Over the past few installments in this series, we saw how the German dye industry became
the Pharma industry around the turn of the 20th century, and then turned a red synthetic
dye, Prontosil, into the first broad spectrum antibacterial drug.
This gave rise to the first class of synthetic antibiotics, the sulfa drugs.
The sulfa drugs saved millions of lives in World War I, but they were by no means perfect
drugs.
Many bacterial strains, especially the Gram-negative ones, were not sensitive to sulfa drugs, and
mortality from bacterial pneumonia at that time was still 15-20%.
The next breakthrough came purely by accident, and it occurred not in Germany for a change,
but in Britain.
Many people know the story of Dr. Alexander Fleming, who coming back to his office at
St. Mary’s Hospital in London after his summer vacation, found that his Petri dishes
containing colonies of Staphylococcus aureus, had been contaminated by a mold which had
drifted into the office through the open window.
Examination of the colonies under the microscope showed that most of the bacteria were dead.
Fleming was quick to speculate that the mold, identified as Penicillium notatum, must contain
some bactericidal substance.
In this way, Fleming’s careless mistake was eventually the key to one of the most
important medical discoveries of the 20th century.
This serendipitous event occurred in 1928.
Fleming then identified the mold, grew it in the lab, and was able to refine the extracts,
proving that they kill Staphylococcus strains in vitro.
His work was published in 1929.
Fleming called the active principle “Penicillin”, but he had neither the competence nor the
means to isolate and identify the new antibiotic.
His work initially raised zero interest.
It wasn’t until about ten years later when Dr. Howard Florey, director of the Sir William
Dunn School of Pathology at Oxford University, focused on Fleming’s results and began researching
the class of molecules we now call penicillins.
In 1940, Florey and his colleague Ernst Chain were able to refine the extraction process
and treated a number of mice that had been infected with a Streptococcus strain.
All the treated mice survived, whereas those left untreated died.
They finally had the active antibiotic synthesized by the mold, which they called Penicillin
G, as the mold also made a few other structurally related penicillins.
The structure of this compound was proposed by chemist Edward Abraham in 1942 and confirmed
via X-ray crystallography techniques by Dorothy Hodgkin in 1945.
It was an unusual, unprecedented structure, which left even illustrious organic chemists
quite skeptical.
To briefly speak the language of the organic chemist, the molecule presented a very strained
4-membered ring called azetidinone, or beta-lactam, fused to a five-membered ring containing a
sulfur atom, also called a thiazolidine.
Its high reactivity was the key to its bioactivity.
The compound has the ability to disrupt the production of bacterial cell walls, a structure
not possessed by our own cells, which is why this drug and others like it can kill bacteria
without harming us, and such reactivity was specifically due to the beta lactam ring.
This gave rise to a new broad class of antibacterials, called beta-lactam antibiotics, which will
be discussed in a moment.
First let’s go back to 1940.
The mold produced a very small amount of penicillin.
Specifically, it took 2,000 liters of mold culture to obtain enough pure penicillin G
to treat a single person.
In September 1940, an Oxford policeman became the first patient.
He had wounded his face in his garden, and the wound had become seriously infected.
Sulfa drugs were not helping and the policeman was dying.
Florey and Chain heard about this by chance, and asked the treating doctor whether they
could try their new drug on the dying policeman.
After five days of penicillin injections, the patient’s conditions improved dramatically,
but Florey and Chain ran out of the drug, so the patient relapsed and ultimately died.
The scientists took the result as a partial success, and at this point they were convinced
that only the mass production of their miracle drug stood in the way of a huge medical breakthrough.
In 1941, the US entered World War II, and the US Pharma industry was tasked with solving
the penicillin supply problem in order to treat US soldiers in view of the European
invasion.
Thus, the war provided the necessary boost to penicillin research.
Florey and colleagues had already flown to the United States, and all of a sudden they
found a receptive environment for their studies.
The key problem was that the mold, Penicillium notatum, was a lousy producer of penicillin
G. Florey became convinced that they should search for a more productive mold.
The story goes that a laboratory assistant bought a cantaloupe at the market, brought
it to the lab, and found that it was covered with a “pretty, golden mold.”
The mold turned out to be the fungus Penicillium chrysogenum, which produced 200 times more
penicillin G than the notatum strain.
The scientists got to work on improving the strain by mutation, and were eventually able
to reach a culture that produced the antibiotic in 1,000 times greater concentrations than
P. notatum, Fleming’s original mold.
The years that followed were a period of feverish activity.
Penicillin G proved far superior to the sulfa drugs in treating infections.
Urged by the military, the US pharma industry worked around the clock to produce enough
penicillin to potentially treat all US soldiers involved in the war.
In 1941, the world supply of Penicillin G was only enough to treat 10 patients.
But the US War Production Board created a plan to accelerate production at all participating
companies, so new fermentation techniques were invented and implemented rapidly.
By the time the invasion of Normandy began in 1944, 2.3 million doses were available.
And with that, the race had been won.
In that same year, Fleming, Florey, and Chain received the Nobel Prize for medicine.
A bit later, chemist John Sheehan at MIT was the first to synthesize penicillin from scratch
in 1957, and he wrote a book on the new wonder drug entitled The Enchanted Ring: the Untold
Story of Penicillin, although there are many books telling the riveting story of this wonder
drug.
Nowadays, penicillin is produced in huge amounts through the fermentation of bioengineered
fungal strains, and the global output is 50 million kilograms per year, at a bulk cost
below $10 per kilogram.
The penicillin story represents a milestone in the history of the fledgling US pharma
industry.
After 1945, there was enough penicillin to treat the entire civilian population and penicillin
G became the key drug for treating bacterial infections.
Physicians, however, were quick to notice a problem.
Bacteria rapidly developed “resistance” to penicillin, meaning that mutated forms
developed which withstood attack by the new antibiotics.
One of the strategies evolved by bacteria was the production of a class of enzymes called
“beta-lactamases”.
These enzymes catalyzed the cleavage of the beta lactam ring, thereby inactivating the
antibiotic.
Some strains had this ability built into their DNA.
Given that bacteria and molds had co-existed for millions of years, it is no surprise that
bacteria had developed a strategy to cope with killer molds.
In addition, strains that had this beta-lactamase ability were able to exchange, or donate this
ability to other bacteria, in a stunning example of horizontal information exchange, meaning
between existing organisms.
Chemists looking to improve on penicillin G noted that the only synthetic modification
one could make to this molecule was on what we call the amide side chain.
This gave rise to some improved semisynthetic penicillins, such as amoxicillin, which is
still in use today.
However, resistance was still a problem, and better antibiotics were needed.
The next Fleming-scale breakthrough did not happen in Germany or Britain, but on the island
of Sardinia, in Italy.
Giuseppe Brotzu was the head of the microbiology institute at the University of Cagliari.
His true passion was research.
However, during World War II his lab was heavily bombed, and in 1945, with the war coming to
an end, there was absolutely no money to fund his research.
Brotzu had noticed that, although the local young men were frequently bathing in the waters
where the sewers of Cagliari were discharged into the sea untreated, none of them developed
the expected infections, like salmonella, or typhus.
He was convinced that the local waters contained a powerful antibiotic.
As unlikely as the idea might seem, it provided a powerful drive for him to spend his hours
collecting samples from the bay and trying to identify some new active principle.
With no research money whatsoever, Brotzu resorted to rather unusual means to support
his studies.
His lab had no electricity, so he worked by candlelight.
To power his few instruments, he was able to secure a large gift of methanol from a
local company, and was able to build an electricity generator using methanol combustion.
To feed his mice, he went out to the countryside every day before dawn and stole vegetables
from the fields of local farmers.
To sustain his fermentation cultures, he found that placentas from women who had just given
birth ensured a protein-rich culture medium for the microorganisms he was breeding, and
once a week he went to the hospitals, which had agreed to save the placentas for him.
On July 20th, 1945, Brotzu found a new antibiotic that is produced by a known mold, Cephalosporium
acremonium.
He worked 16-hour days to produce enough material to treat mice first, and then injected the
drug into himself to make sure it was not toxic.
Finally, in 1947, he produced enough concentrate to treat a few patients.
The first one, a 5-year old boy dying of typhus, was administered the crude solution and recovered
completely in six days.
Convinced that he had made a medical breakthrough, Brotzu tried in vain to obtain research funding
and publish his results, but nobody was interested.
Unfazed, Brotzu created a new journal: Lavori dell’Istituto di Igiene di Cagliari.
In the first and only issue of the first and only volume, his landmark paper appeared,
the only paper.
It was written in Italian, and it did not create any interest whatsoever.
Coincidentally, a few months later a British medical officer was visiting Sardinia, and
Brotzu provided the colleague with samples of the culture, which were later delivered
to Professor Edward Abraham in Florey’s group at Oxford.
In 1949, Abraham filed a patent in his own name, and later sold the rights to pharma
companies Glaxo and Eli Lilly, obtaining huge royalties.
As sometimes happens to dedicated scientists, Brotzu never made a single lira from his own
discovery.
But he received an honorary doctorate from Oxford, and was elected mayor of Cagliari,
which made him happy enough.
Finally in 1960, the Oxford group isolated the active principle, and in 1961 Dorothy
Hodgkin determined the structure.
The new antibiotic was called Cephalosporin C.
Here is the structure of Cephalosporin C. As chemists came to realize, it has two possible
points of chemical modification available, and so theoretically, many more semisynthetic
analogs were possible.
However, two problems remained to be solved.
Cephalosporin C was very difficult to ferment and was still unavailable in large amounts
to serve as raw material for new analogs.
In addition, there was no way to cleave the amide side chain without disrupting the beta-lactam
ring and destroying the molecule.
In 1969, chemist Robert B. Morin at Eli Lilly solved both problems, cleaving the amide side
chain in a clever way.
Noting that cephalosporins have the same number of carbon atoms as penicillins, he hypothesized
a common biosynthetic origin.
By finding a way to open the 5-membered ring of the penicillin and close it back up to
form the 6-membered ring of the cephalosporins, Morin created a huge new field of research.
Newer and more potent generations of cephalosporins emerged, with increased scope and spectrum.
Just to highlight how complex this type of drug design has become, one can look at a
typical 5th generation drug, Ceftobiprole, with the modified side chains in red.
Today, the 70 available cephalosporins represent the most commercially important class of antibacterials,
with over $13 billion in sales per year, or about 30% of the world market for antibacterials.
Penicillins and cephalosporins are by no means the only antibiotics available in the pharmacy.
Over the years, working with new molds, newer classes of antibacterials were discovered,
such as the tetracyclines, aminoglycoside antibiotics like streptomycin, the macrolide
antibiotics like erythromycin, and the ansamycins exemplified by rifampicin.
These are all structurally complex classes of naturally-derived antibiotics.
However, the second most important class of antibiotics, after the beta-lactams, is strictly
synthetic, the class of quinolone antibiotics exemplified by ciprofloxacin.
So why is it that scientists developed all these antibiotics and more?
As we already mentioned, the main problem was bacterial resistance.
Given enough time, it is essentially guaranteed that resistant bacterial strains will emerge
to any antibiotic.
The excessive use of penicillins in the world since 1945 has created a huge resistance problem,
and the former wonder drugs have slowly become more of a mediocre solution.
A description of the multiform and fascinating mechanisms by which bacteria develop resistance
is beyond the scope of this series, but it is important to remark that the battle against
infectious bacteria is an ongoing challenge.
Especially prevalent today are nosocomial infections, meaning originating in a hospital,
because highly resistant bacterial strains are most often found in hospitals.
Every year, almost 2 million Americans contract serious infections in the hospital, and almost
100,000 die from them, with this number increasing each year.
However, while we have already introduced vaccines in the context of viral infection,
it is worth mentioning the excellent progress that has been made in the field of anti-bacterial
vaccines, especially against antibiotic-resistant strains.
Nevertheless, the war against pathogenic bacteria is an ongoing one.
All the battles we have won thus far have not yet led to the end of the war, due to
the extreme biological resilience of our enemies.
This is certainly one of the most fascinating chapters in the history of drugs, but unfortunately,
the widespread threats to our health are not exclusively a bunch of tiny critters.
Let’s now turn to other areas where mankind has made tremendous progress from the 20th
century onwards.