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Course: Scientific Discovery Instructor: Dr. Alexandra Vankley Presentation By: Sri Ram 10/21/03 STORY OF PENCILLIN Interesting Facts The wonder drug The first antibiotic popularly known The first antibiotic produced and used on a massive scale The pioneer of chemotherapy The 2 parts of the story a-the discovery of penicillin as an antibacterial and Alexander Fleming b-the discovery and realization of its chemotherapeutic potential Discovery of Penicillin Background Works of Louis Pasteur and Robert Koch, helped elucidate the connection between infectious diseases and the invasion of the body by bacteria and other microorganisms . Microbial Discoveries Year 1880-82 Microbe discovered Typhoid 1883 Cholera 1884 Tetanus 1886 Pneumonia 1894 Plague Related Events In 1871, Joseph Lister ( antiseptic practice surgery) - a mold in a sample of urine - inhibiting bacterial growth. In 1875 John Tyndall - a species of Penicillium caused some of his bacteria to burst. In 1877 Louis Pasteur and Jules Joubert airborne microorganisms could inhibit the growth of anthrax bacilli . Related Events contd. Ernest Duchesne in 1897 focused on the interaction between E. coli and Penicillium glaucum . Inoculating mold and typhoid bacilli -prevented contraction of typhoid in animals. But, he died of T.B before he could complete his research. Experiments carried out by Emmerich and Loew (1899) and later by Gratia and Dath and others did not give any favorable results. Related Events contd. Instead various forms of vaccination and serum treatment were evolved. The first Nobel Prize for Physiology or Medicine in 1901 was given for serum therapy for diphtheria. Human and animal bodies - produce protective substances in the fight against the invaders. Sir Alexander Fleming Sir Alexander Fleming on August 6th,1881 at Lochfield, Scotland in a farmer’s family . He had his early days spent more in the farms but was sound in his fundamental education. 1895-The untimely death of his father and the success of the medical practice of his step-brother Tom had him relocated to London. Born Sir Alexander Fleming –He attended the Polytechnic School in Regent Street . 1896-1900- He worked as a clerk in a shipping firm. 1900- In 1900, when the Boer War broke out joined the Scottish regiment but never saw the combat. 1895 Sir Alexander Fleming Later encouraged by his brother and supported by the money he received from an Uncle’s demise he looked towards a medical career. 1901-He won a scholarship to St. Mary's Hospital Medical School, London University, Paddington and joined it. He qualified with distinction and received his degree in 1906 Sir Alexander Fleming His switch to bacteriology was even more surprising: if he took a position as a surgeon, he would have to leave St. Mary's. The captain of St. Mary's rifle club knew that and was desperate to improve his team. Knowing that Fleming was a great shot he did all he could to keep him at St. Mary's. He worked in the Inoculation Service and he convinced Fleming to join his department in order to work with its brilliant director -- and to join the rifle club. Sir Alexander Fleming 1906- Fleming joined the Inoculation Department as medical bacteriologist under the direction of Sir Almroth Wright. He made the St. Mary's Hospital Medical School his professional home for the rest of his life. 1914-Flemming became a lecturer at St.Mary’s. 1914-1918- Served as Captain in W W 1 Sir Alexander Fleming 1918 -He returned to St.Mary's. 1928- He was elected Professor of the School in 1928 1943-He was elected Fellow of Royal Society. 1944-Flemming was knighted. 1945-Received his Nobel Prize. 1948-Elected the Emeritus Professor of Bacteriology, University of London. 1951-54-He was Rector of Edinburgh University. Died on March 11th,1955 Fleming’s Ideology Wright believed, as did Fleming, there were substances in the human body that could be used to fight infection, and strengthening the immune system was key. Hence, they believed more on vaccine therapy rather than chemotherapy. Ehrlich’s “Salvarsan” In 1909 German chemist-physician Paul Ehrlich developed a chemical treatment for syphilis. salvarsan - "that which saves by arsenic". Ehrlich’s samples were given to Fleming by Wright and became efficient in its administration. He did so with the new and difficult technique of intravenous injection. He soon developed such a busy practice he got the nickname "Private 606.“ Antiseptics of that period During World War I- wound-research laboratory in Boulogne, France. Chemical antiseptics like carbolic acid (phenol) do not sterilize jagged wounds; rather, pus has its own antibacterial powers. He was able to show that chemical antiseptics in dilutions harmless to bacteria actually damage white blood corpuscles (leukocytes)—the body's first line of defense. Lysozyme Discovery Fleming looked for -a chemical like salvarsan, that could help fight microbe infections. After war in 1920, back in St.Mary’s Fleming searched for an affective antiseptic. He discovered Lysozyme, in nasal mucus . Its an enzyme found in many body fluid, like tears, etc. It is a natural antibacterial not effective against the stronger infectious agents. Lysozyme’s Activity Lysozyme Lysozyme, in its natural state, seemed to be more effective against harmless airborne bacteria than against diseasecausing bacteria. And attempts to concentrate it, proved unsuccessful. Fleming continued his research of finding a better and less toxic antiseptic and antibacterial. The Chanced Discovery Fleming's legendary discovery of penicillin occurred in 1928, while he was investigating staphylococcus, a common bacteria then caused diseases ranging from boils to disastrous infections. Fleming at his usual work The Halo of Pencillin He left a culture plate smeared with Staphylococcus bacteria on his lab bench while he went on a two-week holiday. When he returned, he noticed a clear halo surrounding the yellow-green growth of a mold that had accidentally contaminated the plate. The Halo of Pencillin The culture-plate How This Happened Luckily, Fleming had not stored his culture in a warm incubator. London was then hit by a cold spell, giving the mold a chance to grow. Later, as the temperature rose, the Staphylococcus bacteria grew, covering the entire plate--except for the area surrounding the moldy contaminant. Fleming’s Deduction Fleming correctly deduced -mold must have released a substance - inhibited the growth of the bacteria. He was never clear on his observations. The evidence of the first culture, which he photographed indicated that Fleming observed lysis, the weakening and destruction of bacteria—as in his lysozyme studies. Penicillin and Lysozyme Activity Fleming’s Deduction But sometimes Fleming described it inhibition, or prevention of bacterial evidenced by a clear zone surrounding the mold. Although these two effects occur under quite different conditions, Fleming probably forgot which observation came first. Penicillin Identified He discovered that the antibacterial substance was not produced by all molds, only by strain of Penicillium notatum). Although he could not isolate it, he named the active substance “penicillin.” He studied methods of producing the impure product and determined its stability at different temperatures and over various lengths of time. Penicillin is Non-Toxic Fleming found that penicillin was not toxic to animals and that it did not harm white blood cells (leucocytes) by injecting healthy mice extract from the mold growth. Previous, observations of such injections proved toxic and penicillin was an exception. Penicillin Properties Penicillin would not be absorbed if taken orally. Penicillin taken by injection - excreted in the urine in a matter of hours-well before it could have its effects. Moreover, the unstable penicillin was never available for clinical testing. Fleming’s ways of using Penicillin For Fleming, penicillin's therapeutic potential - topical antisepsis. Fleming did continue to use in bacteriology. Penicillin suppressed the growth of certain bacterial species, allowing one to selectively culture certain others (such as those causing influenza, acne and whooping cough). Selective Inhibition Penicillin in vaccine production Penicillin became a valuable in manufacture of vaccines. The penicillin was crude--good enough for Fleming's purpose, but hardly strong enough to destroy a serious human infection. Paper on Penicillin Little notice was taken by the scientific community of his paper published in the British Journal of Experimental Pathology (June 1929). Explanation for Failure His belief - cure comes from within the body itself rather than from an external chemical agent. Difficulties -he had experienced in isolating and stabilizing penicillin, producing sufficient quantities for clinical trials- prevented him from realizing the full fruits of his research. Fleming-After Penicillin Fleming had turned his research to other chemical antibacterials, the sulphonamides He kept producing and supplying his sample of penicillin to other labs and researchers for various studies and experiments. For more than a decade or so no progress was made in the discovery of penicillin. End of the 1st half The 2nd half The discovery and realization of the chemotherapeutic potential of Penicillin Sir Howard Walter Florey Sir Howard Walter Florey Born on September 24, 1898, at Adelaide, South Australia . He graduated in M.B., B.S. in 1921from Adelaide University. He was awarded a Rhodes Scholarship to Magdalene College, Oxford, leading to the degrees of B.Sc. and M.A. (1924). Sir Howard Walter Florey In 1925 he visited the United States on a Rockefeller Travelling Fellowship for a year, Returned in 1926 to a Fellowship at Gonville and Caius College, Cambridge, received his Ph.D. in 1927. In 1931 he succeeded to the Joseph Hunter Chair of Pathology at the University of Sheffield. Sir Howard Walter Florey Leaving Sheffield in 1935 he became Professor of Pathology and a Fellow of Lincoln College, Oxford. 1936-He was made Director to the Sir William Dunn School of Pathology. He was made an Honorary Fellow of Gonville and Caius College, Cambridge in 1946 Sir Howard Walter Florey Honorary Fellow of Magdalen College, Oxford in 1952. In 1962 he was made Provost of The Queen's College, Oxford In 1944 he was created a Knight Bachelor. In 1945 was awarded the Nobel Prize. Dr. Florey died in 1968. Ernst Boris Chain Ernst Boris Chain Born on June 19, 1906, in Berlin. He next attended the Friedrich-Wilhelm University, Berlin, where he graduated in chemistry in 1930. He worked for three years at the Charité Hospital, Berlin, on enzyme research. In 1933, after the access to power of the Nazi regime in Germany, he emigrated to England. Ernst Boris Chain He first worked on phospholipids at the School of Biochemistry, Cambridge, under the direction of Sir Frederick Gowland Hopkins . In 1935 he was invited to Oxford University where he worked in the Sir William Dunn School of Pathology. in 1936, he was made demonstrator and lecturer in chemical pathology. Ernst Boris Chain In 1948 he was appointed Scientific Director of the International Research Centre for Chemical Microbiology at the Istituto Superiore di Sanita, Rome. He became Professor of Biochemistry at Imperial College, University of London, in 1961 1945-Awarded Nobel prize Dr.Chain died in 1979 Florey’s Team Florey recruited -a interdisciplinary group of scientists- to study pathological evidence of disease and physiological processes by which those symptoms arose, traced to the chemical and even the molecular level. Florey joined by Chain Among his first hires was the biochemist Ernst Boris Chain. With Chain , one of the projects pursued was the crystallization of lysozyme and the characterization of its substrate—the location on bacteria to which it usually attaches. Future Plans In 1938, while the lysozyme research was concluding, Florey and Chain decided to study selected antibacterial substances produced by certain microorganisms. They thought these substances were all enzymes like lysozyme. Chain’s suggestion This process was greatly facilitated by Chain's near-photographic recall of the many scientific papers he had read, including Fleming's 1929 paper on penicillin. Work on Penicillin Begins They originally chose substances from three organisms: pyocyanase, a topical antibacterial, from Bacillus pyocyaneus; extracts from certain organisms in the soil called actinomycetes; and penicillin Work on Penicillin While Florey and Chain were assembling grants and funds, work was begun on penicillin. Fortuitously, there was already a penicillin culture at the William Dunn School The research program rapidly narrowed its focus to penicillin alone. Isolating Pure Penicillin Chain, along with another chemist, E.P.Abraham -technique for purifying and concentrating penicillin. The key- pH of the “juice,” the sample's temperature, freeze-drying it. Later improved on by Norman Heatley and other scientists. Purification Difficulty Gallons and gallons of mold broth were used to produce an amount just large enough to cover a fingernail! First Toxicity Test In March 1940 Chain injected mice with a sample of the penicillin extracted. Far higher dosage than Fleming's - the mice survived apparently unharmed. The more-concentrated penicillin had passed its first toxicity test. Florey Impressed Florey directed that the antibacterial properties of penicillin in mice be tested—the step that Fleming had not taken. Heatley’s Contributions Norman Heatley -technical inventions to produce penicillin on a larger scale. Heatley- contributed a lot to the purification process and different methods of growing mould in various containers. First Therapeutic Test In May 1940, the team had been able to produce enough penicillin to test on infected animals . Eight mice were infected with a deadly dose of streptococci bacteria each. One hour later, four of them were injected with penicillin and four mice were left without treatment. First Therapeutic Test Mice Injected with Bacteria with Penicillin without Penicillin The Miracle The four mice that hadn't been injected with penicillin started to show signs of illness and later died. The four mice treated with penicillin remained fine! Hearing this Howard Florey exclaimed, "It looks like a miracle!" The Miracle The Mice injected with Penicillin survive ! Publication On 24 August 1940 Florey and Chain reported their findings in the Lancet; By then World War II had already engulfed Europe, calling for finding means of combating the diseases and infections of war to hold the advantage. The First Trial on Humans The first patient was a young woman volunteer with non-treatable cancer (January 1941) . She showed an alarming reaction—trembling and sharply rising fever. With paper chromatography- Abraham was able to separate out the impurities -showed that they caused the adverse reaction. The First Clinical Trial On 12 February 1941 a policeman with an invasive infection - first patient with an infection to be treated with penicillin. The dosages and the length of treatment required were being worked out by just trials. The First Clinical Trial First improved and then relapsed. The penicillin supply ran out- even retrieving penicillin from the man's own urine- failed to save him. Florey vowed that from then on he would always have enough penicillin to complete a treatment. Efforts to Mass Produce Increasing production - overriding importance. Penicillium mold requires air to growsurface-cultured in regular laboratory flasks. All manner of vessels usedhospital bedpans, made-to-order ceramic pots,etc Industrial Production Florey approached various British pharmaceutical firms. British pharmaceutical firms-committed to manufacturing other drugs needed for military and civilian populationsdevastated by enemy bombardment. Entry of USA To obtain the assistance of the United States- noncombatant- in increasing production and furthering research, Florey and Heatley flew across the Atlantic in the beginning of July 1941. Joint Action Florey’s contacts helped him get to have the production taken up by the U.S Firms and Government agencies. Soon it was decided to have an expedite unified action on penicillin . Joint Action At the height of the program- the British-American penicillin effortthousands of people and some thirtyfive institutions: university chemistry and physics departments, government agencies, research foundations, and pharmaceutical companies. Only Life Saver in War By D-day there was enough penicillin on hand to treat every soldier who needed it. By the end of World War II, it had saved millions of lives. Efforts to Study Penicillin Some chemists -synthesize penicillin from a few organic chemicals. Efforts to understand the molecular structure of the penicillin. Reacting the substance with various chemical reagents, which resulted in products of known structure. Efforts to Study Penicillin From these bits of structural information -deduce how the original molecule was organized. At Oxford the problem of determining penicillin's structure was given to Chain, Abraham, and Robert Robinson, a senior organic chemist . Penicillin Structure By fall 1943, groups working at Oxford and at Merck had proposed two different structures penicillin molecule. Chain and Abraham as well as by Robert Burns Woodward at Harvard— four-membered beta- lactam ring lay at the heart of the penicillin molecule Robinson instead proposed a structure based on oxazalone Proposed Structures Beta-lactam ring structure for penicillin. The beta-lactam ring is shown in red. Proposed oxazalone structure for penicillin. The oxazalone ring is shown in red. Newer Techniques to Study New techniques for analyzing the structure of organic molecules - X-ray crystallography- practiced by Dorothy Hodgkin In 1945 she was able confirmed Chain and Abraham's deduction. This evidence ran counter to Robinson's proposed structure for penicillin. Production by Fermentation only In 1957 John Sheehan created such a synthesis, but fermentation - the commercial production of penicillin and related antibiotics. But the structural knowledge gained developing penicillin-like antibiotics that were more effective, convenient to give and had fewer side effects. Awards time World War II ended and the Nobel Prizes in physiology or medicine distributed to Fleming, Florey, and Chain for their work on penicillin. The Nobel Prize in Physiology or Medicine 1945 "for the discovery of penicillin and its curative effect in various infectious diseases" Penicillin-The master Drug Pneumonia, syphilis, gonorrhea, diphtheria, scarlet fever and many wound and childbirth infections that once killed indiscriminately suddenly became treatable. Deaths caused by bacterial infections plummeted Words of Caution By this time, even Fleming was aware that penicillin had an Achilles' heel. He wrote in 1946 that "the administration of too small doses ... leads to the production of resistant strains of bacteria." It's a problem that plagues us to this day. References www.nobel.se www.chemheritage.com www.hisortylearningsite.co.uk www.pbs.org www.time.com