Course: Scientific Discovery

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• Course Scientific Discovery
• Instructor Dr. Alexandra Vankley
• Presentation By Sri Ram
• 10/21/03

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STORY OF PENCILLIN
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Interesting Facts

• The wonder drug
• The first antibiotic popularly known
• The first antibiotic produced and used on a
  massive scale
• The pioneer of chemotherapy

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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

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Discovery of Penicillin
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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 micro-organisms .

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Microbial Discoveries
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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 .

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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.

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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.

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Sir Alexander Fleming
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Sir Alexander Fleming

• Born on August 6th,1881 at Lochfield, Scotland in
  a farmers 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.

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Sir Alexander Fleming

• 1895 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.

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Sir Alexander Fleming

• Later encouraged by his brother and supported by
  the money he received from an Uncles 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

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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.

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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.Marys.
• 1914-1918- Served as Captain in W W 1

1906 he joined the staff of the
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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.

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• 1948-Elected the Emeritus Professor of
  Bacteriology, University of London.
• 1951-54-He was Rector of Edinburgh University.
• Died on March 11th,1955

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Flemings 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.

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Ehrlichs Salvarsan

• In 1909 German chemist-physician Paul Ehrlich
  developed a chemical treatment for syphilis.
• salvarsan - "that which saves by arsenic".
• Ehrlichs 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.

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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.

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Lysozyme Discovery

• Fleming looked for -a chemical like salvarsan,
  that could help fight microbe infections.
• After war in 1920, back in St.Marys 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.

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Lysozymes Activity
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Lysozyme

• Lysozyme, in its natural state, seemed to be more
  effective against harmless airborne bacteria than
  against disease-causing bacteria.
• And attempts to concentrate it, proved
  unsuccessful.
• Fleming continued his research of finding a
  better and less toxic antiseptic and
  antibacterial.

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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.

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Fleming at his usual work
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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.

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The Halo of Pencillin
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The culture-plate
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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.

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Flemings 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
  bacteriaas in his lysozyme studies.

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Penicillin and Lysozyme Activity
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Flemings 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.

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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.

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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.

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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.

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Flemings 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).

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Selective Inhibition
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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.

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Paper on Penicillin

• Little notice was taken by the scientific
  community of his paper published in the British
  Journal of Experimental Pathology (June 1929).

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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.

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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.

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End of the 1st half
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The 2nd half

• The discovery and realization of the
  chemotherapeutic potential of Penicillin

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Sir Howard Walter Florey
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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).

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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.

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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

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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.

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Ernst Boris Chain

•  

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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.

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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.

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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

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Floreys 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.

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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 substratethe location on
  bacteria to which it usually attaches.

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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.

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Chains 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.

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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

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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.

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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.

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Purification Difficulty

• Gallons and gallons of mold broth were used to
  produce an amount just large enough to cover a
  fingernail!

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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.

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Florey Impressed

• Florey directed that the antibacterial
  properties of penicillin in mice be testedthe
  step that Fleming had not taken.

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Heatleys 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.

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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.

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First Therapeutic Test
Mice Injected with Bacteria
with Penicillin
without Penicillin
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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!"

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The Miracle
The Mice injected with Penicillin survive !
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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.

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The First Trial on Humans

• The first patient was a young woman volunteer
  with non-treatable cancer (January 1941) .
• She showed an alarming reactiontrembling and
  sharply rising fever.
• With paper chromatography- Abraham was able to
  separate out the impurities -showed that they
  caused the adverse reaction.

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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.

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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.

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Efforts to Mass Produce

• Increasing production - overriding importance.
• Penicillium mold requires air to grow-
  surface-cultured in regular laboratory flasks.
  All manner of vessels used- hospital bedpans,
  made-to-order ceramic pots,etc

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Industrial Production

• Florey approached various British pharmaceutical
  firms.
• British pharmaceutical firms-committed to
  manufacturing other drugs needed for military and
  civilian populations-devastated by enemy
  bombardment.

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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.

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Joint Action

• Floreys 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 .

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Joint Action

• At the height of the program- the
  British-American penicillin effort-thousands of
  people and some thirty-five institutions
  university chemistry and physics departments,
  government agencies, research foundations, and
  pharmaceutical companies.

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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.

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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.

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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 .

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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

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Proposed Structures
Proposed oxazalone structure forpenicillin. The
oxazalone ringis shown in red.
Beta-lactam ring structure forpenicillin. The
beta-lactam ringis shown in red.
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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.

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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.

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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.

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The Nobel Prize in Physiology or Medicine 1945
"for the discovery of penicillin and its curative
effect in various infectious diseases"
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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

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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.

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References

• www.nobel.se
• www.chemheritage.com
• www.hisortylearningsite.co.uk
• www.pbs.org
• www.time.com