It was well recognized by then that the structure and enzyme systems that allow bacteria to thrive are very different from their mammalian counterparts. Scientist devoted considerable effort from the late nineteenth century on to the search of chemicals that would exploit those differences so as to specifically eradicate bacterial cells. A tantalizing early clue lay in the stains that were used by microbiologists to study their prey. By the end of the nineteenth century scientist had identified a variety of dyes that stained bacteria in preference to mammalian cells. The key to finding a drug that would preferentially eradicate bacteria seemed to be to identify a stain that was lethal to the bacteria that bound that dye. The long search seemed to have finally borne fruit in 1932. Gerhard Domagk, working in a laboratory set up by I.G. Farben, discovered that the red dye Prontosil Rubrum protected mice that had been injected with otherwise lethal doses of staphylococci. Use of the dye to successfully treat a human infection (Domagk's daughter) confirmed that this was indeed a therapeutic breakthrough. There had by then however been more than a few reports of seemingly miraculous cures of disease due to bacterial infection. The resulting skepticism from the inevitable failure of those treatments, led to surprisingly slow acceptance of the dye, by now simply called Prontosil. There was however the puzzling fact that the dye was only marginally effective in killing bacteria in the then-standard test tube experiment for antibacterial activity. The mystery was solved by a group of scientists at the Pasteur Institute in France. They discovered that the dye molecule is transformed chemically in animals. Liver enzymes they found split the molecule in two. One of the halves, subsequently named sulfanilamide, turned out to be a fully effective antibacterial compound both in test tubes or when administered to infected mice. The other half showed no antibacterial activity whatever. This work gave birth to the discipline of drug metabolism. Prontosil was to be but the first case of a drug that needed to be modified by the body for activity.
An immediate result of this finding was the abandonment of Prontosil in favor of the chemically much simpler sulfanilamide. This drug can be synthesized by chemists in just a few steps from benzene. This synthesis was in fact for many years a laboratory exercise in beginning organic chemistry courses. It probably enticed more that one student, including the author into a career in pharmaceutical research.
The discovery of sulfanilamide marked the beginning of the search for agents to treat infectious disease among compounds made from a scratch by organic chemists. We come back to that story later. The discovery of the other important source of compounds that selectively kill microbes dates back to 1929 Alexander Fleming well-known serendipitous discovery. He noted a microbe free clear zone around a mold colony that had contaminated a film of bacteria growing in a Petri dish. He correctly ascribed that to an antibiotic substance secreted by the mold. He named this unknown secretion penicillin after the producing mold that he had identified as Penicillium notatum. The imminence of World War II is said to have spurred the transition of what had been considered as simply an interesting laboratory observation into a useful antibiotic drug. Starting in about 1938, Howard Florey led the very major effort to isolate penicillin. This was finally accomplished in 1940, largely through the work of his Oxford collaborator Ernst Chain. The team isolated just enough pure penicillin to ascertain it's near miraculous activity in humans. Production of penicillin was transferred to the US since the British chemical industry was then fully tied up with war production. In its original form Penicillium notatum grew best as a surface mat. Quantity production invoked visions of the use of shallow tanks with enormous surface areas. The project was assigned to the U. S. Department of Agriculture Northern Laboratory in Peoria, Illinois which had experience in industrial fermentation. There they devised a method for growing the mold as a submerged culture. By this and other means they greatly increased the yield of penicillin. The method developed by USDA was then transferred to industry; a large number of companies with expertise and facilities for fermentation were enlisted in the effort. This even at one time included Schenley, better known as a producer of spirits.
Penicillin in the then-used form had a number of very serious shortcomings. The drug had to be administered by injection as it was not orally active. The molecule is also very reactive leading to poor stability. Early research aimed to produce more stable congeners led to several salts with improved stability. Reasons for the sensitivity of penicillins emerged with the determination of the chemical structure. The compounds in essence comprise of two discrete connected pieces. The essential part consists of a ring structure called a beta-lactam. This is the reactive part that in the end kills bacteria; it also contributed to lack of stability. The rest of the structure, which is also required for activity, consists an organic acid connected through a chemical bond. Penicillin obtained from fermentations is actually a mixture of closely related compounds in which the invariant beta-lactam is hooked to slightly different acids. Scientists had noted that they could increase the proportion of one or another congener by adding a large amount of that acid to the culture medium. This allowed them to selectively produce one or another of the congeners. These still however shared many of the same shortcomings of the original drug. This included poor stability and lack of oral activity; the drugs were also not effective against a significant number of classes of bacteria. It had become apparent by 1960 that further improvements would require manipulation of the chemical structure.
It was later established that the selectivity of the beta-lactam antibiotics traces back to the fact that bacteria are more closely related to plants than animals. Individual animal cells are surrounded by a membrane whereas plants and thus bacteria depend on a wall for cell integrity. In bacteria that structure is composed of a dense network of protein filaments that is cross linked by chemical bonds. A significant number of the amino acids that compose the proteins have chemical structures that are mirror images of those found in animals. The beta-lactams (penicillins and cephalosporins bellow) are mistaken by bacterial enzymes as small pieces that will be used to form the cross links. Once they get incorporated they bring the process to dead halt causing the cell wall to rupture. The drugs are thus selective because mammals do not use cell walls and in addition utilize enzymes that do not recognize the mirror image amino acids used to make bacterial cell walls.
By 1940 sulfanilamide had come into widespread use particularly in treating war wounds. The drug was used both as a tablet and sprinkled directly onto open lesions. Though the drug saved numerous lives many types of bacteria were immune to its action. Chemists in a number of pharmaceutical laboratories then tried to make changes in the molecule in attempts to broaden the activity of this class to other classes of bacteria. Systematic work showed that there was only a single place on the molecule that could be manipulated and still retain antibacterial activity. Hundreds of analogues of sulfanilamide may well have been prepared in a number of laboratories between 1940 and the late 1950's when the work was finally abandoned. No fewer than twenty-seven of these were granted non-proprietary names. This is often an indication that the sponsor intends to test the compound in the clinic. At least eight of these so-called sulfa drugs are currently used in the clinic.
The antibacterial activity and selectivity of this class of drugs again depends on the fact that bacteria uniquely rely on biochemical processes that have no counterpart in more complex organisms. Folic acid, perhaps better known as one of the B vitamins, is an essential factor in various metabolic processes such as formation of red blood cells and of DNA itself. Over the course of evolution many organisms have lost the ability to make this compound and rely on obtaining it from foods. Bacteria on the other hand synthesize this vitamin from a scratch. An important biochemical step involves hooking a small molecule, PABA (para-aminobenzoic acid) onto the growing molecule. The chemical structures and properties of the sulfa drug are similar enough to PABA to cause bacterial enzymes to incorporate these molecules. The resulting product can however go no further in effect causing the bacterium to die for lack of folic acid.
One of the sulfa drugs, sulfamethoxazole forms part of a combination tablet that still currently comprises first line treatment for urinary infections. The other active ingredient, trimethoprim, traces back to work carried out by future Nobel winner George Hitchings at Burroughs Wellcome in the late 1950's. Taking their clue from compounds involved in enzyme action he and as associates prepared a congener called pyrimethamine. This agent proved to inhibit bacterial growth. Further work along the same lines led to the synthesis of trimethoprim. The combination tablet exploits the fact that each of the active ingredients inhibits bacterial growth by interfering with different enzymes that bacteria need survive.
Isolated reports of unusual side effects came with widespread use of sulfa drugs. Very high dose caused some patients to excrete water and others to show a drop in blood sugar levels. Chemists in pharmaceutical laboratories seized on these apparent side effects to develop entirely new classes of drugs. By manipulating the chemical structures scientists at Hoechst came up with a compound that normalized blood sugar in adult onset diabetics. This drug, tolbutamide is virtually devoid of antibacterial activity. The only drugs available in the nineteen-forties for treating conditions that required loss of body water, the diuretics, included mercury in their chemical composition. Scientists at Merck led by Sprague, were able to ring changes on the structure of the sulfa drugs to produce a well tolerated diuretic drug. This compound, chlorphenamide, also devoid of antibacterial activity is no longer in use. It has been superseded by hydrochlorothiazide first synthesized by chemists at Ciba. This drug, often better known by its acronym, HCTZ, is still used as first line treatment of patients with mildly elevated blood pressure.
The discovery of penicillin led to the recognition of the ability of fungi to protect themselves against microorganisms by secreting compounds that inhibit bacterial replication and in fact often snuff out those threatening organisms. Penicillin itself showed that such antibiotics may act specifically on enzymes that do not have counterparts in mammals. This property, shared with the sulfa drugs, led to low toxicity to humans. The search for new molecules in this class turned to the investigation of the host of fungi that inhabit the world. The competition between these organisms and bacteria in soils pointed to that domain as a potentially rich source of antibiotics. In the early nineteen-forties, Albert Schatz working at Rutgers University under the supervision of his professor, Selman Waksman, discovered that the mold, Streptomyces Griseus, produced an antibiotic that had a chemical structure that was quite different from penicillin. It was more stable and was effective against as somewhat different set of pathogens. This therapeutic agent, streptomycin, today still comprises one of the indispensable drugs used to combat tuberculosis. This discovery also launched a major effort in the laboratories of many pharmaceutical companies to screen soils from a wide variety of sources. Thus in 1949, Benjamin Duggar of the University of Wisconsin who was a consultant to the Lederle Laboratories, discovered a new antibiotic that he called aureomycin. Scientists at Lederle used this finding to develop a family of chemically closely related antibiotics. These are called tetrayclines after the chemical structure that involves four linked rings. A soil sample from the Phillipines was sent back to the Indianapolis labs of Eli Lilly by one of their local employees at about the same time. Screening of that sample led to the isolation of a new antibiotic with yet another novel chemical structure. The organism, at that time called Streptomyces erythaeus, gave the compound its name. (Many streptomyces species have since been reclassified as Actinomyces for reasons of taxonomic accuracy). This antibiotic was developed into the still widely used drug erythromycin by a team a Lilly led by J. M. McGuire. A soil sample from a much less exotic source led to another new class of drugs. A detail man in the American midwest sent a sample of soil collected in Lincoln, Nebraska back to his employer, the Upjohn Company, in Kalamazoo. There scientist isolated an antibiotics whose chemical structure and range of activity was markedly different from the other hitherto known agents. It was named lincomycin after the producing organism, Streptomyces Licolnensis. That in turn being named after the capital of Nebraska. The drug was eventually largely superseded by the derivative clindamycin produced by modifying the structure chemically.
The foregoing touches on only the highlights of what was a major program in many laboratories in the late forties to early sixties. The screening process became highly efficient with time. An unexpected major stumbling block was the frequent rediscovery of previously known antibiotics. Many laboratories thus maintained extensive dictionaries of the properties of all antibacterial substances produced by soil organism. These tomes, maintained in order to avoid wasting time on known substances, were considered highly confidential and were jealously guarded, particularly on occasions where an employee left the company. These drugs kill bacteria by a variety of mechanisms which are beyond the scope of this narrative.
Penicillin itself had in the meantime not been forgotten. Limited work on attaching unusual acids to the beta-lactam part of the molecule by feeding those to the fermentation tanks had not been particularly successful. In 1959 however scientists at Beecham Laboratories in the UK managed to devise conditions that allowed them to isolate from fermentation broths the beta-lactam portion of the molecule itself without the attached acid part. The availability of this substance called 6-aminopenicillanic acid, or more familiarly 6-APA, offered organic chemists the chance to hook acids, also called side-chains, never found in nature onto the active part of the molecule. The first drug from this research, pheneticillin was developed by Beecham in collaboration with Bristol Myers in Syracuse, New York. This development was eagerly seized upon by a number of competing laboratories. Many of these now launched their own programs aimed at synthesizing and testing analogues of penicillin with novel side chains. The output of these penicillin analogues was limited only by the high skill needed to carry out manipulations on these sensitive and reactive molecules. The work was subsequently facilitated by new methods for converting the much more easily available penicillin-V into 6-APA by either chemical or enzymatic means. These new so-called semi synthetic penicillins included much more stable drugs as well as a number with broader activity and some that were active when taken by mouth. This massive research effort resulted in at least 35 discrete substances that showed enough promise so that their sponsors went through the process of acquiring non-proprietary name. Not all, needless to say made it to the market. The orally active semi-synthetic drug amoxicillin is one of the more widely prescribed antibiotics to this day.
Other environments rich in bacteria and fungi were examined for potential antibiotics as well. The lead for a new series of drugs based on a beta-lactam came from the isolation, in 1945, of a mixture antibiotic from Sardinian sewage sludge by the Italian scientist Brotzu. The active principle, named Cephalosporin C after the producing species, Cephalosporium acremonium, was too weakly active to be considered as a drug candidate. Some of its properties, such as resistance to a bacterial enzyme that destroyed penicillin, however made it an attractive starting point for further research. Though the chemical structure differed from penicillin it shared enough similar features, such as the beta-lactam part, to lead scientists, to apply the same methodology to try to prepare more active compounds. Attempts to introduce different side chain acids by adding those to fermentation were not very successful. Neither were experiments aimed at producing the bare beta-lactam part of the molecule, equivalent to 6-APA, by fermentation. A procedure for obtaining the bare nucleus, 7-ACA (7-aminocephalosporanic acid) from cephalosporin C either chemically or by treatment with enzymes was finally published in 1962 by a team of chemists at Ciba in Basel. The availability of this intermediate now made it possible to launch research programs analogous to those that had led to the collection of semi-synthetic penicillins. The group at Eli Lilly was particularly active in this field. Its first drug from this program was the injectable antibiotic cephalothin (Keflin). The starting material for this and later products, Cephalosporin C was more difficult to obtain than the penicillins. Extensive research on production methods combined with demand had drastically lowered the price of bulk penicillins. This had in fact become a bulk commodity chemical that by the late nineteen-nineties could be bought less that one dollar per gram. The fact that both molecules shared a beta-lactam led to considerable research on the part of chemists to find a way to transform penicillins into cephalosporins. These efforts were rewarded when Robert Morin devised just such a procedure at the Lilly labs. It is likely that the great preponderance of today's cephalosporin drugs start life as penicillin V or possibly its cousin penicillin G. The now ready availability of 7-ACA led to intensified research throughout pharmaceutical company laboratories. This resulted in a virtual flood of antibiotics; no fewer forty four of these were assigned non-proprietary names. The group of drugs that were made available to physicians included many that could be administered orally. Most were quite resistant to bacterial enzymes that destroy the beta-lactam. One of the most significant advances lay in the fact that selected semi-synthetic cephalosporins were active against a great many types of bacteria that were not sensitive to the earlier drugs. As chemical methods and expertise accrued it became possible to make modifications on the beta-lactam portion itself present in the molecules. A number of drugs that included such modifications have been approved for use in the clinic. Carbenicllin (Trade name Geocillin) represents on of the most drastically modified compounds. To prepare this antibiotic chemists at Merck devised a means for virtually destroying one of the two fused rings in 6-APA that contains sulfur; it is then built it up again with a ring that has only carbon. The resulting antibiotic is active against a particularly wide assortment of bacteria.
Pharmaceutical research in the early 1960's relied heavily on the output and imagination of its organic chemists. These individuals as a rule synthesized compounds that were aimed at some specific therapeutic target and were tested in a model for that disease. Surplus amounts of the samples were then tested in a standard screen. This usually comprised array of tests that were designed to identify chemicals that showed some degree of biological activity in other disease models. The rationale for this procedure lay in the observation of the number compounds that had been found over the years to have more activity on some unintended endpoint than their purported target. Some of the compounds discovered by the screening procedure had provided leads for agents that had gone on to become successful drugs. The Sterling Drug labs at that time had a long standing program on tropical disease. A compound, submitted by one of their chemists, George Lesher showed unexpected activity in the ongoing antibacterial screen. According to one account this was a byproduct from the synthesis of an antimalarial drug. The chemical showed enough activity for further development. This drug, nalidixic acid, was developed further an approved for use in treating urinary tract infections. The chemical structure of little heralded antibiotic was very different from any known antibiotics. It incorporated, it should be noted, all the necessary features that would be found in the later quinolones. The activity of this forerunner was quite modest. This was apparently not enough to stimulate research in competing laboratories. Only a few additional entities reached the clinic from an assortment of other firms over the next decade. These analogues were only modestly more effective than the forerunner. Research did apparently continue at a low level in several laboratories. Replacement of hydrogen by fluorine was known to markedly increase activity in several classes of drugs as for example the corticosteroids. Two specific modifications of the chemical structure of the quinolones, one of which interestingly comprised introduction of a fluorine atom, dramatically changed the picture. This compound, norfloxacin, was first synthesized in the late nineteen-seventies in the laboratories on the Kyorin Pharmaceutical Company in Japan. It activity in test models and subsequently in humans was clearly superior to any of its predecessors. It was introduced in the US by Merck. The excellent broad spectrum activity of this novel drug stimulated a virtual race to produce new chemical entities it has been estimated that over a thousand quinolones have been synthesized in various laboratories, twenty two of which have been assigned non-proprietary names. One of these, ciprofloxacin, known to the press as cipro of course gained considerable fame in late 2001. This drug gained wide exposure as a remedy and preventative at the height of the anthrax scare.
The quinolones, it has been determined, act by a unique and intriguing mechanism. Recall that DNA is a very large string-like molecule. The length of this compound must be reckoned in feet rather than millimeters. This entity must obviously be tightly coiled for one to be packed into each and every cell nucleus. It would take far too long to carefully uncoil what has been compared to a snarl of fishing monofilament in order to read a stretch for DNA to exert its controlling role. Biochemistry then does what any impatient fisherman would do: cut sections in order to pass strands through another. A very special enzyme called topoisomerase then temporarily holds the cut ends together until the section has passed through and then subsequently reconnects the cut pieces. Bacterial DNA floats loose within the cell as microbes have no cell nucleus. Their topoisomerase, possibly because of this difference, is quite different from that in organisms that have a cell nucleus. The quinolones act as quite specific inhibitors of bacterial toposiomerase. This in effect degrades the DNA since the cut ends are not marked for reconnection. The lack of an intact DNA template prevents the affected organism from replicating.
The very large selection of very effective antibiotics has led to a significant diminution of research on new antibiotics. The increasing incidence of antibiotic resistant organisms may lead to a revival of the field. A drug introduced within the past five years may be an early harbinger of this. Reports occasionally appear in the scientific literature of unusual chemical structures that seem to have a bit of antibacterial activity. Scientists at Upjohn launched a program, probably in the late nineteen eighties to synthesize a series of compounds based on one of those reports. All the analogues contained a common structural feature called an oxazolidone. Two of the compounds from the collection of new analogues synthesized at Upjohn showed very promising antibacterial activity. One of these, linezolid, which has been approved for human use, is quite effective against classes of bacteria resistant to known antibiotics. The drug also acts by a quite novel and unique mechanism.
This brief account also reflects of some of the plate tectonic-like shifts within the pharmaceutical industry over the past half century. The great majority of companies named in this chronicle have ceased to exist as independent entities. To take these one at a time in order appearance:
- The pre-World War II German chemical cartel I.G. Farben was broken up after hostilities. Its pharmaceutical division was more or less reconstituted as Hoechst. After some intermediate mergers Hoechst is now a part of Aventis.
- Burroughs-Welcome is now part of Glaxo
- Lederle was a division of the chemical concern Cyanamid when the work described in this account was carried out. It was acquired by American Home Products. That firm changed the name of its pharmaceutical division to Wyeth within the past few years.
- Upjohn merged with the Swedish firm Pharmacia in 1995. This conglomerate in turn was recently acquired by Pfizer.
- Beecham became part of Glaxo after some years as part of Smith-Kline Beecham
- Bristol Myers merged with Squibb to become Bristol Myers Squibb
- Ciba first merged with Geigy; the combine later acquired the remaining Swiss concern Sandoz. After a few more acquisitions the firm changed its name to Novartis.
- Sterling drug underwent a particularly convoluted sojourn. The company was first acquired by Kodak. It was then sold off to Smith Kline Beecham. What remained became part of Glaxo with the rest of it owner.
- Eli Lilly, Merck and Kyorin still continue as free-standing concerns as of this writing.
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