Antimicrobial Chemotherapy, 5th Edition

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 USA 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 that 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 hexamine, 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, as 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 exhibitselective toxicity.

Early tests of this hypothesis met with limited success, although certain aniline dyes were found to have some useful effects in trypanosomiasis of animals. Interest in dyes as chemotherapeutic agents continued, and was later to pay off in several directions, notably in helping to transform some firms in the European dyestuffs industry into large multinational pharmaceutical companies. However, Ehrlich was deflected from the study of dyes and turned to arsenicals.

Arsenicals

Arsenicals and other metallic compounds have been used in medicine at least since the time of the sixteenth century Swiss physician and alchemist, Paracelsus (Philippus Theophrastus Bombastus von Hohenheim). The explorer David Livingstone was among those who used arsenic in the treatment of nagana, a disease of ungulates, 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 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 (Neosalvarsan), 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.

Dyes

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).

Antimalarial agents

Remarkably, the very discovery of aniline dyes was sparked off by an antimalarial compound, as 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, plasmochin (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.

Sulphonamides

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, an experimental pathologist 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 was liberated from the dye. It was this colourless compound—hitherto unsuspected of possessing any antimicrobial activity—that was responsible for the astonishing properties of Prontosil.

As a chemical sulphanilamide had been synthesized and described as early as 1908 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 USA, ‘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 USA.

Such was the situation when penicillin appeared on the scene as a potential therapeutic agent in 1940 (see Table 1).

Antibiotics

Penicillin

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, pyocyanase, produced by the bacterium Pseudomonas aeruginosa, had actually been used therapeutically by instillation into wounds, at the turn of the twentieth century.

 

Table 1 Some chemotherapeutic agents (and their indications) that predate the introduction of penicillin

Date

Agent

Disease/pathogen

Pre-1890

Extract of male fern

Tapeworm

 

Santonin

Intestinal roundworm

 

Oil of chenopodium

Intestinal roundworm

 

Cinchona bark (quinine)

Malaria

 

Ipecacuanha root (emetine)

Amoebic dysentery

 

Mercury

Syphilis

 

Chaulmoogra oil

Leprosy

Arsenicals and antimonials

1905

Atoxyl

Trypanosomiasis

1909

Arsphenamine

Syphilis

1912

Neoarsphenamine

Syphilis

1912

Tartar emetic

Leishmaniasis

1917

Tartar emetic

Schistosomiasis

1919

Tryparsamide

Trypanosomiasis

Dyes and dye derivatives

1891

Methylene blue

Malaria

1904

Trypan red

Trypanosomiasis

1906

Trypan blue

Trypanosomiasis

1916

Suramin

Trypanosomiasisa

1926

Plasmochin

Malaria

1932

Mepacrine

Malaria (etc.)

1932

Prontosil red

Bacteria

1934

Chloroquineb

Malaria

Other substances

1895

Hexamine

Bacteria (urine)

1899

Pyocyanase

Bacteria (topical)

1925

Tetrachloroethylene

Hookworm

1939

Tyrothricin

Bacteria (topical)

Later (1940s) also used in onchocerciasis.

Chloroquine was first synthesized in 1934 in Germany, but developed in the USA in 1945.

 

Thus, when Alexander Fleming interrupted a holiday to visit 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% 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 USA 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.

 

 

Cephalosporins

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 that 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

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initiated a systematic search for non-toxic antibiotics produced by soil micro-organisms, notably actinomycetes, a group that includes theStreptomyces species 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 filtrates 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 real breakthrough came in 1943 with the discovery by Waksman's research student, Albert Schatz, of streptomycin, the first aminoglycoside 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. It remained the staple treatment for tuberculosis (together with some synthetic compounds; see below) until the antibiotic rifampicin—named after the 1955 French gangster filmRififi—was introduced in 1968.

The success of streptomycin stimulated the pharmaceutical houses to join the chase in the years following the end of the Second World War. 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.

Since 1960 few truly novel antibiotics 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 semisynthetic compounds with improved properties.

Non-antibiotic antibacterial compounds

Alongside developments in naturally occurring antibiotics, chemists and microbiologists have also been successful in seeking synthetic chemicals with antibacterial activity. Most have emerged through an indefinable mixture of biochemical know-how and luck rather than the rational targeting of vulnerable processes within the microbial cell.

The first important advance came with the discovery of para-aminosalicylic acid and isoniazid as effective antituberculosis drugs in the early 1950s, ushering in the era of reliable triple therapy (with streptomycin) for tuberculosis. Among the rest, the nitrofurans have a long history stretching back to before the Second World War, but attracted little attention until the development of nitrofurantoin in the early 1950s. The diaminopyrimidine, trimethoprim, was first synthesized in America by George Hitchings and his colleagues in 1956.

Commercially the most successful synthetic antibacterial agents have been the quinolones. The original compound, nalidixic acid, discovered in the 1960s as a by-product of the synthesis of the antimalarial agent chloroquine, was of minor importance, but after an unpromising start quinolones began to assume a more significant role in the 1980s when derivatives such as norfloxacin and ciprofloxacin emerged that exhibited much better activity against a broader spectrum of bacteria.

Antifungal, antiparasitic, and antiviral agents

The revolution in therapy brought about by the numerous antibacterial ‘wonder drugs’ was not mirrored in infections caused by other microbes, but considerable progress in the treatment of non-bacterial infection was nevertheless made in the second half of the twentieth century.

Treatment of fungal infections was the first to benefit. In 1949 at the height of the search for naturally occurring antibacterial compounds, Elizabeth Hazen and Rachel Brown discovered an antibiotic with surprisingly good antifungal activity. They named it nystatin after the New York State Department of Health in whose laboratories they worked. The related polyene, amphotericin B, was developed by scientists at Squibb in the 1950s. Another antifungal antibiotic, griseofulvin, had been described as early as 1939, but not used in human medicine until 1958, following the work by James Gentles in Glasgow on dermatophyte infections in experimental animals. A major step forward occurred in the 1970s with the discovery in Germany and Belgium of the unexpectedly broad-spectrum antifungal activity of certain nitroimidazole derivatives such as clotrimazole and ketoconazole (which offered the added advantage that it could be given orally) leading to the later development of the triazoles, fluconazole and itraconazole.

Among antiprotozoal agents, most progress was made among antimalarial agents, starting with the development in America after the Second World War of a pre-war German discovery, chloroquine, and culminating in the successful testing of artemisinin, the active ingredient of an ancient Chinese herbal remedy qinghaosu in the late 1970s. Many other protozoal diseases fared less well, but patients suffering from amoebic dysentery, giardiasis, and 
trichomonal vaginitis (and those with infections caused by anaerobic bacteria) benefited from the development in France around 1960 of metronidazole, a synthetic compound based on the structure of a naturally-occurring antibiotic, azomycin.

Even helminthic disease treatment was to profit from the intense research activity, albeit through investigations into drugs for worm infections of farm animals. Remarkably, by the early 1980s, three anthelminthic compounds—praziquantel, albendazole, and ivermectin—were available, which between them offered safe and effective treatment for a diverse range of human worm infections.

For many years, antimicrobial agents played a very minor part in the treatment of viral infections, although prevention of a number of viral diseases through the use of vaccines was outstandingly effective. The first real breakthrough came with the nucleoside analogue aciclovir as an offshoot of anticancer research in the mid-1970s. Even this drug, the first antiviral agent to display selective toxicity, was restricted to the treatment of herpesvirus infections. Antiviral chemotherapy was not to come of age for another decade, when the emerging AIDS pandemic stimulated the pharmaceutical industry into a flurry of activity that has produced an array of drugs offering palliative if not curative therapy.

The discovery of the first ‘miracle drugs’ was sanguinely declared by some to herald the virtual conquest of infection. More than 60 years of use have prompted a more sober assessment of the limitations of antimicrobial therapy. Microbes have shown amazing versatility in avoiding, withstanding, or repelling the antibiotic onslaught, while parallel medical advances have provided a large and increasing group of vulnerable patients for them to attack. Antimicrobial agents are essential tools of modern medicine, but the battle against infection is far from won. The challenge now is to preserve the remarkable achievements of the twentieth century by learning to use these powerful drugs more judiciously.