Continued from page 36 found in a bacterium, such as Esche richia coli), "It's amazing this big piece is stable in yeast," Szalay says. The whole piece is integrated into a yeast chro mosome and is carried along (and replicated) during the yeast's normal growth cycle for many generations. The nitrogen-fixation genes are pre served when yeast cells undergo meiosis, during which duplicate cop ies of genetic material are halved in preparation for the sexual reproduc tion side of the yeast life cycle. The available means for manipu lating such complex packages of genes are more sophisticated for yeast cells than for mammalian cells. The plasmids that carry the nitrogen fixation genes are, in effect, programed to in tegrate into the yeast chromosome. The Cornell team's work is even a bit more fanciful than that, involving a kind of of flying trapeze effect, during which one set of genes on a plasmid was used to swing into the chromo some another set of genes on a second plasmid. Regardless of such high-flying techniques, the question remains whether the genes can work inside the yeast cells. And, the answer to that question is the premise for going on to the eventual challenge of transferring those genes into green plants, such as corn, that currently depend on an expensive outside supply of nitrogen fertilizer. "If we can't get expression [of the nitrogen fixation genes] in yeast, it's not worth spending the public's money to get it to work in plants," Szalay says. But he's confident that "we can push yeast to do it." Jeffrey Fox, Washington
Iron chelators may aid in anemia treatment The National Institutes of Health's six-year-old project to develop a new drug for use in treating patients with the genetic blood disease Cooley's anemia is coming up with some unexpected findings. As its initiators had hoped, the project is beginning to produce some very promising drug candidates. But it also is showing both aca demic researchers and governmental administrators that the task of de veloping a successful drug is an enormously complicated one in which successful chemistry is by no means the only critical factor. The project's aim is to develop a safe, effective, inexpensive, and easy-to-take drug to remove the ex cess iron that builds up in patients 42
C&EN Sept. 29, 1980
with Cooley's anemia (C&EN, May 2, 1977, page 24). These people produce inadequate amounts of the beta chain of the hemoglobin molecule so that their own blood is not an effective oxygen carrier. They require regular blood transfusions to survive, but this treatment eventually overloads their bodies with iron. The iron accumu lates particularly in the kidneys, liver, and heart, where it leads to organ malfunction and death, often when the patient is in the mid-teens or early twenties. The current project does not aim at curing Cooley's anemia, but rather at controlling a potentially fatal side effect—the buildup of iron in critical tissues. An early success of the program, most involved in it agree, was getting a drug approved for removing excess iron from Cooley's anemia patients. This was accomplished in 1977 when deferrioxamine, a drug used for treating acute iron poisoning, was approved for use in this disease. Deferrioxamine is not an ideal drug, however. It must be given by injection, and, since its half-life in the body is very short, it works best when it is slowly infused for six to 12 hours each day. Such treatment is prohibi tively expensive: Elmer B. Brown, professor of medicine at Washington University school of medicine in St. Louis, and a participant in the NIH project, estimates that drug costs alone are more than $4500 per year for this therapy. However, better drugs may be on the way. The one that is presently causing the most excitement is being developed by Colin G. Pitt, a chemist at Research Triangle Institute in North Carolina. Pitt has several compounds that remove more iron from rat and mouse models than does deferrioxamine. His most promising compounds are ester derivatives of N,iV / -bis(2-hydroxybenzyl) ethylenediamine-iV^iV'-diacetic acid or HBED. This compound itself is a
better remover of iron from these animal models than is deferrioxam ine. However, when it is made into the dimethyl, dipropyl, or dipentyl ester derivative, it becomes even better. These ester derivatives are orally ac tive and less toxic than HBED in acute toxicity tests in animals, but like HBED they are better than de ferrioxamine at removing iron from animals. The derivatives are an order of magnitude better at removing iron from the animals than anything pre viously tested, Pitt says. In fact, they are able to remove nearly 30% of the iron theoretically available in the animal, he says. "If the efficacy of the esters of HBED is confirmed clinically, the goal of identifying an orally active chelating agent capable of controlling iron overload will have been achieved," Pitt told a symposium on the development of iron chelation drugs held in San Francisco last month. Finding these compounds was a combination of logical chemical in vestigation and pure luck, Pitt points out. The compounds are phenol de rivatives, a class of compounds ex pected to bind ferric ion tightly based on the stability constants of iron(III) complexes of phenol. Furthermore, many bacteria contain phenolate compounds called siderophores, which the bacteria produce in order to scavenge iron from their environ ment. In the molecule HBED, carboxylate and amino groups also are present to assist the compound in binding iron. These groups, too, often are found in naturally occurring siderophores. However, the chemical behavior of these compounds is not completely predictable from their structures and the stability constants of their iron complexes. Pitt and colleagues tested many compounds expected to have a high affinity for iron based on these criteria, and found them to be inac-
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tive. In fact, there was found to be no general relationship between activity and stability constants, suggesting that other factors, such as solubility, biostability, and kinetics, also are important in determining iron binding efficiency. For example, a phenolate ligand very similar to HBED, ethylenediamine-N,N / -bis(2-hydroxyphenylacetic acid) or EHPG, is more effective than HBED in removing iron from mice, even though HBED has the greater iron binding constant. Even among the esters of HBED there are unexplained differences in activity. The even-numbered esters—ethyl, butyl, and decyl, for instance—are not orally active. This may reflect differences in the ease of formulation or metabolism of these compounds, Pitt su2£ests. The next step for the HBED derivatives is long-term testing in animals to determine whether these compounds are safe for long-term use in humans. Their acute toxicity is very low (LDÔQ'S of greater than 800 mg per kg body weight). Long-term toxicity testing—the phase II and phase III studies required by the Food & Drug Administration before a new drug can be marketed—still must be carried out. Despite obvious enthusiasm for the HBED derivatives among synthetic chemists and clinical physicians involved in the drug development project, there seems to be a growing sense that even these promising compounds are a long way from a marketable drug. For one thing, several drugs that looked promising earlier in the program now have been tested in humans, and all of them are either more toxic or less effective than deferrioxamine. Rhodotorulic acid, for example, is a naturally occurring iron chelating agent produced by a yeast. Its production costs in large scale likely would be less than those for deferrioxamine, and early studies in man seem to indicate that the compound can draw iron from a larger pool within the body than can deferrioxamine. However, intramuscular or subcutaneous injections of this compound into two patients with Cooley's anemia produced intense pain that lasted for several days, and tenderness and swelling that lasted for more than a week. Since deferrioxamine does not produce these effects, there seems little potential for clinical use for rhodotorulic acid unless these side effects can be controlled. A similar disappointment was the fairly simple phenolate ligand, 2,3dihydroxybenzoic acid. Like the esters of HBED, this compound showed promise of being effective when given
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C&EN Sept. 29, 1980
orally. It also trapped more iron from an overloaded rat than did deferrioxamine. But preliminary studies in 13 patients with Cooley's anemia, al though they showed no toxic effects, also showed no effect in removing iron from the serum or liver. In fact, the more the researchers learn about the way iron chelators work in vivo, the more they realize how poorly understood is the whole field of iron metabolism. Iron seems to be present in the body in at least four pools—within the cells in heme proteins and in a storage protein called ferritin, in the serum in a pro tein called transferrin, and probably in some sort of intracellular labile iron pool, explains Allan Jacobs, a hematologist at the Welsh National School of Medicine in Cardiff. Although not all investigators agree, one explanation for the ex tremely low toxicity of d e f e r o x a m ine is that it cannot enter the cells and extracts iron only from the labile pool. Because this pool is in some sort of equilibrium with iron in ferritin and transferrin, deferrioxamine can in directly extract iron from cells and serum as well. The most effective iron chelators, however, and the only ones that would be orally effective, would be those that can enter the cell. But such agents run a much greater risk of in terfering with vital iron pools that are necessary for good health. An iron chelator that took iron from hemo globin would make Cooley's anemia patients even sicker. Thus, repeated failures to come up with a better iron chelating drug than deferrioxamine have led some researchers to question whether it is its very awkwardness of administration that makes defer rioxamine so safe to use. Similarly, it is the iron that even tually builds up in critical organs, particularly the heart, that is fatal to Cooley's anemia patients. But re searchers do not know which pool this iron comes from, or whether defer rioxamine or any of the other iron chelators under study are attacking the "right" iron to keep these patients alive. Finally, there is the question of whether other research programs that aim at curing Cooley's anemia more fundamentally—perhaps by manip ulation of the defective gene that is responsible for the condition, for ex ample—may progress fast enough to make a full-scale, and expensive, drug development program unreasonable. Such solutions are still likely to be a long way off (see page 35) but they seem very much more feasible now than they did when the NIH drug development program began. Rebecca Rawls, Washington
