ELION, GERTRUDE BELLE

(b. New York, New York, 23 January 1918;

d. Chapel Hill, North Carolina, 21 February 1999),pharmacology, antimetabolites, immunosuppressors, anticancer drugs, antiviral drugs.

Elion shared the 1988 Nobel Prize in Physiology or Medicine with James Black, who discovered beta-blockers and H₂–receptor antagonists, and George H. Hitchings, with whom she had collaborated for more than forty years, the two being responsible for the discovery of many major therapeutic agents—anticancer, antiviral, antibacterial, immunosuppressive, anti-gout—whose common characteristic was that they were specifically targeted at nucleic acids. Elion may thus be considered a founder of molecular pharmacology. Although she was the fifth woman to receive the Nobel Prize in Physiology or Medicine, she was the first who was neither a physician nor the holder of a doctoral degree.

Elion was born in New York on 23 January 1918. Her father, Robert Elion, had immigrated from Lithuania at the age of twelve and studied dental surgery in New York. Her mother, Bertha Cohen, had arrived in the United States from the Russian-Polish borderlands at age fourteen. Their daughter graduated from Hunter College in 1937. Academia was scarcely welcoming to women in those days, and she was unable to pursue her studies immediately. She taught for a semester at the New York Hospital School of Nursing and volunteered at a chemistry laboratory. Only in 1939 did she embark on postgraduate studies at New York University, where, two years later, the sole female candidate, she was awarded a master of science degree in chemistry.

When her grandfather died of cancer, Elion began to dream of a career in medical research, but she was obliged to start out as a food analyst for the Quaker Maid Company. The American mobilization for World War II, by opening up many positions to women, gave her a chance to enter the pharmaceutical industry. In 1944, after a few months with Johnson & Johnson, she was offered employment with Burroughs Wellcome as an assistant chemist in the laboratory of Hitchings in Tuckahoe, New York. There she began, ten years before the discovery of the double helix, to investigate modifiers of nucleic-acid metabolism.

Contemporary advances in antineoplastic chemotherapy prompted the reorientation of this research toward cancer. The first concrete results began to appear in 1947, with the formulation of the antileukemics 6-mercaptopurine and 6-thioguanine. Elion and her colleagues subsequently developed azathioprine, a powerful immunosuppressive drug. Another line of inquiry led to allopurinol, a treatment for gout and hyperuricemia.

Elion’s work culminated in a great discovery, that of the strong antiherpetic action of acyclovir. From 1967 on, she headed Burroughs Wellcome’s Experimental Therapy Department. Her name appeared on forty-five patents. She received twenty-five honorary doctorates and was elected president of the American Association for Cancer Research. She entered semiretirement in 1983, but was invited to teach at Duke University (Durham, North Carolina) and at the University of North Carolina at Chapel Hill. She also worked for the World Health Organization and strove to increase support for young researchers, notably through the Wellcome Foundation. The Nobel Prize was a fitting coda to her remarkable career.

Gertrude Belle Elion, known to her friends as “Trudy,” died at the age of eighty-one on 21 February 1999 in Chapel Hill.

Research Program . In the mid-1940s, the idea of producing drugs designed to treat viral conditions seemed incongruous, for hitherto almost all efforts had been bent toward the development of vaccines capable of preventing such conditions. The aim now was to intervene early, at the beginning of the reproductive cycle of viruses. One of the difficulties facing Elion and her coworkers was the relative crudeness of the techniques available: Their physical and chemical apparatus was of the most basic kind, and they had little by way of spectroscopic or radiological equipment.

Antimetabolites . The theory of antimetabolites, conceived in 1940, was the brainchild of the Oxford biochemist Donald Woods, who argued that exogenous substances could compete with the natural substrates necessary to the anabolism of microorganisms and thus inhibit their replication. Woods observed that brewer’s yeast impeded the action of sulfonamides, and concluded that this yeast contained a substance closely resembling sulfanilamide and capable of competing with it. Based on this competition between sulfonamides and aminobenzoic acid (PABA), a natural substrate of bacterial metabolism, the term antimetabolite was applied to any substance capable of exercising such an inhibitory function.

Following a similar line of inquiry, Hitchings showed that different antimetabolites could prevent the growth of microorganisms. Thus pyrimethamine, a dihydrofolatereductase inhibitor in protozoa, was proposed as an anti-malarial agent.

In 1966 other chemical modifications brought about in the benzene cycle of 5-benzyl-2,4-diaminopyrimidine made it possible to obtain a derivative that was active against Proteus vulgaris. This was trimethoprim, an inhibitor fifty thousand times stronger than the human enzyme with respect to bacterial dihydrofolate reductase. A combination of trimethoprim with the sulfonamide sulfamethoxazole (co-trimoxazole) was offered by Burroughs Wellcome.

Hitchings was convinced that it should be possible to arrest the growth of rapidly dividing cells (bacteria or cancerous cells) by means of antagonists of the nucleic-acid bases their division depended on. The object was to exploit the high speed of their proliferation as compared with normal mammalian cells and ultimately to classify the biochemical differences between cell types according to the ways in which they responded to these antimetabolites.

The importance of dihydrofolate reductase inhibitors was recognized because this enzyme is an important rate-limiting step in the de novo folate synthesis pathway, which is an universal step in cell life cycle in eucaryote and procaryote cells as well. That is why the pharmacological targets of those inhibitors are so various: For instance, methotrexate is a cancer chemotherapeutic agent, whereas trimethoprim is an antibiotic. This is explained through the fact that despite both being dihydrofolate reductase inhibitors, methotrexate has a selective affinity for mammalian dihydrofolate reductase, while trimethoprim has a selective affinity for the bacterial enzyme.

Possible targets of research were provided by the various enzymes of nucleic acids then known: nucleases, nucleotidases, nucleosidases, deaminases, xanthine oxydase, and uricase. Hitchings and Elion began by studying guanase and xanthine oxydase in order to learn whether purines acted as substrates or as inhibitors of these enzymes. In 1948 they discovered that 2,6-diaminopurine was a powerful growth inhibitor for Lactobacillus casei, except when adenine was present. A strain of Lactobacillus casei resistant to diaminopurine made it possible to demonstrate that adenine and 2,6-diaminopurine were both modified by the same enzyme, namely adenylate pyrophosphorylase, described in 1955 by Arthur Kornberg (Nobel laureate, 1959).

Antileukemic Drugs . At the beginning of the 1950s, methotrexate was the only anticancer drug available. A leukemic child had a life expectancy of only three to four months once diagnosed, and only 30 percent of children survived for more than a year. In the late 1940s, the administration of 2,6-diaminopurine to leukemic mice (mice that are highly susceptible to development of spontaneous leukemias)and the exposure of tumor cells to it in vitro resulted in strong inhibitory activity. When it was tried out on patients, in 1951, impressive remissions of chronic myeloid leukemia were observed, as well as an in vitro inhibition of vaccinia virus. Unfortunately an excessive toxicity put an end to the clinical application of 2,6-diaminopurine.

In 1951 Elion and Hitchings synthesized and evaluated more than one hundred purine derivatives, finding that the replacement of the oxygen atom by a sulfur atom at the 6-position of guanine and hypoxanthine produced inhibitors of purine utilization, namely 6-thioguanine (6-TG) and 6-mercaptopurine (6-MP). Following toxicological testing of these agents on animals, Joseph Burchenal of the Sloan-Kettering Institute in New York carried out clinical trials with children suffering from acute lymphoblastic leukemia. Some complete remissions were recorded, though most patients relapsed in the medium term. Consequently, in 1953, the U.S. Food and Drug Administration approved the use of 6-MP for acute leukemia in patients of this type, and the median survival rate for children thus treated rose from three to twelve months. A combination of thioguanine and cytosine arabinoside was later used to treat acute leukemias in adults.

Immunosuppression and Transplantation . In 1958 a team of Boston researchers led by William Dameshek and Robert Schwartz achieved an immuno-pharmacological breakthrough when they showed that prolonged and simultaneous administration of 6-MP and an antigen prevented the development of antibodies against the antigen in question. An immunological screening test allowed Schwartz and Elion to identify a new agent, 1-methyl-4-nitro 5-imidazolyl, or azathioprine (Imuran®), a prodrug for the 6-MP produced by the red blood cells under the influence of glutathion. At the suggestion of Schwartz and Elion, the British surgeon Roy Calne achieved a very good outcome when he replaced 6-MP by azathioprine in his already successful canine kidney homograft studies: With 6-MP, Calne had achieved a forty-four-day survival in a dog that had received a kidney from an unrelated donor— far longer than the nine- or ten-day graft survival expectation for control animals—furthermore, azathioprine proved to be even more effective in preventing rejection.

In humans, kidney transplantation from unrelated donors became possible in 1962, thanks to the use of immunosuppressive treatments combining azathioprine and cortisone derivatives. In the early twenty-first century, azathioprine was still a mainstay of transplantation. Its immunosuppressive properties are also called on in the treatment of a variety of autoimmune disorders.

Gout and Hyperuricemia . It was while searching for additional antileukemia agents that Elion and Hitchings came upon a treatment for gout. The biosynthesis of 6-thiouric acid, a product of the catabolism of 6-MP, is inhibited by xanthine oxydase. To evaluate the inhibition of xanthine oxydase in vivo, Elion and Hitchings used an analog of hypoxanthine, namely 4-hydroxypyrazolo-(3,4-d)-pyrimidine (allopurinol). They found that when allopurinol and 6-MP were administered simultaneously to mice, oxidation of the 6-MP was indeed inhibited. The antineoplastic and immunosuppressive properties of 6-MP were multiplied by a factor of three or four, whereas its toxicity was “only” doubled. Wayne Rundles studied leukemic patients on this basis, but found that in humans, unfortunately, toxicity increased in proportion to antitumoral action.

Another line of research with xanthine oxydase concerned the formation of uric acid from hypoxanthine and xanthine. It was found that treatment with allopurinol (a xanthine oxydase inhibitor) brought about a significant reduction in uric acid in blood and urine. This finding raised several questions. Would the pharmacokinetics of allopurinol ensure a durable impact on the metabolism of purines and uric-acid production? Would other metabolic products accumulate, such as hypoxanthine or xanthine? Did excess hypoxanthine and xanthine induce increased enzyme synthesis, thus indicating the administration of higher dosages of allopurinol? What would be the long-term effects of this drug?

All these questions were carefully explored, first with animals and then with human subjects. Allopurinol was determined to be a competitive inhibitor as well as a substrate of xanthine oxydase. Its oxidation produces the corresponding analog of xanthine, oxypurinol (or alloxantine), which is also a potent xanthine-oxydase inhibitor. Whereas allopurinol was found to have a plasma elimination half-life of just 90–120 minutes, that of oxypurinol was 18–30 hours: with a single daily dose of allopurinol, it was possible after a few days to maintain a roughly constant oxypurinol concentration. By adjusting the dosage, uric-acid concentration could be kept at the desired level. Completely absorbed after administration by mouth, allopurinol could become an ideal “prodrug.” It was the first radical treatment for gout.

Antiviral Drugs . In 1968, Hitchings and Elion and their team turned their attention once more to a working hypothesis—framed twenty years earlier but prematurely set aside—concerning the antiviral action of 2,6-diaminopurine. The idea of using this agent had been abandoned in view of its high toxicity, but the discovery of the antiviral properties of adenine arabinoside (ara-A) justified revisiting it and also considering 2,4-diaminop-urine-arabinoside, synthesized by Janet Rideout and subjected to virological study by John Bauer at the Wellcome Research Laboratories in the United Kingdom. This new derivative was effective not only against the vaccinia virus but also against herpes simplex, implicated in a variety of pathologies of the skin and mucous membranes. Furthermore, it was less cytotoxic than ara-A for eukaryotic cells. Hitchings and Elion had been right on target, and this was the beginning of what Elion called their “antiviral odyssey.” For several years they worked with purine arabinosides, investigating the relations between their structure and their action, searching for the best syntheses, and studying not only their metabolism but also their antiviral effectiveness. Among the most intriguing derivates of diaminopurine arabinoside (ara-DAP) were guanine arabinoside (ara-G), a more potent antiviral than the deami-nation product of ara-A, hypoxanthine arabinoside. Did this superiority warrant a full-scale development of araDAP? The answer was soon to come.

When Hitchings and Elion’s research laboratory moved to North Carolina in 1970, Howard Schaeffer, a specialist in analogs of adenosine, joined them as head of the organic chemistry department. Schaeffer had studied the effect of various changes on acyclic side-chains at the 9-position of adenine, and found that 9-(2-hydroxyethoxymethyl)-adenine was a constant substrate of adenosine deaminase. It followed logically that other enzymes too might likewise be able to mimic such side-chains and that nucleoside analogs of this kind might have antimetabolic properties. Research was therefore focused on acyclic nucleoside analogs. Schaeffer’s syntheses were followed by antiviral testing by Bauer and Peter Collins, while Elion’s group explored the mechanisms of action and conducted in vivo enzymological and metabolic studies. This was the context in which the potency of acycloguanosine (acyclovir) came to light.

Acyclovir . Elion’s most celebrated article was published in Proceedings of the National Academy of Sciences (PNAS) in December 1977, barely two months after she submitted it. The advent of acycloguanosine amounted to a therapeutic revolution. Acyclovir proved effective with respect to herpes simplex, just as, in the case of the purines, the analog of 2,6-diaminopurine had done. The big surprise was that acycloguanosine was more than one hundred times as active as the “diamino” compound. Four days after the Elion team’s pioneering article was sent to PNAS, they submitted to Nature a contribution that was largely concerned with clinical issues and already included, as well as recommendations on inhibitory dosages, some discussion of experimental pathology (experimental herpetic encephalitis and herpetic keratitis) and of pharmacokinetics. This was the start of the pharmacological career of acyclovir, an analog of guanine whose performance far surpassed that of 2,6-diaminopurine.

Acyclovir’s spectrum of action was found to cover herpes simplex virus types 1 and 2 and varicella zoster virus. It also had interesting properties in connection with Epstein-Barr virus (EBv). Unfortunately, it turned out to be less active with cytomegalovirus (CMV). Its great virtue was its very low toxicity. Acyclovir was manifestly more effective than all previously proposed antiviral agents. The utility of this new inhibitor resided not only in its potency but also in its very high selectivity, for it was not cytotoxic to mammalian cells in which herpes viruses grow.

The reasons for this selectivity were explored by the virologist Philip Furman. Although acyclic substitution at the 9-position did not, properly speaking, confer the structure of a nucleoside on it, acyclovir nevertheless behaved like an arabinoside-type derivative. Its chief chemical property lay in its ability to be triphosphorylated, an indispensable trait for any antimetabolite.

Ordinary derivatives can be phosphorylated by cellular kinases; in contrast, a viral kinase was solely responsible for the activity of acyclovir. Vero cell cultures, uninfected or infected in advance with strains of herpes simplex virus type 1 were incubated with radioactive acyclovir labeled with carbon 14 (¹⁴ C) at the 8-position of the guanine and with tritium (³ H) in the side-chain. Extracts of the cells were examined, after separation, by chromatography. The uninfected cells were found to contain only nonphosphorylated acyclovir. By contrast, three new radioactive compounds appeared on the chromatograms of the HSV-infected cells: these were identified as the monophosphate (ACV-MP), diphosphate (ACV-DP), and triphosphate (ACV-TP) of acyclovir.

Once the first phosphate had been added, the second was formed by a cellular guanylate kinase, while the third could be formed by several other thymidine kinases. Since the cellular thymidine kinase could not use acyclovir as a substrate, very little ACV-TP formed in noninfected cells. Once formed, the ACV-TP became a potent inhibitor of the viral DNA polymerase, which was also inactivated by the formation of an enzyme-template-acyclovirmonophosphate complex (an inactivation that did not occur with cellular DNA polymerase).

Pharmacokinetic and metabolic studies of acyclovir in several animal species and in humans revealed that it had a remarkable metabolic stability. Because its plasma half-life was about three hours, intravenous infusion of acyclovir (Zovirax®) was generally ordered on an eight-hourly basis. It was also possible to administer it via the genital, ophthalmic, or labial mucous membranes, or by mouth. Since the mid-1990s, the drug has been widely used to treat varied conditions in which herpes virus is implicated: first episodes of genital herpes infection, herpes zoster, prevention of herpes infection in immunode-pressed patients, herpetic encephalitis, and so on.

The discovery of acyclovir was a major therapeutic breakthrough per se, but the lessons learned from its history have also turned out to be extremely fruitful for subsequent research on antivirals, most notably in connection with AIDS. In-depth study of its mechanism of action has led to a better grasp of the enzymatic differences between healthy and virus-infected cells and helped assess the impact of the specific properties of enzymes on their therapeutic applications.

The introduction of acyclovir was undoubtedly the high point of Elion’s career. It is a remarkable fact that, more than fifty years after she began her work, the discoveries of this extraordinary pharmacologist were still indispensable and unsurpassed therapeutic tools.

BIBLIOGRAPHY

WORKS BY ELION

With George H. Hitchings, Gertrude B. Elion, Elvira A. Falco, et al. “Antagonists of nucleic acid derivatives. I. The lactobacillus casei model.” Journal of Biological Chemistry 183 (March 1950): 1–9.

With Sandra Callahan, R. Wayne Rundles, and George H. Hitchings. “Relationship between Metabolic Fates and Antitumor Activities of Thiopurines.” Cancer Research 23 (1963): 1207–1217.

With R. Wayne Rundles, James B. Wyngaarden, George H. Hitchings, et al. “Effects of a Xanthine Oxidase Inhibitor on Thiopurine Metabolism, Hyperuricemia, and Gout.” Transactions of the Association of American Physicians 76 (1963): 126–140.

“Enzymatic and Metabolic Studies with Allopurinol.” Annals of the Rheumatic Diseases 25 (1966): 608–614.

With S. Singer and George H. Hitchings. “Resistance to Inhibitors of Dihydrofolate Reductase in Strains of Lactobacillus casei and Proteus vulgaris.” Journal of General Microbiology 42, no. 2 (1966): 185–196.

With Vincent Massey, Hirochika Komai, and Graham Palmer. “On the Mechanism of Inactivation of Xanthine Oxidase by Allopurinol and Other Pyrazolo [3,4-d]-pyrimidines.” Journal of Biological Chemistry 245 (1970): 2837–2844.

With Paulo de Miranda, Lowrie M. Beacham III, and Teresa H. Creagh. “The Metabolic Fate of the Methylnitroimidazole Moiety of Azathioprine in the Rat.” Journal of Pharmacology and Experimental Therapeutics 187, no. 3 (1973): 588–601.

With Janet L. Rideout, Paulo de Miranda, Peter Collins, et al. “Biological Activities of Some Purine Arabinosides.” Annals of the New York Academy of Science 255 (1975): 468–480.

With Phillip A. Furman, James A. Fyfe, Paulo de Miranda, et al. “Selectivity of Action of an Antiherpetic Agent, 9-(2-hydroxyethoxymethyl)guanine.” Proceedings of the National Academy of Sciences of the United States of America 74, no. 12 (1977): 5716–5720.

With Howard J. Schaeffer, Lilia Beauchamp, Paulo de Miranda, et al. “9-(2-hydroxyethoxymethyl)guanine Activity against Viruses of the Herpes Group.” Nature 272 (1978): 583–585.

With Karen K. Biron. “In Vitro Susceptibility of Varicella-Zoster Virus to Acyclovir.” Antimicrobial Agents and Chemotherapy18 (1980): 443–447.

With Brenda M. Colby, James E. Shaw, and Joseph S. Pagano. “Effect of Acyclovir [9-(2-hydroxyethoxymethyl)guanine] on Epstein-Barr Virus DNA Replication.” Journal of Virology 34 (1980): 560–568.

With Marty H. St Clair, Phillip A. Furman, and Carol M. Lubbers. “Inhibition of Cellular Alpha and Virally Induced Deoxyribonucleic Acid Polymerases by the Triphosphate of Acyclovir.” Antimicrobial Agents and Chemotherapy 18 (1980): 741–745.

With Phillip A. Furman, Paulo de Miranda, and Marty H. St Clair. “Metabolism of Acyclovir in Virus-Infected and Uninfected Cells.” Antimicrobial Agents and Chemotherapy 20 (1981): 518–524.

With Paul M. Keller, James A. Fyfe, Lilia Beauchamp, et al. “Enzymatic Phosphorylation of Acyclic Nucleoside Analogs and Correlations with Antiherpetic Activities.” Biochemical Pharmacology 30 (1981): 3071–3077.

With Goerge H. Hitchings. “Layer on Layer: The Bruce F. Cain Memorial Award Lecture.” Cancer Research 45 (1985): 2415–2420.

“Nobel Lecture: The Purine Path to Chemotherapy.” In The Nobel Prizes 1988, edited by Tore Frängsmyr. Stockholm: Nobel Foundation, 1989.

With F. Chast, C. Chastel, N. Postel-Vinay, et al. Virus Herpès et pensée médicale, de l’empirisme au Prix Nobel [Herpes Virus and Medical Thought: From Empiricism to the Nobel Prize]. Paris: Imothep-Maloine, 1997.

OTHER SOURCES

Chast, François. Histoire contemporaine des médicaments [Contemporary History of Drugs]. Paris: La Découverte, 2002.

“Gertrude Belle Elion: A Lifeline.” Chemical Heritage Foundation. Available from http://www.chemheritage.org/EducationalServices/pharm/chemo/readings/lifeline.htm

François Chast