Fleming’s work had provided impetus but Florey and Chain were also inspired by the earlier work of Gerhard Domagk, a German pathologist and bacteriologist who, inspired himself by Paul Ehrlich, showed in 1935 that the injection of a simple compound, the sulfonamide Prontosil, cured systemic streptococcal infections. It was the first drug to be effective against bacterial infections. Domagk treated his own daughter and saved her from having her arm amputated. The discovery of Prontosil earned Domagk the Nobel Prize in 1939 but he was unable to accept the award until 1947 because of opposition from the Nazi regime.
Florey and Chain had different explanations for why they decided to pursue antibacterial substances. According to Florey, while they were working on lysozyme it was Chain who suggested it and he agreed to go along with it. Chain’s explanation was that the project evolved, that in science there is no blueprint and each discovery is as individual as art. The scientist looks for interesting biological phenomena which it might be possible to explain in chemical terms. Again we are reminded of Pasteur’s adage, that chance favours the prepared mind.
Chain began enthusiastically conducting a thorough review of related scientific literature. In 1938, while looking at old articles written about lysozyme, including those written by Fleming in the 1920s, Chain happened across the one Fleming had written on Penicillium notatum in 1929. While Chain was reading Fleming’s paper, the memory of a woman walking along a corridor in the Dunn School with a dish of mould in her hand came to him. Chain sought her out and when he questioned her about the mould she told him it was Penicillium notatum. Ernst Chain exclaimed that this was the species of mould that Fleming found in 1928. The woman confirmed this. Among the various samples that Fleming had given out at the time, one had gone to the Dunn School and the mould had been kept alive ever since.[25] How often do science and serendipity go hand in hand.
Chain approached Florey, who gave the go-ahead to investigate penicillin. Over the course of the penicillin project individual members of the team concentrated their attention on areas in which they had the most knowledge, but they often met to exchange ideas. Norman Heatley, whom Florey had invited to join the Dunn School interdisciplinary team in 1936, devoted his energies to the production of penicillin by improvising methods for extraction, his specialty being microchemical methods. The team grew with Arthur Gardner taking up the bacteriological investigations, assisted by Jena Orr-Ewing for the studies on the anaerobic bacteria. Dr Margaret Jennings (an Oxford-educated doctor who joined the team as a junior research assistant, and whom Florey would eventually marry after Ethel’s death) was responsible for some bacteriological work and assisted in the pharmacological and biological investigations into the impact of penicillin on animals. The group was joined by A.G. Sanders, who later set up and operated one of the large-scale extraction plants, and by E.P. Abraham, who collaborated with Ernst Chain on the chemical and biochemical aspects of the work. Ethel Florey joined the team to work with her husband and Charles Fletcher on the clinical trials. There were also a number of devoted technical assistants who saw the project through.
Initially Chain and Florey worked to identify the active ingredient in the penicillin. It then took Chain almost two years to extract it because it is so unstable. In early 1940 he delivered the first tiny amount of yellow powder. Florey’s team was ready to perform one of the most important medical experiments in history on 25 May 1940. The team was so eager that everyone came into the laboratory on a Saturday morning to test penicillin for the first time.[26] Eight mice were injected with a lethal dose of a virulent strain of streptococci bacteria—110 million of them, more than enough to kill the mice within a day. After an hour four of the mice were injected with a penicillin solution and the other four were used as controls. Norman Heatley kept an hour-by-hour vigil.[27]
By late afternoon the four control mice were sick and they began to die soon after midnight. By 3.30a.m., sixteen and a half hours after being injected with the bacteria, all four were dead. The four treated with penicillin exhibited no symptoms whatsoever. Heatley cycled back to his rooms through wartime, blacked-out Oxford to sleep for a couple of hours before returning to the lab. Howard Florey, when told of the success, uttered what must have been the understatement of the century: that things looked quite promising.[28] The results were in fact phenomenal because by the end of World War II there was enough penicillin to treat every soldier who needed it.
Four small mice had provided the proof that Howard Florey, Ernst Chain and Norman Heatley needed. Chain apparently was almost dancing with delight and the usually laconic Florey telephoned Margaret Jennings and with a little more exuberance said that the outcome looked like a miracle. The results of the mouse experiment were published in The Lancet on 24 August 1940, including a stated claim that the team members were the discoverers of penicillin. The names included were Florey, Chain, Heatley, Gardner, Orr-Ewing, Jennings and Ethel Florey.[29] This article was considered to be the most important ever published in medical history.
The next major hurdle as Florey saw it was to prepare penicillin for a human trial. It had taken two years to produce enough for the mice and they now needed 3000 times that amount. Factories were on a war footing and military needs took precedence, so Florey decided to set up a clandestine mould factory at the Dunn School. First they needed containers in which to grow the fungus, so anything that was functional was used: laboratory glass, biscuit tins, trays and enamelled bedpans. Every warm spot was located (mould grows at around 24°C). Heatley then worked in a cold room when the mould was ready, as ice was needed for the extraction process. Throughout the Dunn School there were dangerous machines full of highly flammable substances and bedpans filled with mould.
Progress was slow and large quantities of mould produced only minuscule amounts of penicillin. Production had to be scaled up. Funding would help. After a surprise visit by Sir Edward Mellanby from the Medical Research Council in London, Florey wrote and asked for £600 but was awarded only £25.
During a meeting between Florey, Chain and Heatley to discuss their progress, or lack thereof, in March 1940, Heatley listened as Chain and Florey hotly debated why penicillin vanished. In his modest way Heatley put forward what he later called a ‘laughably simple’ idea, which involved extracting penicillin from a neutral buffer of water into ether and then transferring it out of the ether into water made alkaline by passing the mould broth back and forth between acid and alkaline to purify it.[30] Florey accepted Heatley’s idea but it led to vehement argument between Florey and Ernst Chain, who felt his control over the chemical work had been usurped. From that time on Chain was suspicious of Florey and the incident created difficulties between Chain and Heatley as well.
It was largely due to the technical ingenuity of the unassuming Norman Heatley that enough penicillin was produced for the first hospital tests. On leaving university, having completed a PhD in biochemistry, Heatley’s aim had been to set up his own commercial analytical service, but when Florey offered him the job at Oxford University he changed his plans. Heatley had an inventive mind and engineering skills that he now put to work in March 1940 to make his automated extraction apparatus. Because of war shortages Heatley improvised: the frame was made from a discarded oak bookcase from the historic Bodleian Library and was about 6 feet (1.8 metres) high and 3 feet (0.9 metres) wide. He used glass tubing, assorted pumps, laboratory bottles, an old doorbell to signal when a bottle was about to become empty or full, coloured warning lights, nozzles and copper cooling coils.[31] This ingenious continuous reverse extraction contraption was ready for use early in 1941.
In one hour 12 litres of crude penicillin broth could be extracted and it was ten times more powerful than any previously produced. Chain admitted that he had been wrong but relationships were never really restored. The machine did not survive either.[32] The early industrial producers of penicillin used Heatley’s extraction and purification process for several years but they spent thousands of pounds building their apparatus.<
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Heatley had also turned his resourceful mind to finding the best way to grow the most penicillin in the shortest time. He meticulously recorded the details of every ingredient in his lab notebook, jotting pertinent notes in red. In something akin to Paul Ehrlich’s testing of 900 arsenical compounds, Heatley added substance after substance to the penicillin broth: nitrate, sodium, aluminum salts; he put in glucose, sucrose and lactate; a reduction of cow and horse muscle; extracts of malt and various meats; greater and lesser amounts of phosphate, glycerol, peptone, oxygen, carbon dioxide; he even added Marmite.[33] But to no avail. Finally the addition of yeast halved the growing time to ten days but did nothing to increase the yield.
The Blitz also thwarted Florey’s plans. The sustained bombing of the United Kingdom by Nazi Germany began in September 1940 and continued until May 1941. Although the main attacks were focused on London, the Luftwaffe carried out air raids on towns and cities all over Britain, night after night, killing 43,000 people and destroying more than a million houses. It came as no surprise to Florey that pharmaceutical companies that had the equipment to culture mould in the amount he needed were unwilling to divert vital resources from the war effort into producing an untested drug.
Professor Gus Fraenkel, the founding Dean of the School of Medicine at Flinders University in Australia, was a medical student at the Dunn School during World War II. He later recalled those strange times for medical students who were studying with Florey. One of Fraenkel’s duties was to patrol around the School of Pathology. Students were equipped with a tin hat, a civilian gas mask and a wooden cudgel inscribed ‘Oxford University Police’ which may not have been that effective against the Luftwaffe and German parachutists.[34]
As it was highly likely that Oxford would be bombed, the team interrupted their work to dig air-raid shelters. Highly flammable liquids used for the extraction process were buried in the grounds. Florey, concerned for the survival of the mould rubbed some into his, Chain’s and Heatley’s coats. Stocks could then be destroyed if the Nazis invaded.[35] Penicillin could be a valuable weapon and there were rumours that the Germans had tried to acquire Penicillium mould through neutral Switzerland.
The fact, however, remained: more penicillin was needed. The make-do containers that were used as fermentation vessels were now completely inadequate for the teams’ needs. Unable to find an alternative, Heatley designed his own, based on the bedpan and shaped so that they could be stacked in the department’s small autoclaves for sterilisation. They were to hold 1 litre of culture medium at a depth of 1.7 centimetres, the optimum for fungal growth and penicillin yield.[36] The Pyrex Glass Company was approached but was unable to produce them because of war commitments, and another firm that could wanted an up-front fee of £500 to prepare a special mould, plus there was a waiting time of six months.
The team was frustrated, production was held up. It was Heatley to the rescue again, suggesting ceramic vessels glazed only on the inside would be easier to handle. Florey sent Heatley’s designs to a contact in an area called the Potteries located in the English Midlands, where fine tableware had been made for hundreds of years. The firm of James MacIntyre and Co. agreed to make the vessels at a very reasonable cost and gloom immediately lifted at the Dunn School. Just before Christmas in 1940 Heatley borrowed a van and brought back the first batch of 174 vessels to Oxford, one of which Heatley kept in his home for years afterwards.[37]
On Christmas Eve everyone at the Dunn School pitched in washing, sterilising and filling the new fermentation vessels. On Christmas Day Heatley seeded them with the spores of the fungus Penicillium notatum and stacked them for their ten-day incubation period, at the end of which time Florey’s team hoped the liquid on which the fungus was growing would contain enough penicillin to begin tests on humans. Assisting the team with the mass-production were three women who were dubbed the ‘Penicillin Girls’. Although they were not formally trained, they quickly became efficient at setting up the cultures and harvesting the penicillin fluid. Within a month there were 80 litres of crude penicillin solution, with around 1–2 units of penicillin per millilitre. Later developments in commercial production would increase the yield of penicillin to an astonishing 40,000 units per millilitre.[38]
By February 1941 Howard Florey had enough penicillin to conduct a human trial. It was thought unwise to inject this unknown substance into a healthy member of the team. Florey, however, gargled with some liquid penicillin to see if it would have an effect on his sore throat but there was no noticeable improvement. A young doctor at the Radcliffe Infirmary, Charles Fletcher, was asked to select a patient. In the septic ward (a ward which no longer exists in hospitals) was a 43-year-old policeman, Albert Alexander, who had been injured in a bombing raid two months earlier and was near death with a staphylococcal and streptococcal infection. Alexander’s face and scalp were swollen, his head was covered with suppurating abscesses, one of his eyes had been removed and the other had to be lanced to relieve the pain of the swelling. Abscesses on Alexander’s arm had to be drained and his lung was also abscessed.[39] He had been treated with sulfonamides but these are ineffective once a patient is saturated with pus.
Given Alexander’s desperate situation, Florey and his team had nothing to lose yet everyone had something to gain. On 12 February Charles Fletcher administered the first dose of penicillin and Alexander immediately started to improve. More penicillin followed with more improvement. But Florey had no idea how much penicillin would be needed, and for how long, for the patient to make a full recovery. Despite the production efforts there was precious little of the wonder drug. After the first day, Fletcher collected the patient’s urine so that the penicillin could be extracted and reused. Staff members from the Dunn School took turns in riding their bicycles to the laboratory to deliver the urine to Florey and Chain as quickly as possible. Someone was available every two hours to take their turn on what had been christened the ‘pee patrol’.[40] This included Ethel Florey, who ironically had been able to return to her medical career because of the war.
The circumstances under which Ethel was able to resume work were not ideal for a mother. In 1938 she had begun to help organise teams of doctors for the new blood transfusion service in Oxford which was essential because of the war. Late in 1940, she and Howard and other Oxford academic families had taken the decision to evacuate their children and send them to the safety of America; 127 children were accompanied by 27 mothers.[41] Fortunately the Floreys had friends in the United States with whom their children could stay, but it was a wrench and Ethel was devastated. The children were so young—Paquita was ten and Charles only just five.
By 19 February Albert Alexander was well on the way to recovery—his temperature had come down and he was sitting up in bed—but each day a little less penicillin could be retrieved from his urine. When the supply was exhausted and the injections ceased, the bacteria began to regain their hold. Alexander relapsed and died on 15 March. It had been harrowing for the Oxford team and they were deeply affected by Alexander’s death. A necessary decision was made to concentrate their efforts on sick children, who would not require such large quantities of penicillin.
Their second patient was a boy, fourteen-year-old Johnny Cox who had a staphylococcus infection in his eye socket. He was not expected to live. Within hours of his first injection Johnny improved. The next day the boy was well enough to play and the penicillin was stopped. Totally unexpectedly, four days later Johnny went into convulsions and died. The team was devastated, heartbroken. Florey convinced Johnny’s parents to agree to an autopsy. No bacteria were found but the infection had weakened one of Johnny’s vital arteries, which had consequently burst.
And then success! A fifteen-year-old boy, Arthur Jones, whose wound had become septic after a hip operation to insert a pin, had not responded to sulfonamides. His temperature had been over 100°F (37.8°C) for two weeks. He was given penicillin, and within two days his temperature was normal. Four weeks later, Arthur was fit enou
gh to undergo another operation to remove the pin.[42] Four more patients were then successfully treated and the results were described in a landmark paper modestly entitled, ‘Further observations on penicillin’, which was published in The Lancet in August 1941.
***
Success with the trials meant the Oxford team had to find some way to get adequate funding to ensure that penicillin production progressed from the manufacture of a scarce, impure brown powder manufactured at the Dunn School to the commercial production of a purified and powerful antibiotic that could have widespread benefits. Heatley was still trying to improve the yield and with each batch he set aside a few vessels, varying the culture medium or the conditions, but his efforts were in vain.
Florey first approached British firms to take up the production of penicillin but heavy bombing closed that avenue even though the need for an antibacterial was more critical than ever. British people were living in dangerous times and suffering great privation. A two-fold plan was adopted. The Oxford team helped the government set up a network of ‘minifactories’ for penicillin production while their leader, urged to do so by contacts he had established at the Rockefeller Foundation, travelled to the United States to try to interest pharmaceutical companies there to take on largescale production of penicillin. Accompanied by Norman Heatley, Florey left England in July 1941 and flew to neutral Portugal while battles were being waged all over Europe. From there the two flew to New York on the Pan-Am Clipper seaplane. The windows were blacked out as the plane crossed the dangerous Atlantic Ocean.
Even though Ernst Chain opposed the trip, believing that they should first patent penicillin in Britain, he felt that he should have accompanied Florey, not Heatley. Chain and Florey had argued bitterly about seeking a patent for penicillin. Chain understood the importance of patenting and put his suggestion to Sir Edward Mellanby in London. His entreaty met with severe criticism and he was accused of wanting to profit personally. Ernst Chain was told that in England patenting medical discoveries was unethical and not the British way.[43] But it was the American way.
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