First to discover medically useful antifungal antibiotics

by King-Thom Chung, Department of Biology, The University of Memphis

Fungal infections can cause terrible diseases in humans. It affects millions of people world wide. Before 1950, there was no effective drug to deal with them. Fungi thrive in or on the earth, in soils and plants of all kinds. They are so pervasive, it is likely that practically everyone has been exposed to one or more at some point in their lives. Many of those exposed have developed one (or more) of the mycoses but have not been aware of it because their natural defenses produced antibodies to ward off the microorganisms and provided a degree of immunity against further infection. Others whose defenses were not able to repel the pathogenic fungus contracted one of the milder forms of the disease. They were not seriously ill but rather embarrassed, inconvenienced or temporarily incapacitated by something like athelete's foot or ringworm. The most prevalent fungal disease usually experienced by age group 30-65 is acne. Once the pathogenic fungi have gone out of control and enters the lymphatic system of the body, they disseminate, spreading to other parts and producing infections that are all but impossible to arrest. Fungal disease may cause nonspecific symptoms of mild upper respiratory infection such as low-grade fever and cough or chills, sweating and headaches. Further probing may reveal symptoms of pneumonia, tuberculosis, meningitis, rheumatoid arthritis, brain tumors or other afflictions. Unless the physician directs (and the laboratory performs) the highly specialized and time-consuming tests for pathogenic fungi and the possible cause of the disease solutions may not be found.

In 1950 Elizabeth "Lee" Hazen, a female American scientist, along with Rachel Brown, discovered Nystatin as an antifungal antibiotic which provided an effective and safe treatment of human disease. Elizabeth Hazen led the search at Albany laboratory for New York State's Health Department. She studied the useful antibiotics in the control of fungal disease. She faced the same problems as Alexander Fleming on how one can extract and chemically define an active substance. She found Rachel Brown, an exceptionally talented organic chemist who was familiar with extraction of active ingredients from bacterial cultures. This collaboration resulted in finding the active principle in Hazen's streptomycete and provided the evidence that it was highly active against a number of important fungi and only slightly toxic in experimental animals. They first called their agent "fungicidine" but renamed it "nystatin" after " New York State".

Nystatin, the Brown-Hazen discovery has proven to be the most effective agent against Candida and Aspergillus species infections in the intestines, the vaginal tracts, the skin, as well as the mouth. If applied locally or taken orally, nystatin should help rid mucous membrane and other body surfaces of this disease.

Elizabeth "Lee" Hazen's career is a good example for us to learn. Her discovery of the useful antibiotics, benefits us immensely. She was a pioneer medical mycologist and also a good example of pioneer women scientist.

In the 1940's, Hazen found that training in diagnosis of fungal-diseases---the mycoses---was meager or non-existent. Diagnostic trials were primitive as were the available tools and specific antifungal drugs did not exist. In spite of the difficulties, Elizabeth Hazen was still determined to explore this new frontier, regardless of repeated discriminations based on her sex.

Before Lee Hazen began to study medical mycology there were several other microbiologists testing for cures of fungal diseases. However, she met the growing need to arrest the disease and accomplished her goal "to develop the practical side of the sciences without sacrificing complete and accurate knowledge of principles," a motto she lived through her life.

Elizabeth "Lee" Hazen was born on August 25, 1885 in Rich, Coahome County, Mississippi, which is a small farming community. It is a few miles east of the Mississippi River and some sixty miles South of Memphis. Her given first name was Lee. Her father was William Edgar Hazen and mother Maggie Harper. Both parents died at an early age. Lee was the second daughter of the family since her younger brother died at the age of four. Being without parents, Lee and her sister were raised by an uncle and his wife. They lived in the county in which the Hazen family had been established before the Civil War. Lee's grandfather, Munson Hazen, a native of Vermont had chosen to settle at nearby Friar's Point. The seventeen year old purchased a 320 acres farm called "The Hazen Place" for less than fifty cents an acre. He later passed this land to Lee's parents when she was born. Robert Henry "Lep" Harper and Laura Crawford Hazen reared the orphaned children. The family was deeply religious so the children always attended church. This interest in the Baptist religion also continued throughout Lee's college years. The Lula School that Lee attended in 1891 was a one-room, one-teacher public school with about fifty students ranging from

pre-school to high school age. Her academic record was outstanding and her teachers were "committed" persons. They would have given her more than 100 points due to her determination to excel in schoolwork had this been possible. She was a zealous reader, concentrating on history and biography, a preference she changed later as she entered science classes. Conway Dickey, a cousin, donated Lee's Library to the Mississippi University for Women after her death in 1975. He reported that there was not a single novel in the collection. Despite her bookish attitude, she had a sparkling sense of humor and was very outspoken, not like the stereotype of a "bookworm. " Clara Hazen, her "sister-cousin" recalled the Valedictorian address Lee gave in 1904 about Lucius Quinctius Cincinnatus Lamer, a revered Mississippi statesman who served as representative, senator, secretary of the interior and associate justice of the United States Supreme Court from 1888-1893. Clara believed Lucius inspired Lee to set high goal for her life.

Upon completion of high school, she received private tutoring sessions in Memphis. In 1905, she enrolled in the Mississippi Industrial Institute and College in Columbus (Later Mississippi University for Women), where tuition was free and room and board cost as little as ten dollars per month. Her interest in science blossomed in physiology, botany, zoology, physics, plant physiology and anatomy. Her grades were also excellent. This knowledge was perhaps the turning point in her life whereby she decided to explore science (with the most practical of ends--the alleviation of human suffering.) One of the thirty-five graduating students from college on May 27, 1910, Lee was noted in the yearbook as having been secretary and treasurer of the Baptist Missionary Society and assistant business manager of the campus newspaper, the Spectators; she was also a member of the cast of an all-girl theatrical production "Men and Maids from Gay Paris." Her picture depicted a sweet, handsome young woman, immaculately dressed and with a serious look that typified her deeply held determination.

With her degree in hand, she moved to Jackson, Mississippi where she taught physics and biology at Central High School for six years. During that period, she attended summer sessions at the University of Tennessee, where she studied biology and also the University of Virginia, where she took lecture and laboratory classes. In 1916 she entered Columbia University to continue her study and received a Master 's degree in biology in 1917. During her enrollment at Columbia's College of Physicians and Surgeons, she offered her services to the U.S. War Department during World War I. By this time she received the nickname "Elizabeth" from her new friends and associates, and since then 'Elizabeth" was used. While in the Army, she worked as a technician in diagnostic laboratories in Camp Sheridan, Alabama in 1918-1919 and in Camp Mills, New York in 1919. This position gave her a lot of practical experience. She became assistant director of the Clinical and Bacteriological Laboratory of Cook Hospital in Fairmont, Virginia, where she remained until 1923. In the Fall of 1923 Hazen decided to go back Columbia University for further graduate work in organic chemistry and to continue her study and research in the Department of Bacteriology and Immunology at the College of Physicians and Surgeons. On one of her research projects she wrote a report entitled Unsuccessful Attempts to Cure or Prevent Tuberculosis in Guinea Pigs with Dreyer's Defatted Antigen. This thesis on general and local immunity to ricin, a toxic substance occurring in the castor bean contained a list of fifty-nine references to the previous work along with accurate summaries of the earlier findings by microbiologists. In 1927 she published this article and received her Ph.D. in Microbiology. She continued her research at Columbia until 1928, when she was appointed resident bacteriologist at the college-affiliated Presbyterian Hospital. She left that post to become a member of the teaching staff in the Department of Bacteriology and Immunology at the College of Physicians and Surgeons. This move allowed her to interact with many eager students.

Possibly another major turning point in her career came in 1931 when Augustus Wadsworth put her in charge of the Bacterial Diagnosis Laboratory in Albany, New York. This position was much to her liking and her performance led to increasing responsibilities, such as direct supervision of a large number of technicians, who were engaged in the examination of pathological specimens for the diagnosis of infectious diseases such as diphtheria, septic sore throat, typhoid fever, tuberculosis, gonorrhea, spinal meningitis and bacillary and amebic dysentery. In addition, she supervised the work of the serum diagnosis department in which the test for syphilis is performed on thousands of blood and spinal fluid specimens. She also found time to trace an outbreak of anthrax, a fatal disease in animals that is transmissible to man; pinpoint sources of tularemia, and also reported the first case in the United States of Clostridium botulinum, Type E, a cause of poisoning from improperly preserved foods. In spite of her strenuous schedule, she found time to attend lectures in organic chemistry and to study mycology in the Department of Dermatology. She studied in the Mycology Laboratory that was established by J. Gardner and Rhoda W. Benham (1895-1957). Rhoda Benham was an authority on pathogenic fungi. Under Dr. Benham's supervision, Hazen collected and examined specimens taken from patients. Hazen also made cultures at bedside and took them to the laboratory for further observation and identification of fungal agents. Elizabeth treasured every positive specimen, slide or culture, carefully preserving it, not only for her own use, but with Dr. Benham's encouragement, to take them to her own laboratory, thus bringing mycological manna as well as gospel to her disciples and associates at the Division of Laboratories and Research. She undertook an independent investigation to study Microsporum audouini, which causes severe itching and loss of hair in school age children.

In 1944 she established her own culture collection of pathogenic fungi which became a state-approved laboratory where special facilities had been made available to the physicians of the state for identification of fungi and related microorganisms. This collection served as a valuable teaching tool and later became the basis of her book, Laboratory Diagnosis of Pathogenic Fungi Simplied a standard reference in the field. Her published works began to reveal her future direction of work. The Mycologia paper stated that in the research reported to date the most promising antifungal preparations had no widespread application because of toxicity or other undesirable pharmacologic properties. "Consequently," it added, "no antibiotic agent approaching the efficacy of penicillin and streptomycin against bacterial infections is available in fungal infections either of the superficial or the deep-seated type." This force investigative screenings of soil samples in the search for microorganisms active against pathogenic fungi. She would eagerly open each new soil sample arriving at the branch laboratory, carefully labeling the place of origin and name of donor. A tiny amount of each was mixed with sterile saline solution and seeded on a nutrient base until any actinomycetes had grown to the stage of visibility. These isolated cultures were then grown in liquid nutrients, placed in mason jars, labeled and shipped to Rachel Brown in Albany where she would extract active antibiotic ingredients from these mycotic cultures. Each mason jar received by Brown contained a whole culture, either static (a mixture of broth and a matted growth called the pellicle) or shaken (a suspension of the growth in the broth). Her first job was to determine the location of the active antifungal substance. Was it in the pellicle, the broth or both? Hazen then tested whether or not the extractions from the fungi would stop fungal growth by inoculating the two pathogenic fungi in the laboratory. After preliminary tests, she mainly focused her research on the antifungal substances produced from just two of several actinomycetes sampled. No. 42705 proved to have good antifungal properties but was toxic to mice. No. 48240 yielded two antifungal substances which Hazen was able to extract and separate. One extraction proved to be active against Candida neoformans. The other extracted, from the pellicle, showed considerable activity against both C. albican and C. neoformans. As she attained purer materials, those that showed the greatest potency against the test fungi were used by Hazen against actual fungus diseases induced by these pathogenic fungi in laboratory animals--mice and rabbits in this case. The animal experiments would show whether the antifungal activity seen in the laboratory was protective in living creatures, and whether the substances were toxic in test animals and in humans. No. 48240 was given the name nystatin. Hazen, having established that actinomycetes from which fungicidin was derived was a previously undiscovered microorganism called Streptomyces noursei, which was isolated from a soil sample from Virginia farm of some friends named Mourse. All the samples Hazen tested were from compost, peat, and manure as well as various forest, field and garden soils.

In the spring of 1951, the U.S. patent Office issued a patent on fungicidin to Hazen and Brown. They signed an agreement to allow Squibb Institute to manufacture and sell fungicidin in the form of an antibiotic. Also, in August of 1954, the Food and Drug Administration approved the sale of Mycostatin tablets in oral dosage form as the first broadly effective antifungal antibiotic available to the medical profession. It was recommended for the prevention and treatment of intestinal monilasis or candiasis. In June 1957, nystatin was placed on the market with the patent

commission's permission. With nystatin effective against Candida infections of the mouth, skin, and intestinal as well as vaginal surfaces. Squibb eventually produced Mycostatin in various forms such as oral tablets, and suspensions, creams, ointments, topical powders, and vaginal tablets. Brown and Hazen received royalties. Their patent expired in 1974, over thirteen million dollars had been generated. Half of the royalties were placed in the Brown-Hazen Fund which were used to support research and training in the medical-biological sciences, provide aid to women scientists in academic institutions, and add grant programs to continue the fight against fungal diseases. Furthmore, this fund also provided the American Type Culture Collection in Rockville, Maryland to collect, preserve, and distribute to the scientific community reference cultures of microorganisms, viruses, animal and human cell lines.

Hazen 's life was not disturbed by the discovery and the money generated. She continued her work vigorously. In 1954, The new York State's Branch laboratory was disbanded, Hazen continued her research and diagnostic screening at the Central Laboratory in Albany. In 1958, Hazen accepted an associate professorship at Albany Medical College, where she retired in 1960. After that she became a full-time guest investigator in the Columbia University Mycology Laboratory. In 1973, Hazen visited her ailing sister in Seattle. However, she was ill and for many years she suffered ulcers and disabilities that prevented her return to New York. She remained at the nursing home with her sister. Two years, later, on June 24, 1975, she died of acute cardiac arrhythmia.

Hazen received many awards in her later part of life. In 1955, she shared with Rachel Brown the Squibb Award in Chemotherapy. In 1968, she received a Distinguished Service Award from the New York State Department of Health. In May of 1975, a month before her death, Hazen and Brown were the first women to receive Chemical Pioneer Award of the American Institute of Chemists.

Hazen's book Laboratory Diagnosis of Pathogenic Fungi Simplified was well received. The first edition was published in 1955. The second edition was in 1960 and the third edition cauthored with Morris A. Gordon was published in 1970.

Hazen came from a poor Mississippi farm. Her journey had met considerable obstacles and personal sacrifice. She never married. She never presented a paper which she coauthored with Brown. She always avoided reporters and photographers in public. However, she was warm , outgoing and opinionated about matters of science and politics, especially she was concerned about women's status in society. She worked extremely hard. Her passion was her work.

Elizabeth "Lee" Hazen and Rachel Brown should be commended for their determination, perseverance, and patience in the microbiology field in which they found cures for fungal diseases that are still being administered worldwide. Their work also reveals the need for scientists to be alert to fields other than their own immediate interests. Her love to human beings and hard working attitude overcame sexual discrimination and other problems of her time. Her hard working attitude is the key to success. Elizabeth Hazen's life is worthy for us to remember. She deserves the respect and emulation of fellow microbiologists.