Antimicrobial Chemotherapy, 4th Edition
Note on the use of drug names
In the UK the British Approved Name (BAN) has traditionally been used for all prescription drugs. A European Union directive now requires member countries to conform to the recommended International Non-proprietary Name (rINN). In the case of most antimicrobial agents that are affected, the change is minor (e.g. cefalotin [rINN] for cephalothin [BAN]; amoxicillin [rINN] for amoxycillin [BAN]). Since these changes are being introduced over a transitional period, and variant spellings are usually self-evident, we have decided to keep the familiar (at least to UK readers) BAN spellings for this edition. Oddly, the antiviral agent officially known in the UK as tribavirin does not feature in published lists of necessary changes, but in recognition of the fact that this name is rarely used, even in the UK, we have accepted the universally used ribavirin in the case of this compound.
Editors: Greenwood, David
Title: Antimicrobial Chemotherapy, 4th Edition
Copyright ©2000 Oxford University Press
> Table of Contents > Historical introduction
Although the ‘antibiotic revolution’ can be accurately dated to the early 1940s when Howard Florey and his colleagues in Oxford seized on Alexander Fleming's penicillin and turned it into a major therapeutic compound and Selman Waksman in the United States began his systematic pursuit of antibiotics from soil micro-organisms, the quest for chemotherapeutic agents active against pathogenic microbes began much earlier. Indeed, hopes of discovering specific antimicrobial drugs were kindled almost as soon as the microbial enemy was definitively identified by Louis Pasteur, Robert Koch, and others during the second half of the nineteenth century. By the end of that century Paul Ehrlich, often called the ‘father of chemotherapy’, had started the work which was to put the quest on a sound scientific footing.
Of course, the search for effective remedies is as old as mankind itself, but before the aetiological agents of infectious disease were identified and made amenable to laboratory investigation, progress had to rely entirely on the vagaries of chance and empirical observation. Not surprisingly, therefore, mankind's earliest therapeutic successes against infecting organisms came in the form of natural products such as honey that could be applied topically to infected lesions, or infusions that expelled worms visible to the naked eye. Herbal anthelminthics known since antiquity include extract of male fern (Dryopteris filix-mas) which is an effective vermifuge for tapeworms, and two compounds that expel intestinal roundworms: santonin (obtained from the seed-heads of Artemisia cina; wormseed), and oil of chenopodium (Chenopodium ambrosioides; American wormseed).
Observations that natural substances controlled the spectacular symptoms of certain diseases no doubt led to the initial recognition of two other ancient remedies: quinine, obtained from the bark of the cinchona tree, and emetine, an alkaloid obtained from ipecacuanha root. These compounds were introduced into Europe from South America in the seventeenth century; both are active against protozoa (the parasites of malaria and amoebic dysentery respectively), and both have survived into present-day use.
So far as antibacterial remedies were concerned, little had been achieved when Ehrlich began his work. Mercury had been used for the treatment of syphilis since the sixteenth century (giving rise to the aphorism ‘one night with Venus—a lifetime with Mercury’), and chaulmoogra oil from the seeds of Hydnocarpus species had been used since ancient times in India for the treatment of leprosy. Otherwise the only useful antibacterial compounds were topical antiseptics that were far too toxic for systemic use. At the end of the nineteenth century hexa-mine, a compound that spontaneously decomposes in acid conditions to release formaldehyde, was described as being useful in urinary tract infection.
The foundations of modern chemotherapy
Oddly, in view of later developments, the foundations of twentieth century chemotherapy were built on a search for antiprotozoal agents, since it was to the newly discovered parasites of malaria and African sleeping sickness (trypanosomiasis) that Paul Ehrlich first turned his attention. He reasoned that, since these parasites could be differentiated from the tissues of infected patients by various dyes in the laboratory, such substances might display a preferential affinity for the parasites in the body as well. In a phrase, such dyes might exhibit selective toxicity.
Early tests of this hypothesis used the aniline dyes methylene blue and trypan red. Neither compound proved to be of much value, but the idea was pursued in France by Félix Mesnil and Maurice Nicolle, who found that two related dyes, trypan blue and afridol violet, had some useful effects in trypanosomiasis of animals. Interest in dyes as chemotherapeutic agents continued, and was later to pay off in several directions; however, Ehrlich was deflected from the study of dyes and turned to arsenicals.
Arsenicals and other metallic compounds, have been used in medicine at least since the time of the sixteenth century Swiss physician, Paracelsus (Theophrastus Bombastus von Hohenheim). The explorer David Livingstone was among those who used arsenic in the treatment of nagana, a disease of horses, later shown by David Bruce (of Brucella fame) to be caused by trypanosomes. Ehrlich had also exhibited a passing interest in these compounds, and this was rekindled in 1905 by a report from the Liverpool School of Tropical Medicine by Anton Breinl and Harold Wolferstan Thomas, who demonstrated that one arsenical, atoxyl, protected mice from trypanosomal infection. Ehrlich resumed research into arsenical compounds and visited the Liverpool School in 1907.
Despite its name, atoxyl was anything but atoxic and Ehrlich, together with his chemist Alfred Bertheim, set about trying to produce safer arsenical derivatives.
A Japanese assistant, Sahachiro Hata, joined the team in 1909 and spirochaetes, which were thought at that time to exhibit similarities to trypanosomes, were included in the screening programme. Later that year Hata showed that arsenical compound number 606 cured rabbits infected with the spirochaetes of syphilis and, equally importantly, displayed an acceptable safety profile. Compound 606, later known as arsphenamine and marketed as Salvarsan, was the first really efficacious antibacterial agent, although its activity was restricted to spirochaetes, which are scarcely typical bacteria. An improved derivative, neoarsphenamine (Neo-salvarsan), was produced in Ehrlich's laboratory in 1912.
Interest in arsenicals and other metals was also pursued elsewhere. Hopes remained of finding a drug for the treatment of African sleeping sickness, and tryparsamide and melarsoprol (Mel B) later emerged as useful compounds. Another ancient metallic remedy, tartar emetic (potassium antimony tartrate) was discovered to exhibit useful activity in two very different tropical diseases: kala azar (a protozoal disease of the reticuloendothelial system) and bilharzia (a worm infection of the blood). Tartar emetic was a familiar nostrum of Victorian medicine, and it is likely that its empirical use in previously untreatable conditions led to the discovery of its efficacy. Antimonials are still used for the treatment of kala azar, but safer drugs have been developed to treat bilharzia.
Although Paul Ehrlich's optimistic hope of exploiting the differential affinities of dyes therapeutically came to nothing, the idea eventually bore fruit when the German dyestuffs industry started to take an interest in antimicrobial compounds. The most direct link with Ehrlich's ideas is provided by suramin (Germanin), a colourless derivative of trypan blue developed by scientists of the Bayer organization during the First World War and marketed in 1924. Like tartar emetic, suramin has proved useful in two quite unrelated parasitic diseases: trypanosomiasis and onchocerciasis (a worm disease of the skin).
Remarkably, the very discovery of aniline dyes was sparked off by an anti-malarial compound, since it was during an attempt to synthesize quinine from coal tar in 1856 that the 18 year-old William Perkin, a student at the Royal College of Chemistry, stumbled on mauve purple, the first aniline dye.
The progression from dye to antimalarial was accomplished, again at Bayer, through attempts to improve the activity of Ehrlich's methylene blue. Modification of the dye produced nothing of value, but information gained on the effects of various substitutions prompted the chemists to try similar modifications on quinine-like compounds. In this way the first synthetic antimalarial drug, plas-mochin (pamaquine), and the acridine derivative, atebrin (later called mepacrine
or quinacrine), were produced. These compounds eventually led to the synthesis of primaquine and chloroquine, which are still in use today.
The German obsession with dyes also paid off with the discovery of the first broad-spectrum antibacterial agents, the sulphonamides. This discovery came about by another of those happy accidents with which the history of chemotherapy is littered. In 1932 Gerhard Domagk, a bacteriologist working in the laboratories of the Bayer wing of the IG Farbenindustrie consortium, tested a number of dyes synthesized by his colleagues, Fritz Mietzsch and Josef Klarer. In one such compound, Prontosil red, a sulphonamide group had been linked to a red dye in the hope of improving the binding to bacterial cells as it was known to do with fibres. Remarkably, mice treated with Prontosil red survived an otherwise fatal infection with haemolytic streptococci, but when the compound was tested against streptococci in vitro it was found to have no antibacterial activity whatsoever. This paradox was explained by the Tréfouëls and their colleagues in France who showed that in the experimental animal sulphanilamide—a colourless compound hitherto unsuspected of possessing any antimicrobial activity—was liberated from the dye.
Sulphanilamide was already in the public domain, and Bayer were unable to protect the discovery by patent. Naturally, many other firms seized the opportunity to market the drug so that by 1940 sulphanilamide itself was available under many different trade names and a start had been made on producing the numerous sulphonamide derivatives that subsequently appeared. One proprietary version marketed in the US, ‘Elixir Sulfanilamide’, was formulated in diethylene glycol and killed over 100 people. This event led to a law giving the Food and Drug Administration power to regulate the licensing of new drugs in the US.
Such was the situation when penicillin appeared on the scene as a potential therapeutic agent in 1940 (see Table 1).
Table 1 Some chemotherapeutic agents (and their indications) that predate the introduction of penicillin in 1941
When Howard Florey and his team at the Sir William Dunn School of Pathology in Oxford first took an interest in penicillin in the mid 1930s, the concept of antibiosis and its therapeutic potential was not new. In fact, moulds had been used empirically in folk remedies for infected wounds for centuries and the observation that organisms, including fungi, sometimes produced substances capable of preventing the growth of others was as old as bacteriology itself. One antibiotic substance, pyocya-nase, produced by the bacterium Pseudomonas aeruginosa, had actually been used therapeutically by instillation into wounds, at the turn of the century.
Thus, when Alexander Fleming returned from holiday to his laboratory in St Mary's Hospital in early September 1928 to make his famous observation on a contaminated culture plate of staphylococci, he was merely one in a long line of workers who had noticed similar phenomena. However, it was Fleming's observation that sparked off the events that led to the development of penicillin as the first antibiotic in the strict sense of the term.
The actual circumstances of Fleming's discovery have become interwoven with myth and legend. Attempts to reproduce the phenomenon have led to the conclusion that the lysis of staphylococci in the area surrounding a contaminant Penicillium colony on Fleming's original plate could have arisen only by an
extraordinary concatenation of accidental events, including the vagaries of temperature of an English summer.
Early attempts to exploit penicillin foundered, partly through a failure to purify and concentrate the substance. Fleming made some attempts to use crude filtrates in superficial infections and there is documentary evidence that Cecil George Paine, a former student of Fleming's, successfully treated gonococcal ophthalmia with filtrates of Penicillium cultures in Sheffield as early as 1930. However, it was left to Ernst Chain, a German refugee who had been recommended to Florey as a biochemist, to obtain a stable extract of penicillin. Chain had been set the task of investigating naturally occurring antibacterial substances (including lysozyme, another of Fleming's discoveries) as a biochemical exercise. It was with his crude extracts that the first experiments were performed in mice and men. Since these extracts contained less than 1 per cent pure penicillin, it is fortunate that problems of serious toxicity were not encountered.
Further development of penicillin was beyond the means of wartime Britain, and Florey visited the US in 1941 with his assistant, Norman Heatley, to enlist the support of the American authorities and drug firms. Once Florey had convinced them of the potential of penicillin, progress was rapid and by the end of the Second World War bulk production of penicillin was in progress and the drug was beginning to become readily available.
The discovery of the cephalosporins (which are structurally related to the penicillins) is equally extraordinary. Between 1945 and 1948, Giuseppe Brotzu, former Rector of the University of Cagliari, Sardinia, investigated the microbial flora of a sewage outflow in the hope of discovering naturally occurring antibiotic substances. One of the organisms recovered from the sewage was a Cephalosporium mould which displayed striking inhibitory activity against several bacterial species—including Salmonella typhi, the cause of typhoid and now considered as a serotype of S. enterica—that were beyond the reach of penicillin at that time. Brotzu carried out some preliminary bacteriological and clinical studies, and published some encouraging results in a local house journal. However, he lacked the facilities to develop the compound further, and nothing more might have been heard of the work if he had not sent a reprint of his paper to a British acquaintance, Dr Blyth Brooke, who drew it to the attention of the Medical Research Council in London. They advised contacting Howard Florey, and Brotzu sent the mould to the Sir William Dunn School in 1948.
The first thing to be discovered by the Oxford scientists was that the mould produced two antibiotics, which they called cephalosporin P and cephalosporin N, because the former inhibited Gram-positive organisms (e.g. staphylococci and streptococci) whereas the latter was active against Gram-negative organisms (e.g. Escherichia coli and S. typhi). Neither of these substances is a cephalosporin in the sense that the term is used today: cephalosporin P proved to be an antibiotic
with a steroid-like structure, and cephalosporin N turned out to be a penicillin (adicillin). The forerunner of the cephalosporins now in use, cephalosporin C, was detected later as a minor component on fractionation of cephalosporin N. Such a substance could easily have been dismissed, but it was pursued because it exhibited some attractive properties, notably stability to the enzymes produced by some strains of staphylococci that were by then threatening the effectiveness of penicillin.
Antibiotics from soil
The development of penicillin, cephalosporin C, and, subsequently, their numerous derivatives represents only one branch of the antibiotic story. The other main route came through an investigation into antimicrobial substances produced by micro-organisms in soil. The chief moving spirit was Selman A. Waksman, an emigré from the Russian Ukraine, who had taken up the study of soil microbiology in the USA as a young man. In 1940, Waksman initiated a systematic search for non-toxic antibiotics produced by soil micro-organisms, notably actino-mycetes, a group that includes the Streptomyces spp. that were to yield many therapeutically useful compounds. Waksman was probably influenced in his decision to undertake this study by the first reports of penicillin and by the discovery by an ex-pupil, René Dubos, of the antibiotic complex tyrothricin in culture fil-trates of Bacillus brevis.
Waksman's first discoveries were, like Dubos's tyrothricin, too toxic for systemic use, although they included actinomycin, a compound later used in cancer chemotherapy. The first real breakthrough came in 1943 with the discovery by Waksman's research student, Albert Schatz, of streptomycin, the first amino-glycoside antibiotic, which was found to have a spectrum of activity that neatly complemented penicillin by inhibiting many Gram-negative bacilli and—very importantly at that time—Mycobacterium tuberculosis.
The appearance of streptomycin stimulated the pharmaceutical houses to join the chase. Soil samples by the hundred thousand from all over the world were screened for antibiotic-producing micro-organisms. Thousands of antibiotic substances were discovered and rediscovered and, although most failed preliminary toxicity tests, by the mid-1950s representatives of most of the major families of antibiotics including aminoglycosides, chloramphenicol, tetracyclines, and macrolides, had been found.
Naturally occurring antibiotics that inhibit pathogenic fungi were also discovered. The first, nystatin, was named after the New York State Department of Health in whose laboratories it was discovered in 1949 by Elizabeth Hazen and Rachel Brown. The related polyene, amphotericin B, was developed by scientists at Squibb in 1960. Another antifungal antibiotic, griseofulvin, was first described in 1939, but not used in human medicine until 1958, following the work by J. C. Gentles on dermatophyte infections in experimental animals.
Since 1960 few truly novel antibiotic substances have been discovered, although a surprising number of naturally occurring molecular variations on the penicillin structure have emerged. A more fruitful approach, especially with penicillins and cephalosporins, has been to modify existing agents chemically in order to derive semi-synthetic compounds with improved properties.
Chemists and microbiologists have been successful, too, in seeking antibacterial activity in non-antibiotic substances, but none has arisen by premeditated attack on known biochemical pathways. In fact, all the synthetic antimicrobial drugs presently used therapeutically, including the diaminopyrimidines, nitrofu-rans, imidazoles, quinolones, and most antituberculosis drugs, have emerged through an indefinable mixture of biochemical know-how, and luck. The relatively small number of antiviral agents in therapeutic use have generally arisen in a similarly unsystematic way, although some of the newer drugs used against human immunodeficiency virus have been developed by precise targeting of specific viral processes.
The scope of antimicrobial chemotherapy
Without question, the discovery of the first ‘miracle drugs’—sulphonamides, penicillin, and streptomycin—in the late 1930s and early 1940s revolutionized the treatment of bacterial infection. Indeed, the appearance of these and subsequent drugs was sanguinely declared by some to herald the virtual conquest of infection. With over 60 years' hindsight and hundreds of new agents at our disposal, we can now take a more dispassionate view of the benefits and limitations of antimicrobial therapy. The truly amazing versatility that microbes have displayed in terms of their ability to avoid, withstand, or repel the antibiotic onslaught, or to take advantage of vulnerable patients, could scarcely have been predicted. Moreover, unwanted side-effects, including disturbance of the delicate bacterial ecology of the body, are more common than might have been supposed.
Finally, it should not be forgotten that the most spectacular triumphs have been achieved in the treatment of bacterial disease. Those numerous infections caused by viruses, protozoa, helminths, and fungi are, with some notable exceptions, less amenable to chemotherapy. Indeed some non-bacterial infections are treatable only by toxic agents of restricted potency, and a few still lie beyond the scope of systemic therapy.