9
Breaking the Shackles of Age
Year after year, ongoing chemical processes are shackling the structural proteins of your body together, holding them back from their vital jobs. Eventually, this leads to a familiar (and ultimately fatal) range of age-related disabilities and diseases—especially in the kidneys, heart, eyes and blood vessels. What if we could break these chemical shackles, and thereby allow those proteins to get back to work, as they did when you were young? Scientists are making progress toward drugs that could achieve just such a goal.
You’re in the last hours before the big holiday feast, and the atmosphere is heavy with the smells and emotional charge of the season. It’s been a long day in the kitchen—the oven running continuously, the matron of the house trying to keep cool by leaving a window left slightly ajar—and at last the hurry and stress are giving way to a more expectant, eager sort of tension. The potatoes are mashed, the cranberry sauce has been spooned into serving dishes, the sweet potatoes are being kept hot in the oven, pumpkin pie is cooling on the windowsill…and now, a single component of the meal dominates the cook’s attention and the appetites of her family.
Every fifteen minutes, like clockwork, for the last hour and a half, the turkey has been lovingly basted with its own fat, and perhaps a little honey; now, to perfect the feast, the broiler is flipped on, to glaze its surface.
All the time that the turkey has been in the oven, complex chemical processes have been imperceptibly proceeding—and now they accelerate. Down at the molecular level, the high heat causes the sugars and fats to attack the proteins in the bird’s skin. Molecular bonds are forged; new chemical products arise and are broken down; neighboring proteins are tied together in shotgun marriages, tightening the outside surface of the turkey and coating it with thick, gooey chains of linked proteins, fats, and sugars.
Finally, the deed is done. Mom flips the oven off and dons her oven mitts as she calls to Dad to get the carving knife. The family looks on her handiwork with eager eyes, gazing with hunger and appreciation at the darkened, crispy, sticky, slightly toughened surface that the chemical maelstrom has made of the turkey’s skin. Dinner is served.
I’m sure that you and your family have played out a similar script at Christmas—or at Hanukkah, browning the latkes or sufganiyot. But, in a profound sense, you have as much in common with the feast as you do with the family.
Every day of your life, the same processes that are involved in the browning of meats and other glazed or fried foods are insidiously at work in your body. In your arteries. In your kidneys. In your heart, your eyes, your skin, your nerves. At this very moment, in all your tissues, the sugar that provides your body with so much of its energy is also performing some unwanted chemical experiments, caramelizing your body through exactly the same processes that caramelize onions or peanut brittle. Slowly but steadily, unwanted bonding by sugars and fats handcuffs your proteins, inactivates your enzymes, triggers unhealthy chemical signals in your cells, and damages your DNA. Aging you.
Make that: AGEing you. And I’m not just reminding you of my nationality by adding that final e.
The Way We AGE
The body relies on sugar as a key energy source. But, like any fuel, sugar can only be “burned” by our cells because it is chemically reactive—and, again like other fuels, that volatility can make it dangerous to work with. Advanced Glycation End-products (or “AGEs,” as they’re appropriately called) are the end results of the complex chemical processes through which the structure of proteins is warped by sugars and other fuels. This same chemistry is the cause of the “browning” you see when you roast a turkey, caramelize a sauce, or pop a slice of bread into a toaster. AGEs accumulate in your tissues, leading to gradual loss of function, then disease, and ultimately an early grave. AGEs transform the supple grace of youth into a “crusty” old age, through exactly the same chemical processes by which they form the crust on a loaf of bread.
The many chemical reactions, intermediates, and stable end products of AGE chemistry have been the subject of an enormous amount of research, first in the food technology and chemical sectors and more recently in biomedicine. Scientific study began with work by a food chemist named Maillard in the 1910s and ’20s, but it took until the 1980s for role of AGEs in the complications that ravage the diabetic body to become a hot topic in diabetes and aging research. Even now it’s clear that we’ve only begun to understand the furious promiscuity of this biochemistry and its impact on the aging or diabetic body.
Though the details needn’t concern us here, you will need to grasp the outlines of how AGEs form if you are to understand the various strategies that have been employed in the search for a way to shield us from their fossilising influence. In the best-understood pathway (the main stream of the Maillard reaction—see Figure 1a), a molecule of sugar opens its structure and glues onto (“glycates”) a protein molecule, forming a Schiff base. This structure is relatively unstable, so the Schiff base will often spontaneously fall apart. Sometimes, however, it will collapse into a more stable structure called an Amadori product. Amadori products are much longer-lived than Schiff bases. (This fact has long been exploited in a lab test that measures levels of glycated hemoglobin or HbA1c, an Amadori product in red blood cells, as an indicator of the average amount of sugar that has been present in the blood over the course of the previous few weeks.)
Relatively stable though they may be, however, Amadori products are still subject to the biochemical hurly-burly around them. They can therefore be put through any number of further chemical transformations, such as rearrangement or degradation of their basic structure, forcible insertion of water molecules or removal of amino groups, or attack by free radicals. Many of these changes lead to the formation of even more stable structures, either directly or via highly reactive intermediate compounds such as oxoaldehydes. These structures are stable enough, in fact, to be called “end products”—they are the advanced glycation end products, or AGEs.
For our purposes, the important outcome of these processes is the formation of AGE cross-links, a subset of AGEs in which proteins that are already working with one arm tied behind their backs because of glycation become shackled to a second, neighboring protein.
Figure 1. The ways we AGE. (a) The “chemical” (Maillard) pathway; (b) The “metabolic” (triosephosphate) pathway; (c) Sources of methylglyoxal.
AGEing happens much more quickly in people with diabetes than in the rest of the population, partly for the simple reason that diabetics’ blood sugar levels are higher: In any chemical reaction, a higher concentration of an active agent will tend to increase the rate of its interactions with its targets, provided those targets are plentiful. But AGE cross-links also accumulate in people with normal blood sugar levels, and it’s quite clear that they are responsible for much of the pathology and increased vulnerability to the insults of daily life that accompany “normal” aging.
Browning to Death
The cross-linking of proteins is similar, at both the molecular level and the functional level, to the processes that cause windshield wipers to lose their flexibility. For people who don’t have diabetes, the most life-threatening locus of the ensuing stiffening of the tissues is the cardiovascular system. AGE cross-links slowly impair the youthful elasticity of your heart and blood vessels, making them rigid and unyielding. The resulting hardening of the arteries is in large part responsible for the increase in systolic blood pressure that everyone suffers with age. (Systolic pressure is the first of the two numbers that you get from a blood pressure reading, like the “110” in “110 over 80.”) Meanwhile, the AGEing of your heart impairs its capacity either to contract to pump blood through your body, or to expand in order to fill up with that blood in the first place. The combination of these two factors increases the workload on the heart, ultimately leading to one of several forms of heart failure if nothing else kills you first. The same lack of plasticity also means that your blood vessels become less able to withstand the constant surges of blood that course through them: they become brittle and eventually break under the pressure like old rubber bands, one potential result of which is a bleeding stroke.
And the damage caused by AGE cross-links extends well beyond the cardiovascular system. They shackle proteins all over the body, accumulating with age in tissues as diverse as the tiny blood vessels in your eye and the supporting myelin sheaths of your nerves. Everywhere they occur, AGE cross-links impair the functioning of those proteins, contributing to age-related dysfunction, disability, and death. In your eyes, they accumulate on the crystallin proteins that make up the structure of the lens. AGEd lens proteins stop allowing light to pass through them, leading to the brown pigmented spots in the lens that we know as cataracts. The combination of this browning with several effects at the cellular level is why age and diabetes are the major risk factors for this, the single greatest cause of vision loss worldwide.
And that isn’t the only way in which AGEs contribute to vision loss. Elsewhere in the eye, AGEs contribute to diabetic retinopathy (vision loss in diabetics linked to damage to the fine blood vessels feeding the light-absorbing tissues at the back of the eyeball), to age-related macular degeneration, and possibly also to open-angle glaucoma.
The kidney, too, suffers badly from AGE assault—again, especially among people with diabetes. Diabetic damage is the single biggest cause of kidney failure in the United States, and a third of all patients who find themselves in the dialysis ward got there because of their diabetes. Indeed, the severity of kidney disease in diabetics tracks the level of renal AGEs, which cross-link the proteins of the kidneys’ biological filter material and trigger an inflammatory process that leads the body to overcompensate by growing too much replacement tissue, in a sort of out-of-control wound-healing response. The net effect of these two processes is a buildup of something similar to scar tissue in the kidney, which accumulates to levels that literally squeeze the tiny blood vessels where filtration is supposed to occur, reducing the amount of filtering surface available and leading to inefficient screening of materials in the blood—as if you had glued a coffee filter paper back on itself before running the machine, leading to a ground-filled mess when the water starts to back up.
AGEs also contribute to diabetic neuropathy, the debilitating damage to the nerves that is suffered by so many diabetics. The severity of this disease can vary, but the most common symptom is an unremitting version of the experience one has after a temporary, pressure-induced reduction in blood flow to the hands or feet (i.e., when the extremity is said to be “asleep”): a sensation of “pins-and-needles,” pain, or numbness in the affected limbs, along with some loss of control or clumsiness in their use. People with diabetic neuropathy also lose some of the unconscious control by their nervous systems of functions such as the regulation of the heart’s rhythm, the digestive process, the bladder, and erectile function; they also often suffer dizziness, and nausea that may extend to vomiting. Whether AGEs play any role in similar, more subtle defects in nerve function with age in otherwise healthy people is unclear, but it seems likely.
Comparisons of the rates of accumulation of cross-links in the tissues of slower-and faster-aging species, and of slower-and faster-aging individuals within a species, suggest that AGEing plays an important role in aging per se, not just in specific diseases or the complications of diabetes. Both the rate of age-related buildup of one of the more easily measured AGEs (pentosidine) and the related toughening-up of the proteins in skin or tail tissue are inversely associated with the maximum lifespan of different mammalian species. This means that the more slowly a species ages, the more slowly its collagen is stiffened by AGEs (see Figure 2). Likewise, calorie restriction—which is, as I’ve mentioned in previous chapters, the best-studied way to slow down aging in mammals—slows these processes down; and in fact, higher rates of tissue AGEing have been shown to predict early death in individual calorie-restricted animals.1 In our own species, studies show that even within the “normal” range (i.e., at values well below those typical of people with diabetes), higher blood levels of either glucose itself2 or of the Amadori product HbA1c3 are associated with a higher risk of death from all causes.
A drug that would slow down or reverse the accumulation of AGEs would thus help people with a wide range of diseases and disabilities. It could potentially improve, or even cure, problems as wide-ranging as the gradual increase in blood pressure over a lifetime; the terrible kidney, nerve, and visual complications of diabetes; and several forms of heart failure. And it could also help us to address a major contributor to aging itself.
Figure 2. Maximum life span as a function of AGE formation rate. Redrawn.4
This idea didn’t just pop into my head recently, of course: a variety of schemes to reduce the tissue AGE burden have been explored over the years, and many of them have even reached relatively advanced clinical trial status. Yet, despite years of work, none of these treatments has been shown to be safe and effective enough to find its way into the drug arsenal of any developed country. The obstacles that have plagued their development and limited their usefulness represent yet another case study in the problems of trying to deal with age-related damage by tinkering with the complex biochemistry of life.
Listening to Parmenion
“Sugar Pills”
The fact that AGE cross-links are often ultimately the result of sugar molecules acting like a glue, gumming up our tissue proteins, immediately suggests one possible solution to the problem: just lower people’s blood sugar levels, and you’ll reduce the formation of Schiff bases (see Figure 1) in their bodies and thereby lower their AGE burden. Of course, this has long been the major focus of diabetes management, and in the 1990s, two massive and widely cited scientific studies—the Diabetes Control and Complications Trial (DCCT) and the UK Prospective Diabetes Study (UKPDS)—were hailed as the clearest proof yet of the effectiveness of this strategy when taken to its limits. These two studies showed that when diabetics take strict steps (aggressive use of blood-sugar-lowering drugs and regular feedback in the form of frequent blood sugar testing) to keep their blood sugar under very tight control, they are at greatly reduced risk of developing the major complications of the disease. The DCCT, in particular, showed that—as compared with the standard of care at that time—a regimen of intensive blood sugar control could reduce a diabetic’s risk of developing nerve disease by close to two-thirds, diabetic kidney disease by about half, and diabetic retinopathy by an astounding three-quarters.
The results of these two studies were trumpeted around the world—by their government sponsors, by patient advocate organizations, and by pharmaceutical companies looking to boost sales of glucose-lowering drugs. The plan was to encourage doctors to prescribe these drugs to patients whose blood sugar control was in the range that made them safe by previous standards but demonstrably at risk based on the new data, and also to increase the doses taken by people with worse control who were already using the drugs.
The benefits that would accrue to patients as a result of such a surge in drug use seemed to be clear-cut: people with diabetes all over the world would enjoy miraculous improvements in the quality and length of their lives through dramatic reductions in their risk of blindness, nerve damage, and kidney failure. But when scientists actually assessed the overall quality of life of people who had undergone the intensive therapy regimens in the trials, the results were surprisingly gloomy. Despite the fact that more aggressive treatment had reduced the risk of all major diabetic complications, the intensive-therapy patients enjoyed no improvement in their net well-being as compared to people who had been assigned to standard care.5,6
Many factors probably contribute to the lack of clear-cut benefits from aggressively lowering blood sugar levels. While diabetic complications clearly have a negative impact on quality of life, the drugs used to lower blood sugar also come with costs that are not included in the sticker price. People on such medications tend to gain weight, which reduces their quality of life—both directly, and by increasing the risk of other diseases such as osteoarthritis. Many patients also find that sticking with the rigid schedule of injections and finger-prick tests required to keep up with these regimens imposes real restrictions on their lives, which some studies report contributes to depression, frustration, isolation, or troubles at work.
And finally, constantly trying to push blood glucose even into the “normal” range carries with it the risk that blood sugar levels will drop too far, leading to a “hypoglycemic crisis” whose consequences can range from dizziness to a coma. This is of particular relevance to normal aging. If pushing blood sugar levels down is a mixed bag for diabetics, you can see that it would be a decidedly dubious solution to the AGE problem for the rest of us, in whom the wiggle room between our normal blood sugar levels and a hypoglycemic crisis is much smaller, making the potential benefits more limited and the risks higher.
And even if we could safely bring our blood sugar down to the lowest possible safe level, we’d be quite far from a complete solution to the AGE problem. All of us must maintain some level of glucose in our blood as an energy source, and some percentage of that glucose will always wind up reacting with tissue proteins, leading to cross-linking.
And on top of that, not all AGEs are even derived from glucose. Blood fats (triglycerides) can also cause the cross-linking of proteins, particularly if there’s a high level of oxidative stress; this is the chemistry that underlies the browning of a turkey skin as it roasts, even without a sweet, syrupy slather on its surface. As with blood sugar, diabetics usually have high triglyceride levels, and even many nondiabetic people would benefit from having their triglyceride levels brought down; but triglycerides also resemble blood sugar in being indispensable to normal function, so there’s only so far that such a strategy can be safely pursued.
Less Is More…Is Worse
And that’s not all: attempts to control levels of both these early precursors of AGEs, even by nonpharmacological means, can have perverse metabolic consequences.
For instance, one established effect of very low-carbohydrate diets of the Atkins type is to bring down both triglyceride levels and the body’s total exposure to carbohydrates, so some advocates have hypothesized that these diets would reduce a person’s AGE burden. Unfortunately, it turns out that the metabolic state that these diets induce (the notorious “ketosis”) has the unfortunate side effect of causing a jump in the production of the oxoaldehyde methylglyoxal, a major precursor of AGEs that is also, ironically, produced within the cells of diabetic patients when they are forced to take in more glucose than they can immediately process (see Figures 1b and 1c). A recent study tested the size of this effect in healthy people who successfully followed the first two phases of the Atkins diet for a month, and who had the ketones in their urine to prove that they were sticking to the diet. These previously healthy people suffered a doubling of their methylglyoxal levels, leading to concentrations even worse than those seen in poorly controlled diabetics.7 Like other oxoaldehydes, methylglyoxal is far more chemically reactive than blood sugar (up to 40,000 times more reactive, in fact), and is known to cause wide-ranging damage in the body, of which AGE cross-links are but one example. This potentially makes the Atkins diet a recipe for accelerated AGEing, not a reprieve from it.
“Radical” Proposal—Lukewarm Results
Even before the counterintuitive results of the DCCT came out, it was obvious that a blood-sugar-lowering strategy would not be a complete solution to the AGE problem. The body needs blood sugar and fats as fuels, and yet no level could be so low as to eliminate all cross-link formation: at best, lowering the concentration of glucose and triglycerides would delay the inevitable. So some scientists turned their attention elsewhere, to cross-link-inducers whose harmful role in the body is less ambiguous.
One such AGE-prevention strategy is the use of high doses of antioxidants to bring down free radical levels. As you can see from Figure 1a, free radicals can accelerate the conversion of some AGE precursors into certain specific full-blown cross-links—a phenomenon called glycoxidation. Based on the effect of adding free radicals to proteins and sugars in test-tube experiments, glycoxidation can be predicted to hit diabetics with a double whammy, because in addition to the excessive levels of blood glucose and fat, the impaired metabolic state of diabetes also causes an overproduction of free radicals in victims’ cells. Put the two factors together and you have a potentially synergistic interaction. Also highlighting the importance of free radicals in AGE formation is the fact that birds have sky-high blood glucose levels that would rapidly kill a human, yet generally live around ten times longer than mammals of the same size; part of how they get away with this is probably by having really good control of oxidative stress.
If glycoxidation were a major reason for diabetics’ high levels of AGE, then sopping up their excess burden of free radicals with antioxidants might considerably reduce the cross-linking of their tissue proteins, resulting in longer life expectancy and reduced risk of crippling complications. And many studies carried out in laboratory rodents have supported this expectation: dosing them with various free radical quenchers typically reduces the cross-link burden in their tissues considerably, cutting back on the incidence and severity of diabetic kidney, nerve, and even retinal damage.
When antioxidants were tried as an anti-AGE therapy in humans, however, the results were disappointing. The effects on AGE levels and symptoms were minor or nonexistent—and even when benefits were observed, the effect was almost exclusively confined to the most severe cases of the disease, with more typical diabetics getting no relief.8,9,10 We now know that there are a couple of major reasons for this. First, human diabetes causes a much less severe increase in oxidative stress than is suffered in the rodent version of the disease, as can be seen by comparing the levels of molecules damaged by free radicals in the two species’ skin. This lower free radical load makes glycoxidation a less important factor in human diabetics’ AGE chemistry, and thus weakens the potential benefit of reducing its impact.
But another, more general reason for the lack of efficacy of antioxidants as an anti-AGE therapy is the sheer riotous promiscuity of the highly reactive precursors of AGE cross-links. One highly revealing animal study11 illustrates the point. Diabetic rodents were given diets fortified with different antioxidant supplements (vitamins or green tea extracts), and the impact on the animals’ AGE burden was assessed by comparison with both healthy animals and diabetics given unsupplemented chow. To tease out the biochemical pathways involved, scientists measured levels of substances produced at several different steps across the spectrum of the glycoxidation process, from initial glycation events to the creation of specific AGE cross-links—some of which are created by glycoxidation, and others by straightforward glycation, i.e., without the involvement of free radicals.
As previous rodent studies had shown, antioxidant treatment did exert some benefits on diabetic complications. Equally predictably, the treatments had no effect on the initial glycation of proteins, since the window of opportunity for free radicals to work mischief in AGE chemistry opens up further along in the process (Figure 1a). But the researchers got a surprise when they began looking at actual cross-links. Antioxidant supplements had no effect on the levels of those AGEs whose formation doesn’t require free radicals, of course—but the intervention actually increased the levels of the two glycoxidation-derived AGEs, so that diabetic animals receiving green tea extracts actually wound up with more total cross-linking than those who simply suffered the “natural” course of the disease.
This remarkable result yet again illustrates the hopeless complexity of the tangled skein that is metabolism. The precursors of these AGE cross-links don’t simply disappear when they aren’t hit by free radicals—they have to go somewhere—and when much of the excess oxidative stress was relieved by antioxidant supplementation, these precursors began to build up until they spilled over into one of the alternative pathways of cross-link formation. It was the same effect you see in traffic jams when a main traffic artery is cut off: a few drivers may indeed just turn their cars around and go home, but most of them turn off onto the local side streets, creating secondary traffic congestion in hitherto sleepy residential neighborhoods.
Collateral Damage
The best-understood pathways of AGE cross-linking are fundamentally random events, not too far removed from what happens in the browning of food, or in a test tube. The fuels of metabolism, dissolved in your blood or in the fluid inside your cells, randomly bump into tissue proteins; depending on factors like temperature, concentration, and the presence of transition metals and free radicals, a series of chemical events may occur; and if they happen in just the right order, an AGE cross-link will form.
But some AGEs result more directly, from the regulated activity of metabolic processes. One recently identified example is the enzyme myeloperoxidase, which is used by macrophages to kill bacteria by generating toxic hypochlorous acid. It has been shown that hypochlorous acid, in the presence of the protein building-block serine, can itself induce AGE-type cross-linking, independent of the usual fuel chemistry of sugars and fats.12
If myeloperoxidase were only ever activated to kill bacteria, it might be a relatively unimportant source of AGEs in people living in the developed world who don’t have chronic infections (although the number of such people is much higher than is generally appreciated). But, as we saw in Chapter 7, macrophages don’t just attack bacteria: they also become aggravated—and crank up their myeloperoxidase activity—in their shortsighted efforts to clear trapped cholesterol from your arteries. Some scientists now believe that myeloperoxidase is probably a major contributor to the high levels of AGE found in the atherosclerotic foam cells of nondiabetic people.
While reducing excess myeloperoxidase activity might be desirable at the sites of atherosclerotic plaque, we probably could never lower its activity pharmacologically without also impairing our ability to defend ourselves against bacteria. As people with AIDS know, when your immune system is suppressed, you’re not just at risk from relatively rare bacterial killers like tuberculosis: you can be felled by infections that most of us shake off before we have even the beginnings of symptoms. Moreover, and surprisingly, one study found that animals bred to produce something like human atherosclerosis, but lacking the ability to produce myeloperoxidase, showed more severe atherosclerosis than animals with normal activity, again illustrating the frustrating complexity of metabolic processes.13
The Drug That Failed
Okay, so trying to lower the levels of the ultimate AGE precursors, like glucose and fat, is difficult—and also unsafe, because they are essential biological fuels. Soaking up free radicals and sequestering transition metals is of limited effect because there are so many alternative routes to AGE formation.
But an overview of Figure 1 may suggest a much more attractive target: oxoaldehydes. For one thing, these reactive compounds are present at much lower concentrations than blood sugar or triglycerides, meaning that you would only have to knock out relatively few molecules to lower the total level in the body by a significant proportion: methylglyoxal, for instance, is several thousand times less concentrated in the blood than glucose. On top of this, oxoaldehydes are very virulent molecules (as I mentioned earlier, methylglyoxal is up to 40,000 times more prone to attacking tissue proteins than glucose is), so that each molecule you take out of circulation is much more likely to translate into the prevention of a cross-link in the waiting. Oxoaldehydes also play their role in cross-link formation relatively late in the process, leaving fewer alternative pathways by which an AGE might form if they could be soaked up. And in contrast to sugars, which are essential molecules for which there is a limit beyond which lowering their concentration in the blood becomes life-threatening, oxoaldehydes are fundamentally toxic molecules, so that one should be able to reduce their concentration drastically without doing any harm to the body.14
Thus, if trying to lower AGE formation with antioxidants or blood-sugar medications is like launching a wide-sweeping crackdown on an entire crime-plagued neighborhood, a drug that mops up oxoaldehydes would be like springing a carefully targeted police raid on known members of a brutal criminal gang.
For a long time, a drug called aminoguanidine (trade name Pimagidine) seemed poised to fulfil this promise, revolutionizing the treatment of diabetes and perhaps landing the first serious punches on the Mike Tyson that is biological aging. The drug enjoyed a lot of buzz in the scientific literature on diabetes, as well as in some of the commentary on life extension in popular magazine articles and Internet discussion groups, because its clearest mechanism of action was precisely its ability to mop up oxoaldehydes.
Over the course of many years, researchers put aminoguanidine to the test—first in test tubes filled with AGE precursors and mixtures of catalysts, then in cell cultures, and eventually in animal studies. And at nearly every juncture, hopes for the drug continued to rise. In diabetic rats, it lowered AGE formation in the cells of the retina and reduced the maladaptive overgrowth of the blood vessels feeding them. In dogs, it prevented the loss of the retinal blood vessel cells and the associated accumulation of dead blood vessels through which blood had stopped flowing. It also kept both species’ hearts and blood vessels more flexible.
Somewhat less consistently, aminoguanidine showed promise against other complications of diabetes. It reduced the total level of kidney tissue that was so cross-linked as to be indigestible by strong acids, and prevented much of the thickening of the kidney’s filtration machinery that accompanies diabetes in rats—although it was unable to affect the course of the disease in dogs. Further, diabetic rodents (but not baboons) given the drug exhibited less loss of blood delivery to nerves, and improved ability to conduct nerve impulses.
Most important for those of us looking for interventions against AGEs’ role in the degenerative processes of aging, aminoguanidine even seemed to reduce heart and kidney AGE levels (and the resulting loss of those organs’ function) in animals that were suffering from purely age-related AGE accumulation rather than diabetes.
After a few small human studies designed to test for any obvious toxicity seemed to go well, the company that had patent control over aminoguanidine for use as a drug for diabetes attracted the attention of the biotech giant Genentech, which partnered with them to launch two full-scale clinical trials. Each one was to involve about six hundred people with the beginnings of diabetic kidney disease, in medical centers spread out all over North America. The first trial (ACTION I) recruited people with type I (autoimmune) diabetes; the other trial (ACTION II) was to involve patients with the more common type II (late-onset) diabetes, which usually develops as a result of lifestyle, albeit sometimes overlaid on genetic vulnerability. Ambitiously, patients in both trials were to be well medicated to control both blood sugar and blood pressure before being put on aminoguanidine, so that differences between the groups would be entirely the result of the test drug’s direct anti-AGE effects.
But when ACTION I was completed in 1996, the best spin that one could put on the results would be to say that they were disappointing. On the positive side, risk factors like blood pressure, LDL (“bad”) cholesterol, and triglycerides went down in people who had received the drug. And some crunching of the data suggested that the drug might improve some indicators of kidney function. Plus, in a tiny subgroup who had been tested before and after the trial, diabetic damage to the retinas seemed less serious in patients taking aminoguanidine than those taking placebo—though these observations were suspect, because they were made as an afterthought after the trial had been shut down rather than being part of its original design.15
What the study was supposed to show was a direct effect on kidney health, as measured by a standard laboratory test for kidney function—and the data just weren’t strong enough to support that conclusion. The raw numbers looked, on their face, better in aminoguanidine users than in the placebo group, but the difference was so small relative to the number of patients in the trial that it seemed likely to be a statistical fluke, like getting “heads” in six out of ten coin tosses instead of the expected five.
Worse: while the benefit attributable to aminoguanidine was dubious, the risks associated with the drug seemed undeniable. Along with signs of an overactive (and possibly damaged) liver and strange flu-like symptoms that went away when they stopped using the drug, a few people taking aminoguanidine developed signs in their blood of an autoimmune disorder, which—in three patients taking the higher dose—was associated with a form of highly inflammatory kidney disease that leads to complete loss of kidney function in a matter of just weeks or months. Two of the three patients who developed the disease progressed to end-stage kidney failure. Fortunately, this apparent side effect was caught early in the trial, and the safety committee accordingly introduced a monitoring program, after which no one was allowed to progress into clinical signs of the disease.
A FATAL RESEMBLANCE
Even now, we still don’t know for sure what caused aminoguani dine’s severe toxicity, which had not been observed in animal studies. But there’s a good guess—and if it’s correct, it makes aminoguanidine yet one more case study in how trying to repress the dark side of metabolic processes so often has repercussions.
What made aminoguanidine a promising AGE-blocking drug was its mechanism: the sopping-up of oxoaldehydes. These sub stances are in a class of chemical compounds called carbonyls: organic molecules with a carbon atom double-bonded to an oxygen atom. This structure makes many carbonyls highly biologically active, which is why oxoaldehydes are such relentless shacklers of bodily proteins. Of course, metabolism relies on the harnessing of highly active compounds to carry out the biochemistry of life—and so it’s hardly surprising that many essential biological molecules also feature prominent carbonyl groups.
The problem is that aminoguanidine can’t necessarily tell one carbonyl-bearing molecule from another. That might be expected to cause it to sop up some essential carbonyl-bearing molecules along with toxic ones like oxoaldehydes. In fact, we know that it does so in at least one case: vitamin B6. As a result, animals given aminoguanidine can easily develop a deficiency of this vitamin indistinguishable from simply putting a subadequate supply of it in their diet.16
Damningly, a blood pressure drug called hydralazine, which brandishes the same carbonyl-trapping hydrazine surface that aminoguanidine’s business end uses to neutralize oxoaldehydes, is well known to cause a lupus-like autoimmune disorder, the first sign of which is the appearance in the blood of the same antibodies observed in the aminoguanidine users in ACTION I.
If a drug as promising as aminoguanidine can’t safely prevent enough AGE damage to improve the health of diabetics, you can be sure that it won’t do much for the basically healthy among us. Because the concentrations of blood sugar and fats are much lower in people without diabetes, the buildup of AGE is much slower, and thus harder to slow down to a degree that causes a measurable change in their health. Thus, it would take a lot longer for any potential benefits to accrue, whereas the risks remain at the same high levels for each individual year of use.
Indeed, a study published after aminoguanidine’s withdrawal from clinical development17 appears to show that even the initial reports of a reduction in age-related AGE cross-linking in nondiabetic rodents were specific to the strain of rat used in early studies (which is particularly susceptible to kidney disease). Other strains showed little or no benefit from lifelong aminoguanidine administration.
These are just a few illustrations of the known or anticipated ways in which the mechanisms underlying cross-link formation undermine our ability to prevent AGEing. This nightmare of biochemical complexity is so elaborate as to cause even the most dedicated puzzle enthusiast to snap pencil in two and go to bed in frustration; it should raise serious doubts about the wisdom of continuing to invest resources in seeking new ways to interfere with such a poorly understood, multiply-branching network of pathways (Figure 1). In the pell-mell of the body’s biochemistry, a certain quantity of AGEs is simply inevitable, and trying to prevent enough cross-linking from happening to have a real impact on the stiffening of our tissues, without somehow disturbing essential metabolic processes, may ultimately be futile.
If you’ve read the preceding chapters of this book, you probably have a pretty good idea of the sort of strategy I’d like to see used to deal with the problem of AGEs, whether in diabetics or in “normal” aging. Don’t mess with blood sugar. Don’t try to block free radicals. Don’t go chasing after ways to outsmart metabolism. Don’t try to prevent AGEs from forming at all. No, the anti-aging engineer’s solution should be to allow metabolism to proceed in its infamously messy way, and then to remove full-blown AGEs themselves before they build up enough to impair tissue function, robbing us of youthful flexibility of heart and sinew and increasing our risk of death and disability.
In this case, however, I am not playing the role of visionary, so much as of cheerleader. At least two companies have developed such drugs and tested them in animals. One of them has undertaken several clinical trials already.
A SENS Serendipity
In the decade since Drs. Tony Cerami and Peter Ulrich had first suggested that the cross-linking of proteins by glycation might be the link (pun intended) between high blood sugar levels and the complications of diabetes, they had spent a lot of their time working on ways to do something about it.
They had played key roles in the development and early testing of aminoguanidine, but well before the failure of ACTION I they knew that much stronger molecules would be required to help two specific groups of people with quite different AGE-induced disabilities. On the one hand, diabetics whose disease had progressed so far that they had already suffered a lot of cross-linking would be quickly approaching the threshold at which their total level of cross-linking would begin to result in disability and death, and would therefore require much more effective interventions than would people only in the early stages of the disease. And on the other hand, many people who suffer with AGE-derived diseases such as hypertension and heart failure have normal blood sugar levels. In these people, the precursors of AGEs are present at much lower levels and are thus are much harder to intercept: it’s like trying to shoot down a single bird in the sky, whereas taking on AGE precursors in diabetics is like sighting one of the incredible flocks that once blacked out the sun in the early days of European colonization of the Americas, into which hunters could fire off buckshot without even bothering to aim.
So in late 1991, Cerami arranged for a summit at their labs at the Picower Institute for Medical Research in Manhasset, New York. The meeting brought together himself, Ulrich, several other Picower staffers, and scientists working for a company Cerami had helped form named Alteon, to brainstorm on new ways to inhibit AGE formation.
Analysis of what was believed about the chemistry and products of these reactions had already led many researchers to conclude (correctly) that reactive carbonyls (like oxoaldehydes) would be important potential sources of AGE cross-links, and thus targets for anti-AGE drugs. This was exactly the rationale for the development of aminoguanidine. Ulrich saw that, theoretically, a lot of the body’s AGEs might be formed from a class of reactive carbonyls known as Amadori diones and the related Amadori-ene-diones. These molecules would form when Amadori products broke down: carbonyl groups would join hands across the gulf of adjoining proteins, resulting in an alpha-dicarbonyl link—specifically, a type of alpha-dicarbonyl called an alpha-diketone link. On chemical grounds, such a link would not be expected to remain intact for long—but it wouldn’t just disappear, either. Most likely, Ulrich thought, it would rearrange into a more stable, final structure—a molecular marriage which only death would part.
If that were true, Ulrich could see one potential path to the development of novel anti-AGE interventions. The body has enzymes that break down at least some kinds of dicarbonyl compounds, and many of these enzymes share in common the incorporation of the vitamin thiamine. Research by Ukrainian scientists in the mid-1980s had shown that molecules in the same chemical family as thiamine (called thiazolium compounds) break linkages of the same chemical type, albeit embedded in organic chemicals rather than as AGEs in tissue proteins. The inclusion of thiamine in so many of these enzymes, combined with the mechanism of other thiazolium compounds, suggested that thiamine was the essential feature to all of them, like the common head shape of different brands and sizes of Phillips screwdrivers. The active core of the incorporated thiamine would get an electrochemical grip on the carbonyls in the enzyme’s target molecule, whereupon the enzyme would twist its shape, opening up and tearing the bonds apart.
Ulrich wanted to design a new molecular “tool” that would do the same splitting job with the dicarbonyl bonds in Amadori diones, eliminating their cross-link-forming potential. Starting with the concept of a thiamine-like “business end,” the assembled scientists began throwing out suggestions on how different kinds of molecular “levers,” “swivels,” and “sprockets” might behave, predicting their interactions with Amadori diones from their structures. Ulrich stood at a blackboard, drawing out their proposals.
Finally, they came up with a basic template of a class of molecules that might be expected to cleave the kinds of bonds present in the Amadori-diones that they believed would probably be found in the body. Then they threw together a variety of specific variations on the theme by tacking on various “limbs” to the core “backbone” structure.
At the end of a marathon session, the Alteon scientists took the results of their work back with them for preliminary testing. At Alteon, Dr. Jack Egan assigned several junior scientists to synthesize test quantities of each of their various candidate molecules, and also to cook up large batches of some model Amadori diones. From there it was a straightforward series of simple experiments: pipette small quantities of the candidate molecules into test tubes full of the AGE precursors, and see if they could inhibit their conversion into more permanent structures.
As it turned out, however, “straightforward” did not mean “quick.” After having run dozens of tests at different concentrations and still not having nearly exhausted the range of experiments they wanted to do, Egan was looking for a faster way to run the experiment. In collaboration with scientist Sara Vasan, he devised an alternative method. He wasn’t sure this new method would work, but they would certainly save a lot of time and effort if it did.
At first, the new procedure seemed to work fine: a lot of the work was moved from the old protocol to the new one, and soon they had accumulated a broad enough sample of data to expect that the answers they needed would be buried somewhere inside their mountain of notes. Vasan gathered the results together and began writing them up for internal analysis and possible publication.
And at first, it seemed that they had their results: the test tubes contained varying amounts of AGEs, suggesting that the compounds had inhibited their formation from their precursors to varying degrees. But a few of the results seemed to be wildly out of step from the main body of work, with the levels of the expected reaction products being well in excess of what could be accounted for by the small variations in concentrations and other factors as compared with other, similar tests using the same compound. The chemistry just didn’t make sense.
Embarrassed at having to ask her supervisor, and afraid that she had simply overlooked something or that she or one of her coworkers had improperly performed the experiments, Vasan showed the results to Egan. He agreed that the results didn’t make sense, and they began going back to the original lab notebooks to double-check the results.
It didn’t take long to see that the outliers were coming quite consistently from experiments using the new, faster protocol. Egan and Vasan went back over the protocol, looking for a flaw—the kind of “garbage in, garbage out” error that makes an accounting program tell you that you owe twice your annual income in taxes. Eventually, they found a mistake in the last few steps in the initial production of the model Amadori diones themselves. At one key point, the correct procedure is to put a halt to the reactions occurring in the test tube, preserving the compounds that have been produced. Instead, the protocol was allowing further reactions to occur, generating alpha-diketones instead of freezing them at the precursor phase. They had, in effect, been “overcooking” their biochemical soup, generating mature alpha-diketone linkages and leaving few or no intact Amadori diones available against which Ulrich’s carbonyl-busters could be tested.
But while this was clearly a mistake, Egan and Vasan doubted that it was the only one, because it couldn’t fully account for the anomalous results of the inhibition tests. The results of those experiments had initially looked right, because after the inhibitors were mixed in with their test compounds, their quick-and-dirty assay methods had detected the remnants of shattered Amadori diones floating like molecular flotsam in the test tubes. But how would such chemical debris have been produced if there had been no Amadori diones present for the thiazolium inhibitors to destroy?
It was then that it hit them. The explanation was staring them in the face; indeed, the chemistry would’ve been obvious, if they hadn’t walked into the experiment with a preconceived understanding of the reactions that they would be observing. Because they could see the broken carbonyl groups that would be expected to be left behind after the thiazolium compounds had torn apart the Amadori-diones that they thought were present in the test tubes, Egan and Vasan had been assuming that the presence of those broken bonds meant that the inhibitors were doing what they had been designed to do. But what if they were doing something else entirely? What if the carbonyl groups that they were detecting in the final samples were the remnants of alpha-diketone links that had been produced in error during the generation of the test compounds, and then torn apart by their model drugs?
Egan felt no “Eureka!” moment of insight, however—and not only because he still hadn’t figured out how those alpha-diketones could have persisted long enough to be attacked by Ulrich’s model drugs. No, his mood was neither intellectual satisfaction nor ongoing curiosity, but a sinking recognition that they had been wasting their time. The Amadori diones would have to be resynthesized, probably using the original, time-consuming protocol, and the inhibition assays run over again.
There was no sense in covering things up. Egan contacted Cerami and explained the situation, apologizing for the wasted time and emphasising that it was all going on Alteon’s bill.
Egan initially thought nothing of the questions that Cerami asked about the experiment: scientists are nothing if not inquisitive. But it did begin to seem odd as the doctor pressed him for more and more arcane details of the procedure, the reasoning underlying his conclusions, and even speculations about how the thiazolium compounds might have reacted with the alpha-diketones. Unfortunately, Egan had no real idea how such an interaction might have led to his observations: he was a bench scientist, not a medicinal chemist. What a relief, he thought: Cerami’s curiosity seems to have gotten the better of his frustration with this setback.
Cerami hung up the phone and leaned back in his chair, his mind racing. Was he missing something? Did he dare to believe what Egan was telling him—or to accept the implications?
Trying to calm his trembling fingers, he dialed Peter Ulrich. Impatiently, he waited for his partner to pick up the phone. Finally, the click of a lifted receiver. “Ulrich,” said the familiar voice at the other end.
“Peter?” Cerami said, keeping his voice steady. “Can you explain to me how one of these compounds could break an AGE cross-link?”
Reverse-Engineering Serendipity
Over the course of the next few weeks, Ulrich worked backward from Egan’s protocol and Vasan’s results, developing a tentative scenario under which mature AGE compounds based on an alpha-diketone linkage could be broken by their new thiazolium compounds. Finally, he thought he had the chemistry right.
If the result held, then they were really on to something. Thanks to the laboratory flub, Picower and Alteon were at the center of not one, but two breakthroughs in AGE biochemistry: one theoretical, and one of enormous potential medical significance. First, the result implied that alpha-diketone AGEs might be stable enough to persist in the body long enough to contribute to tissue stiffening without any further chemical alteration. And second: they had unwittingly designed a class of molecules that would not merely prevent AGE from forming, but actually buzzsaw their way through them.
The biomedical implications were startling. Imagine being able to take patients whose bodies were already extensively riddled with cross-links, and to give them a drug that would break the AGE apart. AGEd tissues would be rejuvenated. Arteries would dilate outward in response to the pulsing tide of blood; hearts would fill with the incoming flow; even skin could become flexible again. It was just the solution that one would dream of for advanced diabetic cases, or for people whose AGEs had built up because of time, not high blood sugar. The market would be enormous.
The hard-nosed chemist in Ulrich brought him back from this vision to the steps that lay between him and its realization. For starters, Alteon would have to run the previous experiments again, monitoring the reactions at each step to provide evidence to support the theoretical chemistry that he had outlined to explain the original result. Additionally, everything that they had done thus far was with an AGE that had been cooked up in a beaker: he still didn’t know whether any alpha-diketone AGE (let alone the specific molecule that Vasan had accidentally produced) ever actually formed in the body at levels sufficient to impair tissue function. And then there was the question of whether his test compounds would be able to reproduce in human subjects what they were doing under glass: the body’s detoxification machinery might metabolize them into inactive forms, or it might be impossible to take enough of the stuff to have any effect.
The first few questions were answered by a more careful, intentional repetition of Egan’s and Vasan’s initial experiments, which seemed to confirm all his hopes. The results of these studies were consistent with the hypothesis—that the predicted AGE was in fact formed from the model Amadori diones, and that it persisted long enough to react with his thiazolium compounds, which did indeed appear to sever the cross-link at the alpha-diketone bridge. And based on the observed results, one particular thiazolium compound—a chemical known as N-phenacylthiazolium bromide (PTB)—was an especially effective wrench with which to pull these AGEs apart.
But the fact that PTB severed a bond in an artificial AGE didn’t prove that it would break any of the cross-links that actually tie up the arteries, hearts, and other organs of aging and diabetic humans. At this point, it was time for Cerami, the more medically minded member of their tag-team, to get more actively involved. The two decided to put PTB through a graded series of increasingly challenging tests using more and more lifelike model systems, working their way step by step from single cells up to functional and molecular investigations in living, breathing animals.
The Manacles Fall Away
These necessary studies were again farmed out to people working under Jack Egan at Alteon, whose lab scientists first confirmed PTB’s ability to cut through AGE using isolated, cross-linked proteins and tissues. With each successful jump over an experimental hurdle, their optimism grew, until they were ready to move their work into the living laboratory of diabetic laboratory rodents. When Egan’s team injected the animals with their new compound, the results were again positive: levels of glycated proteins bound to the animals’ red blood cells dropped by over a third in the first week, and kept dropping, going down to half of the original level after three weeks and to just 40 percent by the end of the month. It really looked as though they were on to something.
With that evidence to hand, Alteon scientists began giving PTB injections to rodents with hearts, kidneys, and arteries hardened by AGEs, accumulated over a normal healthy lifetime or in the fast-forward mode of diabetes. Here the real excitement began to build, as PTB continued to live up to expectations, restoring supple performance to cardiovascular systems that had previously lost their youthful flexibility, instead of just slowing down an inevitable decay as aminoguanidine had done. Structurally, the tissues of treated animals were softer and more elastic, stretching out like rubber bands fresh out of the pack, and readily melting away when doused with digesting chemicals; functionally, their hearts were expanding to fill with incoming blood like new balloons, and blood coursed through their arteries without the large backward-rippling “echoes” of pulse that are characteristic of old blood vessels.
They did have one problem, though, which was that PTB is too unstable to succeed as a drug for human use: by the time a pill had made its way through digestive system and the complex chemistry of the body’s drug-metabolizing processes, too little would be present to have a meaningful therapeutic effect. But Ulrich was not going to give up on so promising an agent, and with a bit of work he and the Alteon chemists were able to develop a variant on its basic structure that was not only more stable but more active: 4,5-dimethyl-3-(2-oxo-2-phenylethyl)-thiazolium chloride. For convenience, Alteon first shorthanded this mouthful to ALT-711 (because it was ALTeon’s 711th compound); later, the compound would be rebranded to the more marketable alagebrium.
A drug with the ability to cleave AGEs that had already formed in the body would have applications in diabetes as well as in a wide range of diseases of aging, but regulators only approve drugs for one specific indication at a time. Wanting to carve out as exclusive a niche for the drug as they could, Alteon strategists set their sights on developing alagebrium for conditions that were not already being successfully treated with existing medicines, and that would be expected to respond uniquely well to their new treatment.
One of these diseases was isolated systolic hypertension (ISH), the kind of high blood pressure in which a person’s systolic reading (again, this is the first of the two numbers that you get from a blood pressure cuff, like the “110” in “110 over 80”) is high, even though their diastolic pressure (the second number) is fine. Systolic pressure is a measure of how much pressure is applied to the artery wall by the surge of blood into the vessel as the heart contracts, whereas diastolic pressure is the baseline pressure in the arteries at rest (technically, at “diastole.”). Hormonal and other factors can actively tighten up the blood vessel, keeping the pressure inside the artery high even during diastole; such effects raise blood pressure irrespective of the intrinsic flexibility of the artery as a tissue. But when systolic pressure is high despite a normal diastolic pressure, it’s a sign that the vessel itself has become stiff, unable to expand to accommodate the incoming rush of blood from the heart.
This nonatherosclerotic “artery hardening” is not just a concern in people with a diagnosis of isolated systolic hypertension. As people push past middle age, arterial stiffness becomes an increasingly powerful predictor of heart disease and heart attack, and indeed comes to override many conventional risk factors like cholesterol and blood pressure when it comes to risk of actual cardiovascular events (heart attacks and strokes). FDA and other regulators don’t recognize this “normal” effect of the aging process as a “disease” for which they’ll approve a drug, so Alteon knew that they could never get official sanction for the use of alagebrium to treat these people; but they also knew that, once the drug had been proven to buzzsaw through the constricting manacles of AGEs in the artery, restoring flexibility and opening up the vessels to the systolic flow, they could vastly expand the market for it by quietly encouraging its unapproved (“off-label”) prescription to untold thousands of aging people with age-related arterial stiffening.
Another disease whose victims don’t get much benefit from existing drugs and would be expected to respond more specifically to an AGE-breaker is diastolic heart failure (DHF). The more common, systolic form of heart failure occurs when the heart’s lower, pumping chamber loses the strength to push out enough of the blood that it receives from the upper chamber to keep the body supplied with oxygen and nutrients. But about a third of heart failure patients have a perfectly normal capacity to pump blood; their problem is that the same chamber can’t expand sufficiently well to take in the required volume of blood in the first place, so that the body’s needs remain unmet even after it squeezes out nearly all of the load that it first takes in. The result is the same—the body’s tissues are starved for blood—but the cause is different, and treatments that admirably address systolic heart failure leave the bodies of DHF patients still crying out for critical fuels. While the underlying loss of the heart’s filling capacity can be the result of a variety of factors, many cases of the disease are associated with AGE stiffening of the heart. Again, an AGE-breaking drug would be uniquely suited to restoring healthy functionality to these people, and trials showing that it could restore the elasticity of old hearts would also spark interest in its use by large segments of a “healthy” but rapidly aging population.
Alagebrium proved its mettle quickly, doing everything that PTB could do and more. Studies showed that alagebrium in the drinking water of laboratory animals could deliver the same kind of restoration of heart and artery flexibility that PTB had elicited only via injection, and more easily. And there were things that alagebrium could do that PTB had never been able to pull off. For example, PTB had cleaved some of the AGEs that had accumulated in the kidneys of diabetic rodents, but not enough to restore the organ’s functionality. Treat the same animals with alagebrium, and not only does their kidney collagen become more soluble, but also the fibrotic damage to their kidneys recedes, and the organs get better at filtering proteins out of the blood, preventing their spillover into the urine.
And rodents were only the first order of mammals to benefit from alagebrium. Alteon and their collaborators soon proved that alagebrium could rejuvenate the hearts and blood vessels of dogs and monkeys. These studies were much more informative about the prospects for alagebrium as a true anti-aging drug than anything that had come before, for a couple of reasons. First, they were carried out in animals that were undergoing “normal” aging, whereas the rodent alagebrium studies had used severely diabetic animals. Second, dogs and nonhuman primates enjoy longer lives, and the extra years give the forces of aging more time to induce the same kinds of pathological cardiovascular system changes that are observed in elderly humans, making them better models of human disease from a clinical and theoretical point of view.
As in elderly humans, older dogs’ heart chambers stretch less to accommodate incoming blood than do those of younger animals, leading to reduced filling with blood and a simultaneous increase in the pressure within it. In other words, old dogs suffer from mild diastolic heart failure. When older animals were given a moderate dose of alagebrium for a month, their hearts became about 42 percent more flexible, as demonstrated by an increase in the volume of blood taken up in the absence of any increase in blood pressure inside the chamber. The contrast was even more marked when the volume of blood delivered into the heart pump chamber was increased using drugs: just weeks before, this treatment had even further widened the performance gap between old and young dogs in cardiac flexibility, but after alagebrium treatment their hearts were nearly as elastic as those of the young controls.18
The results seen in our fellow primates were even more striking.19 In 2001, scientists from Alteon—working in collaboration with researchers from the National Institute on Aging (NIA) who were studying the effects of aging and calorie restriction on nonhuman primates, as well as with NIA specialists in cardiovascular medicine—published the results of a study on the effects of alagebrium in the cardiovascular systems of rhesus monkeys. Their test group was old, but “healthy” as biologically old monkeys go—and in particular, free of diabetes.
At the beginning of the study, the monkeys’ arterial flexibility was assessed, as was the degree to which their heart chambers ballooned outward during their blood-filling (diastolic) phase, as a measure of the flexibility of the tissue. The monkeys then received alagebrium every other day for three weeks, after which their tissues were retested every few weeks for the next nine months.
Surprisingly, there was no measurable effect on blood pressure—systolic or diastolic. But three weeks after treatment, and even more profoundly at the six-week mark, their cardiovascular systems’ tissues had clearly undergone a restoration to more youthful elasticity. Using one rough, easy-to-administer test of arterial flexibility, their arteries had become an astounding 60 percent more pliable; a more direct assay revealed a 25 percent improvement. At the same time, their hearts were also opening up more easily: they were taking in 16 percent more blood during the diastolic phase, and other measures of cardiac function at least partially dependent on improved diastolic filling likewise improved after alagebrium treatment.
Alagebrium wasn’t expected to prevent new bonds from forming between sugars and proteins, so it was no surprise that the withdrawal of the drug was followed by the loss of these gains once the gradual molecular manacling of the monkeys’ tissues was no longer being counteracted by an even more rapid breaking of those bonds. Within a few weeks of the peak of their alagebrium-induced return to more youthful suppleness, the monkeys’ arteries were once again as stiff as they had been in the initial run-up to the study. Their hearts held onto their gains a little longer than the arteries, but then they too began tending to fall back into their old recalcitrance. Quitting the drug didn’t leave the monkeys any worse than they had been to begin with—but it was clear that the AGE links being broken by alagebrium could be quickly reforged. The implication is that users of alagebrium would have to take the drug on an almost continual basis in order to keep enjoying their newfound arterial plasticity.
But that didn’t much dampen anyone’s spirits. The results of these studies marked a clear landmark in the development of alagebrium. Toxicity was low; no serious side effects had been observed; and the promise of a new treatment for stubborn diseases was clear.
It was time for human trials.
From Darkness, Light
The first human alagebrium trial, published in the prestigious American Heart Association journal Circulation in 2001,20 looked like the tentative beginning of something big. Seventy-three older men and women with signs of vascular stiffening had their blood pressure and arterial flexibility assessed and were then placed at random into one of two groups. For two months, two-thirds of the patients took alagebrium in pill form; at the same time, the remainder received a look-alike pill with no active ingredient as a placebo control. At the one-month mark, and again at the end of the trial, their parameters were reassessed.
The results were not altogether clear, allowing for a range of interpretations, but the study was understood to be preliminary by its very nature, and most researchers were willing to give the drug the benefit of the doubt based on the remarkable results achieved in the animal models. Systolic and overall blood pressure had gone down in both groups, probably because of an unusually strong “placebo effect” in the group getting the dummy pill: the influence of the power of belief on the actual state of the body, which is a notoriously important confounder in hypertension studies. Whatever the reason, the result was that the drug conferred no clear advantage in blood pressure results. At the same time, the arterial stiffness of the people taking alagebrium seemed to have improved in comparison to the placebo group using two different measures, but there were technical objections to the method used in one of these assays and the nature of the comparison between the groups also made its results less than decisive.
Subsequent trials, however, have provided results that, in aggregate, allow us to draw firmer conclusions—and, unfortunately for Alteon, they suggest that alagebrium will never be approved by regulators for clinical use. Over a thousand patients with systolic hypertension, diastolic heart failure, systolic heart failure (with and without an associated, compensatory overgrowth of the heart’s main pumping chamber) and even erectile dysfunction, as well as some healthy individuals, have been treated with alagebrium in early clinical trials,21,22,23,24 and while the results provide enough evidence to suggest that the drug is safe and is breaking AGEs in these patients, the effect is clearly insufficient to have much impact on actual function. The results on diastolic function in the heart have not been impressive; the benefits in improved arterial flexibility have not been clear-cut; and little, if any, effect on hypertension per se has been observed. Often the main objective of the trials has not been achieved, with the benefits mostly accruing in less-important markers of the disease process that are not clearly linked to clinical outcomes (cardiovascular disease, heart attack, or stroke). Moreover, the benefits that have been observed have not been cleanly tied down to any particular dose of the drug. This is paradoxical because one might well expect that, in a drug that breaks AGEs, benefits would increase with the dose: more drug ought to mean more broken AGEs and therefore more youthful cardiovascular systems.
To date, the sum of the data from the animal studies clearly suggests that alagebrium can break AGEs; the question is why this benefit is not translating into improved vascular and cardiac health in human users as they do in so many other species.
Some critics hold that alagebrium is not actually an AGE breaker after all, but an AGE inhibitor, just as Ulrich and his colleagues originally designed it to be. There is a certain superficial plausibility to this view, but these arguments can’t stand up against the irrefutable fact that, in animal studies, alagebrium doesn’t just slow down the development of complications in diabetic rodents or prevent the AGE-related tissue stiffening of the cardiovascular system in normally aging dogs and monkeys: it reverses them. A drug that only inhibited the cross-linking of tissues would be able to reduce the rate at which new cross-links would form, and thereby slow down the degeneration of cross-linked tissues—but it would not have the kind of rapid restorative effects that have been elicited by alagebrium.
The fact that the tissues of alagebrium-treated animals become inflexible again so quickly after withdrawal of the drug also seems to weigh in against the suggestion that the drug is actually just reducing AGE formation, since the underlying cross-links are clearly reforming much more rapidly than they did over the many years that were required for their initial buildup. This observation suggests that the severing of the alpha-diketone bridge in these AGEs exposes a highly reactive carbonyl group, which soon sticks itself back onto an adjacent proteins. Because of the ongoing breaking of other cross-links, users of the drug keep ahead of this problem in “two steps forward, one step back” fashion—but take it away, and they undergo a rapid return to their old, AGEd state.
What about the inability of researchers to find alpha-diketone cross-links in the body? The reason is almost certainly that these structures are, ironically, a bit too easy to destroy. The difficult thing about designing an AGE-breaker drug is not that there’s any lack of chemicals that can break apart a given cross-link; the problem is to come up with something that won’t also tear normal, healthy proteins to shreds in the process. The common ways of finding AGEs in the body involve soaking a tissue sample in strong acids and examining whatever’s left over. This technique catches the most extremely hard-to-destroy AGEs, such as pentosidine, but wipes out all trace of more delicate cross-links.
It was long suspected, and has in recent years been confirmed, that the crudeness of such assays introduced serious distortions in AGE research. In the last decade, new methodologies have been developed to uncover AGE cross-links in tissue through a painstaking process of breaking down the normal, healthy chemical bonds in a tissue almost one by one, leaving behind only abnormal chemical linkages such as AGEs. Using these techniques, researchers have proven that the AGEs we previously thought to be the most abundant are actually just the most resistant to the chemical carpet-bombing that had previously been used to drive them out of hiding. The most readily-assayed AGEs (like pentosidine) are in fact relatively rare in the body (and therefore make little contribution to the overall state of tissue stiffening), while other cross-links that are much more common (and therefore impose a much larger total protein-shackling burden on living tissues) remained invisible to our testing methods.
I believe that this is the explanation for our inability, thus far, to identify the molecular targets of alagebrium. The predicted structure of alpha-diketone cross-links is such that they would be relatively easy to break apart: indeed, you’ll recall that this is why Peter Ulrich originally didn’t think that they would even hang around for long enough to be worthwhile pursuing as a drug target.
In turn, the unfortunate difference in the functional impact of alagebrium treatment in human patients, as compared with lab rodents, dogs, and monkeys, may be the result of alpha-diketone cross-links simply being a much more common kind of AGE in those species than in our own. It’s clear that there are differences in the metabolic pathways underlying AGE formation amongst the species. For instance, as we saw above, diabetic rats’ bodies suffer much more oxidative stress than ours do in response to the disease. This should affect not only how AGEs are generated, but which specific cross-links are formed: structures whose formation involves free radicals will probably be a much bigger burden in rat tissues than in human ones.
Another reason to think that alpha-diketone cross-links may be less important to our own species than to others is the simple fact that we’re much longer-lived than those other organisms. Long-lived, recalcitrant AGEs like pentosidine are very hard for the body to break down, so they tend to accumulate pretty linearly with age: the result is that, while longer-lived organisms accumulate them more slowly than shorter-lived ones, they wind up with higher absolute levels of such AGEs by the time they end their lives, simply because they’ve had many more years to accumulate them. So, if you look at Figure 2, you’ll see that at fourteen years of age, an extremely “elderly” dog has about forty units of pentosidine in a milligram of its collagen, while a minipig of the same age—but with half of its maximum life expectancy still ahead of it—has only fifteen units. A monkey could possibly live for forty years, and at age ten it has only accumulated five units of pentosidine. A human, with a maximum life span of more than hundred, has fewer units still. Yet by age sixty, when AGE cross-links are beginning to weigh in seriously on a person’s chances of surviving for another year, human skin is burdened with some fifty units of pentosidine cross-linking its proteins per milligram of collagen—more than any of the shorter-lived species had time to accumulate.
Now: remember that, in contrast to a supremely tenacious cross-link like pentosidine, alpha-diketone cross-links—the kind broken by alagebrium—are predicted to be relatively fragile as AGEs go, so the level of these cross-links is an equilibrium between rather rapid formation and breakage. Like all AGEs, the decline of metabolic control of fuel as we age would lead to an increase in the rate of its formation with age—but its relative ease of elimination should allow the body to largely keep on track of this increase, leading to a much slower rate of accumulation than the stubborn pentosidine.
The net result of this would be that, late in life when AGE-induced stiffening is becoming rapidly fatal, the contribution that alpha-diketone cross-links would make to the total burden of AGE (and thus, to loss of needed flexibility) in a tissue would be less in a long-lived species like ours than in a monkey or a dog (let alone a mouse), for the simple reason that we would have accumulated so much more of the more resistant types of AGE than shorter-lived creatures ever get the opportunity to do. Thus, an alpha-diketone breaker like alagebrium would, even if highly effective at its specific molecular task, leave a much greater burden of other cross-links behind than would be the case in model organisms, resulting in much less effective restoration of youthful tissue plasticity.
Alagebrium and Beyond
So where does this leave alagebrium in the SENS agenda? Clearly, the lack of clear-cut clinical benefits in humans indicates that this drug itself will not play a major role in the reversal of cross-link damage in our tissues. Its value, instead, is as a proof-of-principle: it shows us that AGEs can be cleaved in the body, and tissues regenerated, long after metabolism has done its dirty work in binding our proteins together. What will be required is a new generation of AGE-breakers that slice through more abundant AGEs, and thus free us of the structures that are really holding our tissues immobile. Such agents will yield the same kind of benefits in humans that alagebrium does in animals—benefits first demonstrable in diabetes, ISH, and diastolic heart failure, and ultimately in aging itself.
It’s important to stress that no single drug will totally save us from tissue cross-linking. As we’ve seen, glycation leads to the formation of many different AGEs, each with a distinct structure. Drugs that will sunder any given AGE will probably leave most others untouched: no one molecule will be able to sever all of these distinct intermolecular linkages. Therefore, as we saw with amyloids in Chapter 8, we will need to develop a range of drugs, each of which cleaves either one specific cross-link structure or at most a small family of similar ones.
But the eventual need to develop drugs to break a number of distinct AGE structures doesn’t mean that we can’t effectively stop AGEs from contributing to the aging of our hearts, arteries, and other tissues until we have a solution for all such cross-links in hand. The insight underlying the “engineering” school of anti-aging biotech tells us that we don’t have to solve all of our problems at once to intervene in the process: We can “rejuvenate as we go,” taking one challenge at a time.
To see why this is so, remember that the molecular damage underlying aging begins accruing while we are still in our mothers’ wombs, yet we remain youthful well into our thirties: it takes many decades of these insults before the amount of damage is sufficient to exert a functional impact on our bodies. Until this threshold level is reached, a given form of aging damage is essentially harmless to us in itself.
Therefore, to restore more youthful flexibility to a tissue, we do not need to sever all the various kinds of AGE cross-links in our bodies, but only the ones that make the greatest contribution to the stiffening of our tissues. For practical purposes, rejuvenation will be effected as soon as we can cleave a sufficient proportion of the AGE structures in our bodies to keep the total amount of cross-linking beneath the threshold level that actually impairs tissue function.
Once we have a drug that breaks a given kind of cross-link, we will be free of its baleful influence. The solution, by its nature, will not be once and for all: we will undergo a new course of treatment once every few years or decades, taking the drug for a few weeks or months—long enough to break enough AGEs to leave our tissues as flexible as they were in our youth. These cross-links will immediately begin to build up again, of course—but we have the luxury of letting them do so until that critical threshold level is approached again. (Note that this scenario assumes that the AGE-breaker leaves the broken AGE in a chemically inert state, which alagebrium evidently doesn’t do to alpha-diketones; if the broken AGEs are reactive, the drug will have to be taken continuously.) However, the first effective AGE-breaker will not solve the entire problem of AGEs. It will probably reach the clinic first by virtue of targeting the most abundant AGE, but it will surely not break all AGE species. Thus, other AGE cross-links will continue forming in our bodies unabated, albeit at a slower pace. These cross-links will first reach pathological levels once our lives have been extended for long enough that they stiffen our tissues on their own as much as they and the first-targeted AGE jointly do in a currently normal lifetime.
So, yes, we will need to identify these AGEs as well, and to develop treatments that target them. But the important point from an engineering perspective is simply that this doesn’t emerge as an actual biomedical problem until the first wave of anti-aging (including anti-AGEing) treatments has extended our lives quite a bit on its own. The first breaker of an abundant AGE will buy us the time to identify such AGEs and to develop new treatments that will free us from them in turn.
Eventually, we will develop a lifelong regimen of AGE-breakers not unlike the childhood vaccine schedule today, under which we will receive a series of specific cross-link-severing drugs, each administered on its own cycle of years, decades, or perhaps even centuries, based on the rates and sites of their targets’ formation in the body. But to achieve the first great leap forward in restoring the suppleness of youth to AGEd tissues, we need only prioritize the development of cross-link breakers that will carve their way through the cross-links that cause us clinical problems within a presently normal lifespan.
The above section only discusses AGEs, but similar logic applies right across the SENS spectrum. I’ll be discussing it in more detail in Chapter 14.
Know Your Enemy
Today, for the first time in history, we are in the position to design such drugs on a rational basis. Just over a decade ago, when Peter Ulrich and the Alteon chemists were doing the work that ultimately led to the development of alagebrium, they were working in the dark. They didn’t even know for sure that the types of AGE that they were targeting actually existed in the body—they were simply guessing, from studies carried out in test tubes, that such links might form and contribute to stiffening of living tissues. But the new enzyme-based methods that I mentioned earlier now allow us to slowly take tissues apart at the molecular level, layer by layer, revealing the presence and levels of pathological cross-links in our body, exposing our previously hidden opponents to the light of science.
Researchers who have taken the time to develop and apply these new, painstaking procedures to aging human tissues have given us reliable targets on which to fix our crosshairs: a complex structure called glucosepane, which was only identified using these new techniques in 1999,25 and probably another AGE called K2P, which is prominent in the lenses of our eyes and possibly other tissues.26 Glucosepane is the single most important contributor to the body’s AGE burden known to date, tying up as much as one out of every five molecules of the key structural protein collagen in old, nondiabetic humans. Glucosepane levels are around one hundred times as high as that of any other AGE that had previously been found in collagen or the lens. A drug that could free our tissues from these AGE shackles would have a much larger impact on total cross-linking burden than alagebrium does, and would thus bring our tissues much closer to their full youthful flexibility and functionality.
Today, while we will still use the same sort of blackboard molecular engineering that Ulrich and his copanelists did in the early ’90s to design new AGE-breakers, we can fashion molecular bolt-cutters that are precisely tooled to break specific AGEs, whose exact molecular structures are in our possession, and whose presence in our bodies and biomedical significance as major contributors to the total cross-linking of our tissues with age are certainties.
We also have the advantage of having faster screening tools in our possession. We can use software to simulate the behavior of AGE-breakers, and to automate the generation of variations on a core molecular theme. We can put robotics to work to synthesize thousands of ampoules of candidate drugs, and use mechanized, high-throughput techniques to assay their effects against a known culprit in the loss of elasticity in our hearts and other tissues.
Being able to look at glucosepane’s exact chemical structure gives us another advantage in developing its nemesis that Ulrich no doubt wishes he had had so many years ago. The hard part of developing a glucosepane-breaker for clinical use is not identifying chemicals that can destroy it: as we saw earlier, the acids we used in our old AGE-assaying techniques did a regrettably excellent job of it. The problem is to create molecules that will do it selectively, without damaging healthy biomolecules that share the same vulnerability exploited by the would-be drug. Having reconstructed glucosepane’s molecular identity, biochemists can now see just how wide is the latitude they have in designing molecular shears for it.
Fortunately, this latitude may be very wide indeed. The structure of glucosepane is so different from any functional structures in ourselves or other mammals that a drug that selectively targets them should be harmless to any molecule that is supposed to be found in our bodies.
As I’ve explained, the resulting glucosepane-breaker should be the first in a series of AGE-cleaving drugs that we will need to unbind our proteins from their molecular shackles. Then we will have achieved for the first time in humans what was observed just a few years ago in dogs and monkeys treated with alagebrium. Old hearts will open wide again, free to fill with life-giving blood. Hardened old arteries will once again readily expand in response to the surge of the blood of life. The stiffened, inflexible tissues of the aged will move with the suppleness of youth. The absurdity and outrage of a body tying itself into molecular knots will come to an end, and we will bend and flex within and without as children in the jungle gym of life.