X-Message-Number: 11225
Date: Sun, 7 Feb 1999 01:07:40 -0500 (EST)
From: Charles Platt <>
Subject: 21st Century Medicine Seminar Summary: Part 1
The following text is the first of three parts of a
summary of the seminar sponsored by 21st Century Medicine in
Ontario, California, on November 8th last year. This seminar
presented preliminary results of research that promises to
eliminate damage in cryopreserved organs caused by ice,
toxicity, and rewarming.
Originally I had hoped to circulate my summary of this
exciting research last December. I was delayed by problems
transcribing the tape, and by conflicting obligations. I
regret the delay but am now able to offer my summary,
accompanied by excellent reproductions of brain electron
micrographs, in a 16-page leaflet. Members of CryoCare
Foundation will receive this publication automatically;
anyone else can receive a copy free by sending a self-
addressed 9" x 12" envelope to Charles Platt, P.O.Box M,
Jerome, AZ 86331.
Alcor members will find a version of this text, with
lower-resolution photos, in the current issue of Cryonics
magazine. An abridged version, with fewer photographs, is
scheduled to appear in The Immortalist.
The complete seminar is available on a set of five video
tapes for $50. Individual tapes are also available for $15
each. Call toll-free 877-277-0322 or send a check or money
order payable to Life Extension Foundation, Box 229120 Dept.
21MED, Hollywood, FL 33022.
--------------------------------------------------------
Part One
21st Century Medicine Announces Unprecedented Results
in Cryobiology and Resuscitation Research
by Charles Platt
After 13 years of unsuccessful attempts to improve his
own best cryoprotectant formula, cryobiologist Gregory Fahy
has found a way to develop a whole new family of
cryoprotectant solutions that should enable organs to be
vitrified successfully in the near future. "Vitrification"
means changing a liquid to a glasslike solid as temperature
falls, without forming ice crystals that damage cells. For
twenty years, cryobiologists have questioned whether
vitrification of human organs will ever be practical. The
investigators at 21st Century Medicine should get the first
glimpse of the answer in 1999.
Concurrently, biophysicist Brian Wowk, a former
President of CryoCare Foundation, has discovered a different
family of cryoprotectant compounds which enable vitrification
at lower concentrations and higher temperatures. Wowk has
also developed synthetic "ice blockers" that enhance many
other cryoprotectants and eliminate problems associated with
rewarming vitrified organs.
Finally, Mike Darwin, founder of BioPreservation, has
led a highly successful initiative to minimize ischemic
injury--the damage that is caused by insufficient blood flow,
typically when the heart stops beating. Darwin's team now
holds the unofficial world record for resuscitating dogs
after up to 17 minutes of "death" at normal body temperature.
These multiple breakthroughs should enable preservation
of human brains with minimal or even zero ice damage, and may
lead to reversible brain cryopreservation within ten years.
If this goal is achieved, cryonics will not have to rely on
future technology to repair damage caused by freezing or
toxicity, and will take a major step toward credibility in
conventional science.
Long before that, however, the research will have
applications outside cryonics that should be highly
profitable for 21st Century Medicine and its stockholders.
Biologist Christopher Rasch, hematologist Nooshin
Mesbah-Karimi, and surgeon Yasumitsu Okouchi collaborated
with Gregory Fahy and Brian Wowk on their work, while Steven
B. Harris, MD, Sandra Russell, Joan O'Farrell, and Carlotta
Pengelley participated with Mike Darwin.
21st Century Medicine was founded in 1993 by Saul Kent
and Bill Faloon, long-time cryonics activists who run a
lucrative vitamin mail-order business and offer information
on dietary supplements via their Life Extension Foundation.
In 1997, after Kent and Faloon won a long legal battle with
the FDA, they purchased a second building for 21st Century
Medicine, hired additional personnel, and are spending
currently almost $2 million a year on research.
At a seminar on November 8th, 1998 in Ontario,
California, the principal researchers from 21st Century
Medicine described some amazing payoffs that have resulted
from the investment by Kent and Faloon, far sooner than
anyone expected.
The presentations were tantalizing, because key
information is being withheld while patents are being filed.
Still, a huge amount of information was communicated, and I
can provide only a partial summary here. 21st Century
Medicine is selling videotapes to anyone who wants the
complete version.
New Cryoprotectants
Brian Wowk began the presentations by describing his
search for cryoprotectant molecules that would bind less
readily with each other, and more readily with water
molecules, thus reducing viscosity and enabling faster
perfusion. "The idea that we came up with was to replace
hydroxyl groups on cryoprotectant molecules with methoxyl
groups," he said.
For example, propylene glycol consists of a chain of
carbon atoms, with two OH (hydroxyl) atomic groups attached
to the first two atoms in the chain. Wowk proposed replacing
one of the hydroxyl groups with an OCH3 (methoxyl) group,
creating a methoxylated version of propylene glycol. "We can
make similar modifications to a variety of other standard
cryoprotectants," he said. "If you do this, you get some
rather dramatic results."
In the case of propylene glycol, the methoxylated
version is almost 100 times less viscous than the regular
version. Ethylene glycol and glycerol can be modified in the
same way, though the improvements are less extreme.
The modified compounds penetrate cells much faster than
conventional cryoprotectants. Ethylene glycol is one of the
most penetrating cryoprotectants known, but the methoxylated
version gets into red blood cells about four times faster.
Better still, the methoxylated compounds inhibit ice
formation and enable vitrification far more effectively. Wowk
showed a cooling curve for a 45 percent glycerol solution,
and another curve for methoxylated glycerol. The former
indicated significant ice formation; the latter showed
virtually none.
Moreover, methoxylated compounds vitrify at higher
temperatures. Wowk predicted that in the future, we won't
need to use liquid nitrogen for long-term storage because a
suitable cocktail of methoxylated compounds should vitrify
above -79 degrees Celsius (dry-ice temperature), which will
reduce storage costs and the risk of structural cracking.
One problem with the new compounds is that they are more
toxic to cells. However, Wowk has found that toxicity can be
mitigated by mixing appropriate compounds. In the lab,
viability of cells has been measured in terms of their
ability to pump potassium and sodium ions across their
membranes after exposure to and removal of cryoprotective
agents. Ultimately Wowk found that if he replaced propylene
glycol with methoxylated glycerol in VS4-1A (the previous
state-of-the-art cryoprotectant developed more than ten years
ago by Gregory Fahy), it produced no more injury than VS4-1A
itself. Given that VS4-1A formerly was the least toxic
vitrifying agent known, Wowk felt that this was "a pretty
impressive result." However, he went on, "Dr. Fahy completely
destroyed these results with new results that surpassed them
by almost an order of magnitude."
Another Cryoprotectant Family
At this point during the presentations, Gregory Fahy
took the microphone from Brian Wowk to describe his own
discovery. He began by noting the mysterious behavior of
cryoprotectants. "We don't understand their toxicity, and we
can't predict their toxicity," he said. He added that "there
is no consensus, no common denominator, no basic grasp of
what it is we are seeking and how to get to a less toxic
solution."
Initially he suspected that solutions which are more
liable to denature proteins would be more toxic--but found
that just the opposite is true, which "makes no sense." He
also thought that a less-concentrated solution would be less
likely to disrupt biological systems, but found inconsistent
correlation between concentration of cryoprotectants and
viability of cells.
In 1998, Fahy came up with a novel idea to make sense of
the data. This led him to a new way to measure concentration
of cryoprotectants, which does correlate properly with
viability of cells. "Suddenly all the data points fall on a
straight line," Fahy told his audience at the 21st Century
Medicine seminar.
He would not reveal the exact nature of his insight, but
claimed it enabled him to understand how to reduce toxicity
in cryoprotectants more effectively than has ever been
achieved before. He came up with a solution which he calls
VX. For thirteen years he had been trying to find something
less toxic than his previous achievement, VS4-1A, a 55
percent solution of DMSO, formamide, and propylene glycol. VX
turned out to be the answer.
Using it as a starting point he developed four new
vitrification solutions, "each of which is statistically
significantly superior to the previous world champion
solution, VS4-1A." One of the new VX mixes should enable 100-
percent survival of perfused rabbit kidneys, according to
Fahy.
Still, this did not solve the problem posed by larger
organs that cannot be cooled as rapidly as rabbit kidneys,
and tend to suffer from increased ice damage as a result.
Fahy said he considered using "some tricks from nature" to
inhibit the ice crystal growth.
The trick he tried was an antifreeze protein found in
antarctic fish. When he added it to conventional
cryoprotectants in a standard salt solution (the solution
that carries the cryoprotectant into and out of organs during
perfusion), it achieved barely measurable results. However,
when he used a new solution to "carry" the cryoprotectant,
and then added the antifreeze protein, he reduced the amount
of ice formed in a dilute version of VS4-1A known as VS4 by a
factor of 1,000.
He also tried a third "vehicle solution" designed to
enhance a different antifreeze protein found in a species of
beetles. This reduced ice formation even more effectively, by
an additional factor of 10 when no protein was present, and
by an additional factor of 1,000 when beetle antifreeze
protein was present. The practical bottom-line result was
that he could achieve vitrification with a slow cooling rate
of 1 degree Celsius per minute--which is practical for human
kidneys--even using a version of VS4-1A that was diluted to
the point of virtual nontoxicity.
Also he found that the beetle protein would eliminate
another intractable problem: ice crystals forming when a
vitrified sample is rewarmed. Typically, a sample has to be
rewarmed extremely fast to get it from its deep subzero
temperature to above freezing point without ice crystals
causing catastrophic damage along the way. Since raising the
temperature of large organs rapidly is quite difficult, zero-
damage rewarming has always been a formidable challege. But
with Fahy's new vehicle and 1 percent beetle protein, he
found he could avoid ice formation at a warming rate of just
1 degree per minute, even with a solution so dilute as to be
essentially nontoxic.
"This is wonderful," he told the audience at his
presentation, "but beetle protein is hard to come by, and is
expensive. We wanted to come up with our own solution, our
own ice-blocking agent, which is dirt cheap. Why not? Let's
ask for the moon, maybe we'll get it. And luckily Brian found
the moon for us, and now Brian will deliver it."
Ice Blockers
Brian Wowk took over from Gregory Fahy at this point and
described his search for "synthetic ice blockers, hoping they
could be made more inexpensively than natural antifreeze
proteins." He mentioned that the beetle protein used in
Fahy's experiments costs about $1,000 per milligram. Some
researchers are working to synthesize a substitute, but Wowk
believes even this will be relatively expensive, plus its
ice-blocking action will be most effective near freezing
point. He wanted a substitute that would work at the much
lower temperatures required for organ storage.
"We were successful in this, almost completely
successful," he said. "We were able to devise a family of
synthetic ice-blocking molecules that are very inexpensive, a
small fraction of the cost of even fish antifreeze proteins."
He showed a graph of vitrification enhancement that
occurred when he added an ice blocker that he referred to as
21CM-X1 to a solution of dimethyl sulfoxide (DMSO). Without
the ice blocker, a 50 percent concentration of DMSO is needed
to avoid ice formation when cooling at 7 degrees per minute.
Adding 1 percent of the ice blocker enabled the same results
with 47 percent DMSO. "That doesn't sound like a lot," said
Wowk, "but in terms of toxicity it is."
Also, X1 turned out to work like beetle protein in
preventing ice damage during rewarming. "Even if you have a
perfectly vitrified system, generally when you rewarm it ice
forms in it like crazy," Wowk said. "However we found that by
adding very small amounts of X1 we may in fact have got the
devitrification problem under control at even very modest
rewarming rates." He showed a videotape of a lab experiment
in which a DMSO solution formed ice crystals when it was
rewarmed, while the same solution with a tiny amount of ice
blocker showed drastically less ice. Another video
demonstrated that a vitrified solution of ethylene glycol
could be rewarmed relatively slowly, without any ice forming,
if the X1 ice blocker was added.
(Part Two of this summary will be posted here tomorrow.)
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