CrossTalk

Photo by Michael Ruff

Candace Pert

Paradigms from Neuroscience: When Shift Happens

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CrossTalk

andace Pert has spoken often in recent years about how lay audiences can
incorporate the results of modern neuroscience into their own daily lives. Her
lectures often touch on material from her 1997 book, Molecules of Emotion,
now in its twelfth printing, and she in fact refers to her lecturing, somewhat
exasperatedly, as the book tour that never ends. Given the frequency of her lectures, it is
therefore somewhat disarming, as she sits down to talk, that she makes a voiced effort to
relax, explaining that she is particularly excited about our interview, because unlike her
airy-fairy lectures, she here feels that she is addressing her people, neuroscientists.
She smiles proudly as she points out that she attended the very first meeting of the Society
for Neuroscience some thirty years ago, and she is excited to be returning to the meeting in
New Orleans this year to speak about the neurotherapeutic effects of a peptide that has
recently shown dramatic efficacy in the treatment of AIDS. The peptide drug was in fact
written up just last month in the Wall Street Journal, and
Pert is hoping that the clinical success will not only make
a contribution to AIDS, but will further underscore the
potential for peptide drugs in a wide variety of clinical
and experimental settings. Perts interest in bioactive
peptides began with her landmark studies, reported
when she was still a graduate student in Sol
Snyders laboratory, in which she first described
the opiate receptor.

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Interview with Candace Pert

MI: You made such a tremendous splash in pharmacology even as

you started out as a graduate student. How did you happen to get
into the field?
CP: When I was an undergraduate, I was initially an English major. I
was the editor of the yearbook and the literary magazine, and I was a
poet. So, I have to give credit for entering into science to my first
husband, Agu Pert, because he made me aware that Id always been
good at science. Agu was thenand still isa psychologist, now at
the NIMH. It was through my love of psychology that I had met him.
And we developed a common dream, which basically was, together,
to study the brain from two perspectives. Wed spend hours thinking
about it, and I feel like I have an honorary degree in behavioral
psychology because I used to go to all these great seminars and meet
the great people that came to speak at Bryn Mawr, where I was an
undergraduate at the time. And I want to say that Agu and I attended
the very first meeting of the Society for Neuroscience, with only a few
hundred people in attendance.
MI: And you said that you were going to be talking at the meeting
this year?
CP: Yes! Im very excited. I have a ten-minute oral presentation, and
so Im like a wreck. Its frightening, though Ive lectured all over the
world.
MI: Can you say why you feel more stress when it comes to speaking
to a scientific audience?
CP: Its the difference between art and science. When I speak about
hard experiments, there has to be the element of scientific rigor. Im
going to be talking about a new study, and its not just mine. Ive had
the privilege of working with Dr. Susan Wilt, a brilliant young woman
scientist. Im going to be talking about a very elegant rat model of
neuroinflammatory disease, caused by murine leukemia virus, in
which all of the components can be visualized and studied, at the
cellular level. The virus causes profound neurodegeneration by
infecting the endothelial cells of the brain and leading to activated
microglial cells, which are very hot in neuroscience, and infiltrating
neutrophils, so the animal has all kinds of rapidly progressive
symptomstremors, ataxia, spasticity, and hindlimb weakness. Its a
beautiful model, because youve got a behavioral time course, youve
got incredibly beautiful opportunities for visualization of cellular
components. And were most excited that Peptide T, the drug that
weve been developing in the distinct context of AIDS, delays the onset
of the baby rats neurodegenerative symptoms by nineteen days!
So when I get up there, I have to honor my collaborator as well
as the science itself, and I cant make any mistakes. I have enormous
respect for scientists and for the whole scientific culture and its truth
seeking. It just has to be right. Im looking forward to returning to
the fold at the neuroscience meeting. Its been a long time since I have
presented at one. Im particularly excited to be going back to Sol

Snyders laboratory reunion, and seeing him and so many of my old

friends and colleagues from over the years.
MI: Is this the first time that youve looked at the effects of Peptide T
with regard to neurological disease per se?
CP: Well, no. Its the first time in a long time because weve had our
hands full, Dr. Michael Ruff and I, in developing Peptide T as an AIDS
drug. But in 1996, we published, along with Gary Arendash in
Peptides, a paper by Socci et al. showing very good reason to think
that Peptide T might be useful in Alzheimers disease. We showed that
when you lesion the basal forebrain nucleus of the ratand to this
day it is still true that this nucleus is damaged in every person with
Alzheimers disease whether by virus, toxic insult, or head trauma
you see cortical shrinkage and neuronal loss that could be almost
completely prevented by chronic post-lesion treatment with Peptide T.
It is very dramatic, stopping the typical post-lesion shrinkage of the
brain.
MI: Now, why would Peptide T be helping in a neuroinflammatory
disorder? The genesis of Peptide T is that it was based on a protein
from HIV, wasnt it?
CP: Well, its all about chemokines, which are the hottest new class of
peptidergic activity in brain and inflammation. They got hot after it
was discovered in the late 90s that HIV uses chemokine receptors to
enter and infect cells. We showed in Nature in 1988, with Doug
Brenneman and Joanna Hill, the HIV envelope (gp120) alone can
kill neurons -you dont need virus to kill neurons, and Peptide T
prevents neurons from being killed by the envelope protein. We now
know that Peptide T antagonizes chemokine receptors, which we
were the first to show modulate neuronal survival in development and
protect against gp120induced neuronal cell death. Chemokines and
their receptors were first found on immune cellsthe name comes
from chemotactic cytokines. But theyre also on glial cells, and even
neurons there are so many chemokines. And we know that
neuronal degeneration itself is supported by chemokines. Peptide T is
a mixed agonist/antagonist, probably on a number of receptors. But
more important, work in neuroAIDS has shown that HIVand
probably many other viruses with envelope proteins shaped to fit
receptors on the loose- cause havoc not by actually infecting and
destroying cells, but by taking up residence, in a tiny percentage of
cells that can move from the periphery into the brain, and usurping
cellular communication. And what were saying is that by targeting
the receptors that viruses exploit, you can have very fine neutralizing
drugs that act beyond merely stopping the spread of infection.
As to the genesis of Peptide T, it came out of one of the very first
computer-assisted database searches. Now you could do such a search
on a laptop, but at the timearound 1985when we did it in the
National Cancer Institute, the computer was bigger than a large room,
and we had to go out for a long lunch while we waited for the
printout. But what we were seeking was sequence homology between
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gp120 and any peptide in the database.

Based on our database search, I ordered four peptides to be
madeI had learned a heck of a lot of peptide pharmacology from
the endorphin-focused international meetings of the late 70s. I started
my career as a receptorologist, and the way that you prove that
something is really binding to a receptor and not to a piece of gunk is
through structure-activity relationships. So, we did a binding assay
with labeled gp120 versus each of the four peptides (including
Peptide T). We found that three, but not the fourth, of the
compounds were really potent inhibitors of gp120 binding! I was so
excited, that I called up Frank Ruscetti, the expert virologist from the
NCI who was collaborating with us then and is still crucially involved,
and he had found that the same three peptidesand again, not the
fourthblocked the growth of HIV in the test tube. At that point, I
was off and running, and it didnt matter to me who would have said
it was wrong, because I knew it was right. I knew it was right.
I say it didnt matter to me who would have said we were wrong,
because in fact there were several very well respected people who said
they couldnt repeat our results with Peptide T, and I think many
people consequently thought that we were crazy and that we had
gone off the deep end. But there was a great book review in Science
magazine recently about the premature discovery. The premature
discovery is something that comes along before the underlying
supporting technology or scientific logic is available for us to make
sense of it all, and so it seems crazy to people.
The idea at the time we discovered Peptide T was that the
envelope protein used specific receptors to enter and infect cells, and
in fact there was support for this idea in the literature at the time. So,
we assumed that Peptide T was interfering with viral entry into the
cell by binding to this receptor, namely, the T cell antigen CD4,
thought then to be the only receptor for HIV. And we identified a
band on a gel with a receptor binding assay, roughly with the same
molecular weight as CD4, and concluded that Peptide T functioned
by binding to the CD4 receptor. We now know of course that it was
really the chemokine receptorreferred to now as the co-receptor for
HIVthat had a molecular weight similar to CD4, and so we had the
right idea, and the right peptide, but the wrong mechanism in our
1986 PNAS paper, eight years before the first chemokine was even
discovered.
We now know much more about HIV infectivity. Most HIV
viruses use either CCR5 or CXCX4 receptorstwo subtypes of
chemokine receptorsand it turns out the CCR5, which is the one
that Peptide T works on, is most important. In the early stages of the
disease, for the first few years, the virus generally uses only CCR5;
later on in the course of the disease, tropism depends on the CXCX4.
The X4 lab-adapted strains of virus, which all labs other than
Ruscettis used at the time, are virtually insensitive to Peptide T. So
when I danced into the 1987 AIDS conference, used to being Miss
Adorable from my graduate student days, and now a Section Chief of
the NIMH, I was in for a very rude awakening: Everyone else was
working with this X4 lab-adapted strain first isolated by Montagnier
and Gallo, and of course they all said that they could not repeat our

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experiments. To get a really good R5-dependent virus, you need a

primary isolate. Frank Ruscetti, who had worked with Gallo to
discover IL-2, was using an R5-dependent virus that he had isolated
himself. Back then, people didnt know that there were two kinds of
viruses, but the ideathat the drug binds to the receptor, and
consequently, the virus cant get inwas right. We have since verified
the fact that Peptide T binds to CCR5 in all sorts of ways, using cell
lines transfected with CCR5 receptors to show that Peptide T is a
mixed agonist/antagonist at CCR5 receptors. And Michael Ruff went
around to three different HIV laboratories and got them to replicate
the potent Peptide T effect using the appropriate virus in cells that
have the CCR5 receptor, publishing the collaborative paper in AntiViral Research in 2000. And I think that thats the theme of life:
Youve got to get it right.
To this day, in 2003, were the only people who have envelopederived peptides that block HIV infectivity at low concentrations.
MI: Having started out as a psychopharmacologist, what made you
first enter research into AIDS and Peptide T?
CP: I had been very interested in the causes of schizophrenia, and I
sensed that there was really an immunological link. I began spending
time with immunologists, and one of them was so brilliant and so
wonderful that I finally ended up marrying him: Dr. Michael Ruff.
And we were fascinated by the continuity between the brain and the
immune system. We published a paper in 1985 called Neuropeptides
and Their Receptors: A Psychosomatic Network. We started

Interview with Candace Pert

exploring that neurons and immune cells have the same receptors and
make the same peptides. People talk today about cellular traffic
between the brain and the immune system, but there is more to it
than just traffic. Its not just brain stem cells that make new neurons
in the adult, although this is an exciting finding that is finally
accepted. But there may actually be bone marrow cells in your
shinbone that can find their way into your brain and become
neurons, and that is incredible: the psychoimmunoendocrine
network. Its the same cells, starting life in the body and winding up
as neurons in the brain. It is truly a paradigm shift.
MI: Has Peptide T made a big splash in AIDS research?
CP: Well, Michael and I have been working on the development of
Peptide T for over seventeen years, during which time we reported
the things that Ive been talking about. Now weve just published, in
Peptides, several important findings from an eleven-person clinical
trial, and the Wall Street Journal article is a great break in helping us to
move it to the next level. Im most excited about helping to create a
business structure and raising the funds that will allow pivotal Peptide
T phase II trials to start, so that the drug can get out. Im also working
on trial designs and on optimizing the formulation of the drug before
we organize the next trial. Ive spent seventeen years on this, and Im
prepared to spend seventeen years more, but I dont think its going to
take that long.
MI: Well, what did you report in the eleven-patient study?
CP: There are a number of positive changes in the patients. One of
the important findings from the trial is that half of the patients treated
had a statistically significant increase in their CD4 cells, which people
are now thinking is the main solid indicator that clinically significant
immunomodulation has occurred. There was also a sixfold increase in
the number of
-interferonproducing T cells, which are the cells that
are charged to kill virus-infected cells. We showed that the actual
cellular viral levels that you can isolate drops drastically, until after six
months of treatment, we couldnt isolate virus from any of the
subjects cells. We used stringent molecular biology methods and
found, for the majority of these eleven people, that Peptide T leads to
undetectable virus levels in monocytes. This type of cellular flushing
of virus is very big, because we already have so many great AIDS
drugs that can make plasma levels of virus disappear, but none of
them touch the virus that is sequestered within the cellular reservoirs.
Thats the reason that the virus can mutate as soon as patients miss a
few doses of their medication or comes roaring back within a few
weeks of stopping medication. Peptide T is completely non-toxic as
previously seen in phase I trials. So were hoping that cellular
reservoirs of virus can be substantially flushed in placebo-controlled
experiments.
MI: Is raising money for your next round of trials something that
comes natural to you?

CP: It can be exhausting, and it is amazing what we all have to do to

pursue our work, right? But the short answer is it comes neither
naturally nor easily, but Michael and I are willing to do what needs
to be done. This meant leaving a tenured position at the NIH, where
I had an annual budget of two million dollars and a bunch of people
working for me. We took the risk, and there have been some really
lean times. I did an interview with someone who asked, Why did
you write Molecules of Emotion? And the answer was, To make
money! The advance that I got put our daughter through college.
So, at times, our work lives with Peptide T have been really
challenging, and I have to say that Michael Ruff, my collaborator and
husband through all of this, has always reminded me that science
leads the wayI just have such respect for him as a scientist. There
really is something about truth, and its just something when people
discover reality together. Its just extremely exciting, the power of
science. And I love being a scientist more than anything.
MI: You speak about the breakthroughs that are finally happening
with Peptide T. Can you talk about your first big breakthrough, that
is, the discovery of opiate receptors? What was the key element from
your perspective that made it work?
CP: The key in the breakthrough from a scientific perspective was
when I read the work of Patton, the British pharmacologist, who had
a theory that he called the ping-pong theoryI still dont know if this
theory is right or not, but it led me to do the right experiment. He
claimed that antagonists tended to stay on the receptor, whereas
agonists bumped on and off, which led me to say, Aha! I need to get
naloxone radiolabelled, since I had tried many radiolabelled
agonistspurifying them myself in the old days- and none had
worked. At the time, there were several papers in the literature that
said, There is no such thing as an opiate receptor because our lab
tried to find it and we couldnt do it. But I was convinced by the
pharmacological literature that there had to be an opiate receptor, and
I kept at it, experiment after failed experiment, playing with the
numerous variables as rationally as possible. When the big eureka
moment finally came after many months it was incredibly exciting,
and the privilege of collaborating with Sol was a true landmark in my
life.
MI: What in your mind is the real value of the 1973 opiate receptor
discovery that launched your remarkable career?
CP: You mean aside from landing us great staff positions at the
NIMH?
MI: (laughter) Right!
CP: Well, it was the very first usefulsimple and reproduciblein vitro biochemical method for studying a drug receptorand as it
later turned out, after the discovery of endorphinsit actually was a
neurotransmitter receptor. In our early papers, Sol and I collaborated
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with many renowned opiate pharmaceutical chemists and showed

that the simple binding method could amazingly, perfectly, predict
pharmacological activity, and so it could be used to design drugs,
getting rapid feedback with tiny amounts of test compounds.
Actually, our discovery that intrinsic activity- whether an
opiate is an agonist, an antagonist, or something in the middle of
the spectrum-could be beautifully predicted from its sodium
response ratio in the in vitro binding assay [Pert and Snyder,
Molecular Pharmacology, 1974]. This assay was as important as the
original discovery because it made it possible to peg a new
chemicals property in a few hours rather than in weeks or months
of tedious and expensive in vivo testing. This was a big deal in the
search for a non-addictive painkiller, which was believed to need
mixed agonist-antagonist properties. It still astounds me how a
binding assay in crude brain membranes can be used to make valid
predictions. For example, an early paper published out of my NIMH
lab correctly surmised that the valium receptor was coupled to the
GABA receptor through the chloride ion channel well before good
neurophysiological or any molecular biological data proved this in
spades.

MI: What was it that brought you to your present position at

Georgetown University?

CP: Yes! In Sols lab, after the opiate receptor showed what was
possible, it was one receptor after another with different ligands and
slightly modified conditions. The brilliant students that he had
attracted and trained, who read like a list of Whos Who in
Psychoneuropharmacology, were starting the receptor-a-month
club. Anne Young, the president-elect of the Society for
Neuroscience, kicked things off back then by finding the glycine
receptor with its radiolabelled antagonist, strychnine. Later, my
NIMH lab focused on finding neuropeptide receptors like
bombesin, VIP, and the f-met-leu-phe receptor on immune cells.

CP: The reason we came to Georgetown is that Dr. Mike Lumpkin,

whos an expert on GHRH [growth hormone releasing hormone],
was at a talk Michael and I gave, and he noticed a close sequence
homology between Peptide T and GHRH. And that led to an
invitation to join his department and a series of collaborations,
related to the wasting and endocrine effects seen in AIDS, that
started with rat, published in PNAS, where we wound up showing
that gp120, at least pharmacologically, seems to compete with
GHRH receptors and block GH release both at the hypothalamic
and pituitary level. And Peptide T restores growth hormone
secretion, not just in rats, but also in a pilot study of children with
AIDS. And Georgetown has been so wonderful to us and I love the
medical school thereit has great science. Georgetown is leading
the way for integrative medicine. Our physiology department has a
major grant from the Complementary and Alternative Medicine
people of the NIH, and there are medical students who are actually
being studied who are doing meditation and doing new-paradigm
practices. CAM is now part of the curriculum, and all departments
are starting to contribute. This all appeals to my own
interdisciplinary interestswhich is why pharmacology is so
interesting in the first place. And then also, Georgetown carries the
Jesuit mission to do good in the world. Its a wonderful place, and
theyve been really good to us.

MI: So what were your NIH days like?

MI: So what do you hope to be doing in the future?

CP: Pure heaven. To quote Joni Mitchell, You dont know what
youve got til its gone. I got to learn about biological psychiatry from
Biff Bunney. Agu and I got to work on Biffs theory that lithium
works in bipolar disorder by damping oscillations in
neurotransmitter receptor sensitivity- this needs to be revisited
with modern techniques. Tom Insel, NIMH director, among other
scientifically based psychiatrists, actually passed through my lab to
learn the latest peptide and receptor techniques! And oh the pleasure
of working with the great scientists that I had as postdocs, who have
gone on to pharmaceutical and neuroscientfic gloryRemi Quirion,
Sandy Moon-Edley, Stafford McLeanto name three. Of course, the
greatest thing about NIH was the chance to collaborate without
stringsfor the sheer science of it allwith the greats, like Jesse
Roth, Steve Paul, and Elliot Schiffman.
My most fun long-term collaboration (excluding Agu and

CP: My dream is to be hanging out at an esthetically gorgeous

Institute for New Medicine at the Georgetown School of Medicine
where, funded by the successful commercialization of peptide T for
AIDS, a team of blissful scientists will be working on cures for
diseases by designing other short, stable receptor-active peptides
from the sequences of polypeptides and viral envelope proteins that
form the intercellular psychosomatic communication network that
modulates health and disease. Whew! Thats a mouthful of a vision!
Id love to be able to personally focus on schizophrenia and autism,
two neurodevelopmental diseases with a lot of interesting
implicatioins. I also have a plan to make a relaxation/wellness CD
that harnesses the twin transformative powers of great music and
sound scientific conditioning principles. And last but not least, I
hope to one day be bouncing a grandchild on my knee when Evan,
Vanessa, or Brandonmy childrendecide to cooperate!

MI: So the opiate receptor binding assay was the jumping off point
for other drug and neurotransmiter receptor assays?

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Michael, of course) was with Miles Herkenham to develop in vitro

receptor autoradiography taking it to practically an art form [see
the cover of this issue] and studying the detailed distribution pattern
of neuropeptides and their receptors. This later provided a scientific
rationale for calling them the molecules of emotion, and led to my
theory of emotions. You know, mapping receptors is where
biochemistry meets behavior.