Searching for Authentic Anti-Aging Therapies

Appendix

Searching For Authentic

Anti-Aging Therapies T

he Life Extension Foundation® understands that the underlying cause of death in older people is aging (the process by which we die). The diseases that kill most people in the U.S., including heart attacks, strokes, cancer, diabetes, Alzheimer’s disease, and Parkinson’s disease, occur with increasing frequency with advancing age. The reason for this is that the greatest contributing factor to the development of these diseases is aging. When our cardiovascular system, immune system, neuroendocrine system, brain, heart, liver, and kidneys age, they suffer progressive damage that renders us increasingly vulnerable to the diseases of aging. While the causes of death most often listed on death certificates are heart disease, stroke, and cancer, the number one cause of death in the U.S. is actually aging.

An authentic anti-aging therapy would lead to an extended healthy and more youthful life span that would prevent or postpone the diseases of aging in addition to extending the length of the human life span. The quality of life is tied inextricably to the length of life. We know this because an experimental technique called radical caloric restriction (CR) has been shown in hundreds

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of experiments in rats, mice, dogs, and non-human primates to extend maximum life span, prolong youth and vigor, and postpone the diseases of aging.

The Methylation Project The Life Extension Foundation has funded research by Craig Cooney, Ph.D. at the University of Arkansas to determine if dietary methyl and zinc supplements or a SAMe (s-adenosylmethionine) supplement would affect longevity, age-related pathology and relevant molecular parameters in rats. This project also aimed to generate knowledge about how various supplements affect s-adenosylmethionine, homocysteine, and DNA methylation in the blood.

When people are methyl deficient, they are at much greater risk of developing cancer, cardiovascular disease, depression, and a variety of neurological and other disorders. Methyl deficiency is characterized by too little s-adenosylmethionine, too much homocysteine or a loss of DNA methylation. SAMe is our chief methyl donor and is used for most methylation in our cells.

Methylating enzymes use SAMe but are inhibited by metabolic products related to homocysteine. The buildup of homocysteine leads to inhibition of methylation reactions and can also cause cardiovascular disease. DNA methylation is absolutely essential for mammalian development and is a key mechanism in aging and carcinogenesis.

Dr. Cooney maintained one-year-old rats for 12 months on their respective supplements. Their age-related pathology, tissue DNA methylation and other parameters were determined by measurements of blood levels of homocysteine, SAMe, leukocyte DNA methylation and standard blood chemistry measures such as cholesterol and glucose.

The methylation studies conducted by Dr. Cooney showed that all the compounds tested raised methylation levels in mice and reduced homocysteine levels. They showed that methylationenhancing therapies likely have a number of health benefits, but did not have any apparent effects on aging and longevity.

Here is a summary of Dr. Cooney’s report about funding provided by the Life Extension Foundation (LEF): “LEF support has also helped us to be in the position to do the following: A. Encourage the NCI to fund studies of diet, methylation, epigenetics and cancer prevention (see: http://deainfo. nci.nih.gov/concepts/DNAMethylation.htm).

B. Initiate an additional life span study of a methyl supplement using mice through an NIA funded collaboration.

C. Make progress on a program and facility for extensive metabolic multianalyte profiling of long-lived and short-lived animals. We anticipate that this program will exceed all currently existing programs for metabolic analysis in aging and that we will be able to analyze over 200 metabolites with just one or a few analyses.

D. Apply to the NIA for funds to study the effects on life span of epigenetic modification of mice through the maternal diet. We expect this will shed considerable light on the role of DNA methylation in life span.”

The Genesis Experiment For many years it has been known that adult animals (including humans) can be repopulated with missing cell types without which they would otherwise suffer death or disease. Perhaps the most common example of this is the administration of bone marrow via intraperitoneal or intravenous injection to repopulate the marrow of a patient who has undergone chemotherapy and/ or radiation treatment for cancer. Similarly, fetal cell transplants have been used to repopulate neuron-depleted areas of the brains of both rats and humans with Parkinson’s disease. Fetal neurons also have been used to seed the forebrains of aged animals with cholinergic neurons in an attempt to improve cognitive function.

Since the 1930s, animal fetuses, principally from sheep, have been homogenized into cells and given to treat aging. This “cell therapy” treatment was invented by Swiss physician Dr. Paul Niehans and has been popularized by European physicians such

as Hans Schmidt in Germany. Many remarkable claims have been made for this treatment, but there are theoretical and practical reasons to suspect that it does not work.

As originally put forth by Dr. Niehans, the fetal cells from sheep are supposed to “take” and colonize the elderly human host, rejuvenating him in the process. The problem is that even in the same species, each individual is genetically and immunologically unique and such foreign tissues are likely to be rejected.

Indeed, even tissues from a brother or sister (unless from an identical twin) are rejected without the use of powerful and dangerous immunosuppressive drugs, such as cyclosporine, which depress immune function.

A more appropriate animal model for cell therapy would be to administer more or less genetically identical fetal tissue to old animals. One way to do this experiment is to use animals that are so inbred that they are nearly identical genetically. In such animals, tissues can be transplanted to any other animal of the same strain without concern about rejection.

The Fischer 344 rat is such an animal. The Fischer 344 has been inbred over hundreds of generations so that all individuals in the strain have histocompatible tissues. Another advantage to the Fischer 344 is that it is a widely used animal in biomedical research (and in gerontology) and thus old animals, or so-called “retired breeders,” are readily available at a reasonable cost. Similarly, “timed pregnant” females can be ordered so that histocompatible fetal tissue is available on demand at precisely the right time.

Funding was provided by the Life Extension Foundation to conduct such an experiment at the Critical Care Research (CCR) laboratory in Southern California. Three scientists worked on the project: Michael Darwin, Steve Harris, M.D., and Sandra Russell, B.A. Thirty Fischer 344 male rats, 20 months of age (the human equivalent of 60 to 65 years of age) arrived at CCR. Each animal was placed in a separate cage, and the animals were randomly divided into three groups of 10 each. One group (experimental) was given complete tissue samples from fetuses removed from time-pregnant females by intraperitoneal administration.

Another group (the control) was given the vehicle solution in which the fetal cells were prepared (Hank’s Balanced Salt Solution), in the same manner as the experimental group. A third group (also a control) was given liver and spleen tissue from a non-pregnant female of the same age, as used to provide the complete fetal tissues. The purpose of this last group was to serve as a control on the sterile preparation technique used to generate the fetal tissues. The spleen of adult animals also contains many immune cells, including stem cells. The only endpoints tested in this experiment were time and cause of death.

The results of the study showed that late-stage fetal cells could be injected into old animals without rejection and that the experimental animals lived as long as control animals, but that there was no extension of life span or apparent rejuvenation effects. Late-stage fetuses were used in this experiment because they were big enough for cell extraction. However, the stem cells found in late-stage fetuses have already begun to develop into differentiated cells, so it is possible that earlystage totipotent embryonic cells might have had rejuvenation effects on the old mice.

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Identifying Good Candidates for Anti-Aging Therapies As the 21st century approached, major scientific and technologic advances made it possible to develop a shortcut to the development of authentic anti-aging therapies. Two of these advances — the characterization of the entire human and mouse genomes and the development of high-density microarrays (or gene chips) that can rapidly measure gene expression in thousands of genes at a time — enabled innovative scientists to search for good candidates for anti-aging therapies in a small fraction of the time needed for life span experiments.

In 1999, Richard Weindruch, Ph.D. and Tomas Prolla, Ph.D. made a remarkable breakthrough. They used gene chips to measure gene expression in the entire genome of both normally aging mice and calorically-restricted (CR) mice, which live up to 50% longer than normal mice. They found major changes in gene expression in CR mice, which are a genetic roadmap to the mechanisms by which CR slows and reverses aging and postpones the degenerative diseases of aging. Weindruch and Prolla first studied gene expression changes in muscle tissue, but later went on to study them in liver, heart, and brain tissue. They had discovered a new approach to the discovery of CR-mimetics, which would likely be authentic anti-aging therapies. Instead of testing compounds or other experimental approaches to controlling aging, such as lowering body temperature — in lengthy and expensive life span studies, you could use them to treat mice for as little as three months and then compare the effects on gene expression in the entire genome to that of CR mice. If the proposed therapy

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mimicked the gene expression profile of CR mice, they would be good candidates for life span studies to determine if they, indeed, could extend maximum life span.

The Life Extension Foundation soon began to fund gene expression studies to search for good candidates for anti-aging therapies at the laboratories of Weindruch and Prolla at the University of Wisconsin, and at the laboratory of Steve Spindler at the University of California at Riverside through a company originally called LifeSpan Genetics, which was later changed to BioMarker Pharmaceuticals.

The initial gene expression studies we funded produced provocative results that were published in peer-reviewed scientific journals. These studies showed that the life span-extending effects of CR involve genes responsible for antioxidant defenses against free radical activity, DNA repair, insulin and glucose regulation, and other functions. Many changes occur in signal transduction-related genes, which regulate other genes.

In 2003, we decided that we needed to conduct new and larger studies because very few animals were used in the initial studies and because they were conducted with gene chips that could measure the expression of only about 6,000 or 12,000 genes. By 2003, more advanced gene chips were available that could measure the entire mouse genome of 39,000 transcripts, including 34,000 genes. With funding from the Life Extension Foundation, BioMarker established a laboratory in Northern California and developed collaborations with Stanford University, Gorilla Genomics, the Chinese Academy of Sciences, and the Children’s Hospital of Oakland Research Institute, where we could have access to sophisticated new facilities and greater scientific expertise.

BioMarker, in collaboration with Andrzej Bartke, Ph.D. of Southern Illinois University, compared gene expression in Ames dwarf mice, which live exceptionally long, to gene expression in normally aging mice and CR mice. Some of the gene expression changes in Ames dwarf mice proved to be the same as those found in CR mice, while others were different. Ames dwarf mice fed a CR diet lived longer than CR mice or normally fed Ames dwarf mice.

These findings suggest that the benefits of anti-aging therapies based on these two models may be additive. The ability to completely mimic the gene changes found in CR mice could enable humans to live to be 150, while adding the ability to mimic the gene changes in Ames dwarf mice could add another 20 years to the human life span. Thus, future therapies based on both models of extended life span might enable humans to live to 170.

Metformin and Grape Seed Extract With Resveratrol The Life Extension Foundation then funded gene expression studies at BioMarker to search for CR mimetics as good candidates for anti-aging therapies. After finding that metformin, a drug used to treat diabetes, produced a gene-expression profile similar to that of long-lived CR mice, we decided to study the effects of long-term administration of metformin on mean and maximum life span and other biomarkers of aging in mice.

In the metformin study, scientists at BioMarker used a large number of mice fed a normal diet and supplemented them with

a dose of metformin comparable to that usually used in humans.

A control group was fed a normal diet without metformin. The results of the study showed a modest increase in both mean and maximum life span in metformin mice compared to the control group. Further research is needed to determine if metformin can extend maximum life span in mice significantly and to identify the mechanisms of action involved.

In another study, a group of normally fed mice was supplemented with either synthetic resveratrol or grape seed extract with resveratrol. The objective was to compare gene expression in the livers of the mice in the synthetic resveratrol and grape extract groups to gene expression in the livers of normally fed and CR mice. The grape seed extract with resveratrol included two standardized extracts from the seeds and skin of whole red grapes (Vitis vinifera), which contain proanthocyanidins, catechin, epicatechin, gallic acid, and 10% resveratrol from whole red grapes and Polygonum cuspidatum root extract. It also included vitamin C and calcium from calcium ascorbate.

When gene expression in the livers of mice fed grape seed extract with resveratrol was compared to that in CR animals, we found that substantial numbers of genes were expressed in a similar fashion. These results suggested that the molecular pathways that these genes regulate are activated in both groups. Later, a life span study with grape seed extract with resveratrol was conducted, with the results showing a modest increase in mean life span and a slight increase in maximum life span.

Groups of these fruit flies received four doses of grape seed extract with resveratrol in their food. Climbing ability was evaluated in each group and the data was analyzed statistically. The results showed a progressive decline in climbing ability with advancing age, and that this decline was reduced in flies supplemented with grape seed extract with resveratrol, an effect that was more noticeable in male Parkinson’s flies than in female Parkinson’s flies. Interestingly, the dose of grape seed extract with resveratrol that most inhibited the decline in climbing ability of the Parkinson’s flies was similar to that used in a life span study in which flies given grape seed extract with resveratrol lived 15% longer than those fed a normal diet. Flies receiving grape seed extract with natural resveratrol showed greater life span extension than those receiving synthetic resveratrol.

In another study, mitochondria from rat livers were exposed to damaging carcinogens, followed by treatment with either grape seed extract with resveratrol or BMcp-2 (a plant extract).

The results showed that treatment with certain doses of grape seed extract with resveratrol or BMcp-2 protected the mitochondria from damage. Mitchondria are the cellular power plants that fuel the body’s life processes.

Medical Benefits of Induced Therapeutic Hypothermia The Life Extension Foundation made its first grant to support induced therapeutic hypothermia in 1983, long before the conventional emergency care and surgeons recognized its clinical utility in saving human lives. Here is a description of therapeutic hypothermia from Wikipedia (January 20, 2010): “Therapeutic hypothermia is a medical treatment that lowers a patient’s body temperature in order to help reduce the risk of the ischemic injury to tissue following a period of insufficient blood flow.1 Periods of insufficient blood flow may be due to cardiac arrest or the occlusion of an artery by an embolism, as occurs in the case of strokes. Therapeutic hypothermia may be induced by invasive means, in which a catheter is placed in the inferior vena cava via the femoral vein, or by non-invasive means, usually involving chilled water blankets in direct contact with the patient’s skin. Studies have demonstrated that patients at risk

Studies at The Chinese Academy Of Sciences The Life Extension Foundation has also funded studies at the Chinese Academy of Sciences examining the effects of grape seed extract with resveratrol in Drosophila (fruit flies) that were bred to contain the human mutant alpha-synuclein gene. This gene is expressed in the fruit fly brain similarly to that in humans, so that the flies replicate the essential features of human Parkinson’s disease — age-dependent loss of dopaminergic neurons and motor impairment — that manifests (in the flies) as a progressive loss of climbing ability.

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called Critical Care Research (CCR) under the direction of Steven B. Harris, M.D., with funding by the Life Extension Foundation.

A long-term research project at CCR has been to develop a new method of inducing hypothermia that can produce rapid internal cooling in a relatively non-invasive way. Right now, the only way to cool patients rapidly in operating rooms is to deploy cardiopulmonary bypass, which requires highly sophisticated and expensive equipment such as a heart-lung machine and a team of highly qualified surgeons, perfusionists, and other technical personnel. This type of cooling can only be performed in a relatively small number of medical centers.

Mixed Mode Liquid Ventilation — A New Method To Induce Hypothermia

for ischemic brain injuries have better outcomes if treated with therapeutic hypothermia.2" Every day, people suffer heart attacks that either kill them or cause severe brain damage. This occurs because, at normal body temperature, after about five minutes without oxygenated blood being pumped through the brain, the brain’s neurons suffer lethal damage. Severe brain damage caused by oxygen deprivation may also be caused by other conditions, including strokes and closed head injuries.

Although neurons may suffer lethal damage in five to 10 minutes, they do not actually die and disappear until after many hours, or even days. This leaves a window of opportunity to save them. For over 50 years, scientists have known that cooling the brain (and the rest of the body) can have dramatic effects. One of these is to protect against the biochemical processes that kill neurons in brains with inadequate oxygen, a state caused by reduced or absent blood circulation, or ischemia. The success of hypothermia in brain protection is demonstrated in drowning cases, where victims have been revived after as long as 60 minutes of immersion in icy water, because their brains were cooled.

Hypothermia slows down life processes. If applied properly after a person suffers from acute oxygen deprivation, hypothermia has the power to slow down the dying process in damaged neurons. This could save the lives of millions of people. Dogs have been resuscitated after as long as 16 minutes without blood flow at normal body temperature. This was done by cooling the animals with a bypass machine and administering drugs and nutrients to inhibit damage from ischemia, excitotoxicity, and other processes. These steps were taken after their hearts were restarted.

This path-breaking research was conducted at a laboratory

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Methods currently used in the field to induce hypothermia, where it is often needed quickly in heart attack victims, stroke victims, and victims suffering major closed-head injuries do not cool quickly enough for ideal therapeutic benefits. External cooling with ice, cooling blankets, and other methods is extremely slow (about 1 °C per hour) and internal cooling methods such as cutting open the chest or abdominal cavity in order to apply ice and other cooling elements is messy, damaging, and still yields too slow a cooling rate. Scientists at CCR have developed a method of internal cooling that requires intubation into the lungs, which paramedics can do, and the controlled infusion of cooled perfluorocarbon liquid and oxygen by a largely automated portable system. This portable system has been developed by Suspended Animation, Inc. (which is also funded by the Life Extension Foundation) in collaboration with CCR. This method, which is called mixed mode liquid ventilation, can cool very rapidly and safely through the lungs in hospitals, clinics, nursing homes, and in the field.

Perfluorocarbons (PFCs) are molecules in which all of the hydrogen atoms which occupy the non-linking surface positions of a “hydrocarbon” molecule (such as the octane molecule used in gasoline, and many others) are replaced by fluorine atoms. After this modification, the bonded fluorines are difficult to remove, and the PFC molecules become chemically inert. Such molecules are liquids at body temperatures, if they are heavy and complex enough. These PFC liquids do not dissolve in either water or oils, but are capable of carrying oxygen and carbon dioxide (CO2), which dissolve in them.

Liquid breathing with perfluorocarbon (PFC) liquids has been investigated since 1965, as a means of allowing gas exchange within the lung by means of a liquid, without removal of the critical surfactant. In the case of fluorocarbon, surfactant is not removed because it is not soluble in the PFC. The lungs can be completely filled with PFC, if it is oxygenated, in a technique called “Total Liquid Ventilation” (TLV). If a liquid ventilator machine adds and removes the PFC from the lungs, and (while outside the lungs) removes the CO2 from it and adds oxygen to it, animals can be ventilated with liquid alone, without bubbles of gas in their lungs.

Another related use of PFCs developed later by Alliance Pharmaceuticals is a technique called Partial Liquid Ventilation (PLV) in which the lungs were filled to one-third of the volume of capacity (about the amount of a normal tidal breath) with PFC,

and this was allowed to remain in place while gas ventilation was then carried out “on top” of it. Ventilation was accomplished in the rest of the lung by normal gas ventilation methods. Alliance tried to obtain FDA approval for use of this method in premature infants, but they failed to get it approved for this purpose.

In 2001, Critical Care Research (CCR) published a paper showing that mixed-mode liquid ventilation (or MMLV, and later called PFC lung lavage) was able to reduce brain temperature in dogs by a rate as fast as 0.5 ºC per minute. Thus, the needed state of mild hypothermia (-4 ºC cooling) could be induced in a time less than 10 minutes (allowing for heat transfer delays). This was a factor of 12 times faster than the two hours required by the intravenous techniques, the fastest cooling technique which had been reported by any method.

CCR normally allowed many of its experimental animals (canines) to survive long term after the procedure. It was found that an asthma-like syndrome was produced by high rate lavage with many PFCs, but that this did not become appreciable until 24 hours after lung lavage-cooling. It was never fatal, but did cause the animals difficulty in breathing for as long as a week after the procedure. Eventually, as with all PFC procedures, the PFC evaporated and disappeared from the lungs, and the animals recovered completely.

Over the next eight years, CCR has made an exhaustive study of the necessary parameters of lung lavage to minimize the postlavage syndrome. These include investigation of six different PFC fluids, and exploration of the optimal lavage volume, timing, temperature, and method of delivery and mode of removal.

Because PFC is heavy and resists suctioning, the optimal methods

to introduce and remove it turned out to require a dedicated apparatus, but once constructed, this apparatus proved easy to use, and well within the abilities of paramedics to employ.

Developing a Portable Liquid Ventilation System In 2007, CCR contracted with outside engineering consultants at Suspended Animation, Inc. to provide a suitable high capacity (high suction) machine, which would be completely portable (able to run on batteries) and also able to cool the body more than -4 ºC. In designing this machine, high-capacity commercial “plate-type” heat exchangers already available on the commercial market for other uses (these are capable of nearly 600 watts/ degree gradient of heat transfer between ice-water and PFC) were used. The bulky peristaltic pumps that CCR had been using were replaced with inexpensive commercial fluid diaphragm pumps, which proved far superior, and able to run on low voltage DC. With the superior suction capabilities of this system, combined with its ability to provide a constant supply of perfluorocarbon cooled to 2 ºC (from a reservoir containing only 7 liters of fluid) CCR performed a series of 30 lung-lavage dog experiments in 2007 and mid 2008. The first results were encouraging enough to apply for a preliminary patent on the device in September 2007, and a full patent application was filed (Platt, Battiano, Harris) for the device in September 2008.

Later experiments with the portable system show that six to seven minutes of application of 60 ml/kg/min lung-lavage with ice-cold perfluorocarbon (2 ºC) results in at least -4 ºC drop in brain temperature in the first five minutes, and that application

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of lung lavage for more than six minutes results in a permanent body core temperature of -4 ºC. All of these results are novel, and should be of great importance to the resuscitation community.

Post-lavage asthma syndrome is exacerbated by wrong choice of perfluorocarbon fluid, and by use of the wrong techniques during lung lavage (which result in needless overpressures). This problem has also been overcome by research conduced at CCR.

Why Liquid Ventilation Is the Best Way of Inducing Therapeutic Hypothermia The advantages of therapeutic cooling have been agreed upon by all. The main question remaining is how best to do it, and how rapidly it should be done. Although the question of what the best rate of cooling and what the critical delay is in providing it cannot be answered without more data, certainly the most conservative answer is that least damage to the brain is likely to be done if the entire cooling “job” can be done in the time frame that we already know is relatively “safe” for the brain to be without any oxygen, which is about 5 minutes. Thus, the most convincing argument for rapid cooling has been made by Lance B. Becker, M.D., a founder and director of the Emergency Resuscitation Center at the University of Chicago and Argonne National Laboratory. If Dr. Becker’s argument for the need for rapidity in cooling

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is accepted, then the method which unquestionably has the best chance to cool the human brain (and body core, since they cannot be separated, due to high connecting blood flow) by -4 ºC, in less than five minutes, is PFC liquid lung lavage (Liquid Ventilation).

This method approaches the rates available in cardiopulmonary bypass cooling, but without the need for circulatory bypass or surgery. Only intubation is required.

Research conducted at Life Extension Foundation’s Suspended Animation laboratory in Boynton Beach, Florida involves the development of equipment and procedures to mitigate the ischemic injury that normally affects cells in the human brain and body after cardiac arrest. Emphasis is focused on improving the clinical delivery of portable therapeutic hypothermia, which is now embraced by the medical mainstream. Suspended Animation works closely with Critical Care Research, another laboratory funded by the Life Extension Foundation that was described in previous pages.

Advanced Cryopreservation for Transplant Medicine Right now, transplantation is a relatively small field because it depends upon drug-induced immunosuppression of the recipients of organs taken from live or recently deceased humans, whose histoincompatible organs would otherwise be rejected by the receipient. These drugs, such as cyclosporine, are expensive, have toxic side effects and must be taken for the rest of the patient’s life in order to prevent rejection of the transplanted organ. Moreover, there is a limited supply of such transplantable organs, which is grossly inadequate to meet the needs of dying patients.

The single most important problem in transplant medicine today is the lack of enough organs to meet the demand worldwide. As a result, billions of dollars have been invested in new technologies to generate cells, tissues and organs for transplant.

Among the new technologies being developed are: tissue engineering (the merging of biologic and artificial systems); organ culture methods to grow transplantable tissues and organs in the laboratory; human therapeutic cloning from embryonic stem cells and the development of induced pluripotent stem cells from differentiated cells, both of which get around the rejection problem; and xenotransplantation, in which it is proposed that tissues and organs from animals such as pigs and monkeys be transplanted into humans.

Whatever the source of cells, tissues and organs for transplantation, there is an across-the-board need for improved methods of cryopreservation for storage. Current methods of freezing are quite poor for cells and tissues and completely inadequate for organs.

The Life Extension Foundation is funding the development of an advanced technology called vitrification at a laboratory in Southern California (21st Century Medicine), which cryopreserves tissues and organs in a glasseous state, without the formation of damaging ice crystals. The cryobiologists at this laboratory have developed the world’s first synthetic ice-blockers and are moving closer to the successful vitrification of rabbit kidneys. Recent advances in this laboratory include the cooling of rabbit kidneys to -45 °C, with subsequent survival and good function after transplantation (after re-warming) of eight out of eight kidneys.

We believe it will soon become common to transplant tissues, organs and other body parts from diverse sources into sick and aging patients. Before long, medical scientists will be transplanting virtually every body part (except for certain brain areas) in order to cure and rejuvenate aging patients. The Life Extension Foundation expects that advanced vitrification technology will be used to set up organ and body-part banks around the world to help keep people healthy and youthful into “old” age.

Vitrifying Brains and Whole Organisms The most ambitious research being performed at the 21st Century Medicine laboratory involves experiments to vitrify rabbit brain slices, entire rabbit brains, and entire rabbits. The short-term potential of this research includes the use of well-preserved brain slices to test potentially therapeutic drugs and antidotes to potential biological and chemical weapons of mass destruction.

This research could also lead to the availability of well-preserved brain sections for neurobiology experiments and the treatment of brain diseases such as Parkinson’s and Alzheimer’s. This research is also essential for achieving suspended animation in humans, which could be instrumental in radically extending the life span of millions of people.

Recovering Viability in Brain Slices

Survival of the First Vitrified Kidney At the annual meeting of the Society for Cryobiology in July 2005, Dr. Gregory M. Fahy, Ph.D., the Director of Research at 21st Century Medicine announced the survival of a rabbit that received a transplanted kidney after the organ had been vitrified to -130 °C and then re-warmed. This result was based on the vitrified kidney’s ability to provide sole renal life support in the recipient animal until it was removed for examination 48 days after it was transplanted. The transplanted kidney sustained some damage from ice formation in its center, but that damage was entirely absent in other parts of the core of the kidney, enabling the organ to support life in a normal fashion.

Scientists at 21st Century Medicine have also successfully vitrified corneas and are conducting research to successfully vitrify hearts, egg cells, stem cells, cartilage, brains, and whole bodies.

Their work is currently performed on rabbits.

Funding for research at 21st Century Medicine is primarily provided by the Life Extension Foundation, with additional grants from the National Institutes of Health.

Today, transplantation is reserved for vital organs such as the heart, lungs, liver and kidneys, which need to be replaced or the patient will die. In the relatively near future, however, when scientists have developed technologies to grow immunologically identical tissues and organs, and/or to manipulate the immune system to prevent the rejection of tissues and organs, a problem which is being worked on at several laboratories, the field of transplant medicine will explode.

One test 21st Century Medicine has used to evaluate brain slice function after vitrification and rewarming is the potassium/ sodium ratio, or ion transport capacity. Another is to determine whether there is normal preservation of slice structure. These tests have shown apparently normal structure and function in hippocampal brain slices. The hippocampus is an area of the brain involved in memory consolidation, storage, and retrieval, and is the part of the brain most sensitive to oxygen deprivation. To further evaluate the effectiveness of its brain studies, 21st Century Medicine has established an in-house neurophysiology laboratory to measure electrical activity in brain slices and sections. This lab is headed by Yuansheng Tan, Ph.D., who is assisted by Joon Chang, Ph.D. In initial studies, it was determined that vitrified, re-warmed brain slices were alive but electrically silent. After several refinements in technique, the 21st Century Medicine brain slice team was able to achieve essentially normal electrical responses in up to seven of nine slices, which compares favorably to the rate of normal electrical responses in uncooled control slices not exposed to vitrification solutions, and exceeds the rate of electrical responses in uncooled control slices in many other laboratories. This is the first time electrical activity analogous to a normal electroencephalogram (EEG) response has been achieved in organized brain tissue after cooling to temperatures low enough to achieve vitrification.

Vitrifying the Entire Brain Like studies of isolated hippocampal slices, studies of entire rabbit brains have found that all regions of the brain appear to be structurally preserved after vitrification and rewarming. Initial

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studies have shown that it was difficult to preserve the brain’s ability to respond electrically after five hours of cold storage or cold perfusion in the absence of cryoprotectant chemicals.

Studies are under way to overcome this problem, however, and the 21st Century Medicine team will eventually look at electrical activity in brains that have been perfused with vitrifiable concentrations of cryoprotectants. It appears as though the entire brain can be vitrified, but further research is required to see whether normal electrical activity in the brain can be preserved after vitrification.

Vitrifying the Entire Body When whole rabbits were vitrified at 21st Century Medicine, scientists collected tissue samples from many brain and kidney regions as well as from the heart, lungs, liver, intestine, muscle, skin, fat, stomach, and other areas. Using a device called a differential scanning calorimeter, scientists tested the samples to see whether ice formed within them. The differential scanning calorimeter can cool tissue samples to vitrifiable temperatures, warm them up, and measure how much ice has melted during warming.

The amount of melted ice is equal to the total amount of ice formed during both cooling and warming, and the temperature at which the ice melts is a measure of how much cryoprotectant was in the tissue before it was cooled.

The results showed that the most difficult tissue to vitrify in the entire body was the center of the kidney, called the inner medulla. Most of the tissue samples showed only small amounts

of ice formation, or none at all. Since 21st Century Medicine is moving toward successful vitrification of whole kidneys, it appears that the entire body can be successfully vitrified as well.

This suggests that whole-body suspended animation is an achievable goal, but a great deal of additional research will be necessary to attain this goal.

Developing Artificial General Intelligence Scientists began working in the field of artificial intelligence (AI) more than 50 years ago. There have been major advances in the field since then, but these advances have been specialized types of AI designed for a specific purpose. For example, many years ago, IBM developed a computer program that learned how to play chess well enough to compete successfully against Grandmasters, but unable to do anything but play chess. Similar AI programs have been developed to improve the performance of specific tasks in various industries. But, again, these AI programs have been restricted to the task they were programmed to perform.

In the early days of AI, there were enormous expectations for the development of artificial thinking systems that could learn in a general fashion, as humans do. But these expectations have yet to be met. In recent years, there have been at least two companies that have made progress in developing artificial general intelligence. The Life Extension Foundation has provided funding for one of these companies — Adaptive AI — which we consider the most advanced in the field.

Adaptive AI Adaptive AI was founded by Peter Voss, an entrepreneur who developed a successful public software company in South Africa, which was sold in 1993. After moving to the United States, Voss worked on a broad range of disciplines — cognitive science, philosophy and theory of knowledge, psychology, intelligence and learning theory, and computer science — which served as the foundation for achieving breakthroughs in artificial general intelligence. In 2001 he started Adaptive AI, with the purpose of developing systems with a high degree of general intelligence and commercializing products based on these inventions.

Computer systems based on Artificial General Intelligence (AGI) technology are specifically engineered to be able to learn.

They are able to acquire a wide range of knowledge and skills via learning similar to the way we do. Unlike current computer systems, AGIs do not need to be programmed to do new tasks.

Instead, they are simply instructed and taught by humans. Additionally, these systems can learn by themselves both implicitly “on-the-job,” and explicitly by reading and practicing. Furthermore, just like humans, they resiliently adapt to changing circumstances. This general ability to learn through natural interaction with the environment as well as from teachers allows them to autonomously expand and adapt their abilities over time so that they become ever more knowledgeable, smarter, and more useful.

In addition to their intrinsic learning ability, AGIs are also designed to function in a goal-directed manner. This means that they automatically focus their attention on information and activities that are likely to help solve problems they have

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been given. For example, an AGI trained and instructed to look for inconsistencies in arthritis medication studies will spend its time perusing relevant articles, news, and background information, and request pertinent additional information or clarification from other researchers. On the other hand, an AGI assigned to be a personal assistant will seek out knowledge and skills necessary for that job, such as learning how to deal with various types of business associates, schedules, priorities, and travel arrangements, as well as its boss’s personal preferences.

AGIs learn both conceptually and contextually. Conceptual learning implies that knowledge is assimilated in a suitably generalized and abstract form: skills acquired for one task are available for similar, but non-identical tasks, while at the same time making the system much more useful and robust when coping with environmental changes. Context, on the other hand, allows the system to utilize relevant background information to appropriately tailor its responses to each specific situation. It can take into account such crucial factors as recent actions and events, current goals and priorities, who it is communicating with, and anything else that affect its current actions.

Other central AGI features include an ability to anticipate events and outcomes, and the ability to introspect and to be aware of its own cognitive states (such as novelty, confusion, certainty, its level of ability, etc.) These design features, combined with the fact that AGIs directly perceive their environments via built-in senses, endow them with human-like understanding of facts and situations.

Note that all these advantages are in addition to computer systems’ natural strengths: large “photographic” memories, high speed, accuracy, upgradeability, seamless interfacing with other systems, etc. Another key feature of such trainable/trained

systems is that, unlike skilled humans, they can be duplicated, and efficiently pool knowledge and experience. These capabilities allow for rapid up-scaling of production. For example, various AGIs, after having been trained in particular specialties, could pool their knowledge and then be duplicated hundreds of times imbuing each one of them with their combined knowledge. From there on these AGIs could pursue coordinated, yet individual paths, while regularly updating each other.

The long-term goal of Adaptive AI is to develop Artificial General Intelligence systems that function at the level of Ph.D. scientists, which could be used in any number to work around-theclock to attempt to solve major problems. Among the problems that Life Extension Foundation would like to see such systems work on are the cure of the diseases of aging and the reversal of human aging (rejuvenation).

The Promise of Nanotechnology The biological changes that occur in our bodies do so at the levels of super-small molecules which are constructed of atoms. In the 20th century, medical technology moved rapidly in the direction of the ability to visualize biological cells at smaller and smaller levels. First, we had the light microscope, then the electron microscope, and finally the scanning tunneling microscope, which enabled scientists to see and manipulate atoms.

At the beginning of the 21st century, we are at the forefront of the development of medicine and manufacturing, which will eventually enable the repair of cells on a molecular level in ailing and aging patients and the construction of material objects, such as buildings, automobiles, airplanes, and computers in an organic, natural way that is as sophisticated as the way in which

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biological organisms are constructed from the plans in DNA. All this will come about because of the emergence of a new, exciting field called nanotechnology. Nanotechnology is the engineering of atomically precise structures and, ultimately, molecular machines. The prefix nano refers to the scale of these constructions. A nanometer is one-billionth of a meter, the width of about five carbon atoms nestled side by side. Nanomedicine is the application of nanotechnology to medicine.

Moving Toward the Development of Nanomedicine The Life Extension Foundation has provided funding to a company called Nanofactory Corporation, which was founded by the two foremost experts in nanotechnology and nanomedicine: Ralph Merkle, Ph.D. and Robet A. Freitas, Jr. The Life Extension Foundation is funding Nanofactory primarily because its research holds unprecedented promise for the development of Nanomedicine — the medicine of the future.

Today, in most cases, physicians must rely chiefly on the body’s ability to repair itself. If this fails, external efforts may be

useless. We cannot yet place the component parts of human cells exactly where they should be and restructure them to ensure a healthy physiological state. There are no tools for working — precisely, and with three-dimensional control — at the molecular level inside the body. If we could do this, we could eliminate a wide variety of disorders, reduce the most elaborate — and expensive — surgical procedures to simple maneuvers, stave off billions of premature deaths, and reach new levels of public health and wellness.

The ultimate tool of nanomedicine will be the medical nanorobot, a machine the size of a bacterium, comprising many thousands of molecule-sized mechanical parts (resembling macroscale gears, bearings, and ratchets), possibly composed of a strong diamond-like material. A nanorobot will need motors to make things move, and manipulator arms or mechanical legs for dexterity and mobility. It will have a power supply for energy, sensors to guide its actions, and an onboard computer to control its behavior. A nanorobot that would travel through the bloodstream will have to be smaller than the red cells in our blood and tiny enough to squeeze through even the narrow capillaries in the human body.

Right now, medical nanorobots are just theory, but there is evidence that such machines are buildable. The first experimental proof that individual atoms could be manipulated was obtained

by IBM scientists back in 1989, when they used a scanning tunneling microscope to precisely position 35 xenon atoms on a nickel surface to spell out the corporate logo “IBM.” To build nanorobots, researchers will need to develop entirely new and much more elaborate molecular manufacturing processes. This is the goal of Nanofactory Corporation.

What might nanorobots look like? Here’s an example: one medical nanorobot called a microbivore could act as an artificial mechanical white blood cell, seeking out and digesting unwanted pathogens like bacteria, viruses, or fungi in the bloodstream.

A patient with a blood-borne infection might be injected with a dose of about 100 billion microbivores (about 1 cc). Microbivores would hunt different bacteria inside the body and digest them into amino acids, mononucleotides, simple fatty acids, and sugars. These basic molecules would then be harmlessly discharged back into the bloodstream through an exhaust port at the rear of the device.

Rather than brewing giant batches of single-action drug molecules, the pharmaceutical industry will be able to shift to manufacturing large quantities of generic nanorobots of several basic types. These devices could later be customized to each patient’s unique genome and physiology, then programmed to address specific disease conditions, right in the doctor’s office just when they’re needed. Nanomedicine would also be able to address the cellular injuries that occur with aging, and repair these cells so that they and the organs and organisms they reside in would be healthy and youthful.

The Timeship Project The Timeship Project was initiated in 1997 and has been aggressively worked on every year since then. More than 800 acres of carefully selected land (free of natural and manmade disasters)

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have been purchased as the site for the Timeship Building. Architectural plans have been drawn up and models of the Timeship Building have been presented in prestigious venues including the Municipal Art Society of New York.

There are two parts to the Timeship mission: biotech research and the cryostorage of biological materials. The first mission, research, will be focused on the design and development of cooling systems and storage devices for the cryopreservation of human organs to allow long term storage for future transplantation, anti-aging and life extension, fertility, and related biomedical research.

In 2007, The Stasis Foundation was granted United States Patent #7,299,641 B2 for the design of a cryogenic storage system with improved temperature controls, a key element to the success of this project. The second mission, cryostorage, will include organ and tissue banking for transplantation; materials to support fertility; tissue for regenerative medicine; DNA, including the DNA of near extinct species; whole mammalian organisms including humans after legal death after all available medical procedures have failed. The critical shortage of organs for transplant results in the needless deaths of tens of thousands of Americans each year. Full scale organ banking at Timeship will help to eliminate this travesty.

An award-winning book authored by architect Stephen Valentine is being promoted at upscale bookstores to enlighten the public about the critical scientific research that will occur at this state-of-the art facility. This book, Timeship: The Architecture of Immortality, is available for purchase from the Life Extension Foundation Buyers Club.

Summary The mission of the Life Extension Foundation is to fight premature aging, prevent disease, and achieve indefinitely extended healthy life spans. We do not base our funding of projects on their probability of immediate financial return. Instead, we identify scientific research that has the potential to radically improve the human condition. Rather than pay for studies that go over the same old ground, Life Extension commits its research to promising areas that are difficult to fund with conservative governmentinstitutional grants or biased drug company dollars.

A review of the 30-year history of our organization reveals that our scientific judgment is often years or decades ahead of the medical establishment. For example, we were the first to recommend the use of low-dose aspirin to prevent heart attack in 1983.

Conventional and alternative doctors ridiculed us and we were threatened with imprisonment by the FDA. By 1998, however, the FDA approved the use of low dose aspirin to prevent heart attack.

Today, the Life Extension Foundation donates millions of dollars a year to research to help develop new therapies and technologies that could lead to cures for the diseases of aging and, eventually, for aging itself. Your product purchases are essential to furthering these goals.

References: 1. Ron Winslow (OCTOBER 6, 2009). “How Ice Can Save Your Life”. Wall Street Journal. http://online.wsj.com/article/SB100014240527487032980045744 55011023363866.html?mod=WSJ_hpp_MIDDLENexttoWhatsNewsSecond.

Retrieved 2009-10-06.

2. Holzer, Michael. “Mild Hypothermia to Improve the Neurologic Outcome After Cardiac Arrest.” New England Journal of Medicine. (2002) Vol. 346, No. 8

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