The Demon Under the Microscope, page 15
One set of compounds they studied contained gold. Gold, like another heavy metal, mercury, had been considered a curative for thousands of years (some gold-containing compounds still are used today in the treatment of joint disease). Koch himself had investigated gold’s ability to kill tuberculosis germs before discarding chemicals containing the metal because of unacceptable side effects, rashes, kidney problems, and an unpleasant condition with the evocative name “gold intoxication.” Klarer and Domagk revisited the field, making and testing a number of new gold-containing compounds. While some worked in vitro, they all proved too toxic in animal tests. They turned to acridine dyes, a group that had started with Perkin’s mauve, a family of dyes that Ehrlich and others had found to have an effect on parasites and bacteria. Some of Klarer’s new acridines worked in the test tube; none stopped disease in mice. They tried a series of new quinine derivatives. Nothing. It was enough to make even the most hopeful give up hope. By 1930, as one American researcher noted, “[I]t was the almost universal opinion of physicians that nothing could be discovered which would be effective against the ordinary diseases produced by bacteria.”
Domagk, however, remained committed to his search. He believed in the test system he had perfected, and he believed in Hörlein’s vision for industrial research into cures. He had seemingly endless supplies of chemicals to test, animals to test on, and—as long as Bayer believed in him—money, support, and time.
Perhaps the most important factor was Hörlein’s optimism. Hörlein, master of the research enterprise at Elberfeld, had set up the system, and Hörlein kept the money flowing to fund it through years of frustration. In his case the optimism was buttressed by proof that his system worked. No breakthrough medicine to use against bacteria had yet been found, but Roehl’s old tropical-disease unit continued to uncover profitable antiparasitic medicines. Fritz Mietzsch had just synthesized a strikingly improved antimalarial, not only more powerful than Plasmoquine but easier to use; it killed the malaria parasite during the point in its life cycle when it was actually causing the main symptoms of the disease, making the timing of the medicine far easier for physicians. Atebrin, as Bayer named it, was released in 1930. It was not a perfect drug: It was expensive, for one thing, and dosing had to be close to toxic levels for the medicine to work well. It could cause diarrhea and vomiting, and it could turn skin yellow. In the army it was rumored to cause impotence, so German soldiers sometimes refused to take it; in military camps in malarial foreign areas, Atebrin often ended up in the toilet rather than in the patient. But when it was taken properly, it worked. It was the best malaria medicine on the market, a terrific sales success for Bayer, and it remained a very profitable drug through World War II.
If his new-drug research system worked for parasitic diseases, Hörlein believed, it would work eventually for bacterial diseases. False starts and dead ends were part of the game. He hewed to his vision, kept the upper administration happy (helped by occasional successes like Atebrin), and encouraged his team. The rewards would come. So his chemists kept pumping out new compounds, the animal staff kept breeding thousands of new mice and rabbits, and Domagk’s operation continued to infect them and try to save them. The goal was simple: a new drug that could generate profits for the firm. Hörlein’s system was going to pay off. He knew it. It was already working.
It just wasn’t working for Domagk.
THE TROPICAL-DISEASE group was housed next door to Domagk’s new laboratory, and a certain amount of communication took place between them. All of Klarer’s chemicals, for instance, were provided not only to Domagk but also to the tropical-disease unit for testing against parasites. Although Mietzsch worked primarily for the tropical-diseases group, many of his chemicals were also tested against bacteria by Domagk. Mietzsch and Klarer worked at neighboring benches up in Workroom 4, and soon they began sharing techniques and approaches.
Domagk also looked to the tropical-disease group for starting points. Roehl had worked through a number of acridine dyes before Domagk and also had looked at another family of dyes, called azo dyes, before his death. Roehl had become interested in azos through his old boss Ehrlich, who found that one of them, called trypan red, had a notable effect against sleeping sickness in mice. Unfortunately, the effect was far weaker in humans. Other researchers had reported minor antibacterial effects of azo dyes as well. One form called chrysoidin had excited some interest. It was especially toxic to bacteria in the test tube, although it also had significantly toxic side effects in animals.
Late in 1930, after the gold and quinine tests had been abandoned, Klarer began working with azo dyes. These chemicals had a number of advantages. They were less toxic than many of the compounds the team had been exploring. Chemical variations were relatively easy to make. The core of the molecule—two carbon rings linked by double-bonded nitrogen atoms (this double bond between the nitrogens, the azo link, gave the family of dyes its name)—was like the frame of a bicycle. A chemist as talented as Klarer could easily change the wheels and gears, customize the handlebars and seat, add a cart in back or a basket in front, make a thousand variations on the core structure. True, azo dyes were a bit difficult to purify, and they had a bad habit of staining test animals various colors. Those were minor setbacks. More important, there were small hints from the beginning that azo dyes could kill bacteria. They were not powerful enough yet, but they might be made stronger. As Mietzsch put it, the chemist’s goal was to put “the right chemical substituents in the right position on the azo group, when it is a question of bringing out the slumbering chemotherapeutic characteristics.” At Bayer they believed that Panacea lived, perhaps within the family of azo dyes. It was just a matter of waking her. A steady stream of new azo dyes began flowing in 1931 from Klarer’s workroom to Domagk’s testing labs.
From the start the results were tantalizing enough—azo dyes sometimes killing bacteria in test tubes, other times having a weak effect in animals—to put Klarer into one of his frenzied states, a rapture of synthesis. Most research chemists did well if they created and purified one or two new molecules a week. Klarer could do that in a day. In 1931, hot on the trail of azo dyes, he began working to his physical limit, creating not only a new azo-dye derivative every few days—sixty-six of them over an eight-month period—but at the same time, because azo dyes were only one of several trails he was following, pumping out twice that many non-azo chemicals. It was a virtuoso performance. Some of his azo dyes began showing activity against tropical diseases, fighting bird malaria, rat leprosy, sleeping sickness. The results with bacteria continued to be spotty.
In the summer of 1931, something bigger began to emerge. When test results came back for the 487th chemical Klarer had synthesized at Bayer (Kl-487 in Domagk’s notebook)—somewhere around the 100th azo-dye derivative the chemist had made, this one with an atom of chlorine attached—there was finally reason for Domagk to put a W in his notes. Wirkung. A definite effect. It wasn’t as strong as they needed—Kl-487 worked against only one kind of bacteria, Domagk’s superstrep, and it worked only in high doses—but something was there. The results were tallied on August 4, when most lab personnel were on summer vacation. When Klarer returned, he attacked the azo dyes with renewed zeal, trimming a side chain here, bonding a new one there, rebuilding and restyling Kl-487. He was doing nothing but azo dyes now. In the first three weeks of September 1931, he delivered fifteen new azos, producing every working day one new substance never before seen on earth. When Domagk tested them, he found the results perplexing. Some of Klarer’s new molecules had a slight effect on bacteria, but others, seemingly close in structure, none at all; some stopped strep, others affected other bacteria; some killed bacteria in the test tube, others did not. There seemed to be no rhyme or reason. Klarer kept at it, but despite his best efforts, he could not seem to make the effects grow stronger.
On the eighteenth of September, Klarer delivered Kl-517 for testing. Like Kl-487, it included a chlorine atom; the chemical difference between the two molecules was slight. But the slight variation somehow made a significant difference. When Domagk saw the animal results in October, he was elated. He wrote “Strepto!” in his lab notebook, followed by several test results marked with a “W!” Kl-517 had protected some mice from a streptococcal infection—afforded not just a longer life, but complete protection. The chemical worked at lower concentrations than others, and it worked even when the substance was given by mouth (the ability to give a drug by mouth was an important sales advantage). Kl-517 again was not perfect—it worked only on his strain of test strep, was still not strong enough for the marketplace—but now Domagk was certain they were moving in the right direction. It was followed by more encouraging results, especially Kl-529, in which Klarer attached two chlorine atoms instead of one, which seemed to extend the effectiveness beyond strep to other bacteria. Klarer was again working at top pace. He was writing progress reports on so many azo dyes that he had a little rubber stamp made with the core azo structure; this he inked and stamped into his reports, then added the side chains by hand. On the eighth of November, 1931, Bayer filed a patent for a process to make the new chemical. As usual at Bayer, it was filed under the names of the chemists who synthesized it, in this case both Klarer and Mietzsch. Domagk’s name did not appear.
Then, for some reason, the positive results stopped.
None of Klarer’s newest variations were working. After a few weeks, Domagk went back and retested Kl-529. This time his results were not as encouraging as they had been in the first round. In December he retested again. This time Kl-529 did not seem to work at all. This was precisely what was not supposed to happen in Domagk’s carefully refined test system, which he had devised specifically to avoid any sort of random variation. His lab notebooks now began to show uncharacteristic question marks along with strings of oWs—ohne Wirkung, without effect.
No matter what they did, Klarer and Domagk could not seem to get the azo-dye effort back on track. By the end of the year, the whole series began to look like another dead end. Not only were they unable to move toward more powerful variations, Domagk could not even replicate his earlier results. By the time they rang in the New Year of 1932, the trail had gone completely cold.
There had been an effect—not a real breakthrough, but at least something—and they had lost it. Klarer, in his year-end research report to the administration for 1931, did not even bother to mention his work on the azo dyes, but something kept Domagk from giving up. On one level he wanted his test system to work perfectly, and the azo results were perplexing enough to make him return simply to see what had happened. Perhaps he saw something in the results, something no one else could, the way he found bouquets of four-leaf clovers where others walked by. Whatever the reason, through early 1932 Klarer kept making azo dyes, and Domagk kept testing them.
The results remained confusingly erratic: an occasional weak effect on some kinds of bacteria, an occasional W, but generally no effect at all. In April, Klarer tried replacing the chlorine with an arsenic atom attached to the azo core, creating Kl-642, a dye that once again seemed to work a bit. Domagk found that it was effective against many kinds of bacteria but, like all arsenicals, was also highly toxic, too poisonous to try on humans. Klarer dropped arsenic and tried moving chlorine to other positions. He tried attaching iodine to the azo core. He tried potassium. He tried varying the length of side chains of carbon, capping them often with a nitrogen group, which seemed to help provide a weak effect. He tried shortening the side chains. He tried moving them around. He tried to find a pattern. None appeared. By the fall of 1932, somewhere around Kl-700, even Klarer was running out of ideas.
Then Hörlein had Klarer in for a chat. Perhaps the older man sensed that his talented young chemist—so much like a racehorse, so fast, so high-strung—was becoming discouraged. Perhaps, given that he himself had started his career at Bayer trying to make azo dyes more colorfast, he took a special interest in the azo work. It could have been that Klarer had asked for the meeting to complain about the progress he and Domagk were not making. At Bayer, after all, a significant portion of any chemist’s income came from a share of money from profitable compounds they patented (all of Bayer’s patents were taken out in the name of the discovering chemists to ensure that they shared in the profits). A percentage of the income from a fast-selling medicine like Atebrin could make a chemist like Mietzsch quite comfortable for years. Klarer, whose work with Domagk had yielded nothing of value, was scraping by on a minimal salary. Whatever the reason, Hörlein and Klarer spoke about the azo dyes. Klarer said later that it was Hörlein who brought up the idea of sulfur. Back in the old days, Hörlein and other chemists had made azo wool dyes more resistant to fading by attaching side chains containing sulfur. They had patented a few. If these sulfur-containing azos stuck more firmly to wool, perhaps they might stick more firmly to bacteria, providing a more reliable medical effect.
In the first week of October 1932, Klarer began attaching sulfur-containing side chains to his azo dyes. One of the first he made was an azo created by attaching para-amino-benzene-sulfonamide, a molecule more commonly called called sulfanilamide, one of the compounds Hörlein and the other chemists had used twenty years earlier. The sulfanilamide molecule itself was nothing special. In fact, it had been common around dye factories ever since a Viennese chemist named Gelmo first made and patented it back in 1909. It was long since out of patent and available in bulk quantities. Sulfanilamide—often referred to as sulfa—was relatively easy to make and cheap to use. It linked easily to other molecules. There was no problem integrating it into as many azo dyes as Klarer wanted.
The first sulfa-containing azo dye to reach Domagk in early October 1932 was Kl-695. Recollections differ as to what happened next. It appears that Domagk took an autumn holiday around the same time that the batch of a half dozen new chemicals, including Kl-695, came into the lab. While he was gone, his assistants kept up a full schedule of animal tests. Margarete Gerresheim, one of Domagk’s top lab workers, remembered testing one of Klarer’s new azo drugs—possibly Kl-695—on mice. The bacterial-disease panel was varied a bit from test to test. Sometimes Domagk’s superstrep was included, sometimes not. Lately Domagk had not been doing strep tests on most of Klarer’s new chemicals. Luckily, the tests were done on this batch. When the results came back, Gerresheim and the other assistants saw one very striking exception to the usual cages full of dead mice: the strep-infected animals who had received Kl-695. This one test batch not only survived but were, she told an interviewer many years later, “jumping up and down very lively.” When Domagk returned from his vacation, Gerresheim proudly presented him with a large table summarizing the latest results. She told him, “From now on, you will be famous!”
Domagk’s lab-notebook page for Kl-695 is different from the thousands of others that preceded it. He was a devil for consistency, always noting the results of tests on his bacterial panel—with its usual depressing list of oWs—down the left side of every page. On the page for Kl-695, however, most of the bacterial-panel results are pushed over to the right. In their place on the left is a long list of results for one kind of infection only: strep. And all down the list are Ws. Ws with plus signs. Ws with double-plus signs. Ws with triple-plus signs. Kl-695 worked like nothing Domagk had ever seen—like nothing anyone had ever seen. The chemical protected mice completely from strep infection. It protected them when delivered by syringe. It protected them when delivered by mouth. It protected them at every dose level he tried. The mice were not only alive but in perfect health. There were no apparent side effects. The results were so perfect it looked more like a mistake than a real experimental finding. Perhaps something had gone wrong in procedure; perhaps the test group had not received the proper dose of bacteria. Domagk immediately ordered retests with a wider range of Kl-695 concentrations. Again the drug kept the test mice alive, even in doses smaller than before. Again there was no apparent toxicity. Strangely, it did not kill strep in a test tube, only in living animals. And it worked only on strep, none of the other disease-causing bacteria. But, given the number and deadliness of strep diseases, it worked where it counted. It kept working through three series of retests. Hörlein was informed; when he heard the good news, he asked Domagk to keep quiet about his findings until they knew more.
Domagk then went back and ran strep tests on every chemical Klarer had given him for the past month or more, azo or not, plus everything every other chemist had sent him. All the animals died. The superstrep test was working perfectly. But Kl-695 continued to protect the mice.
That was standard operating procedure at the firm. Domagk was not much worried about patents. The important thing for Domagk was that nothing had worked for five years and now suddenly everything seemed to be working. Klarer now made variations on Kl-695, finding that as long as sulfa was attached to the azo-dye frame in the correct position, the drug worked against strep. Attaching sulfa to an azo dye—any azo dye—somehow transformed it from an erratic, ineffective chemical into an efficient anti-strep medication. The sulfur-containing side chain simply had to be attached in one particular spot at one end of the azo dye; as long as it was hooked there, it worked—against strep. Klarer kept exploring. Perhaps tweaking the molecule a different way would make it effective against more kinds of bacteria. Perhaps a variation might work against cancer. The guiding principle for both Klarer and Domagk was that the frame in the middle, the azo-dye core, was the power center for the medicine. The side chains, the addons like sulfa, were keys to turn it on. More needed to be known about how this sulfa key unlocked the power of the azo dyes.
