metformin, offering another avenue of
antidepressant exploration.
Return to Page 12
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Best of Neuroscience
INTRIGUING APPROACHES TO
PARKINSON’S DISEASE
Gene therapy, stem cells, the microbiome and more open up a world of novel
approaches to PD
BY JEFFREY BOULEY, DDN CHIEF EDITOR
W ith so many relatively new avenues of potential ther-
apeutics opening up as technologies have improved
and novel tech has entered the scene, it’s no longer just
small molecule vs. large molecule or similarly limited options for
the myriad diseases that vex humanity.
Antibody therapies, antibody-drug conjugates and gene editing
are just a handful of the many choices that recent years have
brought to life-sciences research. In that spirit, we will explore a
few novel arenas that use gene therapy, kinase and protein inhi-
bition, stem cells and microbiome approaches to potentially treat
Parkinson’s disease (PD).
A ‘SUNRISE’ to bring light for PD sufferers
Axovant Gene Therapies Ltd., a clinical-stage company developing
innovative gene therapies, in June 2019 reported six-month
follow-up data from the first dose cohort in the open-label, dose-
escalation portion of the ongoing SUNRISE-PD Phase 2 trial of
AXO-Lenti-PD for the treatment of Parkinson’s disease.
“We continue to be encouraged by the consistency of the data
and improvements in quality of life seen at six months in the two
low-dose cohort patients, as we enroll additional patients in the
second cohort of the SUNRISE-PD study,” said Dr. Gavin Corcoran,
Axovant’s chief research and development officer. “Our patient-
focused goal of improving motor function, reducing dyskinesia,
lowering the requirement for oral levodopa, and improving quality
of life is made possible by the continuous dopamine replacement
strategy of AXO-Lenti-PD gene therapy. These data at six months
highlight the potential for a clinically meaningful improvement
Drug Discovery News
over the currently available standard of care for those patients with
moderate to advanced Parkinson’s disease.”
AXO-Lenti-PD was observed to be generally well tolerated, with
no serious adverse events related to the product or the procedure,
and patients showed continued improvement from baseline across
multiple measurements.
The Parkinson’s Disease Questionnaire-39 (PDQ-39) Summary
Index score, a patient and caregiver reported quality of life
measure, was recorded at baseline, month three and month six.
The average score at baseline was 50 points, which at month three
had improved to an average score of 31 points (a reduction of 19
points from baseline). At month six, the average PDQ-39 Summary
Index score was further improved to 18 points (a reduction of 32
points from baseline).
These scores demonstrate an approximate 37-percent improve-
ment from baseline at month three and an approximate 65-percent
improvement from baseline at month six.
According to Axovant, “These reductions in total score at both
three and six months appear to indicate a substantial clinical
benefit in this quality of life measure.”
“Our focus in this first cohort of the SUNRISE-PD study was
on the safety and tolerability of AXO-Lenti-PD, as well as the
evaluation of efficacy using well-validated, objective measures.
These early data support the safety of the lowest dose of AXO-
Lenti-PD, similar to what was observed with the earlier gener-
ation construct, ProSavin, and also suggest substantially greater
biological activity than the highest dose of ProSavin previously
tested,” said Dr. Gavin Corcoran, Axovant’s executive vice presi-
dent of research and development, when the company reported
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 DDN Best of Neuroscience
three-month follow-up data for SUNRISE-PD in March. “These
findings are highly encouraging, and we look forward to advanc-
ing to higher dose cohorts where we will explore the full clinical
potential of AXO-Lenti-PD in patients with Parkinson’s disease.”
Also with regard to the March update, Dr. Roger Barker, one
of the principal investigators on SUNRISE-PD, noted: “This early
data suggests that AXO-Lenti-PD has the potential to significantly
improve motor function in patients with advancing Parkinson’s
disease. The mechanism of action of AXO-Lenti-PD, which is
designed to deliver all three genes necessary for endogenous
dopamine biosynthesis, as well as our prior clinical experience
with ProSavin, led us to expect that the major benefit would be in
improving the ‘off’ state—and the results so far are very encouraging
in this regard.”
“Off” times are motor fluctuations when medication—usually
levodopa, which is the current gold-standard treatment for PD—is
not working optimally and Parkinson’s symptoms (both motor
and/or non-motor) return. Off periods are more common as the
disease progresses and people take medication for a longer period
of time.
French companies team up on new line
of research
Servier and Oncodesign announced in spring 2019 a strategic
partnership for the research and development of potential drug
candidates for Parkinson’s disease, particularly around LRRK2
kinase inhibitors. These inhibitors are derived from Oncodesign’s
proprietary Nanocyclix platform and are hoped to have potential
to act as therapeutic agents against PD.
The partnership draws on the complementary expertise
of Servier and Oncodesign in the field of neurodegenerative
diseases and macrocyclic kinase inhibitors, the companies said.
Oncodesign was to be responsible for the research program up
to the selection of preclinical candidates, notably at its research
site in Les Ulis, France.
“This partnership is a result of the choices and investments that
we have made over the past 18 months to put together a portfolio
of promising drug candidates derived from our Nanocyclix plat-
form. The pharmaceutical industry is currently showing a keen
interest in new treatments for Parkinson’s disease, particularly
around LRRK2 kinase, which is considered as a high-potential
target for treating this disease,” said Dr. Philippe Genne, CEO and
founder of Oncodesign. “Servier’s expertise will be a key asset in
the early stage of this research agreement with regard to success-
fully carrying out the program which, in the medium term, could
lead to the development of new drug candidates. Moreover, this
partnership means we can channel our own financial resources
into developing our three other proprietary programs: RIPK2,
ALK1 and MNK1.”
Added Dr. Jan Hoflack, scientific director and director of oper-
ations at Oncodesign: “The only therapies currently available for
Parkinson’s patients aim to alleviate the symptoms of the disease.
LRRK2 inhibitors have the potential to act directly on the pro-
gression of the disease, which would result in improved living
standards for patients. This agreement with Servier, a company
Drug Discovery News
that is invested in researching neurological conditions and has
partnered with Oncodesign in the past, represents an important
step towards meeting our goal of offering a real benefit to society
through precision medicine.”
Heading PD off at the pass
Also in the theme of inhibition, University of Dundee researchers
at the U.K. university’s Drug Discovery Unit (DDU) partnered
with Bukwang Pharmaceutical Co. to tackle a new drug treatment
for Parkinson’s disease. In this case, though, the “inhibition” they
are looking for is to keep a certain bad actor from accumulating
in the brain.
A key biological event in the development of PD is the accu-
mulation and misfolding of a small protein in the brain called
α-synuclein, which can kill nerve cells, and research at the Uni-
versity of Oxford has shown that an enzyme, USP8, prevents the
natural breakdown of α-synuclein.
Working in collaboration with Dr. George Tofaris at Oxford, the
DDU identified a series of drug-like molecules that block USP8
and could reduce the levels of α-synuclein in the brain, potentially
providing a true treatment for Parkinson’s disease.
“Finding treatments that target the alpha-synuclein protein
holds promise for one day slowing or stopping the progression of
Parkinson’s—something no current treatment can do,” noted Dr.
Beckie Port, research manager at Parkinson’s UK. “It’s an exciting
time for Parkinson’s research. Our increased understanding of the
biology of the condition means we’re now at a stage to turn our
wealth of knowledge into much-needed treatments for people
with Parkinson’s.”
The partnership with Bukwang Pharm strengthened an exist-
ing Dundee-Oxford relationship, which had been supported by
the Medical Research Council. Bukwang Pharm was to facilitate
a further three-year program of work at Dundee and Oxford to
advance these drug-like molecules toward clinical development.
The aim is to formulate much-needed therapies for PD and other
diseases where α-synuclein pathology is implicated. Bukwang
Pharm holds an exclusive option to acquire worldwide develop-
ment and commercialization rights of resulting novel molecules.
“We are delighted to be announcing this partnership with
Bukwang Pharm. Drug discovery for neurological disorders is
especially challenging and an area where academia and industry
need to be working together,” said Prof. Paul Wyatt, head of the
DDU. “This project brings together the clinical and translational
research expertise in Oxford with Dundee’s professional drug
discovery capabilities allowing us to move one stage further
towards a treatment.”
Stem cells vs. Parkinson’s
A paper that appeared in the Journal of Parkinson’s Disease titled
“Repairing the Brain: Cell Replacement Using Stem Cell-Based
Technologies” suggests that cell replacement therapies in which
dopamine-producing stem cells are transplanted into PD patients
could improve motor symptoms, reducing or eliminating the need
for dopaminergic medicines
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 DDN Best of Neuroscience
Current treatments for PD tend to focus on increasing dopamine
levels in the brain in order to relieve motor symptoms, but the
effectiveness of these treatments declines the longer they are used,
and they come with various side effects.
“We are in desperate need of a better way of helping people
with [Parkinson’s disease]. It is on the increase worldwide.
There is still no cure, and medications only go part way to fully
treat incoordination and movement problems,” said Drs. Claire
Henchcliffe and Malin Parmar, co-authors of the paper, in a news
release about their study. Their thought is that a better long-term
treatment might be to transplant dopamine-producing stem cells
into patients’ brains.
STEMMING PARKINSON’S: Drs. Claire Henchcliffe and Malin Parmar,
co-authors of a paper on the use of stem cells to treat Parkinson’s
disease, believe (despite some conflicting results in previous research
in the field) that stem cells—particularly induced pluripotent stem cells
rather than embryonic ones—could hold potential for becoming
routine Parkinson’s therapeutics.
“If successful ... a single surgery could potentially provide a
transplant that would last throughout a patient’s lifespan, reducing
or altogether avoiding the need for dopamine-based medications,”
Henchcliffe and Parmar noted.
It will likely be a long road to becoming a routine and successful
procedure, though. Data from published studies about such stem
cell transplantation using fetal cells have been promising, the
authors explain, but concerns have been raised regarding the
heterogeneity of clinical outcomes reported. Some of the factors
that might cause this lack of heterogeneity include inconsistency in
cell preparation, cell number and cell composition, issues related
to patient selection, the method used for immunosuppression and
duration of said immunosuppression, as well as varying outcome
measures and study durations.
Also, ethical concerns exist over using cells from aborted fetus-
es, and so methods such as shifting to induced pluripotent stem
cells are likely to be necessary.
Going for the gut in PD treatment
Axial Biotherapeutics, a biotechnology company dedicated to
building a unique class of gut-targeted programs for neurode-
generative and neuropsychiatric diseases, announced in 2019 the
completion of a $25-million series B equity financing. The new
funding was used to advance Axial’s programs in PD as well as
Drug Discovery News
autism spectrum disorder (ASD)—both programs include small
molecules targeting the gut-brain axis.
Axial’s PD program, which evaluates organisms and genes
over-represented in the PD gut-microbiome, has resulted in the
identification of a pathway that is sufficient to induce motor and
GI symptoms and brain pathology in a validated, preclinical PD
model, according to the company.
The program includes both AB-4166 and the AB-4000 series.
AB-4166 is a first-in-class intervention that is currently being
evaluated for safety and tolerability in a subpopulation of PD
subjects. Axial’s PD program has also identified multiple novel
chemical entities in the AB-4000 series which are being developed
to have improved selectivity as potential next-generation drug
candidates for PD and other neurodegenerative diseases.
Axial’s program in ASD is focused on research which
demonstrates that reducing systemic and brain exposure to
problematic microbial metabolites may improve core and non-core
ASD symptoms related to behavior and gut health. The company’s
lead product, AB-2004, is a drug candidate that has demonstrated,
in animal models, the ability to repair leaky gut and improve
repetitive behavior, anxiety and ASD-related sensorimotor gating
deficits by removing key microbial metabolites.
Next-gen Parkinson’s therapy
And finally, as a little bonus, since it falls outside the usual chem-
ical, biological and genetic modalities we cover, we have news
that researchers at the University of Houston recently found neu-
ro-biomarkers for Parkinson’s disease that they believe can help
create the next generation of “smart” deep brain stimulators, able
to respond to specific needs of PD patients.
People with PD sometimes undergo high-frequency brain
stimulation, an established therapy for the progressive nervous
system disorder that affects movement. However, such therapy
has been imprecise.
Currently, stimulators can only be programmed clinically and
are not adaptable to the fluctuating symptoms of the disease. They
newly identified biomarkers are key to improving the technology
to make it responsive—that is, to make it a “smart” system.
“We can now make the closed-loop stimulator adaptive to sense
a patient’s symptoms, so it can make the adjustments to the fluctu-
ations in real time, and the patient no longer has to wait for weeks
or months until the doctor can adjust the device,” said Nuri Ince,
associate professor of biomedical engineering. He and doctoral
student Musa Ozturk, lead author of the paper, published their
findings in the journal Movement Disorders.
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FROM Best of Neuroscience
SPECIAL REPORT ON NEUROSCIENCE
BEYOND ‘REEFER MADNESS’
BY RANDALL C WILLIS
O n some indeterminate date this summer 2018, marijuana
will become legal across Canada, as it already is in a
number of U.S. states. The move is an extension of earlier
acceptance of medicinal marijuana for Canadians suffering a
variety of ailments.
For some observers, this is proof of the slippery slope. For others,
it is a step forward in healthcare and social equalization.
There is yet a third group who see echoes of today’s marijuana
industry in the pre-FDA days of tinctures, potions and snake-oil
remedies. Rather than shut the industry down, however, they are
interested in learning more about the therapeutic potential of the
maligned weed. Their goal is to turn a recreational drug into a
pharmaceutical therapy.
Thus, if there is an anthem for this group, it might be 1967’s
Time Has Come Today:
Time has come today /
Young hearts can go their way /
Can’t put it off another day /
I don’t care what others say /
They say we don’t listen anyway /
Time has come today.
Reefer hangover
Despite growing efforts to legalize Cannabis in several jurisdictions
and a growing medical literature of legitimate clinical trials that
move well past case reports and anecdotal surveys, interest in the
medical application of marijuana—and, more precisely, its two
key compounds cannabidiol (CBD) and Δ9-tetrahydrocannabinol
(THC)—remains controversial.
“I didn’t know anything about cannabinoids a year-and-a-half
ago. They are an interesting mixture of folklore, folk medicine
and really cool bold science,” enthuses Michael Mendez, founder
and CEO of Renew Biopharma. “I was intrigued by the whole
self-pharmacy where people were treating themselves for diseases
using these molecules and, in some ways, having a lot of success.”
Drug Discovery News
He continues to explain that cannabinoids—the 80 or so naturally
occurring compounds from plants (phytocannabinoids), as well as
compounds found within human tissues (endocannabinoids)—
are interesting because they naturally cross the blood-brain barrier
(BBB) and bind receptors, which he says sounds like a trivial feature.
“But you have to understand that medicinal chemists have been
trying to make molecules like this for 30, 40 years,” he explains.
“And for the most part, they have not been very successful to get
molecules across.”
But another feature that drew Mendez’s attention to cannabinoids
was the turmoil and politics of the subject.
“What I mean by that is it is not just the federal laws and the legal-
ity, but the sense of morality around these molecules,” he continues.
“When I first talked about Renew going after cannabinoids,
my colleagues and all the other people that I’ve worked with in
biotech were somewhat taken aback,” Mendez recalls. “Come on,
Mike, you’re not seriously going to go after cannabinoids, are you?”
He finds a degree of amusement in what he describes as soci-
ety’s preconditioning with the Reefer Madness style of thinking,
referencing the American propaganda film released in 1936 to
warn of the dire perils of engaging in marijuana use.
According to Armando Anido, CEO of Zynerba Pharmaceuti-
cals, the social paranoia and need to control the weed has meant
that research into possible medicinal opportunities was signifi-
cantly hampered.
“There has not been a whole lot of basic science that has been
done with CBD throughout the years, because at least in the Unit-
ed States, it is very difficult to get hold of any CBD or THC,” he
explains. “The University of Mississippi has the exclusive farm
that grows it, that is licensed by the Drug Enforcement Agency
(DEA) to supply it to investigators.”
“Investigators will tell you that it is near-impossible to get it,”
he continues. “One, to get the license from the DEA, but two, to
get adequate supplies to do the studies that are necessary.”
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 DDN Best of Neuroscience
Thus, he presses, any knowledge of medicinal efficacy of CBD
and THC has come through anecdotal reporting, a catalog that
is expanding now as CBD and THC have become more widely
available through medical marijuana programs.
But even as social and legal restrictions have started to loos-
en, the challenges of dealing with a natural product—whether
smoking the plant or ingesting extracts—are making it difficult to
study possible therapeutic interventions in a truly clinical manner.
Anido hears about the challenges from clinicians with child
patients coming into California with severe refractory epilepsy.
CANNABINOID INROADS: Cannabinoids—the 80 or so naturally
occurring compounds from plants (phytocannabinoids), as well as
compounds found within human tissues (endocannabinoids)—are
interesting to researchers in part because they naturally cross the
blood-brain barrier and bind to receptors.
“They go one week to the dispensary and get their product
for the next month,” he recounts. “It tends to work very well for
them. And then they go the following month and think they’re
getting the exact same thing. Then all of the sudden, their child
experienced more seizures or more safety concerns.”
Recognizing the significant variability both in yields and molec-
ular profiles of farmed Cannabis plants, companies looking to
develop pharmaceutical-grade therapeutic molecules have had
to move beyond the naturally occurring cultivars to ensure they
can produce enough desired product.
In some cases, this has meant leaving the Cannabis plant entirely.
Seeking abundance
Continuing to see merit in Cannabis, GW Pharmaceuticals has
undertaken a proprietary breeding program that has generated a
variety of plants that offer specific molecular profiles or, as they
describe them, chemotypes.
Steve Schultz, vice president of investor relations at GW,
describes this as a continually evolving library that ultimately
allows GW to tap into more than a dozen of the phytocannabinoids.
“We believe that GW is in a unique position to develop and
manufacture plant-derived cannabinoid formulations worldwide
at sufficient quality, uniformity, scale and sophistication for the
Drug Discovery News
purposes of pharmaceutical development and to meet interna-
tional regulatory requirements,” he adds.
Schultz goes on to suggest that complex extracts can be effective
if you can ensure consistency throughout production. He points to
GW’s original cannabinoid product Sativex, which is approved in
several countries but is still investigational in the United States.
“To manufacture this CBD:THC combination product, we
employ a range of advanced analytical technologies to demon-
strate batch-to-batch uniformity,” he explains. “We standardize the
formulation across the extracts as a whole, not simply by reference
to their key active components.”
This approach, he continues, offers the prospect of developing
a product that enhances the features of one cannabinoid with
the complementary features of another cannabinoid, while being
viewed as a single pharmaceutical product by regulators.
For its part, Renew Biopharma acknowledges the importance
of Cannabis plants as a starting point, but only as a starting point.
“The plant does a lot of interesting things, and again, it’s given
us a lot of drug leads,” Mendez allows. “But that’s not the way to
develop drug leads. You’ve got to pull it out and command the
pathway, which allows you to command the molecules.”
To do that, Renew relies on the emerging approach of syn-
thetic biology.
“How I define that is the ability to synthesize genes cheaply and
to sequence genes cheaply, which allows us to explore really broad
DNA diversity on a lot of these different enzymes,” he explains.
The beauty of synthetic biology, he presses, is the ability to mis-
match, where you can pull in different genes from different organisms.
“I take whatever genes I want; I couldn’t care less if they come
from the Cannabis plant,” Mendez enthuses. “If an enzyme performs
a task that I need it to perform, it’s showing up. I don’t care if you’re
from walrus or wombat or another plant. There are enzymes that
have evolved to just do that job better.”
The company then establishes specific metabolic pathways to
produce specific compounds in microalgae, offering scale and
control that simply is not possible with a farmed plant.
Although the company explored the more traditional microbes
E. coli and yeast—Mendez describing himself as microorganism-
agnostic—microalgae simply made the most sense.
“The reason for that is, simplistically, it is a plant-based pathway
and there are several enzymes in that pathway that prefer to be
expressed within a plant,” he explains.
“It would be a daunting effort just to get them to function in [E.
coli or yeast], whereas with microalgae, it has no issue expressing
those enzymes and they’re highly active.”
The approach, he says, allows the company to produce the naturally
less abundant cannabinoids that researchers have traditionally
struggled to access.
“I don’t have anything against THC or CBD, but a lot of those
molecules are commodities,” he continues. “If they are of interest
for drug development, I’ll have no trouble going after either one
of those—but Renew is interested in all of the other cannabinoids
that are made in such a small amount that researchers haven’t had
a chance to get enough of a pure form of them to study them.”
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 DDN Best of Neuroscience
Just as importantly, the synthetic biology route allows the com-
pany to engineer the enzymes to explore a broader chemical space
than that produced by Cannabis plants.
“The plant molecules are interesting but I don’t think they’re
going to be good enough to really come into their own to play as
a therapeutic,” Mendez cautions. “So, Renew is very interested
in making derivatives.”
“I love the chassis,” he continues. “It gets me across the [blood-
brain] barrier and interacts with the receptor, but there is a lot of
off-targeting by a lot of those natural molecules. That’s not how
you develop a drug.”
But producing non-abundant molecules and derivatives to scale
will serve little purpose if they don’t do anything, which is why in
January, the company announced a preclinical discovery collab-
oration with Ken Mackie at Indiana University’s Gill Center for
Biomolecular Science.
“Ken and his group really have the expertise, not only from the
cell-based assays, but [from] all of the mouse models that they’ve
developed over the years to come back and test these molecules
for us,” Mendez enthuses. “He’s able to quickly assay which of our
molecules he likes, and then go right into a mouse model quickly
and try to demonstrate it.”
And Mackie’s response, says Mendez, has been equally effusive.
“He was completely floored and fascinated by the molecules we
were showing him,” he laughs. “I love seeing how excited people
get just by looking at a structure of a molecule.”
The other advantage the synthetic biology approach offers,
Mendez says, is that it establishes a ready production platform
that can be scaled should a therapeutic be approved.
“There’s been a lot of great development on finding derivatives,
but now no one seems to be able to manufacture these derivatives,”
he adds. “I’ve never done synthesis, but my conversation with the
chemists is that a lot of people talk about the chemistry of synthe-
sizing cannabinoids, but when you get down into the nitty gritty
with the chemists, it is harder than most people think. This is why
companies that have gone down that path are still struggling to
get products to market.”
Researchers at Zynerba might disagree with this sentiment, however.
“I think it is one thing [to have enough product] to do clinical
studies, but it is another thing as you are looking to commercialize
a compound and get it approved by the FDA or any regulatory
agency,” says Anido. “They are looking for consistency in how
you manufacture. They want to know that if you say there is 100
mg of drug in the formulation, it is releasing and giving you 100
mg every single time.”
Despite the best efforts of some of the larger companies that are
trying to simulate Mother Nature, he continues, there are a lot of
factors that can exacerbate an already difficult process—e.g., plant
disease, temperature control, sunlight control, nutrients—and
affect the overall yield.
“It’s pretty hard to make sure that every time you are planting
and harvesting and extracting and purifying, you are getting the
exact same amount and have consistency of dose,” he cautions.
Drug Discovery News
For this reason, the company decided to pursue chemical syn-
thesis in the development of their CBD and THC derivatives,
following the model of classical pharmaceutical development.
That approach not only provides the company with consistent
yield and product, but also allows it to explore alternative
delivery vehicles, choosing transdermal over the more typical
oral formulations.
That decision, Anido explains, was the brainchild of pharma-
cologist Audra Stinchcomb, then at University of Kentucky, who
started a spinoff company called AllTranz.
FROM PARIAH TO POTENTIAL: Cannabinoids still bear a bit of a stigma
in some research circles, but interest is increasing with regard to their
potential efficacy for treating any number of neurological conditions,
from epilepsy to autism spectrum disorder to pain.
As Anido recounts: “Audra had the idea that ‘hey, if I can bypass
the GI tract, improve the bioavailability and get more consistent
blood levels, you probably have a compound that works as well,
if not better, but probably also avoids the peak levels that you get
with an oral delivery that will give you the CNS [central nervous
system] highs.’“
Having spent a decade working on the formulations and
developing intellectual property, Stinchcomb eventually sold the
intellectual property to the investment group Broadband Capital,
which worked with Anido and Terri Seebree to found Zynerba.
Their resulting lead candidates are ZYN001 (transdermal patch
THC) and ZYN002 (transdermal gel CBD).
The availability of these many compounds is leading to a rapid
influx of clinical studies for a variety of neurological conditions,
the predominant target being epilepsy.
Making clinical inroads
“If you go back to what really got companies like GW Pharmaceuticals
to focus in epilepsy, it was primarily reports out of Colorado on the
use of Charlotte’s Web, which was CBD-enriched medical marijuana,
showing that it had a very dramatic effect on kids with Dravet syn-
drome, a rare childhood refractory epilepsy,” suggests Anido.
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 DDN Best of Neuroscience
GW’s current lead product candidate is Epidiolex, which
Schultz explains is an oral formulation of purified CBD for certain
severe, early-onset, drug-resistant epilepsy syndromes, with initial
focus on two rare and particularly difficult to treat forms: Dravet
syndrome and Lennox-Gastaut syndrome (LGS). Both of these
conditions, he says, have been granted Orphan Drug designation
by the FDA.
“All three Phase 3 pivotal studies met their primary endpoints
demonstrating a clinically meaningful reduction in the frequency
of seizures compared to placebo,” Schultz adds. “Overall, Epidiolex
was generally well tolerated and demonstrated a consistent side
effect profile across these studies.”
In a study published early this year in The Lancet, patients with
uncontrolled LGS were given Epidiolex or placebo on top of current
therapy for 14 weeks. Patients in the treatment group not only
experienced a greater reduction in drop seizures, but were also
significantly more like to see a reduction in total seizures, as well as
experience a 50-percent or more reduction in drop seizures.
PROS & CONS: “The cannabinoid pathway itself is not particularly a
super difficult pathway to deal with, but there are key enzymes in the
pathway that present a true problem for molecular biology,” says
Michael Mendez, founder and CEO of Renew Biopharma.
“LGS is one of the most difficult types of epilepsy to treat,
and the majority of patients do not have an adequate response
to existing therapies,” said lead investigator Elizabeth Thiele,
director of pediatric epilepsy at Massachusetts General Hospital,
in announcing the publication. “These results show that Epidiolex
may provide clinically meaningful benefits for patients with LGS.”
Almost a year ago, GW published similarly positive results for
Epidiolex in Dravet syndrome in The New England Journal of Medicine.
At the annual meeting of the American Epilepsy Society (AES)
last December, Zynerba announced the findings of its efforts with
ZYN002 in adults with focal seizures, the Phase 2 STAR 1 study
and open-label STAR 2 extension.
Although STAR 1 did not hit its primary endpoint, according to
Anido, it and the extension offered a number of insights to the company.
“It appears that in adults with focal seizures, it may take
longer for CBD to have its true effect,” he relates. “We’ve had
Drug Discovery News
conversations with a number of cannabinoid specialists and neu-
roscientists who’ve said to us in an adult patient, the neuronal
plasticity has been shot, for the most part. It has been hurt and
depleted or not very malleable, and therefore it may be that with
CBD it just takes a little bit longer.
“And what we have found is that as we’ve followed the patients
from STAR 1 into STAR 2, the more time that they were on drug,
the better the results were.”
Furthermore, although the overall placebo rate in STAR 1 was
within expectations, there were patients within that arm who
achieved unexpectedly high improvement in their seizures.
Parsing the data further, the clinicians noted that these super-
responders tended to have very low baseline seizure rates.
“As we took a look at breaking out those who had high baseline
seizure rates and those who had low, ZYN002 did much better
in those who had a higher baseline seizure rate,” Anido explains.
“Not only did we have better efficacy, but we also saw that the
placebo rate went down considerably.”
Thus, he says, the company will change the minimum number of
baseline seizures for patients enrolled in their next trial, expected
in the second half of 2018.
Also last December, INSYS Therapeutics announced initiation
of a Phase 2 trial of its synthetic CBD oral solution in the treatment
of refractory childhood absence epilepsy. The new study follows on
the heels of success in an earlier Phase 1/2 study—also presented
at the AES conference—that demonstrated a reduction in both
seizure frequency and intensity in the treatment arm.
“Better treatments with greater efficacy and fewer side effects
are needed to improve quality of life and clinical outcomes for
these patients,” according to Steven Phillips, study investigator
and pediatric neurologist at Mary Bridge Children’s Hospital and
Health Center. “CBD holds great promise for this challenging
form of epilepsy.”
Slowly but persistently, however, cannabinoids are being tested
in an increasingly wider array of neurological conditions.
Beyond epilepsy
According to Schultz, GW also is working on various clinical initia-
tives for CBDV (GWP42006) within the field of autism spectrum
disorders (ASD) and received Orphan Drug designation in the
treatment of Rett syndrome.
“A physician-led, expanded-access IND to treat seizures associat-
ed with autism has been granted by FDA for 10 patients, and a num-
ber of patients have commenced treatment,” he says. “In addition,
an investigator-led 100-patient placebo-controlled trial in children
with ASD is due to commence in the second quarter of 2018.”
In Rett syndrome, he continues, the company expects to com-
mence an open-label study in the second quarter of 2018, with hopes
for a Phase 2 placebo-controlled trial in the third quarter of 2018.
Last October, Schultz says, the company presented several abstracts
at a symposium hosted by the Italian Society of Pharmacology.
“These abstracts included science that was developed by GW in
concert with numerous academic partners and highlighted a range
of in-vitro and in-vivo ASD models, including Rett syndrome-like
phenotype rodent models and models related to cognitive and
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 DDN Best of Neuroscience
social impairment,” he explains. “These data will guide and inform
our development program.”
Through their collaboration with Ken Mackie’s group, Renew
has partly focused its interests on the opioid crisis.
“Ken is very passionate about Renew going after opioid addic-
tion,” Mendez says. “[So] our first pass will be opioid craving and
pain management.”
Initial efforts will likely focus on CBD and its derivatives, but
Mendez sees opportunities for other cannabinoids that simply
were not previously available.
Also hoping to enter the opioid addiction space is Aphios Pharma,
which announced its efforts to raise equity capital in late 2017 to
develop its CBD candidate.
Looking to address some of the issues of current therapeutics like
methadone, buprenorphine and naltrexone, which are themselves
opioids, Aphios is leveraging proprietary SuperFluids extraction
technologies to develop cGMP in a DEA Schedule 1 facility.
It is also working to improve delivery of its lead compound by
nanoencapsulating the cannabinoids in biodegradable polymer
nanospheres, according to the company’s president and CEO,
Trevor Castor.
Zynerba has also extended its efforts into the exceptionally rare
fragile X syndrome, a condition to which they were first intro-
duced by Randi Hagerman of the UC Davis MIND Institute, who
was seeing positive results from children using CBD oils.
Working with Hagerman and Nicole Tartaglia of Children’s
Hospital Colorado, the company launched a 20-patient open-label
Phase 2 study of ZYN002 and were impressed with the results.
Once the data were in, says Anido, the company quickly prepared a
meeting with the FDA, held earlier this year, and is hoping to move
into a larger, well-controlled double-blind study.
On the THC side, Zynerba has also developed an interest in
Tourette syndrome. According to Anido, previous double-blind
studies with plant-derived oral THC have demonstrated efficacy
both in the severity and frequency of tics, as well as obsessive-com-
pulsive behavior.
“Today, the only things that have been approved for Tourette
syndrome have been the atypical antipsychotics that are very
difficult to tolerate in patients,” he says. “Most patients don’t like
taking it. So we think there is a nice opportunity.”
The company, Anido continues, is going into Phase 1, where
they hope to find their transdermal ZYN001 can maintain THC in
the blood consistently over a 24-hour period, avoiding the peaks
and valleys often seen with oral delivery.
Another company pursuing this angle in Tourette is Therapix
Biosciences, which in February announced it had completed pre-
IND communications with the FDA for its THX-110 lead candidate,
a combination of synthetic THC and palmitoylethanolamide (PEA).
“We believe that this enables us to continue our clinical program
with minimum risk, which is consistent with our platform of
repurposing and reformulating for unmet and underserved needs
for Tourette syndrome,” said Therapix CTO Adi Zuloff-Shani in
announcing the discussions. “We expect to evaluate THX-110 in a
Phase 2b clinical study in the second quarter of 2018.”
Drug Discovery News
In December, the company announced it had completed enroll-
ment in its Phase 2a single-arm open-label study being conducted
at Yale University.
Meanwhile, Renew is looking beyond simply creating thera-
peutic compounds and moving into prevention.
Hoping to leverage evidence that certain cannabinoids demon-
strate neuroprotective and anti-inflammatory properties, Mendez
and Mackie are eyeing traumatic brain injury and concussions.
“There’s a lot of evidence for a lot of these molecules, and definitely
derivatives of these molecules, that it slows down the progression,”
Mendez explains. “There’s no reason not to believe that if we worked
hard on the derivatives, we could stop the progression.”
More importantly, however, he sees opportunities for such com-
pounds to be used for preventative intervention.
“These molecules should be taken before injury to prevent the
immune response that happens at the point of injury,” he presses.
“We would like to develop these as preventatives, so one would
be able to take these before you went in to play a contact sport
or before you went out on patrol if you’re military. That’s where I
think it’ll have the greatest impact.”
Regardless, the question arises of why there is suddenly so much
activity in this space.
“Why hasn’t this been done before?” Mendez asks. “Why now?
Why 2018?”
A matter of timing
“We knew these compounds had these great effects 20 years ago,
30 years ago,” he continues, yet no one stepped forward as they
have recently to fit this into a traditional pharma model.
“I wouldn’t have been able to start a company like Renew if
California didn’t legalize,” Mendez offers. “It doesn’t matter that
I am developing these things legally. The stigma would still have
prevented anyone from investing in Renew.”
He’s not sure that the stigma has completely disappeared, but
at least, he suggests, people don’t have to worry about jail time.
But even as the industry needed to wait for the social and political
environment to change, companies like Renew also had to wait for
the scientific landscape to change.
“The cannabinoid pathway itself is not particularly a super
difficult pathway to deal with, but there are key enzymes in the
pathway that present a true problem for molecular biology,” Mendez
says. A new approach to the science was going to be necessary.
“[Synthetic biology] allows us for the first time to really modify
these enzymes and really get them into the right place for us to
use in microorganisms,” he explains. “You probably wouldn’t have
been able to do that maybe 10 years ago.”
As society aligns with technology—like Jupiter with Mars—it
seems that time has come today for cannabinoids.
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 DDN Best of Neuroscience
NEXT-GEN NEUROSCIENCE
BY JEFFREY BOULEY, DDN CHIEF EDITOR
F rom an artificial nervous system to uses for artificial intel-
ligence, neuroscience has offered many innovations and
breakthroughs in recent years. We take a look at some high-
lights of what researchers have learned, what they can do now that
they couldn’t before and where they might be headed with all of
that in their armamentarium.
Creating an ‘artificial nervous system’
Researchers at the National University of Singapore (NUS) have
developed something called Asynchronous Coded Electronic Skin
(ACES), an “e-skin” that could enable robots to have a sophisticated
sense of touch for nimbleness and better fine motor movements,
but could also be used potentially in prosthetics for humans.
Essentially, what the NUS scientists did was to create an artificial
nervous system inspired by the human nervous system that that can
detect touch 1,000 times faster than the human nervous system
The e-skin detects signals and differentiates physical contact
like a human being. But unlike nerve bundles in human skin,
the electronic nervous system is made of a network of sensors,
connected with a single electrical conductor.
Pairing ACES with a special transparent and water-resistant
sensor layer also recently developed by the team creates an
electronic skin that can self-repair like the human skin. This type of
electronic skin, NUS notes, could be used to develop more realistic
prosthetic limbs that will help disabled individuals restore their
sense of touch.
Neurodegenerative diseases identified
using AI
Researchers have developed an artificial intelligence (AI) plat-
form to detect a range of neurodegenerative disease in human
brain tissue samples, including Alzheimer’s disease and chronic
traumatic encephalopathy, according to a study conducted at the
Icahn School of Medicine at Mount Sinai and published in the
medical journal Laboratory Investigation. Their discovery may help
Drug Discovery News
scientists develop targeted biomarkers and therapeutics, result-
ing in a more accurate diagnosis of complex brain diseases that
improve patient outcomes.
The buildup of abnormal tau proteins in the brain in neuro-
fibrillary tangles is a feature of Alzheimer’s disease, but it also
accumulates in other neurodegenerative diseases, such as chronic
traumatic encephalopathy and additional age-related conditions.
Accurate diagnosis of neurodegenerative diseases is challenging
and requires a highly-trained specialist.
Researchers at the Center for Computational and Systems
Pathology at Mount Sinai developed and used the Precise Informatics
Platform to apply powerful machine learning approaches to digitized
microscopic slides prepared using tissue samples from patients with
a spectrum of neurodegenerative diseases. Applying deep learning,
these images were used to create a convolutional neural network
capable of identifying neurofibrillary tangles with a high degree of
accuracy directly from digitized images.
“Utilizing artificial intelligence has great potential to improve
our ability to detect and quantify neurodegenerative diseases,
representing a major advance over existing labor-intensive and
poorly reproducible approaches,” said lead investigator Dr. John
Crary, a professor of pathology and neuroscience at the Icahn School
of Medicine. “Ultimately, this project will lead to more efficient and
accurate diagnosis of neurodegenerative diseases.”
Neuroprotective activity from a
cannabinoid?
Emerald Health Pharmaceuticals Inc. (EHP), a clinical-stage company
developing medicines based on cannabinoid science, presented
preclinical data for its drug product candidate, EHP-102, an oral
formulation of a patented cannabigerol (CBG)-derived new chemical
entity (NCE), as well as other novel CBG acid (CBGA) derivatives
for the treatment of Huntington’s disease (HD) and Parkinson’s
disease (PD) at the 29th Annual Symposium of the International
Cannabinoid Research Society (ICRS) in Bethesda, Md.
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 DDN Best of Neuroscience
The data presented compared the in-vivo activity of two of
EHP’s novel CBGA derivatives—CBGA-quinone (CBGA-Q) and
its water-soluble sodium salt (CBGA-Q-Na Salt) —to EHP-102,
the company’s preclinical-stage development candidate, which
has previously demonstrated significant benefits in several models
of PD and HD.
The preclinical findings demonstrated that oral administration
of EHP-102 has potential in PD and HD due to its anti-
inflammatory and neuroprotective properties specific to these
diseases. The CBGA derivatives also showed anti-inflammatory and
neuroprotective effects; however, EHP-102 showed significantly
better effects on various parameters compared to the other two
molecules. The data were reported in a poster titled “Comparison
of the neuroprotective activity of cannabigerol derivatives in
Huntington’s and Parkinson’s disease models.”
AI & NEURO: Artificial intelligence is growing in prominence not just as
a way to screen potential neurological therapies, but also to detect a
range of neurodegenerative disease in human brain tissue samples,
including Alzheimer’s disease and chronic traumatic encephalopathy.
“Patients with Huntington’s disease and Parkinson’s disease
suffer from devastating physical and psychological symptoms,”
said Dr. Jim DeMesa, CEO of Emerald Health Pharmaceuticals.
“There is currently no cure for these diseases, and so the results
of the studies conducted by our scientific team and collaborators,
which demonstrate the possible disease-modifying potential
of EHP-102 and some of our other CBG-derivatives, are very
encouraging as potential therapeutic treatments for these
patients in the future.”
Oral EHP-102, CBGA-Q and CBGA-Q-Na Salt all alleviated
clinical symptoms and the loss of neurons, as well as inhibited
the expression of proinflammatory cytokines in a HD murine
model induced by 3-nitropropionic acid. Oral CBGA-Q and
EHP-102 also improved the behavioral deficits in a mouse PD
model induced by 6-hydroxydopamine and prevented the loss
of neurons in the brain. EHP-102, however, showed statistically
superior effects compared to both other molecules in several of
the parameters measured in each model.
Drug Discovery News
Stem cell line could help recover motor
function after a stroke
Results of a Phase 1 clinical trial reported in 2018 in STEM CELLS
Translational Medicine identified a specific line of human neural
stem cells that shows potential for helping recover motor function
in those who suffer a hemiparetic stroke (where one side of the
body is left weak or paralyzed).
NSI-566 human neural stem cells were implanted adjacent to
the stroke lesion in nine subjects at doses up to 72 million cells
per subject, and subjects were followed for 24 months. Cells
appeared to survive long-term, and patients in the single-arm
trial showed improvements in motor function following cell
transplantation. This indicates the procedure is safe and may
have potential to provide benefit to patients with motor deficits
from stroke.
Strokes are a major cause of prolonged neurological disability
worldwide, and no effective therapies currently exist that will
reverse the damage. Several different cell types have been
proposed and tested in preclinical models and clinical trials to
treat neurological conditions, but few have shown significant
properties to differentiate into genuine brain cells or the
capability to integrate into central nervous system tissue.
One cell type that has been exhibiting some success is the
NSI-566 cell line from Neuralstem Inc. NSI-566 is an epigeneti-
cally expanded line of primary human neural stem cells isolated
from a single fetal spinal cord tissue. Ongoing clinical trials in
the United States for treating amyotrophic lateral sclerosis and
spinal cord injury have provided evidence that NSI-566 can sur-
vive in patients for at least 2.5 years and may have the potential
to provide clinical benefit. To date, there have been no major
side effects reported.
That information prompted the research team—led by Dr. Karl
K. Johe of Neuralstem and Dr. Ruxiang Xu of the Affiliated BaYi
Brain Hospital in Beijing—to ponder whether NSI-566 might help
stroke patients, too.
“To the best of our knowledge, this is the first study to assess the
feasibility and safety of transplanting non-immortalized human
neural stem cells into stroke brain in chronic stage. Another
objective was to determine the maximum tolerated dose into the
peri-infarct sites in these patients,” Johe said.
The patients were divided into three equal groups, with each
group administered a different dose of the stem cell line. The
patients were then followed for 24 months.
“At the end of that period, we saw that transplantation of the
NSI-566 cells was being well tolerated and it suggested prelimi-
nary clinical benefits. Results from imaging studies indicate new
neural tissue formation in the stem cell implantation area of all
nine patients,” noted Xu.
Added Johe: “The results suggest that grafted NSI-566 continue
to survive in the stroke lesion long after cessation of immunosup-
pressant, and that the potential clinical effects persist throughout
the 24-month observation period. Although this was a small, one-
arm study of feasibility, the results are encouraging to warrant
further studies.”
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 DDN Best of Neuroscience
Putting AI to work against neurological
disorders
BullFrog AI Inc., which focuses on artificial intelligence and
clinical data analytics services, and the Lieber Institute for Brain
Development, an independent, not-for-profit medical research
institute working to accelerate efforts to find new cures for
developmental brain disorders, formed a joint collaboration to
apply BullFrog AI’s proprietary artificial intelligence platform
to analyze antipsychotic drug responses.
Choosing and dosing antipsychotic drugs represents one of
the most challenging areas for clinicians and patients alike,
the partners note, with patients experiencing wildly differing
responses to the different drugs on the market, particularly when
it comes to the side effects: “The selection of the best drug for the
particular patient then becomes a matter of educated guesswork,
potentially causing distress and even harm to patients and those
around them.”
To help turn the tide on that, BullFrog AI agreed to utilize
its proprietary artificial intelligence platform to analyze large
multifactorial clinical data sets from patients who received
antipsychotic medication with an eye toward making it possible
to better predict which patients respond best to which medications.
“We are excited to partner with BullFrog AI on this challenging
clinical problem,” said Dr. Kristin Bigos, an investigator at the
Lieber Institute for Brain Development. “Until now there has
been no effective alternative to the trial-and-error approach
for matching patients to the best antipsychotic. Being able to
understand and predict a priori which drugs will work best for
which patients would be of tremendous benefit to both patients
and the physicians that serve them.”
The bfLEAP analytics engine is purpose-built for analyzing
extremely large and complex data sets, and it reportedly has
demonstrated 99.9-percent accuracy in predicting the right
targets across multiple data sets. The key to its success is the
artificial learning, which requires no domain expertise, BullFrog
AI maintains—instead, it uses unsupervised machine learning
coupled with the world’s largest collection of analytical models, all
operating in parallel.
And it’s not just clinical treatment with approved drugs where
this effort could help, but also potentially in clinical trials.
“bfLEAP represents a true leap forward in terms of our ability
to understand what is going on in a complex clinical setting,” said
Vin Singh, CEO of BullFrog AI. “For the first time, we are able to
analyze massive, multifactorial clinical datasets and determine the
root cause of the observed clinical outcomes. Using our platform,
we can examine clinical trial data and identify relationships
between patient-specific factors and clinical response, which
may aid in predicting clinical trial outcomes in the future. The
potential impact of this type of information for the pharmaceutical
industry is enormous, both in terms of the reduced cost and
increased revenue from failed drugs as well as the positive impact
on patients.”
Drug Discovery News
New insight on potential neurological cell
therapies
2018 research from Sanford Burnham Prebys Medical Discovery
Institute (SBP) was reportedly among the first to describe how an
mRNA modification impacts the life of neural stem cells (NSCs).
The study, titled “N6-methyladenosine RNA modification regu-
lates embryonic neural stem cell self-renewal through histone
modifications” and published in Nature Neuroscience, reveals a
novel gene regulatory system that may advance stem cell therapies
and gene-targeting treatments for neurological diseases such as
Alzheimer’s disease, Parkinson’s disease and mental health disor-
ders that affect cognitive abilities.
“Being able to maintain viable stem cells in the brain could lead
to regenerative therapies to treat injury and disease,” says Dr. Jing
Crystal Zhao, an assistant professor at SBP. “Our study reveals a
previously unknown but essential function of an mRNA modifi-
cation in regulating NSC self-renewal. As NSCs are increasingly
explored as a cell replacement therapy for neurological disorders,
understanding the basic biology of NSCs—including how they
self-renew—is essential to harnessing control of their in-vivo func-
tions in the brain.”
NSCs are progenitor cells present not only during embryonic
development but also in the adult brain. NSCs undergo a
self-renewal process to maintain their population, as well as
differentiate to give rise to all neural cell types: neurons, astrocytes
and oligodedrocytes.
The SBP study focused on the self-renewal aspect of NSCs. Using
knockout mice for the enzyme that catalyzes the m6A modification,
Zhao’s team found that m6A modification maintains NSC pool by
promoting proliferation and preventing premature differentiation
of NSCs. Importantly, the researchers found that m6A modification
regulates this by regulating histone modifications.
The importance in this lies in the fact that histones play an
important part of turning genes on or off—these proteins bind and
package DNA to either “hide” a gene from the cell’s protein-making
machinery (off) or “loosen up” DNA for gene exposure (on).
“Our findings are the first to illustrate cross-talk between mRNA
and histone modifications, and may lead to new ways to target
genes in the brain,” says Zhao. “Conceptually, we could use the
modification, which is the methylation of adenosine residues, as
a ‘code’ in mRNA to target histone modifications to turn genes
on or off.”
As SBP notes, drugs that can alter histones are far from a new
concept in psychiatric and neurological care—the problem is they
are often not very specific and affect the entire genome.
“Our current study addressed the interaction between mRNA
and histone modification in a genome-wide scale. In the future, we
plan to study such interaction on a gene-by-gene basis. Ultimately,
by modulating mRNA modification and its interacting histone
modifications at a specific genomic region, we hope to correct
aberrant gene expression in brain disorders with precision,”
explains Zhao.
25
FROM
DDN2018
Best of Neuroscience
The ‘Tao’ of tau?
In a field once dominated by amyloid plaques, Alzheimer’s research related to
tau sees much more attention
BY JEFFREY BOULEY, DDN CHIEF EDITOR
A lzheimer’s has already proven to be a particularly complex
and challenging disease for life-sciences researchers
and pharma/biotech companies. And in a disease where
sticky tangles of proteins seem to atrophy the brain and choke off
cognition, one of the stickiest areas has been the issue of the amyloid
protein vs. the tau protein. The aggregation of the tau protein is
a hallmark of Alzheimer’s disease, but traditionally much of the
energy and effort has gone toward focusing on ways to reduce the
number of amyloid plaques.
As Emily Underwood wrote in a 2016 article in Science, “One of
the telltale signs of Alzheimer’s disease (AD) is sticky plaques of
ß-amyloid protein, which form around neurons and are thought by
a large number of scientists to bog down information processing
and kill cells. For more than a decade, however, other researchers
have fingered a second protein called tau, found inside brain cells,
as a possible culprit.”
The topic Underwood was addressing was an imaging study of
10 people with mild AD that indicated tau deposits, rather than
amyloid, are closely linked to memory loss, dementia and other AD
symptoms. It wasn’t evidence that actually resolved the amyloid-
tau debate—almost certainly both proteins play major roles, and
perhaps other factors as well—but the findings did serve as a
potential jumping-off point for additional effort on tau-targeting
treatments and better diagnostic tools.
And on the subject that tau and amyloid likely represent more
of a “pair of culprits” rather than an “either-or” situation, we
can go back a couple years to a JAMA Neurology paper by G.S.
Bloom that cast ß-amyloid protein and tau in a “trigger and
bullet” metaphor. As the author noted in the abstract, “During
the past dozen years, a steadily accumulating body of evidence
has indicated that soluble forms of Aβ and tau work together,
independently of their accumulation into plaques and tangles, to
Drug Discovery News
drive healthy neurons into the diseased state and that hallmark
toxic properties of Aβ require tau. For instance, acute neuron
death, delayed neuron death following ectopic cell cycle reentry,
and synaptic dysfunction are triggered by soluble, extracellular Aβ
species and depend on soluble, cytoplasmic tau. Therefore, Aβ is
upstream of tau in AD pathogenesis and triggers the conversion of
tau from a normal to a toxic state, but there is also evidence that
toxic tau enhances Aβ toxicity via a feedback loop.”
Druggability of tau
So, fast forwarding to June 28, 2018, we see one company continuing
the trend toward more focus on tau as news came out of Cantabio
Pharmaceuticals Inc. that the preclinical-stage pharmaceutical
company, which is working on therapeutics for AD, Parkinson’s
disease and related neurological disorders, had seen publication of a
peer-reviewed article. Lead-authored by Cantabio’s CEO Dr. Gergely
Toth, along with collaborators at the Hungarian Academy of Sciences
and German Center for Neurodegenerative Diseases (DZNE), the
work appeared in the journal ACS Chemical Neuroscience.
The paper was titled “The structural basis of small molecule
targetability of monomeric Tau protein” and reported structure-based
evidence that native monomeric tau can be a viable target for drug-
like small molecules despite its heterogeneous structure.
As the company noted of the news, the aggregation of monomeric
tau protein is linked to the onset and progression of Alzheimer’s
disease and other tauopathies, and this study and the scientific
team’s previous findings provide theoretical and experimental
evidence for the ability of monomeric tau to be a receptor of small
molecules designed to prevent the aggregation, which leads to
toxicity and cell death.
As per Prof. Eckhard Mandelkow, a co-author of the publication
and group leader at DZNE in Bonn, this is further evidence that
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 DDN Best of Neuroscience
inhibition of tau aggregation by small molecules may be a viable
therapeutic approach for tauopathies such as Alzheimer’s disease.
He noted that “These molecules are currently being evaluated in
animal models of tau-induced pathology.”
“We are excited to publish further scientific evidence that
establishes a structural biology basis for Cantabio’s tau small-
molecule pharmacological chaperone program, which aims to
prevent and reduce aggregation of tau protein as a therapeutic
strategy for Alzheimer’s disease and other tauopathies such as
concussion-related chronic traumatic encephalopathy,” said Toth.
“The tau protein has long been a major target for Alzheimer’s
drug development, but due to the nature of its structure, it has
historically proven to be a difficult target for small-molecule
drug candidates. Our work at Cantabio represents a significant
step forward in developing a therapy that is able to prevent the
formation of the toxic protein aggregates that are associated with
neurodegeneration in these diseases.”
Tau as a therapeutic and diagnostic
target
And, perhaps in a sign of how much tau research remains in
the shadow of amyloid research, there is news from 2017 from
AC Immune SA, a clinical-stage biopharmaceutical company
with a broad pipeline focused on neurodegenerative diseases,
when it shared the top-level insights from a key opinion leader
(KOL) luncheon meeting on the importance of tau as a target in
Alzheimer’s disease and other neurodegenerative diseases. The
meeting featured presentations by KOLs Dr. Khalid Iqbal of the
New York State Institute for Basic Research in Developmental
Disabilities and Dr. Michael Rafii of the University of California,
San Diego, and the University of Southern California.
Iqbal highlighted the critical importance of tau as a therapeutic
target in Alzheimer’s disease and other neurodegenerative dis-
eases, and how inhibition and prevention of the Tau pathology
can potentially rescue the pathology of Alzheimer’s disease and
cognitive impairment, commenting: “Neurodegeneration leads
to tau pathology, and tau pathology leads to neurodegeneration.
Where there is no tau pathology, there is no Alzheimer’s disease.
Tau-based therapeutic approaches have significant potential to
treat a range of neurodegenerative diseases.”
Rafii discussed tau-mediated pathology and the importance of
tau diagnostics in people with Down syndrome, a population with
a genetic predisposition to develop Alzheimer’s-related neuro-
pathological changes, including ß-amyloid plaques and tau tangles.
“Biomarkers of Alzheimer’s, including Tau-PET, can be readily
studied in adults with Down syndrome as in other preclinical AD
populations,” Rafii noted. “By understanding the link between
Alzheimer’s and Down syndrome, we may not only be able to help
the Down syndrome community, but the broader population as
well. People with Down syndrome are an important population
to study as we enhance our understanding of early intervention
and prevention of Alzheimer’s disease in general.”
Drug Discovery News
Potential monotherapy?
Also late in 2017, TauRx Therapeutics Ltd. reported the full results
from its second Phase 3 clinical study of LMTX, a tau aggregation
inhibitor for Alzheimer’s disease, which were published online in
the Journal of Alzheimer’s Disease. The company noted that results
from this study (TRx-237-005) are consistent with those from the
first Phase 3 study, recently published in The Lancet in mild to
moderate Alzheimer’s disease, in supporting the hypothesis that
LMTX might be effective as monotherapy at a dose as low as 4
mg twice daily.
The results of the earlier study showed significant differences
in favor of two higher doses of LMTX (75 mg and 125 mg twice
daily) when taken as monotherapy, compared with the intended
4 mg control dose taken as monotherapy or as add-on therapy to
currently approved treatments for AD in prespecified post-hoc
analyses. In a further analysis, the same difference in favor of
monotherapy compared with add-on treatment was found in
patients taking the 4 mg twice-daily dose.
According to TauRx, in both the LMTX monotherapy and add-
on therapy groups, whole brain atrophy (measured via MRI scans)
initially progressed as expected for patients with mild Alzheimer’s
disease. However, after nine months of treatment, the annualized
rate of whole brain atrophy in monotherapy patients reduced
significantly and became typical of that reported in normal elderly
controls without Alzheimer’s disease. The comparable rate seen
in the add-on therapy group progressed as reported for patients
with mild Alzheimer’s disease.
And early this year, the company reported preclinical study
results, published online in Frontiers in Molecular Neuroscience,
showing that LMTM, the active pharmaceutical ingredient in the
LMTX product developed for the treatment of Alzheimer’s disease,
may also be useful for the treatment of Parkinson’s disease.
It is worth noting that in 2016, there was some significant
disagreement regarding LMTM when Phase 3 results were
presented showing that the drug missed its co-primary endpoints
of slowing cognitive and functional decline in mild to moderate
AD. Some had argued that the placebo and drug results were nearly
identical and, as a commentator on the Alzforum website argued,
a scientist involved in the trial presented “a subgroup analysis that
held no statistical credence yet purported to show a strong benefit
on cognition and brain atrophy.”
Some other takes around the same time contended that LMTM
might not benefit AD patients who are receiving standard of care
but, as a monotherapy, the drug might stabilize cognition and
reduce brain atrophy.
27
 DDN2019
FROM Best of Neuroscience
COMMENTARY
TARGETING α-SYNUCLEIN AND THE
PATHOGENESIS OF PARKINSON’S
DISEASE
BY DR. NEIL CASHMAN OF PROMIS NEUROSCIENCES
P arkinson’s disease (PD) is a progressive neurodegenerative
disorder characterized by the death of dopaminergic neu-
rons in the midbrain (pars compacta area of the substantia
nigra) and the formation of inclusions known as Lewy bodies
(hallmark lesions of PD), which primarily contain toxic, aggre-
gated α-synuclein. Symptoms do not occur in PD until 80 percent
or more of the capacity to produce dopamine is lost. PD is the
second most common neurodegenerative disease after Alzheimer’s
disease, affecting an estimated 10 million people worldwide and
ranging in severity from mild to severe.
Carbidopa-levodopa (L-dopa) and dopamine agonists are the
standard symptomatic treatments for moderate to severe PD.
Administration of L-dopa increases dopamine production by the
remaining midbrain neurons and usually provides effective relief
of motor symptoms for several years. However, treatment with
L-dopa has no beneficial effect on the underlying pathogenesis.
As neuronal death continues unabated, the effectiveness of ever-
higher doses of L-dopa declines, motor symptoms of PD re-emerge
and worsen and new, iatrogenic motor symptoms appear, caused
by chronic L-dopa therapy. No available treatment has been proven
to slow, halt or reverse the progressive neurodegeneration of PD
that ultimately leads to death.
Toxic α-synuclein aggregates in the
pathogenesis of PD
The cause of the neurodegeneration in PD was not understood
until recent years, when specific forms of α-synuclein were found
to be toxic to neurons. Strong genetic, neuropathological and
preclinical model evidence suggests that misfolded α-synuclein
aggregates into oligomers, comprising the toxic species that plays
Drug Discovery News
a central role in the pathogenesis of PD. α-synuclein oligomers are
distinguished from monomers and insoluble fibrils, which are not
toxic to neurons.1 Pathogenic mechanisms of α-synuclein oligomers
include general cellular toxicity, mitochondrial stress, synaptic
dysfunction and compromised cell membrane integrity, among
others. Furthermore, a large and convincing body of evidence also
shows that α-synuclein oligomers act as seeds for the formation of
larger aggregates, which acquire the ability to propagate from cell
to cell in a prion-like manner, spreading the pathology.
Soon after the discovery of its pivotal role in the pathogenesis of
PD and related synucleinopathies, including dementia with Lewy
bodies (DLB) and multiple system atrophy (MSA), α-synuclein
aggregation became a lead target for PD therapeutics development.
A wide range of development strategies have emerged. Because
protein misfolding and formation of α-synuclein aggregates occurs
very early in PD, initiating anti-α-synuclein treatment in prodromal
or early-stage disease is expected to be advantageous and could
prevent the inevitable extensive neurodegeneration.
Early-stage clinical trials
Early-stage human clinical trials with developmental anti-
α-synuclein therapies are underway. Biogen and Prothena
Therapeutics/Roche are developing immunotherapies based on
antibodies aimed at targeting and inactivating misfolded alpha-
synuclein. Both programs are in Phase 2 testing in people with
early PD. AstraZeneca/Takeda are testing an α-synuclein antibody
in Phase 1 in healthy volunteers.
Other approaches have reached early-stage clinical testing. Affiris
has completed a series of Phase 1 trials, including long-term immuno-
genicity assessment, with a vaccine designed to stimulate production
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 DDN Best of Neuroscience
of α-synuclein antibodies. Neuropore and UCB are developing a
small-molecule α-synuclein misfolding inhibitor to interfere with
propagation of protein aggregates and have completed a Phase 1 study
in healthy volunteers. Proclara Biosciences is developing a compound
that binds to α-synuclein, amyloid-beta and tau and is in Phase 1 in
people with mild to moderate probable Alzheimer’s disease. Drugs
that are not specific to α-synuclein but proposed to have mechanisms
of action that decrease α-synuclein toxicity (e.g., inhibit cell-to-cell
transmission, promote autophagic degradation) are in early-stage
clinical development, primarily by academic institution sponsors.
Early-stage clinical trials are undertaken, principally, to
determine the safety and dosing of developmental compounds.
Traditionally, efficacy is not a primary outcome measure until
late-stage trials. However, biomarkers for predicting the efficacy of
developmental α-synuclein therapeutics are currently undergoing
evaluation and validation. If successful, they might soon enable
drug developers to reach go/no-go decisions based on anticipated
efficacy before investing in long and costly Phase 3 trials. Which
of the current drugs in development, if any, might eventually be
approved by FDA will be determined by Phase 3 efficacy trial
results, which will not be available for at least several years.
Preserving normal, physiological α-synu-
clein tetramer
During the last decade, a novel α-synuclein species designated
the helical tetramer was recognized, which is stable and performs
an important physiological function by inhibiting α-synuclein
aggregation.2 More recently, a transgenic mouse model designed
to make mice unable to form the physiological tetramer resulted
in the development of a disease closely resembling human PD.3
These findings have important implications for α-synuclein-based
therapeutics development. If the tetramer must be preserved to
maintain normal α-synuclein homeostasis, what will be the effects
of therapeutics designed to attack all α-synuclein conformations,
including the tetramer? Will these drugs be efficacious and without
adverse effects (AE)? Or will the inactivation of α-synuclein
tetramer compromise efficacy or cause AEs?
It is important to understand that protein misfolding, leading to
the formation and aggregation of toxic oligomeric conformations
and propagation (prion-like, cell-to-cell transmission) of toxic
oligomeric aggregates, is not unique to PD, DLB and MSA. This
pathogenic mechanism is implicated as an underlying cause
of most neurodegenerative disorders, including PD, DLB and
MSA (all three are synucleinopathies), Alzheimer’s disease
(AD, aggregation of Aβ and tau are the characteristic proteins),
amyotrophic lateral sclerosis (ALS, aggregation of TDP43
and SOD1), frontotemporal dementia (FTP; TDP43 and tau
aggregation) and others.
In AD, although amyloid beta (Aβ) exists in many different
conformations, only specific (low molecular weight) Aβ oligomers
(AβO) are toxic.4 The many late clinical-stage failures of developmental
anti-Aβ therapeutics are directly attributable to targeting non-
toxic conformations of the Aβ protein, while failing to target, or
ineffectively targeting, the toxic species, AβO. For α-synuclein
oligomers, it is currently unclear which molecular species are toxic.
Drug Discovery News
It is presumed that avoidance of α-synuclein physiological tetramers
should be avoided, but that other oligomers (including misfolded
tetramers and protofibrils) are contributing to the pathological spread
of the α-synucleinopathies. Therefore, the development of an anti-α-
synuclein therapeutic with maximal efficacy is expected to require
antibodies (or other approaches) that are highly selective for the toxic
forms of α-synuclein (oligomers and/or small soluble fibrils), while
sparing normal, physiological forms (α-synuclein tetramer) and inert
species (monomers) to minimize the likelihood of adverse events.
Discovery of antibodies highly selective
for toxic α-synuclein conformations
The application of classical methods for the discovery of anti-α-
synuclein antibodies yields pan-α-synuclein antibodies that bind
all species of the α-synuclein protein. Discovery research of anti-α-
synuclein antibodies with highly selective binding profiles for only
the toxic forms, while sparing the physiological and inert forms,
is challenging. Toxic oligomers contain unstructured regions and
are unstable. However, misfolding results in the formation of new
epitopes in the misfolded α-synuclein aggregates; and, critically,
the toxic conformations are preserved during the corruptive protein
templating process by which prion propagation and cell-to-cell spread
occur. Scientific literature suggests a complex binding profile for such
an antibody.
For its part, ProMIS Neurosciences has developed a unique
antibody design platform to identify an optimal “disease selective”
binding profile suggested by the scientific literature, “tune”
epitopes to generate antibodies with desired binding profiles, and
then assess functional performance and comparative binding to
select a lead candidate. Application of this discovery platform to
α-synuclein and the synucleinopathies led to the generation and
development of highly selective antibodies that target only the
toxic forms of α-synuclein, toxic oligomers and/or small soluble
fibrils, while avoiding the targeting of physiological tetramers and
inert monomers. These antibodies achieved the targeted binding
profile for treating PD, demonstrating effective neutralization of
toxic oligomers and cell-to-cell spread of soluble fibrils, while
sparing the physiological forms of α-synuclein such as monomers
and helical tetramers. Such antibodies would be expected to treat
PD and other α-syncleinopathies without adverse effects.
References
1. Brundin P, Dave KD, Kordower JH. Therapeutic approaches to
target alpha-synuclein pathology. Exp Neurol. 2017;298(Dec,
pt B):225-235.
2. Bartels T, Choi JG, Selkoe DJ. α-Synuclein occurs
physiologically as a helically folded tetramer that resists
aggregation. Nature. 2011;477(7362):107-10.
3. Nuber, etal. Abrogating Native a-Synuclein Tetramers in
Mice Causes a L-DOPA-Responsive Motor Syndrome Closely
Resembling Parkinson’s Disease. Neuron 2018;100:75–90.
4. Yang T, Li S, Xu H. Large Soluble Oligomers of Amyloid
β-Protein from Alzheimer Brain Are Far Less Neuroactive
Than the Smaller Oligomers to Which They Dissociate. J
Neurosci. 2017;37:152-163.
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 DDN2017
FROM Best of Neuroscience
SPECIAL REPORT ON NEUROSCIENCE
IMAGE-INE THAT
BY RANDALL C WILLIS, DDN FEATURES EDITOR
T he leather chair squeaks as the psychiatrist adjusts her posi-
tion, her legs slowly going numb as she waits for her patient
to open up. An hour into their session and the patient has
yet to say a word.
“I understand that you have been isolating yourself from your
family,” the psychiatrist notes quietly. “Are you afraid of something
or are you perhaps feeling depressed?”
She stares at her patient, willing him to speak.
Instead, he slowly turns his attention to her gaze and twitches
his nose, his small furry body practically swamped by the chaise
on which he is strapped.
Model behavior
Unlike their human counterparts, animal models of neurological
conditions like bipolar disorder, schizophrenia and anxiety
disorders simply cannot tell you how they are feeling or whether
they have suicidal tendencies.
Back in 2010, Mount Sinai School of Medicine’s Eric Nestler
and Harvard University’s Steven Hyman spelled out the challenges.
“Many of the symptoms used to establish psychiatric diagnoses
in humans (e.g., hallucinations, delusions, sadness, guilt) cannot
be convincingly ascertained in animals,” they wrote in Nature
Neuroscience. “When there are reasonable correlates in animals,
(e.g., abnormal social behavior, motivation, working memory,
emotion, and executive function), the correspondence may only
be approximate.”
In part, they averred, this is because little is known about the
pathophysiology of most states contained within the Diagnostic
and Statistical Manual of Mental Disorders, 4th edition (DSM-IVTR)
and there are few, if any, objective diagnostic tests.
“Consequently, diagnoses are based solely on phenomenology;
i.e., on symptoms, signs and course of illness,” the authors explained.
“As a result, the boundaries between DSM-IVTR disorders, and
the boundaries between disorder and normal variation, are often
Drug Discovery News
arbitrary or hazy. This state of affairs creates enormous hurdles for
the development and validation of animal models.”
As the number and variety of organisms modeling human neu-
ropathology increases, the number and variety of techniques used
to validate these models also continues to expand. And as with
behavioral and histological assays, researchers continue to take
their cues from common practices with human patients.
A good example of this is in the growing practice of in-vivo imaging,
including MRI, PET, SPECT, ultrasound and optical imaging.
“The way that we apply imaging is to look how clinical doctors
or neurologists are monitoring pathological events in patients’
brains by using, for example, MRI or PET scanning,” explains Antti
Nurmi, director of science for Charles River Discovery Services at
Charles River Laboratories. They then apply the same processes
to their rodent models.
“This is actually a very strong benefit from a translational point
of view, where we want to think about how well the results from
these rodent models correlate with what is happening in the
human brain,” he continues.
That said, the use of neuroimaging in animal models is still in
its nascent stages, according to Drew Heinmiller, photoacoustics
product manager for VisualSonics, noting that the field lags
significantly behind other therapeutic categories such as cancer or
cardiovascular disease.
“There’s not a lot of in-vivo imaging going on in neuroscience,”
he says. “It’s all histological, neurohistochemistry, microscopy.
It’s really trying to understand the cellular mechanisms at the
cellular scale.”
The imaging systems developed at VisualSonics cannot resolve
single cells, he says, reaching down to about 30 microns—but not
all analysis has to occur at the cellular level.
“We’re imaging function of the brain in a live animal, which I
think is actually an important step, especially in something like
neuroscience where I think it is actually a little bit further behind
30
 DDN Best of Neuroscience
in terms of the field in general because the brain is so complex,”
Heinmiller suggests.
“The challenge from an imaging standpoint, I think, is going to
be sensitivity,” he continues. “Being sensitive enough to any kind
of molecular probe you use is going to be a challenge. There are
plenty of groups working on that.”
One company that is working closely with neuroscientists is
PerkinElmer, which extended and expanded its relationship with
PET system developer Sofie Biosciences last summer.
“We are particularly interested in the use of our G8 PET/CT
system to advance the investigation of Alzheimer’s and Parkinson’s
disease research,” says Olivia Kelada, PET imaging applications
scientist for PerkinElmer.
A HUMAN APPROACH: As the number and variety of organisms
modeling human neuropathology increases, the number and variety
of techniques used to validate these models also continues to expand.
And as with behavioral and histological assays, researchers continue
to take their cues from common practices with human patients.
Examples of this are found in the growing practice of in-vivo imaging,
including MRI, PET, SPECT, ultrasound and optical imaging with
animal models.
“One advantage of our system is the ability to image very low
activities of radiotracer due the high sensitivity of the PET detectors,”
she says. “This can be immensely helpful to detect drugs or probes
that struggle to cross the blood-brain barrier.”
Researchers have also used the system to validate novel tracer
biodistribution and specific uptake, moving CNS imaging away
from challenges associate with the use of [18F]FDG and [18F]
DOPA, she suggests.
Also working in the PET sector, Canada’s Cubresa recently
launched its NuPET platform, a MR-compatible PET scanner that
can work with an existing preclinical MRI to do simultaneous PET
and MRI imaging in small animal subjects. The goal with this unit
is to give researchers the best of both imaging worlds.
“You have MRI, which is a phenomenal modality for anatomical
and functional neuroimaging,” explains Michael Simpson, director
of marketing at Cubresa, whereas the PET modality allows you to
“directly image a neurotransmitter or directly image the binding
potential of a particular drug.”
Drug Discovery News
Thus, he says, within a single short imaging study, you can
monitor the effect that drug might be having on a functional
endpoint if you are, for example, using functional MRI (fMRI) to
look at brain activation or blood flow.
“You start to be able to look at or tease away cause-and-effect,
to look at a mechanism of action, a little bit,” he adds.
Cubresa is not alone in their interest.
At the World Molecular Imaging Congress last September,
Aspect Imaging and Seoul National University announced a part-
nership to offer a complete PET/MRI platform for simultaneous
preclinical imaging.
At the same meeting, Bruker similarly introduced its
MR-compatible PET scanner insert that, according to University
of Leuven researcher Uwe Himmelreich, “enables us to produce
improved PET resolution through MRI-based motion correction,
and to guide external interventions in real time.”
Months later, Bruker expanded its portfolio further by
completing its acquisition of the preclinical PET imaging business
of Oncovision.
For Ross Nakatsuji, Cubresa manager of marketing communi-
cations, this understanding of mechanism isn’t just important for
new, experimental therapies, but also for existing, marketed drugs.
“A lot of times, historically, we’ve had these treatments and
we don’t fully understand the mechanism by which they work,”
he suggests. “This is a great opportunity to be able to see cause
and effect.”
“If you’re a drug company, you want to know how it works and
why,” he presses. “And then, you could take it a step further and
develop treatments for particular phenotypes, particular groups
of patients that have characteristics that you can test for. Then
the therapy becomes all the more effective at least for that subset
of patients.”
Beyond the traditional workhorses of the preclinical and clinical
labs however, technological advances are allowing researchers to
literally shine a light on the neurological spaces long kept dark by
the bones of the skull.
“An increasingly wide range of techniques make use of the
light absorption and/or scattering properties of brain tissue, and
specifically the hemoglobin present in the vascular system, in
order to obtain high spatial and temporal resolution readouts of
hemodynamic changes,” University of Sheffield’s Chris Martin
noted in 2014. “These techniques usually require visualization of
the brain either through a craniotomy or a thin cranial window
(the skull is thinned to translucency over the imaged brain tissue).”
Even with this invasive treatment, however—which potentially
complicates the experimental results—Martin also noted the
significant disadvantage of many standard optical imaging
approaches: limited depth penetration to just the first few hundred
microns of cortical tissue due to light scattering and absorption
by tissue.
Layering optical imaging atop its long-held expertise in ultra-
sound, VisualSonics may have a response to Martin’s challenge
in their newly launched photoacoustics platform.
“We essentially use pulsed laser light to generate an ultrasound
signal,” Heinmiller explains. “You pulse the laser in and anything
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 DDN Best of Neuroscience
that absorbs the light will heat up a little bit and give this ther-
moelastic expansion. That creates a pressure or sound wave that
comes through the tissue and then we listen to that.”
The system uses light in the near-infrared (NIR; 680-970 nm)
and NIR II (1200-2000 nm) ranges.
“In that range, hemoglobin in blood is the main absorber,”
he continues.
He explains that although researchers could already visualize
blood in the form of a Doppler signal in ultrasound or through
the use of contrast agents such as microbubbles, those techniques
could only image moving blood.
“If the blood’s not moving or is stagnant, which is often the
case in some tumors, you have hemorrhaging or stagnant blood
or nonfunctional vasculature, we wouldn’t be able to see it,”
Heinmiller explains.
This is not an issue for photoacoustics.
“Combining it with ultrasound for the anatomy, Doppler for
the blood flow, microbubbles for the perfusion, and now you’ve
got oxygen saturation, you combine all of these things and now
you’ve got a pretty powerful tool for looking at a variety of different
parameters,” he enthuses.
“And because it doesn’t use ionizing radiation, is relatively easy
to use, fairly high-throughput, now you can do these longitudinal
studies where you watch disease progression and measure all of
these different parameters as you go.”
And it is in the ability to do longitudinal studies that these new
platforms may find their greatest utility, as Heinmiller describes in a
collaboration with Emory University’s Alex Kuan, who studies strokes.
“He had this model of stroke where he’d ligate the common
carotid artery and then have the animal breathing lower oxygen
for about half an hour,” Heinmiller explains. “You [then] release
the ligation and you put the animal back on air or breathing
100-percent oxygen.”
“On the ligated hemisphere, you actually see a stroke forming,
but not on the non-ligated hemisphere.”
Looking for signs of stroke, however, required that some ani-
mals be sacrificed, the assumption being that all animals within
the study experienced the same degrees of impairment.
With Doppler and photoacoustic imaging, however, the
researchers could perform live imaging during the course of sur-
gical intervention.
“We were able to actually ligate the common carotid, watch the
drop in perfusion on the one side, and simultaneously measure
things like oxygen saturation, watching the drop in oxygen satu-
ration,” Heinmiller says.
Charles River’s Nurmi likewise sees the benefit.
“One of the big justifications for us to invest in small-animal
MRI units, PET/CT and SPECT/CT units for this preclinical
imaging was to minimize the use of animals in the experiments,”
he explains. “It is a massive opportunity for any study if you can
monitor longitudinal progression of the disease or potential
alleviation of the disease or therapeutic reversal when you’re
developing new drugs.”
Rather than needing 250 animals for a study, he suggests, the
longitudinal opportunities might reduce that need to 40 animals.
Drug Discovery News
Heinmiller adds that “you’re essentially using the same animal
as its own control.”
That said, there are still many reasons to move forward cautiously,
as neuroimaging in humans and model animals is not identical.
“Technical differences in performing the neuroimaging in
animals and humans can influence our interpretation of in-vivo
neuroimaging data,” says PerkinElmer’s Kelada. “For example,
there are both temporal and spatial constraints that need to be
considered during the interpretation of both neuroimaging signal
and neurovascular coupling.”
“In addition, the use of anesthesia in animal research studies
can restrict the translational implications of findings especially
for fMRI data,” she continues. “Also, many of the major neuro-
psychological questions that are investigated in humans cannot
be explored in current experimental animal models, such as the
sleep-wake cycle.”
Move to true multimodal
Because each of the imaging modalities offers both strengths and
weaknesses in the types of data it produces, researchers have long
taken a multimodal approach to studying both human patients
and animal models.
“In general, CT, MRI, and [ultrasound] are anatomic imaging
methods but they have low sensitivity,” highlighted Third Affiliated
Hospital of Guangzhou Medical University’s Zhi-Yi Chen and
colleagues in 2014. “Radionuclide imaging and optical imaging
are functional imaging techniques, while they suffer from low
resolution, which often lack structural parameter.
“The combination of different molecular imaging techniques—
namely multimodality imaging—can provide synergistic advantages
over any modality alone and compensate for the disadvantages of each
imaging system while taking advantage of their individual strengths.”
According to Heinmiller, part of the interest is simply improv-
ing experimental efficiency.
“We go to shows and you see more and more of these multimodal
things because people want to get more data out of the same
experiment,” he recounts. “Getting just one form of dataset, it’s
almost not enough anymore.”
But the serial application of these technologies offers its own
constraints on data acquisition and interpretation.
“The theme of multimodal imaging is not new,” Cubresa’s
Simpson acknowledges. “And in the preclinical setting, microPET
or microPET/CT scanner and a preclinical MRI system would be
two tools that have really been around for a while and are mature
technologies that a lot of core imaging facilities have today.”
But as he further explains, the “multimodal” image for many
such experiments comes as a coregistration of the data after the
imaging sessions have been completed and the data is merged.
But coregistration is no easy task as many changes may occur
in the shift from one modality to another, including the physical
movement of the test subject. Thus, there is a growing interest in
platforms that can perform multiple imaging approaches simulta-
neously—true multimodality—such as those offered by Cubresa
and VisualSonics.
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 DDN Best of Neuroscience
Says Simpson, this offers researchers “the ability to simultaneously
acquire that data by essentially having this single combined
instrument with a single overlapping PET and MRI field of view that
can acquire the data very accurately in that small animal, and that
registration is not happening after the fact, but is kind of happening
implicitly in the combined scanner.”
And this can be especially important when neuroimaging very
small animals, Simpson presses.
“Their brains are very small,” he explains. “The emphasis on
higher resolution is very important in the preclinical setting, so
that you can actually have sufficient resolution to look at compo-
nents of the rodent brain in sufficient detail.”
Simpson further goes on to explain that animal physiology—
typically much faster than that of a human—is a further complicat-
ing factor, adding to the need for simultaneous image acquisition.
“[With] sequential registration of two separate systems, you’re
losing a lot of confidence in being able to correlate what you
see on MRI with what you see on PET,” he suggests, adding that
researchers have a “much better ability to elucidate the relation-
ships between some of these processes by virtue of looking at
them together.”
Shortening the window of the imaging time, he continues, offers
many benefits for the precision of the preclinical imaging study.
“It lowers the amount of imaging time,” he says. “It lowers
the window for physiological changes to happen, so you have
maybe a little more control over the measurement system, which
is animal physiology.”
And it lessens the cumulative effect to anesthesia, which is
known to impact neuroimaging, as Kelada noted earlier.
But it’s not just about the instrument, presses Cubresa’s Nakatsuji.
“It’s about developing radiotracers that are designed to target
certain things, certain neurotransmitters, and then having the
ability to see it in great detail with the MRI component to study
the response,” he argues.
Chen and colleagues said much the same thing.
“It is still difficult to carry out multimodality imaging due to
existing problems regarding the accuracy of coregistered image, extra
ionizing radiation, the extra dosage of contrast agent and the toxicity
of fused contrast agents,” the authors wrote. “Therefore, it is in urgent
need of developing multimodality molecular imaging agent.”
Agents of change
Traditionally, contrast agents have been used to monitor gross ana-
tomical and physiological changes within an organism, highlight-
ing, for example, disruptions of blood flow and the blood-brain
barrier or identifying accumulations of neurofibrillary tangles
and plaque in the brain.
In recent years, however, there has been a steady growth in the
use of ligands, whether small molecules or peptides, to monitor
change at the molecular level, such as the presence and activity
of neurotransmitters or signaling itself.
As though hearing Chen’s call, Stanford University’s Sanjiv
Gambhir and colleagues recently reviewed the growth of
nanoparticle-based imaging agents, focusing their discussion on
those already approved for clinical use.
Drug Discovery News
“Nanoparticles are a new and exciting class of imaging agents
that can be used for both anatomic and molecular imaging,” the
authors wrote, suggesting that the small size and unique properties
of nanoparticles offer:
• Intense, longitudinally stable signals;
• Different targeting strategies, whether passive via the mono-
nuclear phagocyte system or active via ligand targeting;
• High avidity as multiple ligands can be added per particle;
• Theranostic capabilities, as a single nanoparticle can be used
for both diagnostic and therapeutic purposes;
• Multimodal signal capabilities as, for example, one nanopar-
ticle can be detected by MRI for deep tissue imaging and
screening as well as optical imaging for intraoperative guid-
ance; and
• Multiplexing, as a nanoparticle can be functionalized to
detect various molecular targets simultaneously.
Speaking directly to the optical space, Heinmiller describes
agents that now can be used with photoacoustic imaging because
they absorb light: NIR fluorophores, for example, and gold or
oligomeric nanoparticles that absorb NIR light very strongly.
“So now we can have a nanoparticle, attach some kind of targeting
moiety to it, inject it systemically and see it accumulate in various
disease models,” he says.
From his perspective, how these molecular contrast agents
evolve will in many ways dictate how photoacoustics evolves.
“It’s sort of like the situation with fluorescent imaging,” he says.
“You have to follow the optical probes, how good they are and
how sensitive you are to them. I do see the two fields developing
in parallel and with help from each other.”
He recounts his recent experiences at the SPIE Photonics
West meeting.
“They had a whole section on PA imaging, and there was some
work presented by these calcium-channel signalling molecules,
where they would basically change their optical properties based
on the calcium concentration,” he recalls excitedly.
“There are significant limitations there,” he recognizes. “For
example, depth of penetration and the sensitivity. So right now,
they’re looking at things like fruit flies and zebrafish. But they are
using photoacoustic imaging to do it.”
“You can see the firing of neurons noninvasively,” he enthuses.
“That is absolutely huge.”
“Is it here yet in the mouse model?” he says with a shrug. “I
would say not yet. But that’s where people want to go with it.”
Although the new and advancing in-vivo imaging modalities are
unlikely to replace existing methods used to characterize animal
models of human disease, their increased use is likely to provide
ever-expanding insights with ever-clearer sights in.
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 DDN2018
FROM Best of Neuroscience
COMMENTARY
BRINGING PRECISION MEDICINE TO
THE EVALUATION AND TREATMENT
OF MENTAL ILLNESS
DR. ANTONY LOEBEL OF SUNOVION PHARMACEUTICALS INC.
M ental illnesses are qualitatively different from other
diseases. Most of the time, psychiatrists can’t use bio-
chemical tests or MRIs to diagnosis mental illnesses;
instead, they observe how patients behave and ask questions
about thoughts and feelings. Psychiatric medications are com-
monly developed, approved and prescribed without a precise
knowledge of how they may work in individual cases. We simply
don’t understand the underlying genetics and biology of mental
illness in the same way as other medical conditions or even other
brain disorders, such as multiple sclerosis or Parkinson’s disease,
for example.
However, we can use what we do know about the genetics,
neurobiology and behavioral science of mental illness to improve
diagnosis and treatment. The National Institute of Mental Health
has pursued this strategy since 2013, when then-director Tom Insel
launched its Research Domain Criteria (RDoC) project. The idea is
to move away from using the lists of symptoms describing specific
mental disorders, as noted in the Diagnostic and Statistical Manual
of Mental Disorders (DSM), and toward an approach that empha-
sizes what is known about the biological roots of mental illness.
This transition is essential if we are ever to bring mental illness
into the precision medicine era. In its purest form, precision
medicine relies on genetic data or biomarkers to develop and tailor
treatments for individual patients. We’re still a long way from being
able to do that for mental illness. But in the case of schizophrenia,
an analysis we recently performed lays the groundwork for further
steps in that direction.
Schizophrenia is an incredibly diverse disease, with a broad
spectrum of symptoms that vary from one person to the next.
One patient may hear voices and harbor delusions, while another
Drug Discovery News
may show disorganized behavior and have difficulty learning or
comprehending tasks. Yet another may become extremely isolative
and close him or herself off from the world.
But the reality is that antipsychotic drugs are only indicated
for the overall treatment of schizophrenia, not for specific types
of symptoms. As a result, psychiatrists rely on past experience
and professional expertise when needing to treat specific types of
symptoms. This may work well in many cases, but it’s not exactly
precision medicine.
This problem is also encountered in developing new medicines to
treat patients with schizophrenia. The most widely used assessment
of schizophrenia severity, called the PANSS (Positive and Negative
Syndrome Scale), is used in most schizophrenia clinical trials.
However, the scale broadly encompasses many areas affected by the
illness and, 30 years after its introduction, lags behind our growing
understanding of the nature of schizophrenia symptoms.
Because the PANSS total score is a composite—like the
often-criticized IQ measure of intelligence—it is difficult to
understand which specific symptoms a treatment addresses. For
researchers, this limitation makes it nearly impossible to assert
that any improvements are in the areas of current unmet need
we hope to address.
To overcome this, our team recently published a study that
used a novel method to analyze the PANSS score. Using this new
statistical analysis, we were able to build and improve upon PANSS
to isolate different clusters of schizophrenia symptoms.
This is just one example of how improving psychiatric assess-
ments could pave the way toward more precise evaluations of new
treatments with more specific clinical endpoints. It also poten-
tially allows regulatory bodies to include these updated scales
and their results in treatment labels, so physicians have more
information to guide prescribing.
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 DDN Best of Neuroscience

Unlocking the full potential of precision medicine in mental

illness will require not only refining our psychiatric assessment
tools, but a better understanding of the myriad of factors that
influence how these disorders emerge and develop.
For example, researchers at Massachusetts General Hospital
and Harvard Medical School have developed a new method to
extract symptom information from electronic health records that
predicts length of hospital stays or readmission risk in neuropsy-
chiatric illnesses. Researchers at Stanford University are using
brain imaging, along with other clinical and social information, to
identify “biotypes” of depression and anxiety that can be studied
with different treatments in clinical trials.
Ultimately, these efforts will enhance the efficiency of clinical
development and empower physicians, researchers and patients
with more reliable and comprehensive information about the
nature of serious mental illness. Through better diagnostic tools
and deeper insights into the biological, psychological and social
variables contributing to each person’s mental health, we can
begin to bring precision medicine approaches to this area of great
unmet need.

Drug Discovery News 35