The search SubbaRow directed at Lederle Laboratories for antibiotics with wider range of cures than the then available penicillin and streptomycin led to the discovery of polymyxin, widely used even today in cattle-feed, and Aureomycin, the first of tetracycline antibiotics which all of us have had some time or the other in our lives. Tetracyclines have saved millions of lives over the last 50 years.
Aureomycin was presented to medicine in 1948, the year SubbaRow died. It was the first broad-spectrum antibiotic, that is, one effective against both gram-positive and gram-negative germs. It was thus more powerful than either Fleming's penicillin or Waksman's streptomycin.
When SubbaRow's centenary year began in 1994, tetracyclines --especially Doxycycline -- helped confine and then eradicate the plague epidemic that broke out in Gujarat and Maharashtra. It was a debt SubbaRow paid to his motherland almost half a century after death which claimed him soon after the unveiling of Aureomycin before a medical gathering at the New York Academy of Sciences.
Along with pheno‑sulphazole on trial in the Texas polio epidemic of 1948 was an antibiotic called Aureomycin. It too did not make the grade in the fight against the crippling disease but it was one of the first drugs to be effective against viruses and rickettsiae. Rickettsiae occupy a position between viruses and bacteria.
The discovery and development of Aureomycin came from a screening programme SubbaRow instituted in the spring of 1944 simultaneously with the programme that got him Hetrazan. It was an antibiotic SubbaRow had acquired with the help of "amateurs", men with no pre-conceived ideas who investigated the unknown with a truly scientific spirit.
SubbaRow had by then completed his work on fermentation and isolation of penicillin and wanted to look for sources of new antibiotics. Since filamentous moulds, Aspergilli and Penicillia, had all been surveyed extensively, he wished to institute a more thorough examination of Actinomycetes. Actinomycetes are close to bacteria in appearance but resemble fungi in their growth. His decision was influenced by the recent announcement of streptomycin obtained from an actinomycete and by the feeling that bacteria had not received due attention as antibiotic producers because penicillin had overshadowed gramicidin.
SubbaRow got his first amateur when he looked for a plant physiologist for his antibiotic screening programme. Remembering his pleasant meeting with an old plant physiologist at the University of Wisconsin, he asked Ed Backus on his mycology group to write to his brother at Madison to meet the professor and secure the services of one of his students. Benjamin Minge Duggar, when approached by the elder Backus, said his own services were available for SubbaRow. A distinguished authority on fungus diseases of plants, he had retired some six months earlier with the reputation as one who had made mushroom cultivation in the United States independent of European spawn.
SubbaRow had reservations about "prima donnas" but was touched by the fact that Duggar had to retire because of the age rule. Duggar was not only active but anxious to dedicate the evening of his life to something of more significance to human welfare than mushroom cultivation. He was willing to screen plant extracts and mould broths. It was a job he had never done before and he would not be handicapped by experience and theories.
Duggar came over on April 1, 1944. He began to screen soil and other samples received from everywhere. He prefixed A to code numbers for actinomycetes that he isolated and B for bacteria.
His first break came with B-71, the 71st bacterial isolate, found in a sample of Colorado soil. To let Duggar concentrate on actinomycetes, SubbaRow handed over the isolate identified as Bacillus polymyxa to John Porter, a mycologist on his staff. Porter and his associates were able to secure from this the antibiotic polymyxin. Polymyxin was found by physicians to be effective against undulant fever, whooping cough and meningitis. But SubbaRow withdrew it as it was somewhat harmful to kidneys.
Meanwhile in August 1945, Du-ggar noticed a beautiful growth on agar in test tubes to which he had transferred actinomycetes isolated from some University of Missouri soil samples. The conspicuous grey‑brown spores at the top were followed by a pink and a white zone ending in a moist golden area at the bottom. On agar plates, the actinomycete produced an antibiotic which arrested the growth of a broad spectrum of deadly disease germs ‑-those susceptible as well as those resistant to the then known sulphas and antibiotics.
Exciting as the versatility of A‑377 (the 377th actinomycete isolated), Duggar was disappointed it was not active against the tubercle germ. An anti‑TB drug more potent but safer than streptomycin was one of the objectives set for him.
So when he sent A-377 along with other promising actinomycetes to Edwin Ball for fermenting antibiotics in bottles, Duggar expressed his real enthusiasm for A-491 which had given a very sharp zone against the tubercle germ in agar plates.
SubbaRow did not share Duggar's enthusiasm when the report came from the bacteriology group on Ball's mould broths. They were having deaths with animals to which A-491 injections had been given. Right away, he realised it was nothing to fool with. It cleared the lungs of tuberculosis but itself killed the animals.
He was on the other hand very happy when six guinea pigs which should have been dead with deadly germs injected into them were all alive because they had been given the partially purified A-377 broth.
Deciding that further work should be with A-377, SubbaRow picked up another amateur to solve the first of many production problems he would now face. Joseph Niedercorn was a synthetic chemist who had protested, “What do I know about microbes?”, when he was asked earlier to join the fermentation group. SubbaRow had then settled the issue by telling him that he could learn, and now silenced him by saying, “You can at least try,” when Niedercorn protested about the assignment to grow the yellow mould in five-gallon stirred bottles. This was June 1946.
The yellow mould turned out to be quite individualistic. It was so unlike penicillin and streptomycin moulds in the manner it produced the antibiotic, and experience with the production of earlier antibiotics served as no guide. It virtually killed itself with the antibiotic it produced in the medium found so suitable for penicillin production. For in that medium the mould also produced lactic acid which combined with the antibiotic to form a salt that got dissolved in the medium and destroyed the mould mycelia. Whereas the penicillin medium had to be kept slightly acidic for that antibiotic to remain stable, the A-377 medium had clearly to be neutralised to prevent the antibiotic dissolving in the medium and attacking the mould. But the neutralisation of the medium was not done because antibiotic production was supposed to have gone up by ten per cent earlier when the medium was acidified.
Niedercorn tried every possible change in the nutritional composition of the medium and as weeks passed he accumulated -- a great deal of statistics on how much is produced in what medium.
When Niedercorn did not take the hint from his indifference to statistical experiments, SubbaRow lost temper and scolded him in the presence of assistants: “If you want to do those statistics, sit down with Dr Albert Einstein.” He refused to read any report with statistics and sent them all back to Niedercorn with a big “X” on them.
Niedercorn was forced to make a more systematic study of the mould’s nutrition, discovered what was happening in the medium and found high antibiotic concentrations in the medium when it was neutralised or made alkaline. He got a precipitate on February 26, 1947 when he added calcium carbonate (lime water) to the medium. The antibiotic formed an insoluble calcium salt, and the mould lived longer to produce more antibiotic.
Before moving on to production in big tanks, SubbaRow had to choose between A-377 and five other yellow moulds all of which produced the antibiotic more or less equally well in Niedercorn's five-gallon bottles. The choice was ultimately between A-377 and A-406. A-406 showed some toxicity in egg embryo experiments. SubbaRow decided on A-377 despite its inactivity against the tubercle germ when Ed Backus reported that it was more responsive to mutations. He remembered how artificial strains of Penicillium chrysogenum produced by mutation had helped boost penicillin production. Backus came up with a mould strain, A377-899 (the 899th mutant), which produced two to three times more antibiotic than the natural mould, and his A377-2920-3 produced 15 times more antibiotic.
The pilot antibiotic plant got ready within SubbaRow’s department in April 1947. It took a year to straighten out problems in recovering the antibiotic from the fermentation mash and isolating the pure antibiotic in crystalline form.
Charles Pidacks, who was given the task of making a solvent extract of the antibiotic from the wet cake taken out of fermentation tanks, was an organic chemist but had worked for a while on penicillin and streptomycin purification. He could not quite cope with the procedures and his yields of the new antibiotic were poor. SubbaRow brought in George Krupka, his personal hatchet man, and he was able to boost yields by salting the antibiotic with sodium chloride into acetone. The addition of equal volumes of acetone and sodium chloride forced the antibiotic into the acetone layer.
The next step in purification was effected when the antibiotic was separated from water insoluble material in the fermentation mash by addition.
The hydrochloride of the antibiotic thus obtained was still amorphous with lot of ash in it. Try as he did, Pidacks could not get rid of the ash.
SubbaRow was in the mean time in a desperate battle with the management to save the antibiotic project. One of the big Cyanamid executives wanted SubbaRow to scrap this single most expensive project on his research programme because money supply was tight with the country going through an economic recession. SubbaRow fought the suggestion like a bear and persuaded Bell to let him keep the project going by promising that everyone would work harder on new discoveries that would make more money for the company.
But it looked for a while as though the unpleasant task had to be done. They could not get the near-pure product to crystallize, and SubbaRow gave his men just two weeks more. “Either we get it or we drop it,” he said bringing Krupka back into the Pidacks group. Krupka found the solution in just four days. He used picric acid to get the pure antibiotic and Pidacks put it in alcohol, and it crystallised.
SubbaRow was there that night on the first floor of Building 100 when under the microscope they got the first indication that the antibiotic was crystallising. They spent rest of the night working and left for home at dawn when finally it went into gram quantities, leaving everything in the cold room for crystallisation to complete.
By then A-377 had been pronounced safe by the pharmacologists. Testing a material that was only 25 per cent pure, they found that oral doses produced almost no side-reactions in animals. It however created irritation and inflammation at the site of injections. When the pharmacologists began shaking their heads, Krupka asked, “Well, why don't you give it by mouth?” SubbaRow said light-heartedly, “The physician will not allow it. He's got to get his five dollars for the injection.” Once its wide-ranging action was proven, physicians were not slow in prescribing A-377. But physician acceptance of the new antibiotic at that time, when penicillin injections were in wide practice, appeared to be difficult.
SubbaRow initiated another series of animal trials. A-377 was effective against the typhoid-paratyphoid group of microbes as well as erysipelas infection which had killed his only child. But could it match a rival company claim that it had an antibiotic which was active against viral and rickettsial infections? Harold Cox and his associates said yes. It was effective against viral pneumonia and a venereal disease caused by a virus when tested in mice and guinea pigs. It also controlled rickettsial diseases like the “Q” and the spotted fevers.
SubbaRow took A-377 in November 1947 to Johns Hopkins University's Perrin Long who was the first to recognise the merit of sulpha drugs in wound infections, had supervised war-time clinical-testing of penicillin and was currently giving polymyxin its clinical trial. Test-tube and animal studies in Johns Hopkins department of preventive medicine and paediatrics of A-377’s action against bacterial infections were discouraging. Dr Long did not share SubbaRow’s faith in A-377 and his belief that the new antibiotic might be more successful against infections in man.
SubbaRow therefore decided to have the viricidal properties of A-377 tested. He invited to Pearl River the distinguished Negro surgeon, Louis Tomkins Wright, and discussed with him Herald Cox’s reports on the antibiotic’s effectiveness against viral and rickettsial infections of animals. Dr Wright was impressed and agreed to test it at his Harlem Hospital in New York in patients suffering from the viral venereal disease called lymphogranuloma venereum (LV).
The clinical trial of A-377 began on January 22, 1948. Twenty-five patients received the antibiotic and showed astonishingly fast disappearance of symptoms. Fourteen cases followed for two to sixteen weeks showed no relapse.
Returning from Harlem, SubbaRow called the boys who had worked on A-377 and announced: “We have a million-dollar drug.” He wished to share his happiness over the subjugation of a viral disease with those whose dedicated work had saved A-377 from the dust-bin. The management had to wait till the next day for the news.
The mysterious “Q” fever broke out in a big way in California in May, and A-377 effected some dramatic cures of this rickettsial infection.
SubbaRow went with these clinical reports again to Johns Hopkins and Dr Long was now willing to listen to him since he was disturbed by the high incidence of toxic reactions he was having with streptomycin.
Johns Hopkins doctors first used A-377 in persons suffering from rickettsial diseases and got dramatic cures of the Rocky Mountain spotted fever. They were emboldened to try it in undulant fever, an occupational disease among butchers. A-377 succeeded where other drugs had earlier failed.
SubbaRow then went to Boston City Hospital and gave the antibiotic to Dr Maxwell Finland who first tried it in the test-tube against 186 strains of disease germs isolated from patients. It was effective against many of them including those resistant to penicillin and streptomycin. Finland and associate physicians gave A-377 by mouth to a hundred patients suffering from a variety of bacterial infections. It exerted “a definite beneficial effect” in many bacterial diseases.
Drug trade journals got scent of these clinical trials and began to complain about “this bad man SubbaRow” who would not tell them anything about his new antibiotic.
By now SubbaRow had enough data from his frontal attack on the entire field of infectious diseases and set July 21 as the date for unveiling the drug before the medical world at a conference in New York City.
Duggar had long since identified A-377 mould as a member of the same streptomyces group of actinomycetes to which the streptomycin mould belonged. He named it Streptomyces aureofaciens or the golden mould. The antibiotic it produced was named “Aureomycin” the golden antibiotic. The antibiotic crystals were golden yellow in colour, and Aureomycin heralded a golden era in the history of antibiotics.
Aureomycin was the first of tetracycline antibiotics, which have cut down the need for hospitalisation in many cases because they can be taken by mouth and have made elaborate laboratory tests unnecessary because they check any of a wide range of bacterial, rickettsial and viral diseases.
Aureomycin is chlor-tetracycline because it has a chlorine atom hooked to the tetracycline molecule. Tetracyclines as a group are the nearest things to the “panacea” – universal remedy – that SubbaRow sought.
Aureomycin, with the tetracycline molecules, was derived from the fungus Streptomyces sp. and proved for the first time that a single drug can be used for controlling infections caused by both gram-positive and gram-negative bacterial germs. The previously available penicillin could battle only the former and streptomycin only the latter. Also unlike penicillin and streptomycin, Aureomycin could be taken orally.
The second generation antibiotics with the tetracycline molecule helped eradicate the plague which broke out in Gujarat and Maharashtra just when SubbaRow's centenary year began in 1994. It was a debt SubbaRow paid to his motherland almost half a century after death which claimed him soon after the unveiling of Aureomycin before a medical gathering at the New York Academy of Sciences. People then said death was jealous of SubbaRow who was coming out with a new potent drug every year and was set to conquer cancer. Death, Be Not Proud! we can say with John Gunther. For the four molecules he presented to the world in four years continue to battle ever new diseases.
Aureomycin was sent soon after its medical release to the Haffkine Institute in Bombay (now Mumbai). There the famed Dr Sahib Singh Sokhey was able to save nine out of ten experimental animals suffering from septicaemia caused by the plague germ Pasteurella pestis. Sokhey incidentally was SubbaRow's senior at Harvard Medical School biochemistry department.
After Aureomycin proved itself in a plague case later that year in the American state of Arizona, it was tried in 1951 in Latur, a hyper-endemic plague area then in Hyderabad state but became part of Maharashtra later. Of the 15 plague victims treated by Dr K Ramachandran at the Isolation Hospital, 12 were cured and discharged. The three who died had been brought in a serious condition and the drug had no time to act. Tetracycline was therefore ready for plague in Latur and Surat in 1994. It cannot resolve the debate whether the epidemic was plague or falciparum malaria as it, particularly Doxycycline, fights both the diseases.
Doxycycline, the third generation tetracycline, has now been cleared by the US Food and Drug Agency (FDA) for prophylaxis of malaria, especially the malignant variety caused by Plasmodium falciparum. Unlike the traditional chloroquine and the new mefloquin, Doxycycline is least toxic and is effective if taken just 24 hours before exposure. Mefloquin needs at least 7 days to impart immunity against malaria. In September 1999 when the United Nations Assistance Mission in East Timor (UNAMET) was ordered into the region wrested from Indonesia, its international staff packed Doxycycline in their survival kits.
Tetracycline is even today the only drug effective in Rickettsial group of diseases, like scrub typhus and the Rocky Mountain spotted fever, known as the scourge of war because of the heavy toll they took of soldiers in the trenches of the First World War.