Antibiotics

Antibiotics
Masaji Ohno, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan (Chap. 1, 2, 3 and 4) Masami Otsuka, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan (Chap. 1, 2, 3 and 4) Yoshinari Okamoto, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan (Chap. 1, 2, 3 and 4) Morimasa Yagisawa, Japan Antibiotics Research Association, Tokyo, Japan (Chap. 1, 2, 3 and 4) Shinichi Kondo, Institute of Microbial Chemistry, Tokyo, Japan (Chap. 1, 2, 3 and 4) Heinz Oppinger, Hoechst Aktiengesellschaft, Frankfurt, Germany (Chap. 5, 6 and 7) Hinrich Hoffmann, Hoechst Aktiengesellschaft, Frankfurt, Germany (Chap. 5, 6 and 7) Dieter Sukatsch, Hoechst Aktiengesellschaft, Frankfurt, Germany (Chap. 5, 6 and 7) Leo Hepner, L. Hepner and Associates, Ltd., London, United Kingdom (Chap. 8) Celia Male, L. Hepner and Associates, Ltd., London, United Kingdom (Chap. 8)

1. 1.1. 1.2. 1.3. 2. 2.1. 2.2. 2.3. 2.4. 2.5. 2.6. 3. 3.1. 3.1.1. 3.1.2. 3.1.3. 3.1.4. 3.1.5. 3.1.6. 3.1.7. 3.1.8. 3.1.9. 3.1.10. 3.2. 3.3. 3.4. 3.5. 3.5.1. 3.5.2. 3.5.3.

Introduction . . . . . . . . . . General Denition . . . . . . Historical Development and Classication . . . . . . . . . . Nomenclature . . . . . . . . . Chemotherapeutic Use of Antibiotics . . . . . . . . . . . . Microbial Pathogens . . . . . Tumor Cells . . . . . . . . . . . Chemotherapeutic Uses . . . Use in Agriculture . . . . . . . Units . . . . . . . . . . . . . . . Analysis . . . . . . . . . . . . . Classication of Antibiotics -Lactams . . . . . . . . . . . . Natural Penicillins . . . . . . . Semisynthetic Penicillins . . . Natural Cephalosporins . . . . Semisynthetic Cephalosporins Cephamycins . . . . . . . . . . 1-Oxacephems . . . . . . . . . . -Lactamase Inhibitors . . . . Penems . . . . . . . . . . . . . . Carbapenems . . . . . . . . . . Monocyclic -Lactams . . . . Tetracyclines . . . . . . . . . . Anthracyclines . . . . . . . . . Aminoglycosides . . . . . . . . Nucleosides . . . . . . . . . . . N-Nucleosides . . . . . . . . . . C-Nucleosides . . . . . . . . . . Carbocyclic Nucleosides . . .

.... .... .... .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 2 2 4 4 4 4 4 4 5 5 5 5 5 7 7 10 11 13 14 14 15 16 16 18 18 23 24 24 26

3.6. 3.6.1. 3.6.2. 3.6.3. 3.6.4. 3.7. 3.8. 3.9. 3.10. 4. 5. 5.1. 5.2. 5.3. 5.4. 5.4.1. 5.4.2. 6. 6.1. 6.2. 7. 7.1. 7.2. 8. 9.

Macrolides . . . . . . . . . . . . . . . . 12-Membered Ring Macrolides . . . 14-Membered Ring Macrolides . . . 16-Membered Ring Macrolides . . . Polyenes . . . . . . . . . . . . . . . . . Ansamycins . . . . . . . . . . . . . . . Peptides . . . . . . . . . . . . . . . . . Enediynes . . . . . . . . . . . . . . . . Other Important Antibiotics . . . . Antibiotic Resistance . . . . . . . . . Fermentation . . . . . . . . . . . . . . Screening . . . . . . . . . . . . . . . . Selection, Mutation, and Maintenance of Strains . . . . . . . Process Development Leading to Large-Scale Production . . . . . . . Fermentation Technology . . . . . . Maintenance of the Strain and Production of Inoculum . . . . . . . . Treatment Before and During Fermentation . . . . . . . . . . . . . . . Isolation and Purication of Antibiotics; Quality Specications . . . Isolation . . . . . . . . . . . . . . . . . Purication Techniques, Sterile End Products, Ofcial Regulations Analytical Measurements and Quality Control . . . . . . . . . . . . Microbiological Analysis . . . . . . Isotopically Labeled Antibiotics . Economic Aspects . . . . . . . . . . . References . . . . . . . . . . . . . . . .

26 26 26 27 28 28 29 32 33 34 35 35 36 36 39 39 40 42 42 45 46 46 48 49 50

c 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 10.1002/14356007.a02 467.pub2

Antibiotics the next decade. Instead, synthetic chemotherapeutics, such as sulfonamides, became objects of general interest after the discovery of prontosil by Domagk in 1935 [23]. Only the outbreak of the Second World War in 1939 led to an intense worldwide search for drugs to treat infections and wounds. Toward the end of the 1930s, Florey, Chain, and co-workers began to investigate penicillin in the course of their systematic study of antibacterial substances. They demonstrated the marked activity and therapeutic value of penicillin in 1940 [24]. The production of penicillin had until that time been unsatisfactory, and favorable conditions for the effective formation of the antibiotic were explored in the United States. An active culture of the penicillinproducing organism was sought and submerged fermentation was developed. The use of lactose as a carbon source and the addition of cornsteep liquor to the nutrients were found effective. Irradiation of the culture with X-rays or ultraviolet light produced mutant strains. These ndings set the stage for the industrial production of penicillin. After the discovery of penicillin, Brotzu began a search for antibiotic-producing organisms and examined a culture of Cephalosporium spp. isolated from the sea near a sewage outlet in Sardinia. It secreted substances active against gram-positive bacteria. In September of 1948, Brotzu sent his organism to Abraham at Oxford for detailed inspection. Several antibiotics were isolated from the culture and named cephalosporin [25]. Particular attention was attracted by cephalosporin C, crystallized from the crude mixture of antibiotics, because of its low toxicity and its resistance to penicillinase. Penicillins and cephalosporins, both of which possess a -lactam ring as a structural characteristic, are designated -lactam antibiotics. Extensive attempts to improve their antibacterial spectra through chemical modications led to the development of many kinds of semisynthetic penicillins and cephalosporins. Moreover, nontraditional -lactams, namely penems, carbapenems, and monobactams, have been discovered and commercialized [26 29]. Other novel antibiotics were discovered among the products of fungi, bacteria, and actinomycetes [30]. As a result of a search for water-soluble and heat-stable substances which would be active against gram-negative bacte-

1. Introduction
1.1. General Denition
In 1942, Waksman dened antibiotics as chemical substances produced by microorganisms and capable of inhibiting the growth of microorganisms [20]. Great effort has been devoted to the worldwide search for new antibiotics, and numerous compounds possessing various biological activities, that is, antibacterial, antiviral, antifungal, antitumor, and enzyme-inhibiting activities, have been discovered. These substances are mostly of microbial origin but are also semisynthetic or totally synthetic in some cases. They have a wide variety of structural characteristics. The area dened by the term antibiotics is therefore expanding.

1.2. Historical Development and Classication

In 1877, Pasteur observed that saprophytic bacteria inhibited the growth of pathogenic anthrax organisms. His was the rst scientic description of the antagonism phenomenon. Production of a certain metabolic substance seemed to be responsible for the inhibition. Pasteur suggested the therapeutic potential of this type of growth repression. Vuillemin used the term antibiosis to describe the inhibition of the growth of one organism by another. The potential utility of bacteriotherapy was recognized and enormous experimental efforts were made to investigate the antagonism phenomenon. In 1894, Metchnikoff reported the repressive effect of Pseudomonas on Vibrio cholerae. From the culture of a Penicillium, Gosio isolated an antibacterial crystalline substance, mycophenolic acid, in 1896. Other results also demonstrated the ability of various microbes to produce antibacterial substances [21]. It was in 1929 that Fleming observed that a culture of a Penicillium inhibited the growth of bacteria [22]. He demonstrated the production of an antibacterial substance in the culture broth and named it penicillin. Although he suggested the promising therapeutic utility of penicillin, none of the attempts to isolate penicillin were successful and attention was not attracted for

Antibiotics ria, Waksman isolated actinomycin in 1940 and streptomycin in 1944 from cultures of actinomycetes. Various other antibiotics were isolated from microbes in France, Germany, Japan, the United Kingdom, the United States, and other countries. A yellow substance showing antibacterial activity was found in 1948 among products of Streptomyces aureofaciens and was named aureomycin. Terramycin was isolated from the fermentation broth of Streptomyces rimosus. The chemical and structural similarities of the two soon became apparent; they each have a linearly fused tetracyclic structure of sixmembered rings. This parent skeleton is designated tetracycline, and aureomycin and terramycin are now called chlortetracycline and oxytetracycline, respectively. In 1950, rhodomycin was isolated from the culture broth of Streptomyces purpurascens by Brockmann and his co-workers. Structural analysis disclosed that rhodomycin is a glycoside, combining an amino sugar and a 7,8,9,10-tetrahydro-5,12-naphthacenequinone moiety. Structurally similar antibiotics have since been discovered, and the generic name anthracycline has been assigned. The aglycone of anthracycline is called anthracyclinone. Daunorubicin and doxorubicin are representative anthracycline antitumor antibiotics. Aclarubicin possesses three sugars and is of interest because of its low toxicity. Streptomycin is used for infections of grampositive and gram-negative bacteria and as a specic medicine for tuberculosis. Kanamycin, discovered by Umezawa in 1957, is especially effective against resistant bacteria. Paromomycin, Spectinomycin, and ribostamycin were isolated later. These antibiotics are called aminoglycosides because their structural units are amino sugars, sugars, and amino acids. They are water-soluble and basic in nature. The mechanism of resistance to aminoglycosides has been closely investigated and derivatives for use active against the resistant bacteria have been developed. The rst nucleoside antibiotic, cordycepin, was isolated in 1950. Various nucleoside antibiotics of unusual structure have subsequently been isolated and are important as antibacterials, antineoplastics, and agricultural chemicals.

Macrolides are macrocyclic lactones to which sugars are attached. Various clinically important antibiotics are macrolides. Erythromycin, isolated in 1952, has two sugars connected to different sites on the 14-membered ring aglycone. Dimethylamino sugars are often found in macrolide antibiotics. Macrolides are classied as 12-, 14-, or 16-membered ring macrolides. Ansamycin has an aliphatic ansa bridge spanning two nonadjacent positions of the aromatic system. Rifamycin is a representative ansamycin possessing a 1,4-naphthoquinone moiety. Polyene antibiotics have a conjugated olenic structure in the macrocyclic lactone moiety, as is the case with amphotericin B, and sometimes lack the amino sugar moiety. Polyenes are produced mainly by Streptomyces and show antifungal activity. Actinomycin is a peptide antibiotic effective against tumor cells as well as bacteria. Peptide antibiotics, one of the major antibiotics groups, possess diverse activities, i.e., antibacterial, antifungal, and antitumor activities. Their structures are varied and feature complicated modes of connection of unusual amino acids as envisaged in bleomycin. Ring peptides, linear peptides, lactonic peptides, and peptides containing hydroxy acids have been isolated. The group includes actinomycins, gramicidins, polymyxins, and colistins. Enediyne antibiotics have a medium-sized ring containing a double bond and two triple bonds that generate carbon radicals to induce DNA damage. Neocarzinostatin was the rst enediyne discovered in 1957. More recently, calicheamicin and esperamicin were isolated. Chloramphenicol was rst isolated as a product of Streptomyces venezuelae; it showed a broad antimicrobial spectrum. Chloramphenicol is an unusual natural product because it possesses both chloro and nitro groups in its structure. It is the only antibiotic produced commercially by an entirely chemical synthesis. In addition to the antibiotics described above, various unclassied antibiotics have been isolated. The most important antibiotics and derivatives are discussed in this article. Some representative compounds that have recently been introduced in clinics are also described.

Antibiotics mally, escaping growth regulation. Antibiotics occupy an important position among the various agents for cancer chemotherapy. Doxorubicin, daunorubicin, mitomycin C, bleomycin, actinomycin D, and neocarzinostatin are used clinically. They interact with DNA to inhibit polymerases of DNA and RNA, or to cause DNA strand breakage. Normal cells are also damaged to some extent; the selective toxicity is generally based on the unusually rapid multiplication of the tumor cells. Some of these antibiotics also possess anti-neoplastic activity because they inhibit the synthesis of DNA.

1.3. Nomenclature
In principle, it is the privilege of the discoverer to name to his or her new antibiotic. Usually antibiotics are named after the producing organisms or some aspect of their chemical and biological nature. In accordance with the suggestions of the Nomenclature Committee of the American Society of Microbiology [31], names of antibiotics should be based on (a) the family to which the antibiotic belongs, (b) the chemical structure of the compound, or (c) some property of the antibiotic. If, for some reason, a name cannot be given to the new antibiotic, a code designation may be given.

2.3. Chemotherapeutic Uses

2. Chemotherapeutic Use of Antibiotics

2.1. Microbial Pathogens
Microorganisms that can be treated by chemotherapy include bacteria, fungi ( Antimycotics), viruses, rickettsia, and protozoa ( Chemotherapeutics). Outside the cytoplasmic membrane, bacteria have a rigid shell called the cell wall that is not seen in mammalian cells. The main constituent of the cell wall is peptidoglycan, a cross-linked structure of long parallel chains of polysaccharides and short peptide chains. Bacteria are separated into two classes based on the results of a Grams stain. Gram-positive bacteria hold the color of the primary stain and gram-negative ones are decolorized and are stained by the counterstain. The structure and constituents of the cell walls of gram-positive and gram-negative bacteria are slightly, but distinctly, different. Some antibiotics are effective only against gram-positive bacteria whereas others are active against gram-negative ones. Streptomycin and kanamycin are effective against mycobacteria as well as gram-positive and gram-negative bacteria. Chloramphenicol and tetracyclines are broad-spectrum antibiotics active against not only the usual bacteria but also rickettsia.

Chemical and bacteriological diagnoses are especially important for successful chemotherapy because the choice of drug depends primarily on the sensitivity of the microorganism to the drug. The antimicrobial activity of an antibiotic is expressed as the minimum inhibitory concentration (MIC) measured by the dilution method. Antibiotics are administered by hypodermic, intramuscular, or intravenous injections, or as internal medicines. For external applications, they are given according to the nature of the antibiotic and the characteristics of the disease. Doses large enough to maintain a sufcient drug concentration in the blood and tissues are prescribed. Various undesirable side effects of antibiotics have been reported. One of the serious side effects is the allergic reaction to penicillins. Oto- and nephrotoxicity result from the long-term use of aminoglycosides in quantity. Aplastic anemia caused by chloramphenicol also has been reported. Antitumor and antiviral antibiotics are generally highly toxic.

2.4. Use in Agriculture

Although antibiotics were originally developed for use against microbial diseases in humans, they are also applicable to agriculture. Several antibiotics are used in the treatment of animal and plant diseases. Kasugamycin and blastcidin S are effective against rice blast disease. Polyoxins are selectively effective against certain

2.2. Tumor Cells

Certain antibiotics are effective for clinical treatment of cancer. Cancer cells function abnor-

Antibiotics species of phytopathogenic fungi. Antibiotics are used for veterinary purpose as therapeutic agents to prevent breeding disease and fodder additives to stimulate growth of animal. However, there is the danger of developing a resistance resulting from the veterinary use of antibiotics. WHO recommended to reduce the use of antibiotics as an additive in animal fodder because it may raise the number of drug-resistant bacteria in animal meat. Therefore, worldwide efforts are directed at avoiding the use of the therapeutically important antibiotics, particularly the penicillins and the tetracyclines, as feed additives. Instead, these antibiotics are used only for genuine veterinary treatment in compliance with certain controls and quarantine guarantees.
1 Mega U = 1106 I. U. = 600 mg benzylpenicillin sodium = ca. 1 g benzylpenicillin procaine = 11012 I. U. = 600 kg benzylpenicillin sodium = ca. 1 t benzylpenicillin procaine

1 Mio Mega U

The activities of some older penicillins are given in I. U.

phenoxymethylpenicillin: phenethicillin: penicillin O: 1 mg free acid 1 mg d-potassium salt 1 mg l-potassium salt 1 mg potassium salt 1699 U 1476 U 1470 U 1612 U

All other penicillins that are used therapeutically can be made very pure and the preparations are dosed and traded in mass units (g, mg, g, kg).

2.5. Units
The production, isolation, and processing of commercial products require careful control for all pharmaceuticals. Because of the extremely high sensitivity and the danger of diminished activity, this consideration has always been particularly important. The Oxford unit (O. U.) is dened as the amount of penicillin that just prevents the growth of a certain Staphylococcus aureus species. Very pure crystalline penicillin salts generally have constant biological activities and the Oxford unit has been replaced by the international unit: 1 mg of pure benzylpenicillin sodium contains 1670 O. U.; the O. U. specic to this salt was declared to be the international unit (I. U., usually abbreviated U). Conversely, 0.6 g of benzylpenicillin sodium has the activity of 1 I. U. Because the biological activity comes from the penicillin nucleus, the change to another cation leads to a change in activity proportional to the molecular mass. This change can be calculated. The activities of the chief penicillin salts are:
benzylpenicillin sodium benzylpenicillin potassium benzylpenicillin procaine penicillin-2-hydroxyprocaine penicillin-N,N -dibenzyl-ethylenediamine penicillin-N-ethylpiperidine 1670 U/mg 1598 U/mg 1011 U/mg 1008 U/mg 1213 U/mg 1328 U/mg.

2.6. Analysis
The practical determination of active substances in penicillins and other antibiotics can be divided among three types of methods [25]: 1) Microbiological testing (see Chap. 7). 2) Determination of the contents by chemical or enzymatic conversion followed by a physical method, such as colorimetry. 3) Purely physical methods, such as UV or IR absorption.

3. Classication of Antibiotics
3.1. -Lactams
The -lactam group includes natural penicillins, semisynthetic penicillins, natural cephalosporins, semisyntheric cephalosporins, cephamycins, 1-oxacephems, -lactamase inhibitors, penems, carbapenems, nocardicins, and monobactams. 3.1.1. Natural Penicillins Penicillin was discovered in 1929 by Fleming [22]. At rst it was obtained as a mixture of several similar compounds, but these were later

The mass of 1 U, for benzylpenicillin sodium, 0.6 g, is extremely small. The following larger units of mass are used in production and trade:

Antibiotics

separated from each other. The -lactam structure of penicillin was proposed by Abraham and Chain and supported by Woodward, but it was opposed by those who believed in the alternative thiazolidine-oxazole structure [33]. The lactam structure was nally established by an X-ray crystallographic analysis performed by Hodgkin and Low [34]. Several penicillins have been isolated from the fermentation broths of Penicillium notatum or P. chrysogenum. Of these, benzylpenicillin (penicillin G) shows good stability, activity, and rate of production by microorganisms. Benzylpenicillin benzathine was developed as a dilatorily acting benzylpenicillin that maintains an effective serum concentration for 2 days after a single intramuscular injection. Total synthesis of penicillin V was achieved by Sheehan and Henery-logan in 1957 [35]. Biogenic syntheses of penicillin cephalosporin antibiotics also have been reported [36, 37].

Ampicillin

[69-53-4]

Amoxicillin

[26787-78-0]

Ciclacillin

[3485-14-1]

Piperacillin

[61447-96-1]

Name

Benzylpenicillin (Penicillin G)

[61-33-6]
Aspoxicillin

[63358-49-6]

Phenoxymethylpenicillin [87-08-1] (Penicillin V)

Mecillinam

[32887-01-7]

Phenethicillin

[147-55-7]

Methicillin

[61-32-5]

Benzylpenicillin benzathine [41372-02-5]

Antibiotics 3.1.2. Semisynthetic Penicillins Several limitations have become apparent concerning the antibiotic activity of benzylpenicillin. This drug is not very active against gramnegative bacteria; it is inactivated by penicillinase produced by resistant organisms, and it is not suitable for oral administration because it breaks down under acidic conditions. Penicillins having variously altered side chains have been made by adding appropriate precursors to the fermentation [33]. Various penicillins have been obtained biosynthetically. Among these is phenoxymethylpenicillin (penicillin V) the rst used by oral administration. In contrast, 6-aminopenicillanic acid (6-APA) can be prepared by either enzymatic or chemical means. Penicillin amidase or penicillin acylase cleaves the side chain of penicillin to produce 6-APA. The amide bond of the side chain is also efciently cleaved by treatment with phosphorus pentachloride [25, p. 27]. Penicillins with modied side chains have been synthesized from 6-APA in order to improve the antibacterial spectra and increase the stability against penicillinase [33, p. 59]. Ampicillin is active against gram-negative bacteria. Ampicillin and amoxicillin are suitable for oral administration. Methicillin was developed as lactamase-resistant penicillin. However, emergence of methicillin-resistant Staphylococcus aureus (MRSA), rst identied in 1961, is a current major problem. Mecillinam has an unusual amidino side chain and is relatively stable and effective against gram-negative bacteria.

Name Talampicillin

R1

R2

[47747-56-8]
Bacampicillin

[50972-17-3]
Lenampicillin

[86273-18-9]
Pivmecillinam

[32886-97-8]

3.1.3. Natural Cephalosporins

6-Aminopenicillanic acid (6-APA) [551-16-6]

Modication of the carboxyl group has been found to be effective for the purpose of oral administration, and penicillin esters talampicillin, bacampicillin, lenampicillin, and pivmecillinam have been developed [33, p. 59]. These are absorbed and hydrolyzed by the small intestine to release free acids of the parent penicillins.

The fermentation broth of Cephalosporium spp. isolated by Brotzu contained several antibiotics: cephalosporin P, penicillin N, and cephalosporin C. Cephalosporin P was shown to be an acidic steroidal substance. Cephalosporin C was active against gram-negative bacteria, resistant to -lactamase, and much less toxic than penicillin. The chemical structure of cephalosporin C was determined by Abraham and

Antibiotics

Name R1 Cephalosporin C

R2

Name Cefmenoxime

R1

R2

[61-24-5] [65085-01-0]
Cefalexin Ceftizoxime

[15686-71-2] [68401-81-0]
Cefaclor Cefotaxime

[53994-73-3] [63527-52-6]
Cefroxadine Cefuroxime

[51762-05-1] [55268-75-2]
Cefadroxil Ceftriaxone

[50370-12-2]
Cefalotin

[73384-59-5]
Cefodizime

[153-61-7] [69739-16-8]

Antibiotics
Name Cefsulodin R1 R2 Name Cefdinir R1 R2

[62587-73-9] [91832-40-5]
Cefatrizine Cexime

[51627-14-6] [79350-37-1]
Cefoperazone Ceftibuten

[97519-39-6] [62893-19-0]
Cefotiam Cefpiramide

[61622-34-2]

[70797-11-4]
Cefazolin

[25953-19-9]

10

Antibiotics Cephalosporin esters are also developed for oral administration. These are cefotiam hexetil, cefteram pivoxil, cefditren pivoxil, cefcapene pivoxil, cefpodoxime proxetil, and cefuroxime axetil.

Newton [25]. It consists of 7-aminocephalosporanic acid (7-ACA) and D- a-aminoadipic acid. Woodward and his co-workers synthesized cephalosporin C in a fully stereospecic manner [38].

Name Structure Cefpirome

7-Aminocephalosporanic acid (7-ACA) [957-68-6]

3.1.4. Semisynthetic Cephalosporins

[84957-29-9]

Because the antibacterial activity of cephalosporin C itself is relatively low, the development of a more active derivative is desirable. The phosphorus pentachloride method has been applied to the cephalosporin system to produce 7-ACA in high yield [25, p. 27]. The 3 -acetoxy group of cephalosporin is easily replaced by various nucleophiles [25, p. 134]. Modication of the 7-amino group and the 3 group make possible the various cephalosporin derivatives [33, p. 59]. Cefazolin is active against gramnegative bacteria. Cefuroxime, cefotaxime, and ceftizoxime have a methoxyimino group and a 2-aminothiazole ring in common and are resistant to -lactamase. Cefoperazone is particularly active against Pseudomonas. All of these are used by injection. On the other hand, several cephalosporins are used only by oral administration. These include cephalexin, cefadroxil, cefaclor, cefroxadine, and cefatrizine. Cephalosporins are classied into four generations according to their antibacterial properties. The rst generation includes cefalexin, cefadroxil, cefotiam, and cefazolin. -Lactamase stability is improved in the second-generation cephalosporins that includes cefaclor, cefuroxime, and cexime. The third generation includes cefsulodin, cefoperazone, cefotaxime, ceftriazone, ceftibuten, and ceftazidime. Frequent use of the third generation resulted in the emergence of MRSA. The fourth generation includes cefpirome and cetime.

Cefepime

[88040-23-7]
Cefopiran

[113359-04-9]
Ceftazidime

[72558-82-8]

Antibiotics
Name Cefotiam hexetil Structure Name Cefcapene pivoxil Structure

11

[95761-91-4]
Cefteram pivoxil Cefpodoxime proxetil

[105889-45-0]

[82547-81-7]
Cefditoren pivoxil Cefuroxime axetil

[87239-81-4]

[117467-28-4] [64544-07-6]

Cephalosporins are also obtainable via the ring expansion reaction of penicillin sulfoxide rst devised by Morin [25, p. 183]. Thus, cephalexin is produced by chemical conversion of phenoxymethylpenicillin or benzylpenicillin.

3.1.5. Cephamycins Substances similar to cephalosporin C were found among the products of various streptomycetes and were characterized by the presence of a 7-methoxy group. They are named cephamycins after their cephalosporin skeleton

12

Antibiotics
Name Cefminox Structure

and their production by streptomycetes [39], [29, vol. 1, p. 199]. They are strongly resistant to -lactamase and effective against gramnegative bacteria and bacteria that have acquired resistance to penicillins and cephalosporins. Semisynthetic cephamycins with improved activities are obtained by chemical transformations. These include cefmetazole, cefotetan, cefminox, and cefbuperazone.
Name Structure Cephamycin C

[84305-41-9]
Cefbuperazone

[34279-51-1]
7-Aminocephamycinoic acid (7-ACMA)

[76610-84-9]

[34279-51-1]
Cefmetazole

[56796-20-4]
Cefotetan

[69712-56-7]

Chemical modication of cephamycins requires special devices for the following reasons. 7-Aminocephamycinoic acid (7-ACMA), which corresponds to the 7-ACA of cephalosporins, is not easily isolated because of its instability. It has methoxy and amino groups on the same carbon atom of the -lactam ring and the elimination of the protonated amino group is quite facile, because of the electron-donating nature of the methoxy group. Moreover, the usual phosphorus pentachloride method cannot be applied to the side chain cleavage of cephamycin C because a strong NP bond is formed by the reaction of the carbamate moiety of cephamycin C with phosphorus pentachloride [40]. Instead, exchange of the -aminoadipoyl side chain for another acyl group is achieved by treating the fully protected cephamycin C with the appropriate acyl chloride in the presence of a neutral acid scavenger. This is followed by the simultaneous removal of the amino protective group and the -aminoadipoyl group [41]. The sidechain transformation is also effected using an acyl chloride and partially hydrated molecular sieves [42] (see top of page 13, scheme 1). Chemical conversion of cephamycin into 7ACMA ester has been reported [43] (see top of page 13, scheme 2).

Antibiotics

13

scheme 1

scheme 2

3.1.6. 1-Oxacephems In addition to the modication of the side chains of natural -lactam antibiotics, totally or partially synthetic nuclear analogs of penicillins and cephalosporins have been explored extensively [28 33, p. 59]. In 1974 Wolfe reported the rst 1-oxacephem derived from penicillin, but its antibacterial activity remains unknown because the amino and carboxy protective groups have not been removed. Racemic 1-oxacephalothin, synthesized by Christensen and his co-workers in the same year, was found to be antibacterially active, suggesting that the sulfur atom is not always necessary for the expression of antibiotic activity. Nagata developed latamoxef which exhibits strong activity against pathogenic anaerobes, such as Bacteroides fragilis, as well as gram-negative bacteria, including Pseudomonas [29, vol. 2, p. 1]. It is completely stable against various -lactamases and has low toxicity. A high plasma-peak level and long duration are maintained. Latamoxef is a nuclear analog of cephamycin; it is produced on an industrial scale by a totally chemical process starting with epipenicillin S-oxide [44, 45]. Flomoxef is another clinically used 1-oxacephem that shows wide ac-

tivity against gram-positive and gram-negative bacteria [46].

Name Structure

1-Oxacephem (Wolfe, 1974)

[54997-17-0]
1-Oxacephalothin

[54214-83-4]
Latamoxef

[64952-97-2]

14
Name Flomoxef

Antibiotics
Structure Name Tazobactam Structure

[89786-04-9] [99665-00-6]
Sultamicillin

3.1.7. -Lactamase Inhibitors In the course of screening the substances inhibiting -lactamase, which is responsible for bacterial resistance to penicillins and cephalosporins, a potent -lactamase inhibitor, clavulanic acid, was isolated from Streptomyces clavuligerus [47]. Clavulanic acid is characteristic for its 1oxadethiapenem ring system and the lack of the side chain at position 6. The antibiotic activity of clavulanic acid is not strong, but it has a broad antibacterial spectrum. Instead, it is effective synergistically when used with -lactamasesensitive penicillins and cephalosporins against -lactamase-producing organisms. Clavulanic acid is used in combination with amoxicillin. Sulbactam is a semisynthetic inhibitor of lactamase having a penam sulfone structure [48]. Combinations of sulbactamampicillin, sulbactamcefoperazone are clinically used. Sultamicillin is an ester-linked chimeric drug comprising sulbactam and ampicillin releasing the two constituents when administered [49]. Tazobactam is a derivative of sulbactam used in combination with piperacillin [50].
Name Clavulanic acid Structure

[76497-13-7]

3.1.8. Penems The penem ring system has not been found in nature; it has been designed articially by WOODWARD [26, p. 167], [28 29, vol. 2, p. 315]. It is widely accepted that the antibacterial activity of -lactam antibiotics is based on their ability to acylate enzymes. In penicillins, the rigid, nonplanar bicyclic system enhances the reactivity of the -lactam ring by diminishing the delocalization of the unshared electron pair of the amide nitrogen onto the adjacent carbonyl group. On the other hand, in cephalosporins, where the -lactam nitrogen is bonded almost planar, the double bond of the six-membered ring interacts with the unshared electrons of the -lactam nitrogen, diminishing the delocalization to the amide carbonyl. Therefore, the -lactam ring of cephalosporins is cleaved easily. Penems combine the two structural elements, the ve-membered ring and the double bond. 6-Acylaminopenem-3-carboxylic acids, 6-unsubstituted penem-3-carboxylic acids, and 6-alkylpenem-3-carboxylic acids have been synthesized. Faropenem is a clinically used penem active against penicillin-resistant pneumonococcus [51].

[58001-44-8]
Sulbactam

[68373-14-8]

Antibiotics
Name Structure 6-Acylaminopenem-3-carboxylic acid Name Structure Thienamycin

15

[159995-64-1]
Penem-3-carboxylic acid Imipenem

6-Alkylpenem-3-carboxylic acid

[64221-86-9]
Cilastatin

Faropenem

[82009-34-5]
Meropenem

[106560-14-9]

3.1.9. Carbapenems Cabapenems are a family of antibiotics having the 1-azabicyclo[3.2.0]hept-2-ene system [27 29, vol. 2, p. 227]. The rst carbapenem antibiotic, thienamycin, was discovered at Merck in 1976 among the fermentation products of Streptomyces cattleya [53]. Antibiotics of this type have been isolated one after another in the search for inhibitors of bacterial cell wall synthesis and -lactamase. Imipenem, a semisynthetic derivative of thienamycin, is the rst carbapenem brought into clinical use [54]. It is used in combination with cilastatin, which is an inhibitor of dehydropeptidase-I that inactivates imipenem. Cilastatin also suppress the nephrotoxicity of imipenem. Meropenem and biapenem are currently developed carbapenems [55, 56]. Panipenem is used with betamipron, which reduces the nephrotoxicity of panipenem by suppressing the transportation into kidney [57].
[96036-03-2]
Biapenem

[12410-24-4]
Panipenem

[87726-17-8]

16

Antibiotics
Name Structure Nocardicin A

Name Structure Betamipron

[3440-28-6]

[39391-39-4]

3.1.10. Monocyclic -Lactams A mutant strain of Escherichia coli showing specic supersensitivity to -lactam antibiotics has been and used to isolate nocardicins from Nocardia uniformis by a screening procedure [58]. The nocardicin structure has been elucidated by spectroscopic analysis and chemical degradation. The noncardicins are monocyclic -lactam antibiotics [27], [29, vol. 2, p. 165], [52, p. 281]. The clinical application of was not successful because of hepatotoxicity. Sulfazecin was isolated in 1981 as a product of Pseudomonas acidophila by screening using organisms highly sensitive to -lactams [59]. The structure was shown to be a monocyclic -lactam. In the same year, another research group independently reported on a series of monocyclic -lactams produced by Agrobacterium, Chromobacterium, and Gluconobacter, including a compound identical to sulfazecin [60]. A name monobactam was proposed for compounds characterized by the 3-acylamino-2-oxoazetidine-1-sulfonic acid group. In monobactams, the -lactam ring presumably is activated by the electronic effect of the sulfonate moiety alone, in contrast to the case of penicillins and cephalosporins. Because the antibacterial activity of sulfazecin is not satisfactory, many derivatives have been synthesized chemically [29, vol. 3, p. 339]. Among them, aztreonam, synthesized from threonine, has been found highly effective [61]. Carumonam is also a -lactamase-resistant nomobactam active against gram-negative bacteria including Pseudomonas [62].

Sulfazecin

[77912-79-9]
Aztreonam

[78110-38-0]
Carumonam

[87638-04-8]

3.2. Tetracyclines
The rst tetracycline aureomycin (chlortetracycline) was was found in the culture broth of Streptomyces aureofaciens by Duggar [63]. The linear four-ring-system skeleton is characteristic of the tetracyclines and has given the whole group its name. The strongly conjugated system of keto and enol groups is of particular signicance for the biological activity.

Antibiotics

17

The tetracyclines are bright yellow compounds, amphoteric, and with the exception of rolitetracycline and similarly constructed derivates insoluble in water at the isoelectric point. Their salts, e.g., hydrochlorides, are soluble in water and can be administered either parenterally or orally, although the low pH of the solution causes some problems in the latter instance. Chlortetracycline (aureomycin) shows a wider range of antibiotic activity compared with the earlier antibiotics, penicillins, and streptomycins. Its activity covers gram-positive and gram-negative bacteria as well as Rickettsia and Chlamydiae. Chlortetracycline has been replaced by other tetracyclines in clinical use. Tetracycline was rst obtained from chlortetracycline by reductive dehalogenation in 1953

[143]. It was also obtained either by fermentation of the chlortetracycline-producing organism, Streptomyces aureofaciens, under conditions of chlorine limitation, or by fermentation of a mutant of the organism lacking the chlorinating enzyme. Tetracycline is more stable than chlortetracycline in aqueous solution. Its antimicrobial activity is the same as that of chlortetracycline and oxytetracycline, but its serum concentration after oral administration is considerably higher and more enduring. Demethylchlortetracycline shows more positive antimicrobial activity in vitro and protective effect in vivo compared with tetracycline [144]. Oxytetracycline has had wide clinical use as a substitute for chlortetracycline, even after the introduction of tetracycline [145].

18

Antibiotics

Doxycycline was synthetic compound showing antibacterial activity four times stronger than tetracycline against a variety of pathogens [146]. Oral absorption of doxycycline is higher than that of tetracycline achieving higher tissue concentration in tissue that is maintained longer. Minocycline was also a synthetic compound active against tetracycline-resistant bacteria showing higher activity compared with tetracycline against a variety of pathogens [147]. Minocycline is widely used by oral administration and by drip infusion for serious infections.

3.4. Aminoglycosides
Waksman discovered the rst useful aminoglycoside, streptomycin, in 1944 [67 71]. After wide use of penicillin, streptomycin, chloramphenicol, and tetracycline, resistant organisms appeared in hospital patients. In 1957, staphylococci and gram-negative organisms resistant to all the known antibiotic drugs caused serious infections; kanamycin was discovered at that time by Umezawa and was introduced clinically. More than 150 naturally occurring aminoglycosides have been isolated from culture ltrates of Streptomyces, Streptoverticillium, Nocardia, Micromonospora, Streptoalloteichus, Dactylosporangium, Saccharopolyspora, and other bacterial strains. Most aminoglycoside antibiotics that are important for chemotherapy contain 1,3- or 1,4diaminocyclitols named actinamine, 2-deoxystreptamine, fortamine, or streptidine. Among these naturally occurring aminoglycosides, kanamycin A, kanamycin B, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin, gentamicins, neomycins (fradiomycins) are clinically used. Hygromycin B and destomycin A are used as animal anthelmintics. Kasugamycin is used for the prevention of plant diseases. Among resistant bacteria of clinical origin, the most important mechanism of resistance to aminoglycoside antibiotics is the inactivation by O-phosphorylation, Onucleotidylation, or N-acetylation of specic sites of the antibiotic. The gene for these enzymes is located on a plasmid. Organisms with resistance resulting from permeability barriers to drugs have been isolated, but ribosomal resistance to aminoglycosides is very rare in organisms isolated clinically. Studies of the enzymatic mechanism of resistance to aminoglycosides have been reviewed [73 76]. Semisynthetic aminoglycosides have been developed based on the enzymatic mechanism of resistance. 3 -Deoxykanamycin A has been synthesized and used to inhibit the growth of resistant strains having aminoglycoside-3 phosphotransferase enzymes. Dibekacin (3 ,4 dideoxykanamycin B), synthesized from kanamycin B, shows a strong activity not only against resistant staphylococci and gram-negative organisms but also against Pseudomonas [72].

3.3. Anthracyclines
Anthracycline antibiotics are structurally glycosylated tetracyclines. The aglycone is characterized by the fused cyclohexanebenzene p-quinonebenzene system having hydroxyl and/or methyl substituents. Although anthracyclines show antibacterial activity, they have not been used as antibiotics because of their relatively high toxicity and strong side effects. The antitumor activity of rhodomycin was discovered by Arcamone et al. in 1961 [64], and various antitumor anthracyclines were subsequently isolated [65]. Daunorubicin and doxorubicin are representative anthracyclines. Daunorubicin was found in 1963 in the mycelium of Streptomyces peucetius and the culture broth of Streptomyces coeruleorubidus, respectively [148]. It was the rst anthracycline antibiotic clinically used for therapy of cancers, especially leukemia. Doxorubicin was found in the culture broth of Streptomyces peucetius var. cesius in 1967 [149]. It shows stronger activity against a variety of tumors and leukemia than daunorubicin, and its clinical application in the therapy of cancer is wider than that of daunorubicin. Aclarubicin was found by Umezawa in the culture broth of Streptomyces galilaeus MA144-M1 in 1975 and evaluated its strong antileukemic activity and low cardiac toxicity [150]. Pirarubicin is a tetrahydropyranyl derivative of doxorubicin. Epirubicin, idarubicin, and amrubicin are currently in clinical use. Anthracyclines exert their effect by intercalating DNA [66].

Antibiotics

19

Name Daunorubicin [20830-81-3] Doxorubicin [23214-92-8] Rhodomycin A [1404-50-8] Rhodomycin B [1404-52-0] Aclarubicin [57576-44-0] Pirarubicin [72496-41-4] Epirubicin [56420-45-2] Idarubicin [20830-81-3] Amrubicin [20830-81-3]

R1 OCH3 OCH3 OH OH OH OCH3 OCH3 H H

R2 H H H H H H H H H

R3 OH OH OH OH H OH OH OH OH

R4 H H B OH CO2 CH3 H H H H

R5 COCH3 COCH2 OH CH2 CH3 CH2 CH3 CH2 CH3 COCH2 OH COCH2 OH COCH3 COCH3

R6 OH OH OH OH OH OH OH OH NH2

R7 A A B B C D D A E

These proved the enzymatic mechanism of resistance. Streptomycin is the rst aminoglycoside antibiotic and the second antibiotic introduced clinically after penicillin, isolated from Streptomyces griseus by Waksman [161]. The early structural studies have been reviewed [77]. The two anomeric congurations were found to be l by application of Hudsons rules of isorotation and NMR spectral analysis. The absolute structure of streptomycin has been conrmed by Xray analysis of its oxime selenate [78]. Streptomycin has been synthesized by oxidation of dihydrostreptomycin [79]. Streptomycin shows strong activity against a wide range of grampositive and gram-negative bacteria including Mycobacterium. Streptomycin is the rst choice

among antituberculotic antibiotics and has been used for the therapy of Spirochaeta and Treponema infections.

Streptomycin [57-92-1]

20

Antibiotics M. echinospora as a mixture of at least 16 structurally related components [154]. The major components used in clinical preparations are gentamicins C1a , C1 , and C2 . Gentamicin shows high activity against a variety of grampositive and gram-negative bacteria, including Pseudomonas aeruginosa, Proteus, and Serratia. It has been widely used for the clinical treatment of serious infections. Gentamicin is used alone or in combination with -lactam antibiotics and is being replaced gradually by the newer, less toxic aminoglycosides. Sisomicin was found in the culture broth of Micromonospora inyoensis in 1970 [164]. The structure and activity of sisomicin are very similar to those of gentamicin C1a , the major component of the gentamicin complex. Sisomicin shows stronger bacterial activity and lower renal and ototoxicity than gentamicin C1a .

Kanamycin was found by Umezawa in the culture broth of Streptomyces kanamyceticus in 1957 [151]. It is produced with other components, kanamycin B (bekanamycin) and C. Kanamycin shows toxicity much lower than that of the earlier aminoglycosides, and strong activity against a wide range of gram-positive and gram-negative bacteria, including Mycobacterium. Kanamycin has been used clinically for treatment of such serious infections as dysentery, salmonellosis, and tuberculosis. Bekanamycin (kanamycin B) is one of the two minor components that have been isolated from the culture ltrate of kanamycin-producing Streptomyces kanamyceticus [152]. It shows the same antibacterial spectrum as kanamycin but with stronger activity. Dibekacin (3 ,4 -dideoxykanamycin B) was the rst drug developed on the basis of the enzymatic mechanism of aminoglycoside resistance. It was synthesized by the removal of the 3 and 4 -hydroxyl groups of bekanamycin [155]. Dibekacin shows excellent activity, as expected, against a variety of bacteria, including kanamycin-resistant strains. It shows higher activity than kanamycin against Pseudomonas aeruginosa, Proteus, and other pathogens. Amikacin was synthesized in 1970 by selective N-acylation of kanamycin [156]. Amikacin was designed based on the mechanisms of bacterial resistance to kanamycin and related compounds in which the 3 -hydroxyl group of the antibiotic is phosphorylated enzymatically. The N-acyl moiety prevents this enzymatic inactivation. Tobramycin was found in 1967 in the culture broth of Streptomyces tenebrarius [153]. Structurally it is closely related to kanamycin, i.e., it is a naturally produced 3 -deoxy derivative of bekanamycin. The 3 -hydroxyl group was found to be the target of enzymatic phosphorylation by resistant bacteria. As expected, tobramycin shows strong activity against resistant bacteria, including Pseudomonas aeruginosa, having this phosphorylating enzyme. Gentamicin was found in 1963 in the culture broths of Micromonospora purpurea and

Kanamycin [59-01-8]

Bekanamycin [4696-76-8]

Dibekacin [34493-98-6]

Antibiotics

21

Amikacin [37517-28-5]

Sisomicin [32385-11-8]

Netilmicin [56391-56-1] Arbekacin [51025-85-5]

Micronomicin [52093-21-7] Isepamicin [58152-03-7]

Tobramycin [32986-56-4]

Netilmicin was synthesized in 1976 by introducing an ethyl group into the 1-amino group of sisomicin [165]. The molecular design was based on the biochemical mechanisms of bacterial resistance to the gentamicinsisomicin antibiotic group. The modication at the 1-amino group is known to prevent adenylation at the 2 hydroxyl group and acetylation at the 3-amino group, and to deter acetylation at the 6 -amino residue. Netilmicin shows almost the same activity against a variety of gram-positive and gram-negative bacteria as sisomicin and strong activity against gentamicin - sisomicin-resistant bacteria. Micronomicin was found in the culture broth of Micromonospora sagamiensis var. nonreducans in 1974 [157]. The methylated amino group at the 6 -position is not subjected to the enzymatic acetylation caused by resistant bacteria, including Pseudomonas aerugi-

Gentamicin C1a [26098-04-4]

22

Antibiotics

nosa. Micronomicin is less toxic than gentamicin to the renal and aural systems. Fradiomycin (Neomycin) is produced by Streptomyces fradiae and by Streptomyces albogriseolus [159]. It consists of two closely related components, fradiomycin B and C, and shows strong activity against a wide range of grampositive and gram-negative bacteria, including Serratia and Pseudomonas aeruginosa. Because of its renal and ototoxicity, it is given by oral or topical application. Because it is not absorbed orally, as are other aminoglycoside antibiotics, it is used orally only for the purpose of suppressing intestinal ora, i.e., in treating dysentery, salmonellosis, and diarrhea in the pediatric eld. Fradiomycin also has been used topically in the treatment of bacterial infections of the eye and skin. Ribostamycin was found in the culture broth of Streptomyces ribosidicus in 1970 [158]. It is structurally related to fradiomycin but lacks the diaminoidose (glucose) moiety substituted on the ribose moiety. Ribostamycin is much less toxic than fradieomycin and shows strong activity against a variety of gram-positive and gramnegative bacteria except Pseudomonas aeruginosa. It is used parenterally for therapy of urinary tract, respiratory tract, surgical, and other infections. Paromomycin was found in the culture broth of Streptomyces rimosus forma paromomycinus and of S. Crestomyceticus in 1959 [160]. Whereas the antibacterial activity is weaker than that of fradiomycin, it is much less toxic. Paromomycin is used by intramuscular injection for therapy of respiratory, urinary, and surgical infections and by oral administration to treat dysentery and salmonellosis.

Ribostamycin C [25546-65-0]

Paromomycin I [7542-37-2]

Spectinomycin was found in the culture broth of Streptomyces spectabilis in 1961 [166]. This antibiotic was intended for use in treating various infections, as are the other aminoglycoside antibiotics, but its activity is insufcient for clinical efcacy except against gonorrhea. Spectinomycin does not develop crossresistance with any other antibiotic and shows low toxicity; it is used by deep intramuscular injection for single-session, bolus-injection therapy of gonorrhea.

Spectinomycin [1695-77-8]

Astromicin (fortimicin A) was found in the culture broth of Micromonospora olivoasterospora in 1976 [167]. It has a unique conformation with an acylated diamino inositol moiety different from other aminoglycoside antibiotics. Astromicin is produced with one major byproduct, fortimicin B, and several minor components.

Fradiomycin B [119-04-0]

Antibiotics

23

Astromycin (Fortimicin A) [55779-06-1]

Hygromycin B [31282-04-9]

Fortimicin B [55779-06-1]

Kasugamycin was found in the culture broth of Streptomyces kasugaensis by Umezawa in 1965 [168]. It has a cyclitol structure and an amidinocarboxylic acid moiety, showing strong activity against phytopathogenic fungi, especially Pericularia oryzae, the pathogen causing rice blast. Kasugamycin also shows activity against Pseudomonas, and its toxicity is very low. Kasugamycin has been used in agriculture to protect rice plants against rice blast and for animal infections.

Destomycin A [14198-35-5]

3.5. Nucleosides
The biological effects associated with metabolic processes and specic enzyme control mechanisms are diverse in naturally occurring nucleosides and their synthetic analogs. Nucleosides exhibit several biological effects, including antibiotic, anticancer, and antiviral activities. They possess antimitotic and immunosuppressive activities and cardiovascular and other effects [80 82]. Moreover, it should be kept in mind that nucleoside analogs can assume other functional roles not as yet recognized, and that further therapeutic applications can be expected in the future. These analogs are obtained predominantly from microbial sources. The nucleoside antibiotics consist of a heterocyclic base and a sugar or a carbocyclic sugar linked by a carbon nitrogen (N-nucleoside) or a carbon carbon bond (C-nucleoside). The nucleoside antibiotics fall somewhat outside the normal eld of antibiotics with respect to their activity spectra and hence to their use. They are important for use against fungi, viruses, and certain types of cancer cells. Some typical nucleoside antibiotics are mentioned here.

Kasugamycin [6980-18-3]

Hygromycin B was found in the culture broth of Streptomyces hygroscopicus in 1953 [163]. It showed activity against a variety of gram-positive and gram-negative bacteria as well as fungi. Hygromycin B has been used for therapy of helminthic infections in swine and poultry. Destomycin A was found in the culture broth of Streptomyces rimofaciens in 1965 [162]. It shows activity against a variety of gram-positive and gram-negative bacteria as well as fungi and, more interestingly, against helminths. Destomycin A has been used to treat helminth infections in swine and poultry.

24

Antibiotics Polyoxins were found in the culture broth of Streptomyces cacaoi var. asoensis in 1965 [87 89]. It consists of several closely related components, polyoxin A to polyoxin O, and shows activity against phytopathogenic fungi by inhibition of cell-wall chitin synthesis. Polyoxin has been used in agriculture against fungal infections, especially Alternaria leaf spot in vegetables and fruits.

3.5.1. N-Nucleosides Bredinin, produced by Eupenicillium brefeldianum, shows marked immunosuppressive activity in mice, interferes with replication of Vaccinia virus in vitro, and inhibits leukemia L 5178 cells and Candida albicans [83]. It is used as an immunosuppressive agent for kidney transplantation, nephrotic syndrome, lupus nephritis, and rheumatoid arthritis. Coformycinis isolated from Nocardia interforma along with formycin. Coformycin shows a synergistic effect with formycin on Yoshida rat sarcoma cells because of its strong inhibition of adenosine deaminase, which inactivates formycin. Coformycin, having a characteristic seven-membered-ring base moiety, is thought to be a typical example of a transition-state analog in the adenosine deaminase reaction [84 86]. Cordycepin, 3 -deoxyadenosine, was one of the rst nucleoside antibiotics isolated from Cordyceps militaris. It inhibits Bacillus subtilis, Mycobacterium tuberculosis, KB cell cultures, and Ehrlich ascites tumor cells.

Polyoxin C [21027-33-8] R = OH

Polyoxin I [12886-33-5]

Polyoxin N [37362-29-1] R = OH Polyoxin O [37362-28-0] R = H

Bredinin [50924-49-7]

Blasticidin S was found in the culture broth of Streptomyces griseochromogenes by Yonehara in 1958 [97]. It is a pyran-containing nucleoside and shows strong activity against phytopathogenic fungi, especially Pericularia oryzae, the pathogen causing rice blast. Blasticidin S has been used to protect rice plants.

Coformycin [11033-22-0] Blasticidin S [2079-00-7]

3.5.2. C-Nucleosides
Cordycepin [73-03-0]

Formycin is isolated from Nocardia interforma and from Streptomyces lavendulae [90,

Antibiotics

25

Name Polyoxin A [19396-03-3]

R1 CH2 OH

R2

R3 OH

Polyoxin B [19396-06-6] Polyoxin D [22976-86-9] Polyoxin E [22976-87-0] Polyoxin F [23116-76-9]

CH2 OH COOH COOH COOH

HO HO HO

OH OH H OH

Polyoxin G [22976-88-1] Polyoxin H [24695-54-3]

CH2 OH CH3

HO

H OH

Polyoxin J [22976-89-2] Polyoxin K [22886-46-0]

CH3 H

HO

OH OH

Polyoxin K [22976-90-5] Polyoxin M [34718-88-2]

H H

HO HO

OH H

91]. The antibiotic is effective against Xanthomonas oryzae and Pellicularia lamentosa. Its activity against Yoshida rat sarcoma cell is enhanced by coformycin. Formycin B inhibits Xanthomonas oryzae and interferes with multiplication of inuenza A virus in the cells of chick chorioallantoic membrane [92]. Showdomycin, isolated from Streptomyces showdoensis, is very active against Streptococcus hemolyticus. It is moderately active against other gram-positive and gram-negative bacteria and also effective against Ehrlich ascites tumor in mice and HeLa cells [93].

Formycin [6742-12-7]

Formycin B [13877-76-4]

26

Antibiotics 3.6.1. 12-Membered Ring Macrolides Methymycin, produced by Streptomyces venezuelae, was rst shown to be a 12membered lactone, comprising the aglycone or methynolide and D-desosamine [100, 101].

Showdomycin [16755-07-0]

3.5.3. Carbocyclic Nucleosides Since the pioneering synthesis of the racemic carbocyclic analog of adenosine by Shealy and Clayton and the subsequent isolation of aristeromycin from Streptomyces citricolor, the interest in this class of compounds has been renewed by the isolation of a new carbocyclic nucleoside, neplanocin A. The latter exhibits remarkable antitumor activity against L 1210 leukemia in mice, and its synthetic analogs are now being studied extensively [94 96].

Methymycin [497-72-3]

3.6.2. 14-Membered Ring Macrolides Erythromycin, the rst of the macrolide antibiotics, was found in the culture broth of Streptomyces erythreus in 1952 [173]. Its chemical structure and synthesis have been studied extensively by Woodward. Erythromycin shows activity against gram-positive bacteria and gramnegative cocci as well as Mycoplasma and Leptospira. Erythromycin esters are used for oral administration. An ester, lactobionate, is used by drip infusion for serious infections. Erythromycin derivatives with a modied 14-membered ring, clarithromycin [170] and roxithromycin [171], and ring-expanded derivative, azithromycin [172], are also in clinical use.

Aristeromycin [19186-33-5]

Neplanocin [72877-50-0]

3.6. Macrolides
Macrolide antibiotics are polyfunctional macrocyclic lactones and the majority of them contain at least one amino sugar, which is the cause of the basicity of the molecules. Neutral macrolides containing only a neutral sugar moiety are also known. These antibiotics have become targets in the aldol strategy of organic synthesis to construct their polyhydroxy functions stereoselectively [98, 99]. The antibiotics are classied as either 12-, 14-, or 16-membered ring macrolides and polyenes.

Erythromycin [114-07-8] R1 = H, R2 = O Clarithromycin [81103-11-9] R1 = CH3 , R2 = O Roxithromycin [80214-83-1] R1 = H, R2 = NOCH2 OCH2 CH2 OCH3

Antibiotics

27

Azithromycin [83905-01-5]

3.6.3. 16-Membered Ring Macrolides Kitasamycin, formerly called leucomycin, was found by Hata in the culture broth of Streptoverticillium kitasatoensis in 1953 [102]. This was the rst macrolide antibiotic with a 16membered lactone constituent. Kitasamycin is a complex of leucomycin A components and the ester forms are used for the treatment of grampositive bacterial and gram-negative coccal infections, as well as infections of Mycoplasma, Spirochaeta, and Treponema by injection or by oral topical administration. Josamycin was found in the culture broth of Streptomyces narboensis var. josamyceticus in 1964 [174]. Its identity with leucomycin A3 was conrmed by structural study. Midecamycin was found in the culture broth of Streptomyces mycarofaciens in 1971 [176]. Under specic culture conditions, it is produced by the organism as a single component. Midecamycin shows almost the same antimicrobial spectrum and activity as those of kitasamycin. Although its serum and urine concentrations are low, it distributes in tissues at high concentration after the oral administration. Rokitamycin is a propanoyl derivative of leukomycin.

Leucomycin A1 [16846-34-7] R1 = H, R2 = COCH2 CH(CH3 )2 , R3 = H Leucomycin A3 (Josamycin) [16846-24-5] R1 = COCH3 , R2 = COCH2 CH(CH3 )2 , R3 = H Leucomycin A4 [18361-46-1] R1 = COCH3 , R2 = COCH2 CH2 CH3 , R3 = H Leucomycin A5 [18361-45-0] R1 = H, R2 = COCH2 CH2 CH3 , R3 = H Leucomycin A6 [18361-48-3] R1 = COCH3 , R2 = COCH2 CH3 , R3 = H Leucomycin A7 [18361-47-2] R1 = H, R2 = COCH2 CH3 , R3 = H Leucomycin A8 [18361-50-7] R1 = COCH3 , R2 = COCH3 , R3 = H Leucomycin A9 [18361-49-4] R1 = H, R2 = COCH3 , R3 = H Leucomycin A13 [78897-52-6] R1 = H, R2 = CO(CH2 )4 CH3 , R3 = H Midecamycin [34547-80-8] R1 = COCH2 CH3 , R2 = COCH2 CH3 , R3 = H Rokitamycin [74014-51-0] R1 = H, R2 = COCH2 CH3 , R3 = COCH2 CH3

Spiramycins were found in the culture broth of Streptomyces ambofaciens in 1954 [103] and their acetates were synthesized in 1965. Spiramycin shows almost the same antimicrobial spectrum and activity as the other macrolides, but its acetate, acetylspiramycin, has much better pharmacokinetic properties and activity in vivo.

Acetylspiramycin II [87111-42-0] R = COCH3 Acetylspiramycin III [112501-15-2] R = COCH2 CH3

Tylosin was found in the culture broth of Streptomyces fradiae in 1959 [104 106]. Its antimicrobial spectrum is the same as that of the other macrolide antibiotics, and its activity against Mycoplasma is the widest and highest of any member of its group. Against gram-positive bacteria it is slightly weaker than erythromycin.

28

Antibiotics Nystatin was found in the mycelium of Streptomyces noursei in 1950 [184]. Nystatin was used orally and topically as the rst clinically applied polyene macrolide with antifungal properties. Nystatin shows activity against Candida and lamentous fungi and is used to treat Candida infections of the mouth, digestive organs, and vagina. The application of nystatin in combination with gentamicin and vancomycin to sterilize the gut in perioperation of bonemarrow transplantation has been developed.

Tylosin shows a very high and long-term tissue concentration when administered subcutaneously. It is used by injection or oral administration to treat Mycoplasma and gram-positive bacterial infections in poultry, swine, and other livestock. Ivermectin was synthesized starting with avermectin, which was found in the culture broth of Streptomyces avermitilis in 1977 [175]. Unlike other antibiotics, its activity is strictly against insects, mites, and animal parasites. Ivermectin has been used against pests, mites, and other parasites in domestic animals and livestock.

Amphotericin B [1397-89-3]

Tylosin [1401-69-0]

Nystatin A1 [34786-70-4]

3.7. Ansamycins
Ivermectin B1a [70161-11-4] R = CH2 CH3 Ivermectin B1b [70209-81-3] R = CH3

3.6.4. Polyenes Amphotericin B was found in the mycelium of Streptomyces nodosus M-4575 by Gold in 1956 [183]. It is produced with another polyene macrolide antibiotic, amphotericin A, and separated by solvent extraction. Amphotericin B shows strong antimycotic activity against a variety of fungi and pathogenic yeasts (Candida) and is used by injection and as a vaginal suppository.

The ansamycins have an aliphatic ansa bridge that connects two nonadjacent positions of an aromatic system [107]. The rifamycins are produced by Nocardia mediterranei [108, 109]. Rifamycin B, rifamycin O, and rifamycin S were found in the fermentation products. Rifamycin B was moderately effective against gram-positive bacteria. The oxidation of rifamycin B gave rifamycin O, which can be hydrolyzed to the more active rifamycin S. The latter can be reduced to rifamycin SV with ascorbic acid. Rifamycin SV is converted to the therapeutically important rifampicin. Rifampicin has strong biolog-

Antibiotics ical activity against gram-positive microorganisms and mycobacteria, particularly Mycobacterium tuberculosis and Mycobacterium leprae [110 112].

29

Rifampicin [13292-46-1]

3.8. Peptides
Rifamycin B [13929-35-6]

Rifamycin O [14487-05-9]

Rifamycin S [13553-79-2]

Various low molecular mass peptides, oligopeptides, and protein-like substances are found among the antibiotics of microbial origin. Peptide antibiotics differ from the proteins and peptides of higher animals and plants in many respects [113]. The following characteristics frequently are found in the peptide antibiotics: 1) Molecular masses of the peptide antibiotics are smaller (in the range of 500 1500) than those of peptide hormones. 2) Peptide antibiotics contain some uncommon amino acids that are not found in proteins and peptide hormones of animal or plant origin. 3) Lipids and other non-amino acid structures are found in many peptide antibiotics. 4) Peptide antibiotics frequently contain d-amino acids. 5) Virtually all of the peptide antibiotics resist hydrolysis by proteolytic enzymes. 6) The antibiotics are often cyclic peptides. 7) Families of closely related peptide antibiotics are frequently produced by the same microorganism. Uncommon amino acid constituents of peptide antibiotics are: Dab = ,-diaminobutyric acid; Orn = ornithine; MeVal = Nmethylvaline; MeGly = N-methylglycine; Sar = sarcosine; MOA = 6-methyloctanoic acid; IOA = isooctanoic acid. The arrow indicates the direction of the amide bond between the amino acids. Thus, the arrow begins where the carbonyl group attaches and ends where the amino group attaches ( CO NH). Gramicidin S was found in the culture broth of Bacillus brevis [118]. It is a basic peptide showing strong activity against gram-positive

Rifamycin SV [6998-60-3]

30

Antibiotics Bacitracin was found as a polypeptide complex in the culture broth of Bacillus subtilis and B. licheniformis in 1945 [178]. It was rst used as a mixture of at least nine bacitracin components. The structure of bacitracin A was determined in 1966 [119].

bacteria and considerable activity against gramnegative cocci and Mycobacterium. Gramicidin S is rather toxic but is not orally absorbed; it is used topically as an ointment or as eye or ear drops in combination with other antibacterial drugs.

Gramicidin S [113-73-5] Bacitracin A [1402-99-9]

Polymyxin B was isolated from Bacillus polymyxa [177]. It was later separated into the major component polymyxin B1 and the minor component polymyxin B2 . Polymyxin B is a basic polypeptide and shows strong activity against gram-negative bacteria, but its activity against gram-positive bacteria, Mycobacterium, and fungi is weak. Because of its toxicity, it is used carefully by intramuscular injection for resistant Pseudomonas aeruginosa infections, e.g., sepsis. Polymyxin B is used orally to sterilize the gut in leukemic patients, intraspinally for meningitis, or topically. Colistin was found in the culture broth of Bacillus polymyxa var. colistinus in 1950 [179]. It is closely related to polymyxin and shows strong activity against gram-negative bacteria including Pseudomonas aeruginosa. Colistin and its methanesulfonic acid derivative have been used to treat urinary tract infections caused by Escherichia coli and P. aeruginosa. They have also been used parenterally to treat dysentery and abdominal infections and topically for ophthalmic and otorhinolaryngological infections.

Vancomycin was found in the culture broth of Streptomyces orientalis [180]. It is a large molecular glycopeptide showing bactericidal activity against gram-positive bacteria and anaerobes. It is used to treat resistant infections of Staphylococcus, to sterilize the gut in the perioperation of bone-marrow transplantation, and in leukemic patients. Recently its efcacy has been demonstrated against pseudomembrane colitis, which is caused by Clostridium difcile. Teicoplanin is used for the treatment of aerobic and anaerobic gram-positive bacteria [181].

Vancomycin [1404-90-6]

Polymyxin B1 [4135-11-9] Polymyxin B2 [34503-87-2] Colistin A [7722-44-3] Colistin B [7239-48-7]

R MOA IOA MOA IOA

X Phe Phe Phe Leu

Y Leu Leu Leu Leu

Z l-Dad l-Dad l-Dad l-Dad

Antibiotics

31

Actinomycin D [50-76-0]

Teicoplanin A21 [91032-34-7] R3 = Teicoplanin A22 [91032-26-7] R3 = Teicoplanin A23 [91032-36-9] R3 = Teicoplanin A24 [91032-37-0] R3 =

Bleomycins constitute a group of glycopeptide antibiotics containing unusual amino acid residues and a disaccharide of uncommon sugars produced by Streptomyces verticillus, effective against squamous cell carcinoma and malignant lymphoma [114]. The complete structure has been elucidated by chemical studies and Xray crystallographic analysis of a biosynthetic intermediate [115, 116]. This structure has been veried by total synthesis. The naturally occurring bleomycins are obtained in copper-chelated form as a mixture of congeners that differ only in the C terminus substituents. Metal-free bleomycin can be prepared by treatment with hydrogen sulde. A mixture of metal-free bleomycin A2 (55 70 %) and B2 (25 32 %) has been used for clinical treatment because the mixture has an effect superior to that of A2 alone on human squamous cell carcinoma. The copper ion in bleomycin is replaced by iron after administration to form a bleomyciniron complex that exerts antitumor activity. More than 300 bleomycin analogs have been prepared by chemical modications or fermentations. Pepleomycin possessing improved properties has been brought into clinical use [117].

Teicoplanin A25 [91032-38-1] R3 = Teicoplanin A31 [93616-27-4] R2 =H

Actinomycins (mixtures of A, B, C, and other components) were rst found by Waksman in 1940 in the culture broth of Streptomyces antibioticus [182]. Actinomycin D was found by the same group in 1954, obtained as a single component from S. parvullus. Its strong activity against Wilms tumor and other cancers has been evaluated.

32

Antibiotics
Ala Ala Pro Thr Ala Thr Val Thr Pro Ser Ser Gly Leu Ser Asp Gly Thr Val Val Lys Val Ala Gly Ala Gly Leu Gln Ala Gly Thr Ala Tyr Asp Val Gly Gln Cys Ala Ser Val Asn Thr Gly Val Leu Trp Asn Ser Val Thr Ala Ala Gly Ser Ala Cys Asx Pro Ala Asn Phe Ser Leu Thr Val Arg Arg Ser Phe Glu Gly Phe Leu Phe Asp Gly Thr Arg Trp Gly Thr Val Asx Cys Thr Thr Ala Ala Cys Gln Val Gly Leu Ser Asp Ala Ala Gly Asp Gly Glu Pro Gly Val Ala Ile Ser Phe Asn Neocarzinostatin [9014-02-2]

Bleomycin A2 [11116-31-7]

Bleomycin B2 [9060-11-1]

Pepleomycin [68247-85-8]

3.9. Enediynes
A class of antitumor antibiotics possessing characteristic enediyne chromophores have been isolated [131]. These include neocarzinostatin [132, 133], calicheamicins [134, 135], esperamicins [136, 137], dynemicin A [138]. Neocarzinostatin was isolated from Streptomyces carzinostaticus Var. F-41 by Ishida in 1957 as a complex of a chromophore and an apoprotein [132, 133]. It showed strong cytotoxicity against sarcoma 180 ascites, tumor cells, and leukemia SN-36. A polymer-conjugated derivative of neocarzinostatin was prepared and administered via the tumor-feeding artery showing increased stability in blood and the immunogenicity was much lower than the parental neocarzinostatin [142]. The role of the apoprotein is to stabilize the enediyne chromophore that has a 9-membered ring with a double bond and two triple bonds.

The anticancer activity of neocarzinostatin is due to its capability to cleave DNA. The DNA damage is initiated by nucleophilic attack at C12 of neocarzinostatin chromophore by a sulfur nucleophile, which leads to the formation of a labile cumulene intermediate that undergoes a facile cycloaromatization [139]. The resulting biradical abstracts hydrogen atom of the deoxyribose of DNA (see top of page 33). Similar biradical formation via the Bergman cyclization of the other enediyne class antiI biotics, calicheamicin 1 , esperamicin A1, and dynemicin A, has been proposed [131]. Mylotarg, a CD33 antibody linked to calicheamicin, is useful in targeting therapy for acute myeloid leukemia [140, 141].

Calicheamicin 1 [108212-75-5]

Antibiotics

33

3.10. Other Important Antibiotics

Cycloserine, d-4-amino-3-isoxazolidine, was isolated from many Streptomyces species [120]. Cycloserine is now produced solely by chemical synthesis and used particularly for tuberculosis of the lungs and for leprosy with p-aminosalicylic acid or isonicotinic acid hydrazide.

Esperamicin A1 [99674-26-7]

Cycloserine [68-41-7]

Griseofulvin is produced by Penicillium griseofulvum, P. janczewskii, and Nigrospora oryzae. It is unique in possessing the spirocarbon moiety [121, 122]. Griseofulvin is very active against fungi, and it is used orally to treat fungal infections of human skin.
Dynemicin A [124412-57-3]

34

Antibiotics Fosfomycin was found in the culture broth of Streptomyces fradiae in 1967 [128]. Its chemical structure is simple and unique among antibiotics in having a CP bond. Fosfomycin shows antibacterial activity against gram-positive and gram-negative organisms, including Pseudomonas aeruginosa and Serratia marcescens, and -lactam-resistant Staphylococcus aureus. It shows no cross-resistance with other classes of antibiotics.

Griseofulvin [126-07-8]

Chloramphenicol, the rst of the so-called broad-spectrum medicinal antibiotics, was originally obtained from Streptomyces venezuelae in 1947 [123]. It is now manufactured by a chemical process. Chloramphenicol is active against rickettsia, chlamydiae, and mycoplasmas, as well as a wide range of gram-positive and gram-negative bacteria. However, use is limited by the risk of bone marrow damage or aplastic anemia at too high or too prolonged application [124].

Fosfomycin [23155-02-4]

Chloramphenicol [56-75-7]

Mitomycins have unique chemical structures in which three different carcinostatic functions aziridine, carbamate, and quinone are arranged around a pyrro[1,2-a]indole nucleus [125]. The rst mitomycins were discovered in 1956 by Hata in a culture ltrate of Streptomyces caespitosus. These compounds, designated mitomycins A and B, show highly potent antibacterial activity and moderate antitumor activity, but they are quite toxic in mice. In 1958, mitomycin C, an extremely valuable antitumor drug, was isolated from Streptomyces caespitosus [126, 127]. In 1960, another mitomycin, porromycin, was isolated from Streptomyces ardus.

Fusidic acid was found in the culture broth of a fungus imperfectus, Fusidium coccineum, in 1962 [129]. It has a steroidal structure but shows no hormonal activity. Fusidic acid shows very strong activity against Staphylococcus aureus and weak activity against other gram-positive bacteria and gram-negative cocci and Mycobacterium. Its clinical use is restricted to staphylococcal infections resistant to other classes of antibiotics.

Fusidic acid [6990-06-3]

4. Antibiotic Resistance
An organism becomes resistant to an antibiotic if it survives continued contact. Antibiotics repress the growth of the sensitive organism in a culture, which results in the survival of naturally resistant organisms. Microbial resistance can be acquired through a spontaneous or induced mutation. Various examples of cross-resistance have been observed and several cross-resistant groups of antibiotics are recognized. Combined use of

Name Mitomycin A [4055-39-4] Mitomycin B [4055-40-7] Mitomycin C [50-07-7] Porromycin [801-52-5]

X OCH3 OCH3 NH2 NH2

Y OCH3 OH OCH3 OCH3

Z H CH3 H CH3

Antibiotics two antibiotics retards the appearance of resistant organisms. Microorganisms develop antibiotic resistance by several mechanisms. Microorganisms inactivate or modify antibiotics. -Lactam antibiotics are inactivated through the production of -lactamases, such as penicillinase or cephalosporinase. Aminoglycosides are inactivated by Ophosphorylation, O-nucleotidylation, and Nacetylation. Semisynthetic -lactams and aminoglycosides have been developed to overcome the resistance. Microorganisms alter the proteins that are target of the antibiotics. Methicillin-resistant Staphyllococcus aureus (MRSA) was rst reported in 1961 in UK and spreaded widely in hospitals. MRSA acquired a gene that encodes an altered penicillin-binding protein to which -lactam antibiotics bind with reduced afnity [130]. Glycopeptide antibiotics vancomycin and teicoplanin are used for the treatment of MRSA.

35

Source of Sample. Worldwide screening endeavors to isolate the individual microorganisms not only from soil samples from different sources, but also from other microbe-containing materials. Samples from unusual sources often show the occurrence of selection and adaptation. For example, thermophilic microorganisms are examined in samples taken from deep caves, the sea bottom, hot springs, or geysers. Examination Technique. There are several factors that determine the conditions under which a certain microorganism not only lives and grows but also efciently produces its antibiotic. These factors include the composition and pH of the culture medium, the additives, the air supply, and the temperature. These factors are also of prime importance for any later industrial fermentation. The isolation and testing of the new antibiotic, rst in vitro and then in animal experiments, and the indisputable proof that the new compound is not identical to one of the numerous known antibiotics are part of the examination technique. Purpose of the Examination. In the early days of antibiotic screening, any organism that showed antibiotic activity was screened, but later denite objectives were set and appropriate examination techniques were developed. The factors mentioned in the previous paragraph narrow the choice to certain bacteria and fungi. Further restrictions are brought about by the selection of organisms used to test the efcacy of the antibiotic. Such specic screening methods are used to nd, e.g., antifungal agents, antibiotics active against cancer or viruses, or antibiotics effective against bacteria resistant to other antibiotics. A general overview of the successes and failures experienced during the search for antibiotics has been presented [185]. Goulden [186] reported that in the United States from 1955 to 1966, about 90 000 synthetic compounds, 20 000 plant extracts, and 120 000 culture solutions of microorganisms were tested against different types of neoplasms. About 1600 substances showed sufcient activity to justify their purication; 31 fermentation products reached the rst clinical test, but only ve of them got as far as the second step. Of these only two products, mithramycin and streptonigrin, are clinically used today. It can be concluded that, starting with

5. Fermentation
Fermentation is considered here from the following points of view: 1) Biological development, which includes screening and selection, mutation, and maintenance of the strain. 2) Process development leading to large-scale manufacture. 3) Improvements in fermentation technology.

5.1. Screening
Technical developments in the production of penicillin have given the eld new momentum and have stimulated the search, not only for more efcient strains, but also for microorganisms that produce completely different antibiotics. This process is called screening because valuable antibiotic producers are separated from the large number of organisms found in nature. The screening process and the expected results are inuenced by several factors.

36

Antibiotics Another technique used to obtain improved strains is mutation. Cultures are exposed systematically to mutagens, such as ultraviolet radiation or specic chemical compounds. The dosage is chosen so that of a very large number of treated individuals only a few survive, and these are genetically altered. The mutants obtained in this way are generally valueless. In a few cases, however, it is possible to separate a single organism that possesses properties, such as increased antibiotic production and strain stability, superior to those of the untreated strain. Penicillin production has been perfected to such an extent that todays industrial strains produce at least 35000 U/mL of culture medium. On a smaller scale, still higher production rates have been obtained. The maintenance of the strain, i.e., the production, choice, testing, and storage of efcient antibiotic producers, plays a very important part in the manufacture of antibiotics. The yield of antibiotic tends to decrease through many successive rounds of selection. This tendency must be monitored using tests in culture plates, which include methods using the agar diffusion test, photocytometry, and tests in shaken erlenmeyer asks or in small fermenters with volumes ranging from 10 to 3000 L.

a limited number of samples, the probability of obtaining a therapeutic agent is extremely low. This also applies to other screening objectives, e.g., the search for antibiotics more effective against tuberculosis, resistant microbes, or fungi and yeasts. In order to realize a denite goal, new test methods had to be introduced. Asteromycin was discovered in the process of introducing new tests against mycoplasmas. A search for antibiotics active against bacteriophages led to the discovery of a strain producing dextrochrysin. Dienomycin was found when testing nucleotides with Woods reagent. The leucopeptines, which are active against phytopathogenic microorganisms, gram-positive bacteria, and mycobacteria, were discovered as a result of their antiplasmin activity. Although all these antibiotics have not been approved for use, they show the importance of new screening methods.

5.2. Selection, Mutation, and Maintenance of Strains

The biological production of antibiotics is carried out predominantly by microorganisms. The discovery and isolation of the microorganism are the rst steps of a long process leading to the production of the antibiotic. A yieldimprovment program, a very time-consuming process, is needed to raise the yield of the strain to an economic level [187]. This is done primarily by developing optimal cultivation conditions, keeping in mind that the deep-tank and submerged methods are the only ones technically applicable. Even so, the concentration of antibiotic in the culture medium is generally not enough to start production. For this reason selection is necessary. A large number of single individuals belonging to a strain are isolated. These are bred, and the antibiotic production in the cultures is quantitatively measured. New individuals with good, average, poor, or even no productivity usually develop. Hence, selection is carried out from generation to generation in an effort to develop a strain with as high an antibiotic productivity as possible, one that produces few interfering byproducts (dyes, toxins, other antibiotics, etc.), and one that remains stable over a long period of time, i.e., one whose antibiotic production does not decline.

5.3. Process Development Leading to Large-Scale Production

After an efcient microorganism has been found in the laboratory, the strain must be brought into large-scale production. This process, known as scaling up, is undertaken in steps and poses tremendous technical problems. Several factors are important in scaling up. Transition to Larger Volumes. Microbial growth, begun in the laboratory on culture plates, transferred subsequently to shaking asks having a maximum volume of one liter, and later to small industrial fermenters having a working volume of 3000 L, must ultimately be carried out in production fermenters having a total capacity of about 150 m3 or more. The main problem involved in this process of scaling up is to modify the fermentation conditions in such a way that the same yields are obtained in the larger fermenters as in the smaller ones.

Antibiotics Changes in the Fermenter Geometry. The structure of the fermenter, its construction, and its dimensions, greatly affect the yield obtained. The height versus the diameter of the fermenter, the stirring and aeration systems, the cooling system (jacket, spiral, or inserted cooling), and the protection of the inlets and outlets of the fermenter against infection (pressure sealing) are all important factors in the fermentation process. Experiments in large-scale fermenters are time consuming and expensive. Critical comparisons of similar experiments conducted by different institutes or companies must take into account the fact that the research laboratories have only a few types of fermenters at their disposal. In addition, almost every researcher handles similar fermentation problems using different strains of a microorganism. Hence, a real comparison cannot be made and only reserved conclusions can be drawn. Variations in the Culture Medium or Nutrient Solution. The culture medium inuences the growth of the microorganism and, independently of this, the amount of antibiotic it produces. The growth media or nutrient solutions required for the prefermentation treatment, which primarily must support the rapid multiplication of the microorganisms as a monoculture, have a different composition than the nutrient solution used during fermentation. For example, in fermentation the carbon source should not be too plentiful. As a result of rapid consumption, nutrients must be resupplied to prevent a nutrient shortage. In the choice and supply of nutrients, factors other than the achievement of an optimal antibiotic production must be considered. A nutrient suitable for improving the yield of an antibiotic may simultaneously hinder its recovery. Only an accurate comparison of the yield with the effort required, from the prefermentation treatment to the nal product, can decide whether an apparently good fermentation raw material is also suitable for production. The addition or removal of certain substances has a direct effect on the antibiotic production. In the manufacture of penicillin, the addition of building blocks or precursors to the fermentation broth causes, depending on the addition, a preferred production of benzylpenicillin (addition of phenylacetic acid) or of phenoxymethylpenicillin (addition of phen-

37

oxyacetic acid). On the other hand, if chloride ions are largely removed from the culture medium, e.g., by pretreatment with silver salts [188, 189], or by ion exchange [190], the production of chlortetracycline is suppressed in favor of tetracycline. Certain inhibitors, e.g., inorganic additives, such as bromides and thiocyanates [191], and a great number of organic compounds, also suppress the production of chlortetracycline. Variation of Other Fermentation Conditions. Strict control must be maintained during fermentation. The temperature, the pH (including the effects of nutrients and additives on the pH), the stirrer speed, the air supply, and the duration of fermentation must be monitored constantly. Control of Fermentation by Means of Additives. A resting surface culture or a simple shaken culture contains a denite nutrient medium, which is required to support the growth of the microorganism. The growing culture eventually slows down and ceases growth, usually because the medium is spent. A submerged fermenter allows the sterile addition of additives during the course of fermentation. In this way important changes can be made. A sterile air supply and its generally continuous distribution are vital to all aerobic microorganisms. Any interruption of the air supply must be avoided and the air must be evenly distributed throughout the fermenter. This is achieved by a mixing and air-distributing system. The air supply is often limited in large fermenters as a result of high viscosity and foaming, which makes the addition of antifoam substances necessary (oils, silicones). Mechanical foam destroyers can also be used but they consume large amounts of energy and are not applicable in production plants. The addition of nutrients, in portions or continuously, permits the supply of nutrients to be adjusted at each stage in the fermentation process. The added nutrients may be organic (e.g., sugar as the carbon source) or inorganic (e.g., ammonia as the nitrogen source). The pH can be adjusted during fermentation by the addition of acid or base. However, experience has shown that the addition of certain nutrients causes a simultaneous change in the pH. Slower adjustment is physiologically preferable

38

Antibiotics as possible, using automatic analyzing instruments for sugar, nitrogen, phosphates, biomass, product, etc. Methods that permit the fully automatic withdrawal of samples during fermentation and the automatic transfer of the samples to different analyzers have been perfected. Scale Down. When a fermentation procedure is carried out for the rst time on an industrial scale, scale-up problems occur. After their start-up problems have been overcome, large fermenters often produce disproportionately larger amounts than the previously used smaller fermenters. It is difcult to explain this phenomenon when the same strain and approximately the same fermentation conditions are used. This leads to the scale-down problem, i.e., the problem of increasing the yield obtained from the smaller fermenter to that of the larger. Solution of the scale-down problem is very important for the further development of a strain. A new, promising mutant or variant developed during a large-scale fermentation must rst be evaluated in a small fermenter. The yield thus obtained must be comparable to the yield obtained using the original production strain, also in a small fermenter. Continuous Fermentation. Continuous fermentation has proved to be feasible in breeding yeast, in the activated-sludge purication of waste water, and in the brewing of beer [192, 196]. Continuous industrial production of antibiotics suffers from several difculties, and it has made little progress in displacing batch methods. 1) It is very difcult to keep the yield constant. The highly efcient strains used today tend to degenerate; i.e., the antibiotic production declines. This process makes the maintenance of the strain very important. 2) Maintaining the sterility of the fermentation environment and the additives is much more difcult than in a batch process. 3) A purely technical problem is the continuous accumulation of culture solutions; this generally necessitates continuous further processing. It therefore becomes necessary to convert a factory normally working only days into one working round-the-clock shifts. 4) The main saving in introducing a continuous process lies in the reduction of the volume of

in this case. The addition of sugar often causes a fall in pH because of carbon dioxide formation; peptides and amino acids cause an increase in pH (via the formation of ammonia or other nitrogen bases). The addition of building blocks, precursors, and inhibitors during the course of fermentation has proved useful, especially for the production of penicillins and tetracyclines. In such cases, a single addition at the beginning of the fermentation procedure leads to a concentration toxic to the fungus, but because of its rapid consumption, the precursor concentration should be maintained at a certain level. Measurement and Control Techniques; Analytical Measurements during Fermentation. The process of fermentation is relatively long, and the antibiotic production is very sensitive to disturbances. Precise analytical measurements and rapid and accurate control mechanisms are therefore required. The monitoring of conditions during the course of fermentation can be divided into direct measurements in or at the fermenter and indirect testing in the laboratory of samples withdrawn at regular intervals and under sterile conditions. The direct measurements are immediate and can sometimes be automated. For example, an electrode could be installed to monitor the foam level and automatically release an antifoam additive as required. The formation of foam is inuenced, within certain limits, by changes in the air supply. Equipment to monitor and control the temperature (by adjusting the amount of cooling water) and the pH of the medium is common. Some other direct measurements are the determination of the weight of the full fermenter, e.g., with the help of a pressure gage (especially at the start of fermentation and at harvesting), the measurement and control of the stirring speed and of the air supply, and the determination of the partial pressures of oxygen and carbon dioxide in the fermenter and their concentrations in the exhaust gas. Computer monitoring is of great help in industrial production because of the large number and size of the fermenters. The results of measurements on indirect samples, which are available after hours or a few days, also are fed into the computer. The tendency today is to analyze numerous fermentation samples, taken at as short intervals

Antibiotics the equipment. However, the extent of space saving is directly proportional to the rate of the reaction. When the time required for fermentation is two weeks or less, then the volume of equipment required for a continuous process is scarcely less than that needed for a batch process. In addition, the construction of the equipment required for a continuous process is more complicated and more expensive. Even in the case of antibiotics produced by fast-growing bacteria, e.g., tyrothricin [197] and gramicidin S [198], reasons 1, 2, and 3, along with higher equipment and factory costs, speak against continuous production.

39

Figure 1 shows a schematic outline of the large-scale production of penicillin, an example of a fermentation process. 5.4.1. Maintenance of the Strain and Production of Inoculum The strain of microorganism is maintained as a pure culture in a microbiological laboratory. The underlying principle is preservation; i.e., a form of the microorganism that is as stable as possible must always be available. The microorganism is stored in a large number of small, separate ampules or vials that are used successively. Cultures of good colonies are constantly restarted so that the strain is never depleted. If the microorganism forms spores, its storage is relatively easy. The spores, a resting form, are dried, usually mixed with sterile soil, and stored in ampules. Spores can be stored for months or years. The application of frozen inoculum, stored in the vegetative state, has advantages. This form is easy to prepare in large amounts and germination is no longer necessary. However, the storage times are limited. The inoculum for penicillin fermentation is produced by placing the spore-containing soil in a sterile agar nutrient medium in Roux bottles and incubating at 24 C. The spores germinate in one or two days (vegetative form). A rich mycelium network is formed, from which new spores develop in a few days. These young, freshly formed spores are removed from the fungal network under sterile conditions and with water or normal saline. They are then transferred to erlenmeyer asks containing a suitable sterile nutrient solution. The suspension of spores is shaken at 24 C, enabling them to undergo multiplication. The inoculum is transferred to another shaken ask and is allowed to grow in nutrient solution until a submerged culture can be started. The next steps (Fig. 1) lead to rapid growth and an increase in volume until nally enough mass of mycelium is obtained to inoculate the production fermenter. Besides breeding the inoculation material, the microbiology laboratory has the equally important task of guaranteeing the maintenance

5.4. Fermentation Technology

The essential prerequisites for the production of antibiotics using either submerged fermentation or other fermentation methods are the same. 1) A strain of microorganism should produce the desired antibiotic in satisfactory amounts and, as far as possible, without unwanted byproducts that are difcult to remove. It strain should be as stable as possible; i.e., the production of antibiotic should not decrease with time. The strain should also be resistant to other microorganisms, phages, etc. 2) Complete industrial facilities, which include laboratories for the preparation of inoculum and for the maintenance of the strain and vessels for the prefermentation treatment and for fermentation, must be available. The vessels must be equipped with devices such as temperature regulators, automatic foam destroyers, and appliances for the addition of nutrients and for the supply of sterile air. In addition, a sufciently large recovery plant and enough storage space for raw materials, fermentation aids, intermediates, and nished products must be available. 3) The fermentation process and its optimal operation, the properties of the antibiotic formed, its isolation, and its efcient purication must be known in detail. 4) Analytical equipment and methods to monitor the operation of the fermentation and recovery processes and to control the raw materials, intermediates, and end products must be available.

40

Antibiotics

Figure 1. Schematic outline of the manufacture of penicillin a) Antifoam substance; b) Steam; c) Precursors for penicillin formation; d) Condensate; e) Air; f) Air lter; g) Air-ow recorder; h) Cooling brine; i) Cooling water; k) Spore culture (lled into l); l) Fungal culture with spores; m) Spore suspension; n) Prefermenter (inoculation culture for o); o) Intermediate fermenter (inoculation culture for p); p) Production fermenter; q) Cooling tank; r) Filtration unit; s) Filtrate container; t) Starting vessel for nutrient solution; u) Pump.

and care of the strain, which insures a steady supply of a microorganism with a constant efciency. If the laboratory limited itself to breeding, storage, and regular reinoculation, the antibiotic activity of the fermentation cultures would very soon decrease because these highly productive strains tend to mutate and degenerate. To avoid a decrease in the antibiotic production in industrial fermentation, efcient strains must be subjected annually to several thousand single selections, and the resultant colonies must be tested. The single strains thereby isolated show considerable differences in their stability, i.e., in their tendency to develop into good or bad producers or even into nonproducers. Only after the minimum number of selections necessary for the maintenance of a strain has been exceeded, do the chances of surpassing the efciency of the original strain increase. Then an improvement in the factory productivity becomes possible.

5.4.2. Treatment Before and During Fermentation The manufacture of antibiotics by means of fermentation is always carried out in closed, sterile vessels constructed of stainless steel or of steel lined with stainless steel. Figure 2 shows a typical construction. The supply of sterile air for fermentation is very important. Foreign organisms are ltered from the air by means of glass wool, a ltering candle, or other methods. The composition of the nutrient solutions must meet the nutritional needs of the microorganism; these needs vary depending on the stage of fermentation. The solutions are produced in separate vessels and are sterilized therein, in the fermenter itself, or in a continuous-ow heat exchanger. This heater is also used to sterilize the additives [195].

Antibiotics

41

only minor additives, e.g., trace element salts, are weighed in the normal way. Balance Studies. The balance of energy and materials in the particular case of benzylpenicillin has been described [199] The manufacture of 100 kg of the sodium salt of penicillin requires 1.2 t carbohydrates, 60 kg animal and vegetable fats, 770 kg cornsteep liquor, 220 kg inorganic compounds (buffer, sources of sulfur and phosphorus), and 100 kg phenylacetic acid as precursors. The amount of product and the distribution of energy are shown in Table 1. The energy requirements for the production of 100 kg of benzylpenicillin sodium are:
Electrical energy: Steam: Fermentation air: Cooling water: 10.8 GJ (mainly for stirring) 4 t (sterilization, sealing) 50 000 m3 (at STP) 900 m3

Figure 2. Schematic outline of a fermenter

The amounts of raw material required may only be transported and stored in silos. Hydraulic transport and weighing with a pressure gage have replaced conventional methods, and
Table 1. Balance of energy and materials Mass, kg

Waste Materials. The accumulation of substantial amounts of fermentation waste materials, such as the fungal mycelium and the culture

Raw materials 2350 Products benzylpenicillin sodium 100 4 2453 Fungal mycelium 825 35 11 744 Remaining substance in culture medium 660 28 8051 Carbon dioxide b 765 33 13 176 Total 35 424 a as heat of formation or combustion. b Heat of combustion released during transition to carbon dioxide (carried off with the spent air or cooling water). Table 2. Fermentation residues used as raw material for further fermentation Fermentation residue Penicillium (from benzylpenicillin) Condition moist mycelium moist mycelium moist mycelium mycelium hydrolyzate mycelium hydrolyzate moist mycelium moist mycelium moist mycelium moist or dry mycelium

Mass fraction, %

Energy distribution a , MJ

Penicillium or other mold mycelia from fermentation Penicillium (from benzylpenicillin) Streptomyces (from tetracyclines) Penicillium, Aspergillus, Rhizopus, or yeast Penicillium, Aspergillus, Actinomyces, Rhizopus, yeasts, and activated sludge (from water treatment plants)

Raw material for subsequent fermentationReference oxytetracycline [200] chlortetracycline [201] streptomycin, vitamin B12 , [202] or riboavin phenoxymethylpenicillin nutrient medium (e.g., for [203] Lactobacillus bidus) calcium gluconate [204] tetracyclines, etc. [205] nisin, in connection with lactic [206] acid fermentation (silage) moenomycin (avophospholipol) [207]

42

Antibiotics the fermentation is interrupted and the culture harvested. Microscopic control of the growth of the microorganism. Sterility tests, i.e., tests for the absence of foreign microorganisms. Measurement and correction of the pH of the culture. Determination of sugar. Figures 3 A and 3 B graphically show two possibilities. Figure 3 A shows the sugar consumption. The amount of sugar consumed, plotted against time, is the difference between the total amount of sugar added and the analytically determined sugar concentration at that particular time. Figure 3 B shows the sugar content of the nutrient solution. Here, the shape of the curve is a measure of the sugar consumption (provided no more sugar is added). Determination of nitrogen in the mycelium and in the culture solution, possibly combined with the addition of a nitrogen-containing nutrient solution.

solution freed of antibiotic, is a real problem. After the removal of organic solutions, e.g., by distillation (stripping), the spent medium must be fed into a biological water treatment plant. Seepage is no longer allowed. The fungal mycelium can be processed or disposed of in several ways. 1) It can be fed to animals, directly or after drying. The proceeds cover only a part of the costs, especially if the mycelium has been dried. Also, the presence of antibiotics in the lter cake must be avoided, and ltering aids, e.g., kieselguhr or activated charcoal, may not be used. 2) It can be incinerated after the addition of liquid fuel; this is a clean but very expensive procedure. 3) It can be disposed of by dumping and humus production along with sludge depositing. This alternative often must be considered, although it entails high transportation costs. 4) It can be recycled. The mycelium can be used, directly or after intermediate processing, as a raw material for further fermentation. Mycelium is usually used as a nutrient for another microorganism. This approach is economically attractive (see Table 2 [200 207]). Control of the Fermentation Process. At regular intervals of several hours, samples of the culture solution are withdrawn through the sample port for analysis. Important data are obtained by means of chemical, physical, and biological tests. The values are plotted and curves that present a good picture of the fermentation process are obtained. The most important analyses are: 1) Determination of the amount of antibiotic. (Biological assay is described on 7.) 2) Determination of the weight of the mycelium as an indication of the growth of the microorganism. After inoculation of the fermenter and an initial slow phase, rapid multiplication occurs. This slows down later and nally almost comes to a standstill. The point in time at which the antibiotic production decreases and falls short of economic viability can be empirically determined. At about this point

3) 4) 5) 6)

7)

6. Isolation and Purication of Antibiotics; Quality Specications

6.1. Isolation
When fermentation is completed, i.e., when a sufciently high amount of antibiotic has accumulated in the culture solution, the antibiotic must be separated from the spent medium. The contents of the fermenter are transferred to a harvesting tank so that the fermenter can be turned immediately to the production of the next batch. The aeration is stopped, the solution cooled if necessary, and recovery of the product is begun as soon as possible. If permitted, a disinfectant, e.g., formaldehyde, is added or heat is applied to prevent further proliferation of the microorganism. The recovery of the antibiotic can be carried out in several different ways, depending not only on its properties, but also on its subsequent processing. Drying Process. Technically speaking, the easiest and cheapest process is to dry the entire culture, the culture ltrate, or the lter cake. Drying is employed on a large scale only in the

Antibiotics

43

Figure 3. A. Penicillin formation with continuous glucose addition after development of the mycelium [208] B. Streptomycin formation by Streptomyces griseus [209]

manufacture of antibiotics used to supplement animal feed, e.g., tetracyclines and moenomycin (avophospholipol, avomycin) or salinomycin. Spray drying is the method most often used. Other methods, such as roller drying (possibly under vacuum) are also used. It is advisable to concentrate the solution, e.g., using a downdraft evaporator, before it is actually dried. In any case, the antibiotic must be resistant to higher temperatures. Because of its very short heating time, the spray-drying method is one of the most gentle procedures. Filtration Followed by Extraction and Precipitation. The mycelium is separated from the liquid medium by passing the entire culture solution through a lter press, using ltering aids if necessary. A rotating lter can also be used e.g., an Oliver lter, which has three zones, intended for suction, washing, and peeling. If the culture

solutions contain small amounts of mycelium, separation can also be carried out in a centrifuge. Extraction is the method used to separate most antibiotics contained in the ltrate. A classic example is the extraction of benzylpenicillin (and phenoxymethylpenicillin) with butyl acetate (Fig. 4). It leads to a 120- to 150-fold enrichment. The penicillins are then precipitated from the extract as salts. Only those organic bases that preferentially form sparingly soluble salts with penicillin G or V but highly soluble salts with other penicillins can be used for precipitation from either water or organic solvents. Tertiary morpholines, N-ethylhexahydropicoline and N-ethylpyrrolidine, besides Nethylpiperidine, can be used for precipitation from butyl acetate, amyl acetate, and similar esters. N-Ethylhexamethyleneimine is used for precipitation from chloroform. The salts thus obtained generally are easily crystallized easily and are quite stable in the dry

44

Antibiotics

Figure 4. Penicillin extraction and the sterile nal stage a) Rotameter; b) Mixer

state. They can be stored until further production steps are carried out to give the product that is used in clinical practice. Filtration and Direct Precipitation. After ltration the aqueous culture ltrate can be subjected to direct precipitation. This method was once important in the isolation of streptomycin as a highly insoluble, colored salt, but this use has long been abandoned. Direct precipitation from the culture ltrate is of interest now because the amount of antibiotic produced by todays highly developed, efcient strains is so large that the traces of antibiotic remaining in the aqueous solution after precipitation are negligible. This method has acquired importance in the isolation of, e.g., tetracycline, which can be precipitated at its isoelectric point (pH 4.8) [210]. Another example is the direct precipitation of 5-hydroxytetracycline using a long-chain quaternary ammonium salt [211]. Filtration Followed by Extraction from the Filter Cake. If the antibiotic is present entirely or almost entirely in the mycelium, its isolation is greatly facilitated by ltration, which causes

a considerable decrease in the volume of material. The moist or dry lter cake bearing the product is extracted with a solvent. The resulting solution is ltered again and processed further [212]. Examples are griseofulvin and moenomycin (avophospholipol, avomycin). Adsorption Methods. Adsorption on activated charcoal following ltration is no longer used industrially. However, this method is used with some success in the developmental stages of new antibiotics. Direct adsorption, e.g., on resins, without prior ltration, is still industrially important for the separation of such basic antibiotics as streptomycin, kanamycin, neomycin, and paromomycin. Filtration of the culture solution, especially because of the slimy substances produced by actinomycetes, is laborious and requireds large amounts of ltering aids. The real breakthrough came when the adsorption material (cation exchanger) was brought into contact with an ascending stream of the culture solution, without prior ltration. In this case the antibiotic molecules leave the solution and attach themselves to the surface of the adsorption material.

Antibiotics

45

6.2. Purication Techniques, Sterile End Products, Ofcial Regulations

Antibiotics are fermentation products and are isolated either as unnished products or as intermediates, generally solid substances of limited stability. They are puried by methods normally employed in organic chemistry, which include chromatography, crystallization, and precipitation. A major requirement is that the antibiotics intended for parenteral administration be free of pyrogens (fever-producing substances) and histamine-like compounds. These unwanted substances can be carried over from the fermentation, but such impurities must not appear in the nal product. Special purication steps, e.g., treatment with elemental chlorine (destruction of pyrogens associated with streptomycin), ltration through activated charcoal, or deep-bed adsorption ltration, must be carried out. Precise tests must conrm the absence of all unwanted substances. In the manufacture, purication, and preparation of antibiotics, special measures also must be taken to prevent penicillin contamination. In the processing of active substances to give pharmaceuticals, a strict spatial separation is necessary to avoid mutual contamination through the air. Even minute amounts of antibiotics, especially penicillin, can lead to sensitization in humans and to the formation of resistant microorganisms. These antibiotics are then ineffective in the treatment of diseases because either the patient is allergic or the pathogens have become resistant. Therefore, during the processing and ltering operations the air supply must be monitored very carefully to insure the protection of the operating personnel and to guarantee that no patient unintentionally receives even traces of penicillin with another drug. Production of Sterile Bulk Drug Substances. If sterile bulk drug substances are required, then aseptic conditions must be maintained from the start of the process. Many antibiotics are chemically unstable and cannot tolerate sterilization by heat or other agents. Generally, the solution is sterilized before the nal crystallization, precipitation, spray drying, or freeze drying. This is done by ltering through

porcelain lters, sintered metal lters, layers of lter paper, or graded-porosity lms. Work is carried out in specially equipped sterile rooms, which are fully air conditioned with practically germ-free air. Air-ltering devices similar to those used for the production of fermentation air are used. The rooms are disinfected using, e.g., gaseous formaldehyde, before work is commenced. The oor is kept damp with a solution of a disinfectant (phenol, quaternary ammonium bases) mixed with glycerol, to control dust. The air pressure in these sterile rooms is higher than atmospheric pressure. This prevents the entrance of unclean air. One enters the sterile rooms only after carefully washing and donning sterile clothes and through an airlock equipped with UV lamps and foot mats soaked in disinfectant. Small objects (tools, etc.) are brought into the sterile room through smaller air locks, in which they are disinfected using intense UV radiation. Containers, e.g., stainless steel cans, are rst washed thoroughly and then sterilized in an autoclave. The autoclave is provided with one door that leads to the unsterile washing room and a second door leading to the sterile room. The different steps in the course of further processing the antibiotics, such as centrifugation, drying, pulverization, sieving, and packaging, are performed in sterile glove boxes, which are provided with sterile air at a slightly elevated pressure and are equipped with UV irradiators. Laminar-Flow Technique. In this technique, only one part of a clean or sterile room is maintained as a clean bench. This area is surrounded by a sterile box provided with a working access. To avoid the penetration of unclean air into this conned clean space, a displacing, turbulence-free air stream is created in the box. A continuous stream of sterile, ltered air enters the box from the top or from one side at a xed speed. This air is distributed uniformly and is then sucked out the opposite side at the same speed. Special devices insure that there is minimum turbulence in the area of contact between the owing, sterile air and the stationary, unclean air. Laminar-ow (LF) units can also be used for work with substances that should not escape to the outside world. In this case the air is recirculated through a lter.

46

Antibiotics regulations, including the GMPs, and FDA inspections. The FDA has published detailed rules in the Federal Regulations [218], especially for the registration of imported products. Ofcials in other countries also demand a detailed description of the manufacture, quality, and safety of any drug they import. In many cases, sales depend on a prerequisite inspection of the factory, similar to the one conducted by the FDA. Canadian ofcials, American military forces purchasers, and the British Department of Health and Social Security all have this requirement.

Clean Packing. Antibiotics packed in bulk, in large containers, and intended for therapeutic use, usually are present in a sterile, highly pure state. Products such as tetracycline hydrochloride, intended for oral administration, must be very pure but not necessarily sterile. Ofcial Quality Requirements, Pharmacopoeias. The production process and the quality of the end product are subjected to rigorous ofcial controls. The requirements have long been stipulated in the pharmacopoeia of each country and generally possess legal authority. The pharmacopoeias European Pharmacopoeia, International Pharmacopoeia, and Compendium Medicamentorum (standard pharmacopoeia for all Comecon states) are each valid in several countries. In the United States, the inuence exerted by the federal government goes beyond the determination of minimum quality standards for drugs. The Food and Drug Administration (FDA), attached to the U.S. Department of Health and Human Services, has published a Code of Federal Regulations (CFR) that is continuously supplemented with new regulations and reissued annually. The demands on the quality of a drug are substantially stricter and more comprehensive than those stipulated in the U.S. Pharmacopeia. In addition, detailed requirements have been established for the production and encapsulation techniques and rooms, the documentation, and the storage of raw materials, additives, intermediates, and end products. A detailed analysis of the starting materials, process controls, and tests of the end products and preparations must also be carried out. When a product not yet approved by the FDA is to be registered, preliminary tests must be conducted as well. Food and Drug Administration ofcials have the right to inspect production plants regularly. Complaints can lead to the temporary closing of the plant and to the recall of certain preparations or particular batches of a preparation. In order to achieve a uniform standard in the production of drugs, the FDA [213] and the WHO [214 217] have elaborated and published fundamental rules that are now internationally called Regulations for Good Manufacturing Practices (GMPs). Drugs manufactured in countries outside the United States but imported into the United States are also subject to FDA

7. Analytical Measurements and Quality Control

The analyses of antibiotics can be divided into two basic groups: 1) Tests during production, usually process surveillance and control. 2) Quality control, practiced as required by the WHO and the FDA. These tests have been routinely conducted by independent laboratories for a long time. The end product, raw materials, and intermediates all are tested. The analytical measurements can be divided into chemical and physical tests, on the one hand, and biological tests, on the other. For the former, general methods used in synthetic organic chemistry are applied, along with some special methods that have been worked out for antibiotics. Fully or partially automatic techniques have been introduced to handle the large number of samples.

7.1. Microbiological Analysis

Biological Assay. Numerous microbiological methods are available to determine the amount of antibiotic present in a sample. Agar Diffusion Test (Cylinder-Plate Method). See [219] for the original method; improvements are described in [220, 221]. A standard method has been published by the FDA [222]. For supplements, see [223] and [224].

Antibiotics Twenty milliliters of nutrient agar is placed in a at-bottomed petri dish. After this solidies, four milliliters of a second nutrient solution, seeded with the test bacteria, is poured evenly onto the rst layer (at 48 C). As soon as the second layer has solidied, six sterile stainless steel cylinders are placed on the agar, preferably using a cylinder-placing machine. To the open cylinders are added equal amounts of a standard penicillin solution containing 2.0, 1.5, 1.0, 0.5, and 0.25 U/mL. Samples of the test antibiotic solution are deposited analogously on other petri dishes. The dishes are incubated at 37 C for 16 18 h. During this time the penicillin diffuses out of the cylinders into the surrounding agar and suppresses the growth of the test organism. Thus, the cylinders are surrounded by clear zones, free of bacteria. The diameter of each zone provides an index of the activity of the penicillin preparation. The mean values obtained from 10 20 standard plates are used to draw a calibration curve, and the biological activity of the test solution in international units is determined using conversion tables. Antibiotic Disk Method. This is a modication of the diffusion test. The method is widely used to determine whether a denite strain or a mixture of different microorganisms is sensitive or resistant to a given antibiotic [225 230]. The pathogen is freshly isolated from patients and used to inoculate a suitable nutrient agar plate. Filter paper disks 6 or 9 mm in diameter are placed on the petri dish before incubation. These disks are impregnated with a solution of the antibiotic. The amount is chosen so that the concentration of active substance present after diffusion into the agar medium corresponds to the level attainable in the patient (blood or tissue level). Test doses of 0.5 to 20 U are normal for a disk test of penicillins G or V. In order to maintain a certain uniformity in the production of the nutrient medium for the disk method, the following directions for the preparation of peptone casein agar have proved useful:
Peptone Pancreatic digested casein Yeast extract Meat extract Dextrose Agar 6.0 g 4.0 g 3.0 g 1.5 g 1.0 g 15.0 g

47

The components are dissolved in 1000 mL of distilled water and the pH of the liquid agar is adjusted to 6.55 after sterilization. If an agar plate thus prepared is incubated, the growth of the microorganisms seeded on the plate can be observed from the turbidity of the agar surface. If the antibiotic on the disk is effective, a clear zone of inhibition forms surrounding the disk (Fig. 5). Table 3 shows the experimental values for the diameters of the zones of inhibition for Staphylococcus aureus ATTC 6538P, using the pH 6.55 nutrient medium described above.

Figure 5. Antibiotic disk test The diameter of the clear zone depends on the test dose of antibiotic: disk P (left) contained 0.5 U, disk *P (right) 20 U benzylpenicillin sodium.
Table 3. Diameter of zones of inhibition for Staphylococcus aureus ATTC 6538 P Concentration Inhibition per disk zone Ampicillin 5 g 26 Chloramphenicol 10 g 20 Lincomycin 2 g 19 Methicillin 10 g 26 Novobiocin 10 g 26 Oxacillin 10 g 30 Penicillin (P) 0.5 I.U. 26 Penicillin (* P) a 20 I.U. >40 10 g 16 Streptomycin b Tetracycline 10 g 27 a Massive dose of penicillin (see Fig. 5); b A pH of 8.0 is required for the optimal evaluation of the substance. Under these conditions the diameter of this zone is also 26 mm. Antibiotic

48

Antibiotics available instruments (scanning analysis systems). By connecting a laboratory calculator (e.g., HP 85) to such an instrument, the amount of antibiotic in a test solution can be calculated using reference standards and printed out directly.

Tube Dilution Method. Three milliliters of a nutrient solution is put into each of a row of tubes. Three milliliters of a penicillin solution with a dilution of 1 : 100 is pipetted into the rst tube. After thorough mixing, three millimeters is removed and added to the next tube. After mixing, three millimeters is removed and added to the third tube, and so on. The tubes contain successively lower concentrations of the drug. If the initial penicillin solution had a 1 : 100 dilution, the rst tube now contains a 1 : 200 dilution, the second a 1 : 400, and so on. Each tube is inoculated with one drop of a day-old staphylococcus culture (test bacteria). After incubation for one day at 37 C, the end point is determined, i.e., the lowest concentration of penicillin that prevents the development of turbidity. If tube 3, for example, is clear but tube 4 is turbid, the end point is calculated by multiplying the starting dilution of 1 : 100 by 23 . Thus the bacteriostatic units are obtained, here 800 Bact. U. These can be converted into international units with the help of the penicillin sensitivity factor. For instance, if this factor is 0.04, then 25 Bact. U = 1 I. U. and the solution contains 800/25 = 32 I. U. [231 234]. Slight turbidity also can be caused by protein precipitation resulting from a change in pH. For this reason, it is advisable to add a pH indicator to the nutrient broth. Changes in the pH are then clearly visible. Phenol red and bromothymol blue generally are used. A critical assessment of the test is given in [223]. The tube dilution method is also used to determine the sensitivity of freshly isolated single strains or mixtures of pathogens to antibiotics. A comparison of the lowest inhibitory concentration of an antibiotic with the serum levels attainable in vivo indicates which antibiotic is most suitable for clinical administration. Automatic Analyses. Automatic sample removal and preparation have speeded up analyses. However, in cases that involve special preparatory steps, such as dissolution, ltration, or extraction, full automation is not yet possible. Many references discussing automatic biotesting, are available [223, 224, 235 240]. The measurement of the clear zones in the agar diffusion test, which formerly was conducted visually, can now be performed objectively and automatically using commercially

7.2. Isotopically Labeled Antibiotics

Antibiotics containing a radioisotope at a denite position in the molecule are very important for scientic studies. Labeled substances can be used to trace: 1) Accumulation in specic tissues, e.g., tumors, for diagnostic purposes. 2) Metabolism of an antibiotic, i.e., tracing the metabolites and cleavage products in animal and human organs or excretions. 3) Determining the location and partial degradation of the antibiotic during further fermentation, processing, and purication steps. Isotopically labeled antibiotics can be manufactured in two ways. Fermentation Production. If appropriate isotopically labeled compounds are added to the culture solution during fermentation, a corresponding amount of labeled antibiotic is produced and can be isolated with the unlabeled antibiotic. This technique also is suitable for following labeled precursors, nutrients, salts, etc., during the fermentation procedure. In this way, insights into the mechanism of formation of the antibiotic in a microorganism or the mechanism of formation in the presence of an isolated enzyme system can be obtained. Benzylpenicillin, phenoxymethylpenicillin, and 6aminopenicillanic acid have been labeled with 14 C and 35 S in this way [210, vol. 4, pp. 266, 296], [241]. Streptomycin has been labeled with 14 C and 3 H [210, vol. 4, p. 349]. Labeling. Acylation of 6-aminopenicillanic acid with a 14 C-labeled acid yields a product labeled in the side chain [242]. A subsequent isotopic exchange within the antibiotic molecule is also possible. However, only the easily removed 1 H atoms can be replaced by 2 H or 3 H.

Antibiotics

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8. Economic Aspects
Antibiotics nd widespread use in human and veterinary medicine. As yet, agricultural usage is low and generally conned to Asia where they are used for antifungal treatment of rice plants, etc. Over 30 kt/a of antibiotics was produced worldwide in 1984. There are six main categories of antibiotics: 1) 2) 3) 4) 5) 6) -Lactam Tetracycline Macrolide Peptide and glycopeptide Aminoglycoside Polyether

At least twenty other commercial antibiotics are not included within these six categories. They belong to a variety of chemical types, i.e., polyene, ansamycin, anthracycline, nucleoside, etc. Over the past ten years, output has grown by approximately 4 % per year, with the most rapid growth in the -lactams, macrolides, and polyethers. On the other hand, tetracyclines have presented a static or declining market, particularly for human therapy. Dollar volume sales have grown correspondingly, with a successful new human antibiotic product being dened as one commanding minimal sales of $ 100 000 000 worldwide. -Lactam sales account for at least half of the total human antibiotic market, which exceeds $ 5 000 000 000. All the categories except the polyethers nd use in human medicine. The most important veterinary antibiotics belong to the tetracycline, macrolide, peptide, and polyether families although some -lactams, aminoglycosides, and other antibiotics also have veterinary markets. Worldwide, there are over seventy primary producers of antibiotics by fermentation. If companies involved in producing semisynthetic penicillins and cephalosporins from purchased parent antibiotics are included, the number is well over one hundred. Some companies specialize in the production of a single antibiotic, but more generally a number of different antibiotics are produced, e.g., benzylpenicillin, phenoxymethylpenicillin, cephalosporin C, oxytetracycline, and streptomycin. Large multinational pharmaceutical companies frequently operate a number of separate antibiotic

fermentation plants in one or more countries. For technical reasons, it may not be possible to produce two different antibiotics in the same plant. United States and European companies are active in all categories of antibiotics; Japanese, Chinese, and Korean producers have tended to specialize in the aminoglycosides, macrolides, anticancer drugs, semisynthetic second- and third-generation -lactams, and agricultural antibiotics. Some old antibiotics, which are no longer protected by patents, are traded in bulk at the prices quoted in the following paragraphs. The bulk products are purchased for use in specialities or conversion into semisynthetic drugs by companies that do not have their own fermentation facilities or whose fermentation capacities are not adequate to supply their growing needs. Bulk antibiotics are also purchased on tender by government agencies, charities, etc., for use in developing countries. There is usually a signicant difference between a bulk price and that of a nished (branded or generic) speciality. In the following paragraphs the estimated worldwide antibiotic output for the year 1985 is listed. The antibiotics are grouped into the six main categories plus other antibiotics. The production gures include antibiotics for human and veterinary applications. The specic compounds are arranged alphabetically, not according to their commercial importance. The bulk prices are quoted only for the antibiotics that are traded; this price is much lower than the price of the nished specialty product. -Lactams. Total output is 10 20 kt/a. There are over 50 producers. The bulk price for benzylpenicillin is 25 30 $/kg. The following compounds are included: ampicillin, amoxycillin, carbenicillin, cefaclor, cefamandole, cefazolin, cefoperazone, cefotaxime, cefoxitin, ceftazidime, cefuroxime, cephadroxil, cephalexin, cephalosporin C, cephalothin, cephamycin C, cephradine, clavulanic acid, cloxacillin, dicloxacillin, ucloxacillin, oxacillin, benzylpenicillin, phenoxymethylpenicillin, piperacillin, and ticarcillin. Tetracyclines. Total output is 5 10 kt/a. There are 30 40 producers. The bulk price for oxytetracycline is 25 30 $/kg. The following compounds are included: chlortetra-

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Antibiotics
2. D. Gottlieb, P. D. Shaw (eds.): Antibiotics, vol. II, Biosynthesis, Springer-Verlag, Berlin Heidelberg New York 1967. 3. J. W. Corcoran, F. E. Hahn (eds.): Antibiotics, vol. III, Mechanism of Action of Antimicrobial and Antitumor Agents, Springer-Verlag, Berlin Heidelberg New York 1975. 4. J. W. Corcoran (ed.): Antibiotics, vol. IV, Biosynthesis, Springer-Verlag, Berlin Heidelberg New York 1981. 5. F. E. Hahn (ed.): Antibiotics, vol. V, part 1, Mechanisms of Action of Antibiotic Agents, Springer-Verlag, Berlin Heidelberg New York 1979. 6. F. E. Hahn (ed.): Antibiotics, vol. V, part 2, Mechanisms of Action of Antieukaryotic and Antiviral Compounds, Springer-Verlag, Berlin Heidelberg New York, 1979. 7. J. S. Glasby: Encyclopedia of Antibiotics, 2nd ed., J. Wiley & Sons, Chichester New York Brisbane Toronto 1979. 8. G. Lancini, F. Parenti: Antibiotics. An Integrated View, Springer-Verlag, New York Heidelberg Berlin 1982. 9. L. P. Garrod, H. P. Lambert, F. OGrady: Antibiotic and Chemotherapy, Churchill Livingstone, Edinburgh London Melbourne New York 1981. 10. E. F. Gale, E. Cundliffe, P. E. Reynolds, M. H. Richmond, M. J. Waring: The Molecular Basis of Antibiotic Action, 2nd ed., J. Wiley & Sons, London New York Sydney Toronto 1981. 11. Kirk-Othmer, 3rd ed., vol. 2, 3. 12. J. B rdy, A. Aszalos, M. Bostian, K. L. e McNitt, CRC Handbook of Antibiotic Compounds, vol. I X, CRC Press Inc., Boca Raton, Florida, 1980. 13. H. Umezawa (ed.): Index of Antibiotics from Actinomycetes, vol. I, II, Japan Scientic Societies Press, Tokyo, University Park Press, Baltimore 1978. 14. T. Korzylski, Z. Kowszyk-Gindifer, W. Kurylowicz: Antibiotics Origin, Nature, and Properties, American Society for Microbiology, Washington D.C., 1978. 15. M. J. Weinstein, G. H. Wagman: Antibiotics Isolation, Separation and Purication, J. Chromatogr. Libr. 15 (1978) . 16. R. Reiner: Antibiotics, Georg Thieme Verlag, Stuttgart 1982. 17. W. Kurylowicz (ed.): Antibiotics (a Critical Review), Polish Medical Publishers, Warsaw 1976.

cycline, democlocycline, doxycycline, methacycline, minocycline, oxytetracycline, and tetracycline. Macrolides. Total output is 3 5 kt/a. There are 20 30 producers. The bulk price for erythromycin base is 100 120 $/kg. The following compounds are included: erythromycin, ivermectin, josamycin, kitasamycin, midecamycin, milbemycin, miocamycin, oleandomycin, spiramycin, and tylosin. Peptides and Glycopeptides. Total output is 2 3 kt/a. There are 10 20 producers. The bulk price for bacitracin is 15 $/kg. The following compounds are included: avoparcin, bacitracin, colistin, enramycin, gramicidin, nisin, polymixin, and thiopeptin. Aminoglycosides. Total output was 1 2 kt/a. There are 20 30 producers. The bulk price for streptomycin is 30 $/kg. The following compounds are included: amikacin, apramycin, dibekacin, dihydrostreptomycin, gentamicin, hygromycin, kanamycin, lincomycin, neomycin, netilmicin, paromomycin, ribostamycin, sagamicin, sisomicin, streptomycin, and tobramycin. Polyethers. Total output is 3 5 kt/a. There are 5 10 producers. The following compounds are included: laidlomycin, lasalocid, maduromycin, moenomycin, monensin, narasin, and salinomycin. Other Antibiotics. Total output was 1 2 kt/a. There are 30 40 producers. The following compounds are included: amphotericin, anticancer (including bleomycin, daunorubicin, doxorubicin, epirubicin, and mitomycin), blasticidin, clindamycin, cycloserine, avomycin, fusidic acid, griseofulvin, novobiocin, nystatin, pimaricin, pleuromutilin, pyrrolnitrin, rifampicin, spectinomycin, vancomycin, viomycin and virginiamycin.

9. References
General References 1. D. Gottlieb, P. D. Shaw (eds.): Antibiotics, vol. I. Mechanism of Action, Springer-Verlag, Berlin Heidelberg New York 1967.

Antibiotics
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