Sulfa Drugs and Beta-Lactam Antibiotics PDF

Sulfa drugs and lactam antibiotics CHMI3427EL Winter 2024 Dr. S. Montaut 1 Introduction Antibiotics are microbial metabolites or synthetic analogues that, in small doses, inhibit the growth and survival of microorganisms without serious toxicity to the host. Selective toxicity is the key concept. S...

Sulfa drugs and lactam antibiotics CHMI3427EL Winter 2024 Dr. S. Montaut 1 Introduction Antibiotics are microbial metabolites or synthetic analogues that, in small doses, inhibit the growth and survival of microorganisms without serious toxicity to the host. Selective toxicity is the key concept. Synthetic substances that are unrelated to natural products but still inhibit or kill microorganisms are referred to as antimicrobial agents. 2 History 1676: discovery of bacteria by van Leeuwenhoek. 19th century: Robert Koch isolation of microorganisms from infected patients, growth on culture media and administration to healthy individuals so as to reproduce in healthy individuals all of the classic symptoms of the same disease. 1877: Louis Pasteur reported in 1877 that when what he termed “common bacteria” were introduced into a pure culture of anthrax bacilli, the bacilli died, and that an injection of deadly anthrax bacillus into a laboratory animal was harmless if “common bacteria” were injected along with it. This did not always work but led to the appreciation of antibiosis, wherein two or more microorganisms competed with one another for survival. 3 1929: observation of a clear zone of inhibition (lysis) in a bacterial colony surrounding a colony of contaminating airborne Penicillium mold by Alexander Fleming in England. 1936: discovery of the sulfonamides in France and German. late 1930s and early 1940s: purification of penicillin by Florey, Chain, Abraham, and Heatley. 1939: discovery of tyrothricin 1941: first successful clinical trial of crude penicillin. 1943: discovery of streptomycin. 1947: discovery of chloramphenicol. 1948: discovery of chlortetracycline. 1949: discovery of neomycin. 1952: discovery of erythromycin. 4 There is an increasing impact of bacterial resistance. Intrinsic resistance to antimicrobial agents (resistance present before exposure to antibiotics) was recognized from the beginning. Some bacteria are immune to treatment from the outset because they do not take up the antibiotic or lack a susceptible target. Starting in the 1940s, however, and encountered with increasing frequency to this day, bacteria that were previously expected to respond were found to be resistant, many bacteria became resistant during the course of chemotherapy, and others were simultaneously resistant to several different antibiotics. The organisms were found to be capable of passing this trait on to other bacteria, even to those belonging to different genera. The spread of this phenomenon is aided by microorganisms’ short generation time (sometimes measured in fractions of an hour) and genetic versatility, as well as by poor antibiotic prescribing and utilization practices. 5 6 General Therapeutic Approach Drug Nomenclature Penicillins are derived from fungi and have names ending in the suffix -cillin, as in the term ampicillin. The cephalosporins are likewise fungal products, although their names mostly begin with the prefix cef- (or sometimes, following the English practice, spelled ceph-). The synthetic fluoroquinolones mostly end in the suffix -floxacin. Most of the remaining antibiotics are produced by fermentation of soil microorganisms belonging to various Streptomyces species. By convention, these have names ending in the suffix -mycin, as in streptomycin. 7 Some prominent antibiotics are produced by fermentation of various soil microbes known as Micromonospora sp. These antibiotics have names ending in -micin (e.g., gentamicin). In earlier times, the terms “broad spectrum” and “narrow spectrum” had specific clinical meaning. The widespread emergence of microbes resistant to single agents and multiple agents has made these terms less meaningful. It is, nonetheless, still valuable to remember that some antimicrobial families have the potential of inhibiting a wide range of bacterial genera belonging to both gram-positive and gram-negative cultures and so are called broad spectrum (such as the tetracyclines). Others inhibit only a few bacterial genera and are termed narrow spectrum (such as the glycopeptides, typified by vancomycin, which are used almost exclusively for a few gram-positive and anaerobic microorganisms). 8 Gram Stain - method for staining bacteria so that they are more readily visible under the microscope Gram-positive microorganisms are stained purple by contact with a methyl violet-iodine process. This is largely a consequence of their lack of an outer membrane and the nature of the thick cell wall surrounding them. Gram-negative microorganisms do not retain the methyl violetiodine stain when washed with alcohol but rather are colored pink when subsequently treated with the red dye safranin. The lipopolysaccharides on their outer membrane apparently are responsible for the staining behavior of gram-negative cells. 9 Since Gram stain is dependent on the outer layers of bacterial cells and this also strongly influences the ability of antimicrobial agents to reach their cellular targets, knowing the Gram staining behavior of infectious bacteria helps one decide which antimicrobial might be effective in therapy. Not all bacteria can be stained by the Gram procedure. These often require special staining processes for visualization. Among the more prominent of these for our purposes are the mycobacteria (the causative agents of tuberculosis, for example). These very waxy cells are called acid-fast and are stained by a carbol fuchsin mixture. 10 The Importance of Identification of the Pathogen Experimentally Based Therapy. The selection of an appropriate antibiotic involves sampling infectious material from a patient before instituting anti-infective chemotherapy, culturing the microorganism on suitable growth media, and identifying its genus and species. The bacterium in question is then grown in the presence of a variety of antibiotics to see which of them will inhibit its growth or survival and what concentrations will be needed to achieve this result. This is expressed in minimum inhibitory concentration (MIC) units. The term MIC refers to the concentration that will inhibit 99% or more of the microbe in question and represents the minimum quantity that must reach the site of the infection in order to be useful (see figure next slide). 11 In the top tubes (viewed from the top), a serially decreasing amount of antimicrobial agent is added to a suitable growth medium inoculated with a microorganism. Following incubation, microbial growth is detected by turbidity. The last concentration that produces no visible growth is scored as the minimum inhibitory concentration (m.i.c.) (1/8). Next a loopful is taken from each tube and placed in fresh medium (bottom row). In tubes where the organisms were killed by the drug there is no resumption of growth. Where the organisms were inhibited but not killed, removal of drug allows for resumption of growth. The last concentration that produces no visible growth under these conditions 12 is scored as the minimum bactericidal concentration (m.b.c.) (1/2). One of the most convenient experimental procedures is that of Kirby and Bauer. With this technique, sterile filter paper disks impregnated with fixed doses of commercially available antibiotics are placed on the seeded Petri dish. The dish is then incubated for a period of time. If the antibiotic is active against the particular strain of bacterium isolated from the patient, a clear zone of inhibition will be seen around the disk. If a given antimicrobial agent is ineffective, the bacterium may even grow right up to the edge of the disk. The diameter of the inhibition zone is directly proportional to the degree of sensitivity of the bacterial strain and the concentration of the antibiotic in question. 13 Currently, a given zone size in millimeters is dictated above which the bacterium is sensitive and below which it is resistant. When the zone size obtained is near this break point (the break point represents the maximum clinically achievable concentration of an anti-infective agent), the drug is regarded as intermediate in sensitivity, and clinical failure can occur. This powerful methodology gives the clinician a choice of possible antibiotics to use. This method is illustrated next slide. The widespread occurrence of resistance of certain strains of bacteria to given antibiotics reinforces the need to perform susceptibility testing. In outpatient practice, the choice of antimicrobial agents is more commonly made empirically. 14 Looking down upon a Petri dish containing solidified nutrient agar to which had been added a suspension of a bacterial species. Next, six filter paper discs containing six different antimicrobials were added followed by overnight incubation. The antimicrobials in discs 1, 4, and 5 were inactive. Of the active agents in discs 2, 3, and 6, antibiotic 2 was much more active, as the microorganism was not able to grow as near this impregnated disc. 15 Bactericidal Versus Bacteriostatic. Almost all antibiotics have the capacity to be bactericidal in vitro; that is, they will kill bacteria if the concentration or dose is sufficiently high. In the laboratory, it is almost always possible to use such doses. Subsequent inoculation of fresh, antibiotic-free media with a culture that has been so treated will not produce growth of the culture because the cells are dead. When such doses are achievable in live patients, such drugs are clinically bactericidal. At somewhat lower concentrations, bacterial multiplication is prevented even though the microorganism remains viable (bacteriostatic action). The smallest concentration that will kill a bacterial colony is the minimum bactericidal concentration. 16 With gentamicin, doubling or quadrupling the dose changes the effect on bacteria from bacteriostatic to bactericidal. Such doses are usually achievable in the clinic, so gentamicin is termed bactericidal. The difference between bactericidal and bacteriostatic doses with tetracycline is approximately 40-fold, and it is not possible to achieve such doses safely in patients, so tetracycline is referred to as bacteriostatic. If a bacteriostatic antibiotic is withdrawn prematurely from a patient, the microorganism can resume growth, and the infection can reestablish itself because the organism is still viable. 17 When a patient is immunocompetent or the infection is not severe, a bacteriostatic concentration will break the fulminating stage of the infection (when bacterial cell numbers are increasing at a logarithmic rate). In immunocompromised patients who are unable to contribute natural defenses to fight their own disease, having the drug kill the bacteria is more important for recovery. Thus, although it is preferred that an antibiotic be bactericidal, bacteriostatic antibiotics are widely used and are usually satisfactory. 18 Microbial Susceptibility Resistance. Resistance is the failure of microorganisms to be killed or inhibited by antimicrobial treatment. Resistance can either be intrinsic (be present before exposure to drug) or acquired (develop subsequent to exposure to a drug). Resistance of bacteria to the toxic effects of antimicrobial agents and to antibiotics develops fairly easily both in the laboratory and in the clinic and is an ever-increasing public health hazard. In the laboratory, resistance is almost always found to be due to an alteration in the biochemistry of the colony so that the molecular target of the antibiotic has become less sensitive, or it can be due to decreased uptake of antibiotic into the cells. This is genomically preserved and passes to the next generation. 19 The altered progeny may be weaker than the wild strain so that they die out if the antibiotic is not present to give them a competitive advantage. In some cases, additional compensatory mutations can occur that restore the vigor of the resistant organisms. Resistance of this type is usually expressed toward other antibiotics with the same mode of action and thus is a familial characteristic. Most tetracyclines show extensive crossresistance with other agents in the tetracycline family. In the clinic, resistance more commonly takes place by resistance (R) factor mechanisms. In this case, enzymes are elaborated that attack the antibiotic and inactivate it. Mutations leading to resistance occur by many mechanisms. 20 They can result from point mutations, insertions, deletions, inversions, duplications, and transpositions of segments of genes or by acquisition of foreign DNA from plasmids, bacteriophages, and transposable genetic elements. The genetic material coding for this form of resistance is often carried on extrachromosomal elements consisting of small circular DNA molecules known as plasmids. A bacterial cell may have many plasmids or none. The plasmid may carry DNA for several different enzymes capable of destroying structurally dissimilar antibiotics. Such plasmid DNA may migrate within the cell from plasmid to plasmid or from plasmid to chromosome by a process known as transposition. 21 Such plasmids may migrate from cell to cell by conjugation (passage through a sexual pilus), transduction (carriage by a virus vector), or transformation (uptake of exogenous DNA from the environment). These mechanisms can convert an antibiotic-sensitive cell to an antibiotic-resistant cell. This can take place many times in a bacterium’s already short generation time. The positive selecting pressure of inadequate levels of an antibiotic favors explosive spread of R-factor resistance. This provides a rationale for conservative but aggressive application of appropriate antimicrobial chemotherapy. 22 Bacterial resistance is generally mediated through one of three mechanisms: - failure of the drug to penetrate into or stay in the cell, - destruction of the drug by defensive enzymes, or - alterations in the cellular target of the drug. In many cases, a resistant microorganism can still be controlled by achievable, although higher, doses than are required to control sensitive populations. 23 Persistence Sensitive bacteria may not all be killed. Survivors are thought to have been resting (not metabolizing) during the drug treatment time and are still viable. These bacteria are still sensitive to the drug even though they survived an otherwise toxic dose. Some bacteria also can aggregate in films. A poorly penetrating antibiotic may not reach the cells lying deep within such a film. Such cells, although intrinsically sensitive, may survive antibiotic treatment. Bacteria living in host cells, living in cysts, or existing as an abscess are also harder to reach by drugs and thus are more difficult to control. 24 Combination Therapy A common example is the use of a β-lactam antibiotic and an aminoglycoside for empiric therapy of overwhelming sepsis of unknown etiology. Both of the antibiotic families applied in this example are bactericidal in readily achievable parenteral doses. The β-lactams inhibit bacterial cell wall formation, and the aminoglycosides interfere with protein biosynthesis and membrane function. Their modes of action are supplementary. One may also often successfully combine two bacteriostatic antibiotics for special purposes, for example, a macrolide and a sulfonamide. 25 This combination is occasionally used for the treatment of an upper respiratory tract infection caused by Haemophilus influenzae because the combination of a protein biosynthesis inhibitor and an inhibitor of DNA biosynthesis results in fewer relapses than the use of either agent alone. 26 Serum Protein Binding It is considered in most instances that the percentage of antibiotic that is protein bound is not available at that moment for the treatment of infections so must be subtracted from the total blood level in order to get the effective blood level. The tightness of the binding is also a consideration. A highly bound but readily released antibiotic will have a comparatively short half-life (the time it takes for the amount of a drug's active substance in your body to reduce by half) and work well for systemic infections. An antibiotic that is not significantly protein bound will normally be rapidly excreted and have a short half-life. Thus, some protein binding of poorly water-soluble agents is normally regarded as helpful. 27 Agricultural Use of Antibiotics Their use for treatment of infections of plants and animals is not to be discouraged so long as drug residues from the treatment do not contaminate foods. In contamination, problems such as penicillin allergy or subsequent infection higher up the food chain by drug-resistant microbes can occur. Animals demonstrably grow more rapidly to marketable size when antibiotics are added to their feed even though the animals have no apparent infection. This is believed to be due in large part to suppression of subclinical infections that would consequently divert protein biosynthesis from muscle and tissue growth into proteins needed to combat the infection. 28 Under appropriate conditions, antibiotic feed supplementation is partly responsible for the comparative wholesomeness and cheapness of our food supplies. This practice has the potential, however, to contaminate the food we consume or to provide reservoirs of drug-resistant enteric microorganisms, so it is imperative that antibiotics used for agriculture be utilized appropriately. 29 Sulfonamides The antibacterial properties of the sulfonamides were discovered in the mid-1930s. Prontosil rubrum, a red dye, was one of a series of dyes examined by Gerhard Domagk of Bayer of Germany in the belief that it might be taken up selectively by certain pathogenic bacteria and not by human cells, in a manner analogous to the way that the Gram stain works, and thus serve as a selective poison to kill these cells. The dye, indeed, proved active in vivo against streptococcal infections in mice. Curiously, it was not active in vitro. Trefouel and others soon showed that the urine of Prontosil rubrum–treated animals was bioactive in vitro. 30 Fractionation led to identification of the active substance as paminobenzenesulfonic acid amide (sulfanilamide), a colorless cleavage product formed by reductive liver metabolism of the administered dye. Today, we would call prontosil rubrum a prodrug. 1 4 The discovery of sulfanilamide’s in vivo antibacterial properties ushered in the modern anti-infective era, and Domagk was awarded a Nobel Prize for Medicine in 1939. 31 Mechanism of Action The sulfonamides are bacteriostatic when administered to humans in achievable doses. They inhibit the enzyme dihydropteroate synthase, an important enzyme needed for the biosynthesis of folic acid derivatives and, ultimately, the thymidine required for DNA. They do this by competing at the active site with paminobenzoic acid (PABA), a normal structural component of folic acid derivatives. PABA is otherwise incorporated into the developing tetrahydrofolic acid molecule by enzyme-catalyzed condensation with 6-hydroxymethyl-7,8-dihydropterin- pyrophosphate to form 7,8-dihydropteroate and pyrophosphate. Thus, sulfonamides may also be classified as antimetabolites (see next slide). 32 Microbial biosynthetic pathway leading to tetrahydrofolic acid synthesis and major site of action () of sulfonamides as well as site of action seen in some bacteria (), resulting in incorporation of sulfanamide as a false metabolite. 33 The antimicrobial efficacy of sulfonamides can be reversed by adding significant quantities of p-aminobenzoic acid into the diet (in some multivitamin preparations and as metabolites of certain local anesthetics) or into the culture medium. Most susceptible bacteria are unable to take up preformed folic acid from their environment and convert it to a tetrahydrofolic acid but, instead, synthesize their own folates de novo. Folates are essential intermediates for the biosynthesis of thymidine without which bacteria cannot multiply. Thus, inhibition of the dihydropteroate synthase is bacteriostatic. Humans are unable to synthesize folates from component parts, lacking the necessary enzymes (including dihydropteroate synthase), and folic acid is supplied to humans in our diet. 34 Sulfonamides consequently have no similarly lethal effect on human cell growth, and the basis for the selective toxicity of sulfonamides is clear. In a few strains of bacteria, sulfonamides are attached to the dihydropteroate diphosphate in place of the p-aminobenzoic acid. The resulting unnatural product is not capable of undergoing the next necessary reaction, condensation with glutamic acid. This false metabolite is also an enzyme inhibitor, and the net result is inability of the bacteria to multiply when the folic acid in their cells is used up, and further nucleic acid biosynthesis becomes impossible. The net result is the same, but the molecular basis of the effect is somewhat different in these strains. Bacteria that are able to take up preformed folic acid into their cells are intrinsically resistant to sulfonamides. 35 Structure-Activity Relationships The basis of the structural resemblance of sulfonamides to with paminobenzoic acid (PABA) is clear. The functional group that differs in the two molecules is the carboxyl of PABA and the sulfonamide moiety of sulfanilamide. The strongly electron-withdrawing character of the aromatic SO2 group makes the N atom to which it is directly attached partially electropositive. This, in turn, increases the acidity of the H atoms attached to the N so that this functional group is slightly acidic (pKa = 10.4). The pKa of the carboxyl group of PABA is approximately 4.9. 36 It was soon found that replacement of one of the NH2 hydrogens by an electron-withdrawing heteroaromatic ring enhanced the acidity of the remaining H and dramatically enhanced potency. With suitable groups in place, the pKa is reduced to the same range as that of with p-aminobenzoic acid itself. Not only did this markedly increase the antibacterial potency of the product, but it also dramatically increased the water solubility under physiologic conditions. 37 The pKa of sulfisoxazole, one of the sulfonamides in present use, is approximately 5.0. The poor water solubility of the earliest sulfonamides led to occasional crystallization in the urine (crystalluria) and resulted in kidney damage because the molecules were un-ionized at urine pH values. 38 It is still recommended to drink increased quantities of water to avoid crystalluria when taking certain sulfonamides, but this form of toxicity is now comparatively uncommon with the more important agents used today because they form sodium salts that are at least partly ionized and hence reasonably water soluble at urinary pH values. They are poorly tolerated on injection, however, because these salts are corrosive to tissues. Structural variation among the clinically useful sulfonamides is restricted primarily to installation of various heterocyclic aromatic substituents on the sulfonamide N. 39 Pharmacokinetics. The orally administered sulfonamides are well absorbed from the gastrointestinal tract, distributed fairly widely, and excreted by the kidney. The drugs are bound to plasma protein (sulfisoxazole 30%-70%, sulfamethoxazole 70%) and, as such, may displace other protein-bound drugs as well as bilirubin. The latter phenomenon disqualifies them for use in late-term pregnancy because they can cause neonatal jaundice. Sulfonamides are partly deactivated by acetylation at N-4 and glucuronidation at N-1 in the liver. Plasmid-mediated resistance development is common, particularly among gram-negative microorganisms and usually takes the form of decreased sensitivity of dihydropteroate synthase or increased production of p-aminobenzoic acid. 40 Therapeutic Applications. Of the thousands of sulfonamides that have been evaluated, only a few are still available and are often used in combination with other agents. The surviving sulfonamides (Table 29.1 next slide) include sulfisoxazole, which is used in combination with erythromycin. It has a comparatively broad antimicrobial spectrum in vitro, especially against gramnegative organisms, but clinical use is generally restricted due to the development of bacterial resistance. Susceptible organisms may include enterobacteriaceae (E. coli, Klebsiella, and Proteus) and Streptococcus pyogenes, Streptococcus pneumoniae, and Haemophilus. Sulfamethoxazole in combination with trimethoprim (antimicrobial agent) is more commonly seen. 41 42 Adverse Effects. - Allergic reactions are the most common (rash, photosensitivity, and drug fever). - Less common problems are kidney and liver damage, hemolytic anemia, and other blood problems. - The most serious adverse effect is the Stevens-Johnson syndrome characterized by sometimes fatal erythema multiforme and ulceration of mucous membranes of the eye, mouth, and urethra. These effects are comparatively rare. 43 Antibiotics: Inhibitors of Bacterial Cell Wall Biosynthesis The Bacterial Cell Wall Bacterial cells are enclosed within a complex and largely rigid cell wall. The main functions of the bacterial cell wall are: (1) to provide a semipermeable barrier interfacing with the environment through which only desirable substances may pass; (2) to provide a sufficiently strong barrier so that the bacterial cell is protected from changes in the osmotic pressure of its environment; and (3) to prevent digestion by host enzymes. 44 The initial units of the cell wall are constructed within the cell, but soon the growing and increasingly complex structure must be extruded. Final assembly takes place outside of the inner membrane. This circumstance makes the enzymes involved in late steps more vulnerable to inhibition because they are at or near the cell surface. 45 Gram-Positive Bacteria. Schematic of some features of the gram-positive bacterial cell wall. 46 On the very outside of the cell is a set of characteristic carbohydrates and proteins that together make up the antigenic determinants that differ from species to species and that also cause adherence to particular target cells. There may also be a lipid-rich capsule surrounding the cell (not shown in the diagram). The next barrier that the wall presents is the peptidoglycan layer. This is a spongy, gel-forming layer consisting of a series of alternating sugars (N-acetylglucosamine and N-acetylmuramic acid) linked (1,4)-β in a long chain (see next slide). To the lactic acid carboxyl moieties of the N-acetylmuramic acid units is attached, through an amide linkage, a series of amino acids of which l-alanyl-d-glutamyl-l-lysyl-d-alanine is typical of Staphylococcus aureus. One notes the d-stereochemistry of the glutamate and the terminal alanine. This feature is presumably important in protecting the peptidoglycan from hydrolysis by host peptidases, particularly in the gastrointestinal tract. 47 Schematic of cell wall cross-linking. Pentaglycyl group replaces terminal D-alanine. 48 The peptidoglycan layer is traversed by complex glycophospholipids called teichoic and teichuronic acids. These are largely responsible for the acid mantle of gram-positive bacteria. Beneath the peptidoglycan layer is the lipoidal cytoplasmic cell membrane in which a number of important protein molecules float in a lipid bilayer. Among these proteins are the βlactam targets, the penicillin binding proteins. These are enzymes that are important in cell wall formation and remodeling. In gram-positive bacteria, the outer layers are relatively ineffective in keeping antibiotics out. 49 The inner membrane and its protein components provide the principal barrier to uptake of antibiotics. The penicillin binding proteins are important in construction and repair of the cell wall. βLactam antibiotics bind to these proteins and kill bacteria by preventing the biosynthesis of a functional cell wall. Various β-lactam antibiotics display different patterns of binding to the penicillin binding proteins. These proteins must alternate in a controlled and systematic way between their active and inert states so that bacterial cells can grow and multiply in an orderly manner. Selective interference by β-lactam antibiotics with their functioning prevents normal growth and repair and creates serious problems for bacteria, particularly young cells needing to grow and mature cells needing to repair damage or to divide. 50 Gram-Negative Bacteria. Schematic of some features of the gram-negative bacterial cell wall. 51 These cells usually contain an additional outer lipid membrane that differs considerably from the inner membrane. The outer layer contains complex lipopolysaccharides that encode antigenic responses, cause septic shock, provide the serotype, and influence morphology. This exterior layer also contains a number of enzymes and exclusionary proteins. Important among these are the porins. These are transmembranal supermolecules made up of two or three monomeric proteins. The center of this array is a transmembranal pore of various dimensions. Some allow many kinds of small molecules to pass, and others contain specific receptors that allow only certain molecules to come in. The size, shape, and lipophilicity of drugs are important considerations controlling porin passage. 52 Antibiotics have greater difficulty in penetrating into gram-negative bacterial cells as a consequence. Next comes a periplasmic space containing a somewhat less impressive and thinner, as compared to gram-positive organisms, layer of peptidoglycan. Also present is a phospholipid-rich cytoplasmic membrane in which floats a series of characteristic proteins with various functions. The β-lactam targets (penicillin binding proteins) are found here. Other inner membrane proteins are involved in transport, energy, and biosynthesis. In many such cells, there are proteins that actively pump out antibiotics and other substances at the expense of energy and that may require the simultaneous entrance of oppositely charged materials to maintain an electrostatic balance. 53 β-Lactam Antibiotics A β-lactam is a cyclic amide with four atoms in its ring. The contemporary name for this ring system is azetidinone. This ring proved to be the main component of the pharmacophore, so the term possesses medicinal as well as chemical significance. The penicillin subclass of β-lactam antibiotics is characterized by the presence of a substituted five-membered thiazolidine ring fused to the β-lactam ring. 54 This fusion and the chirality of the β-lactam ring result in the molecule roughly possessing a “V” shape. This drastically interferes with the planarity of the lactam bond and inhibits resonance of the lactam nitrogen with its carbonyl group. Consequently, the β-lactam ring is much more reactive and thus more sensitive to nucleophilic attack when compared with normal planar amides. The earliest penicillins were produced by fungi from media constituents. 55 The cephalosporins were discovered as secondary metabolites of a different fungal species. Because it was stable to many activity-destroying β-lactamases, its core nucleus, 7- aminocephalosporanic acid was substituted with a wide variety of unnatural side chains, and three generations of clinically useful analogues have resulted. Later work produced the carbapenems, monobactams, and β-lactamase inhibitors. Many thousands of these compounds have been prepared by partial or total chemical synthesis, and a significant number of these remain on the market many years after their discovery. 56 Penicillins. There are 3 asymmetric centers. This absolute stereochemistry must be preserved for useful antibiotic activity. 57 Preparation of Penicillins. The original fermentation-derived penicillins were produced by growth of the fungus Penicillium chrysogenum on complex solid media with the result that they were mixtures differing from one another in the identity of the side chain moiety. When a sufficient supply of phenylacetic acid is present in liquid media, this is preferentially incorporated into the molecule to produce mainly benzylpenicillin (penicillin G in the old nomenclature). Use of phenoxyacetic acid instead leads to phenoxymethylpenicillin (penicillin V). More than two dozen different penicillins have been made in this way, but these two are the only ones that remain in clinical use. 58 The sodium and potassium salts of penicillins are crystalline, hydroscopic, and water soluble. They can be employed orally or parentally. When dry, they are stable for long periods, but they hydrolyze rapidly when in solution. Their best stability is noted at pH values between 5.5 and 8, especially at pH 6.0-7.2. The procaine and benzathine salts of benzylpenicillin, on the other hand, are water insoluble. Because they dissolve slowly, they are used for repository purposes following injection when long-term blood levels are required. 59 Nomenclature. Ring and numbering systems of clinically available β-lactam antibiotic types. 60 Chemical Instabilities. The most unstable bond in the penicillin molecule is the highly strained and reactive β-lactam amide bond. This bond cleaves moderately slowly in water unless heated, but breaks down much more rapidly in alkaline solutions to produce penicilloic acid, which readily decarboxylates to produce penilloic acid (see next slide). Penicilloic acid has a negligible tendency to reclose to the corresponding penicillin, so this reaction is essentially irreversible under physiologic conditions. Because the β-lactam ring is an essential portion of the pharmacophore, its hydrolysis deactivates the antibiotic. A fairly significant degree of hydrolysis also takes place in the liver. The bacterial enzyme, βlactamase, catalyzes this reaction also and is a principal cause of bacterial resistance in the clinic. 61 Instability of β-lactams to nucleophiles. 62 Alcohols and amines bring about the same cleavage reaction, but the products are the corresponding esters and amides. These products are inactive. A reaction with a specific primary amino group of aminoglycoside antibiotics is of clinical relevance as it inactivates penicillins and cephalosporins. When proteins serve as the nucleophiles in this reaction, the antigenic conjugates that cause many penicillin allergies are produced. Small molecules that are not inherently antigenic but react with proteins to produce antigens in this manner are called haptens. 63 Commercially available penicillin salts may be contaminated with small amounts of these antigenic penicilloyl proteins derived from reaction with proteins encountered in their fermentative production or by high-molecular-weight self-condensation–derived polymers resulting when penicillins are concentrated and react with themselves. Both of these classes of impurities are antigenic and may sensitize some patients. In acidic solutions, the hydrolysis of penicillins is complex. The main end products of the acidic degradation are penicillamine, penilloic acid, and penilloaldehyde (see next slide). 64 Instability of penicillins in acid. Hydrolysis involves the C-6 side chain. 65 The intermediate penicillenic acid is highly unstable and undergoes subsequent hydrolysis to the corresponding penicilloic acid. An alternate pathway involves sulfur ejection to a product that in turn fragments to liberate penicilloic acid also. Penicilloic acid readily decarboxylates to penilloic acid. The latter hydrolyzes to produce penilloaldehyde and penicillamine. Several related fragmentations to a variety of other products take place. None of these products has antibacterial activity. At gastric pH (∼2.0) and a temperature of 37°C, benzylpenicillin has a half-life measured in minutes. The less water-soluble amine salts are more stable. 66 Structure-Activity Relationship. The substitution of a side chain R group on the primary amine of 6-aminopenicillanic acid with an electron-withdrawing group decreases the electron density on the side chain carbonyl and protects these penicillins in part from acid degradation. This property has clinical implications because these compounds survive passage through the stomach better and many can be given orally for systemic purposes. The survival of passage and degree of absorption under fasting conditions are shown in next slide. 67 68 In vitro degradation reactions of penicillins can be retarded by keeping the pH of solutions between 6.0 and 6.8 and by refrigerating them. Metal ions such as mercury, zinc, and copper catalyze the degradation of penicillins so they should be kept from contact with penicillin solutions. The lids of containers used today are commonly made from inert materials in part to minimize such problems. 69 The more lipophilic the side chain of a penicillin, the more serum protein bound the antibiotic (Table 29.6). 70 Stability of the penicillins toward β-lactamase is influenced by the bulk in the acyl group attached to the primary amine. βLactamases are much less tolerant to the presence of steric hindrance near the side chain amide bond than are the penicillin binding proteins. The stability of methicillin to β-lactamases is an example of this. When the aromatic ring is attached directly to the side chain carbonyl and both ortho positions are substituted by methoxy groups, β-lactamase stability results (Fig. below). β-lactamase–resistant/–sensitive structural features. 71 Movement of 1 of the methoxy groups to the para position or replacing one of them with a hydrogen results in an analogue sensitive to β-lactamases. Putting in a methylene between the aromatic ring and 6-aminopenicillic acid likewise produces a βlactamase–sensitive agent (Fig. below). These findings provide strong support for the hypothesis that its resistance to enzyme degradation is based on differential steric hindrance. Prime examples of this effect are seen in nafcillin and dicloxacillin (Table 29.4 slide 57). β-lactamase–resistant/–sensitive structural features. 72 Mechanism of Action. The molecular mode of action of the βlactam antibiotics is a selective and irreversible inhibition of the enzymes processing the developing peptidoglycan layer. Cell wall cross-linking and mechanism of action of β-lactams. Path A represents the normal cross-linking mechanism. Path B illustrates the reaction of β-lactam 73 antibiotics with the penicillin-binding protein (PBP). Path A. Just before cross-linking occurs, the peptide pendant from the lactate carboxyl of a muramic acid unit terminates in a d- alanyl-d-alanine unit. The terminal d-alanine unit is exchanged for a glycine unit on an adjacent strand in a reaction catalyzed by a cell wall transamidase. This enzyme is one of the penicillin binding proteins (carboxypeptidases, endopeptidases, and transpeptidases) that normally reside in the bacterial inner membrane and perform construction, repair, and housekeeping functions, maintaining cell wall integrity and playing a vital role in cell growth and division. They differ significantly between species and even individual strains, and this fact is used to rationalize different potency and morphologic outcomes following β-lactam attack on the different bacteria. 74 The cell wall transamidase uses a serine hydroxyl group to attack the penultimate d-alanyl unit forming a covalent ester bond, and the terminal d-alanine, which is released by this action, diffuses away. The enzyme-peptidoglycan ester bond is attacked by the free amino end of a pentaglycyl unit of an adjacent strand, regenerating the transpeptidase’s active site for further catalytic action and producing a new amide bond, which connects two adjacent strands together. Path B. The penicillins and the other β-lactam antibiotics have a structure that closely resembles that of acylated d-alanyl-dalanine. The enzyme mistakenly accepts the penicillin as though it were its normal substrate. 75 The highly strained β-lactam ring is much more reactive than a normal amide moiety, particularly when fused into the appropriate bicyclic system. The intermediate acyl-enzyme complex is rather different structurally from the normal intermediate in that the hydrolysis does not break penicillin into two pieces as it does with its normal substrate. In the penicillins, a heterocyclic residue is still covalently bonded and cannot diffuse away as the natural terminal d-alanine unit does. This presents a steric barrier to approach by the nearby pentaglycyl unit and thus keeps the enzyme’s active site from being regenerated and the cell wall precursors from being cross-linked. The result is a defective cell wall and an inactivated enzyme. 76 The relief of strain that is obtained on enzymatic β-lactam bond cleavage is so pronounced that there is virtually no tendency for the reaction to reverse. Water is also an insufficiently effective nucleophile and cannot hydrolyze the complex either. Thus, the cell wall transamidase is stoichiometrically inactivated. The gaps in the cell wall produced by this covalent interruption are not filled in because the enzyme is now inactivated. The resulting cell wall is structurally weak and subject to osmotic stress. Cell lysis can result, and the cell rapidly dies assisted by another class of bacterial enzymes, the autolysins. 77 Resistance. Resistance to β-lactam antibiotics is increasingly common and is rather alarming. It can be intrinsic and involve decreased cellular uptake of drug, or involve lower binding affinity to the protein binding proteins. This is particularly the case with methicillin-resistant Staphylococcus aureus (MRSA). MRSA produces a mutated PBP-2 (PBP-2a) that does not efficiently bind methicillin any longer. More common, is the production of β-lactamases. β-Lactamases are enzymes (usually serine proteases) elaborated by microorganisms that catalyze hydrolysis of the β-lactam bond and inactivate β-lactam antibiotics to penicilloic acids before they can reach the protein binding proteins (se next slide). 78 β-lactamase–catalyzed hydrolysis of penicillins. 79 β-Lactamases resemble the cell wall transamidase, which is the usual target. Hydrolytic regeneration of the active site is dramatically more facile with β-lactamases than is the case with cell wall transamidase, so that the enzyme can turn over many times and a comparatively small amount of enzyme can destroy a large amount of drug. With gram-positive bacteria, such as staphylococci, the βlactamases are usually shed continuously into the medium and meet the drug outside the cell wall (see Fig. slide 46). They are biosynthesized in significant quantities. 80 With gram-negative bacteria, the β-lactamases are secreted into the periplasmic space between the inner and outer membrane so, while still distal to the protein binding proteins, they do not readily escape into the medium and need not be resynthesized as often (see figure slide 51). Allergenicity. Most commonly, the allergy is expressed as a mild drug rash or itching and is of delayed onset. Occasionally, the reaction is immediate and profound. It may include cardiovascular collapse and shock and can result in death. 81 Benzylpenicillin Group. Benzylpenicillin (Penicillin G, Table 29.4 slide 57). With the exception of Neisseria gonorrhoeae and Haemophilus influenzae, and a few bacteria encountered less frequently, the useful antimicrobial spectrum of benzylpenicillin is primarily against gram-positive cocci. Because of its cheapness, efficacy, and lack of toxicity (except for acutely allergic patients), benzylpenicillin remains a remarkably useful agent for treatment of diseases caused by susceptible microorganisms. 82 Phenoxymethylpenicillin (Penicillin V, Table 29.4). Penicillin V is produced by fermentation where the medium is enriched in phenoxyacetic acid. It can also be prepared by semisynthesis. It is considerably more acid stable than benzylpenicillin as indicated by oral absorption (Table 29.5). This is rationalized as being due to the electronegative O atom in the C-7 amide side chain inhibiting participation in β-lactam bond hydrolysis. In any case, penicillin V was the first of the so-called oral penicillins giving higher and more prolonged blood levels than penicillin G itself. Its antimicrobial and clinical spectrum is roughly the same as that of benzylpenicillin, although it is somewhat less potent and is not used for acutely serious infections. Penicillin V has approximately the same sensitivity to β-lactamases and allergenicity as penicillin G. 83 Penicillinase-Resistant Penicillins. Nafcillin (Table 29.4). Nafcillin has a 2-ethoxynaphthyl side chain. This bulky group serves to inhibit destruction by β-lactamases analogous to methicillin. Increased steric bulk in the side chain leads to a β-lactamase– resistant drug. Nafcillin has a narrow antimicrobial spectrum so is used clinically for infections due to β-lactamase–producing S. aureus and a few other gram-positive organisms. 84 Dicloxacillin. Using a substituted isoxazolyl ring as a bioisosteric replacement for the benzene ring of penicillin G produces the isoxazolyl penicillins (oxacillin and dicloxacillin). These drugs are generally less potent than benzylpenicillin against gram-positive microorganisms (generally staphylococci and streptococci) that do not produce a β-lactamase but retain their potency against those that do. They are somewhat more acid stable; thus they may be taken orally. Because they are highly serum protein bound (Table 29.6). Like nafcillin, the isoxazolyl group of penicillins is primarily used against penicillinase-producing S. aureus. 85 Penicillinase-Sensitive, Broad-Spectrum, Oral Penicillins. Ampicillin (Table 29.4 slide 57). Many common gram-negative pathogens are sensitive to ampicillin. This is believed to be due to greater penetration of ampicillin into gram-negative bacteria. The acid stability is believed to be caused by the electron-withdrawing character of the protonated primary amine group reducing the side chain carbonyl participation in hydrolysis of the β-lactam bond as well as to the comparative difficulty of bringing H3O+ into the vicinity of the protonated amino group. The oral activity is also enhanced, in part, to active uptake by the dipeptide transporters. It lacks stability toward β-lactamases, and resistance is increasingly common. Several β-lactamase inhibitors for coadministration have been developed that restore many penicillinase-producing strains to the clinical spectrum of the aminopenicillins. 86 Amoxicillin (Table 29.4 slide 57). The p-phenolic hydroxyl group in the side chain phenyl moiety adjusts the isoelectric point of the drug to a more acidic value and is believed to be partially responsible, along with the intestine dipeptide transporter, for the enhanced blood levels obtained with amoxicillin compared with ampicillin itself (Table 29.5). Better oral absorption leads to less disturbance of the normal gastrointestinal flora and less drug-induced diarrhea. The antimicrobial spectrum and clinical uses of amoxicillin are approximately the same as those of ampicillin. 87 The addition of clavulanic acid (below) to amoxicillin gives a combination (Augmentin) in which the clavulanic acid serves to protect amoxicillin to a considerable extent against β-lactamases. This expands the spectrum of activity to include organisms and strains that produce β-lactamases. 88 Clavulanic Acid. It is a mold product that has only weak intrinsic antibacterial activity but is an excellent irreversible inhibitor of most β-lactamases. It is believed to acylate the active site serine by mimicking the normal substrate. While hydrolysis occurs with some β-lactamases, in many cases, subsequent reactions occur that inhibit the enzyme irreversibly. This leads to its classification as a mechanism-based inhibitor (or so-called suicide substrate). The precise chemistry is not well understood (slide next slide), but when clavulanic acid is added to amoxicillin and ticarcillin preparations, the potency against β-lactamase–producing strains is markedly enhanced. 89 Speculative mechanism for irreversible inactivation of β-lactamase by clavulanic acid and sulbactam. 90 Sulbactam. It is prepared by partial chemical synthesis from penicillins. The oxidation of the S atom to a sulfone (SO2) greatly enhances the potency of sulbactam. The combination of sulbactam and ampicillin (Unasyn) is also clinically popular. Not all βlactamases are sensitive to the presence of clavulanic acid or sulbactam. 91 Penicillinase-Sensitive, Broad-Spectrum, Parenteral Penicillins. Ticarcillin (Table 29.4 slide 57). It is a S-based bioisostere of benzylpenicillin in which one of the methylene hydrogens of the side chain has been substituted with a carboxylic acid moiety. Ticarcillin is classified as a carboxy penicillin. The addition of this functional group increases the water solubility of the drug and significantly enhances the potency against gram-negative organisms by increasing penetration through hydrophilic porins in these organisms. The drug is susceptible to β-lactamases and is acid unstable and thus must be given by injection. When potassium clavulanate is added to ticarcillin (Timentin), the combination has enhanced spectrum due to its enhanced stability to lactamases. 92 Piperacillin (Table 29.4 slide 57). This ampicillin analogue is referred to as an acylureidopenicillin and preserve the useful anti–grampositive activity of ampicillin but has a higher anti–gram-negative potency. Some strains of P. aeruginosa are sensitive to this agent. It is speculated that the added side chain moiety mimics a longer segment of the peptidoglycan chain than ampicillin does. This cell wall fragment is usually a tetrapeptide, so there is expected to be room for an extension in this direction. This would give more possible points of attachment to the penicillin-binding proteins, and perhaps these features are responsible for the enhanced antibacterial properties. This agent is used on gram-negative bacteria, especially K. pneumoniae and the anaerobe, B. fragilis. Resistance due to β-lactamases is a prominent feature of its use. 93 Piperacillin and Tazobactam Combination (Zosyn). Tazobactam is often coadministered with piperacillin because of tazobactam’s ability to inhibit β-lactamases. Tazobactam, like other β-lactamase inhibitors, has little or no antibacterial activity. This effect is analogous to that of clavulanic acid and sulbactam. This combination is the broadest spectrum penicillin currently available. 94 Cephalosporins. History. Abraham and Newton described the structure of the first cephalosporin, cephalosporin C. The compound was not very potent, it had activity against some penicillin-resistant cultures due to its stability to β-lactamases. Cephalosporin C is not potent enough to be a useful antibiotic, but removal, through chemical means, of the natural side chain produced 7-aminocephalosporanic acid, which, analogous to 6aminopenicillanic acid, could be fitted with unnatural side chains (see figure next slide). 95 5 4 6 7 3 1 2 10 Chemical preparation of 7-ACA and 7-ADCA. 96 Chemical Properties. The cephalosporins have their β-lactam ring fused to a six-membered dihydrothiazine ring, in contrast to the penicillins where the β-lactam ring is fused to a five-membered thiazolidine ring. As a consequence of the bigger ring, the cephalosporins should be less strained and less reactive/potent. However, much of the reactivity loss is made up by possession of an olefinic linkage at C-2,3 and a methyleneacetoxy or other leaving group at C-3. When the β-lactam ring is opened by hydrolysis, the leaving group can be ejected, carrying away the developing negative charge. This greatly reduces the energy required for the process (see figure next slide). 97 7 4 3 2 Thus the facility with which the β-lactam bond of the cephalosporins is broken is modulated both by the nature of the C-7 substituent (analogous to the penicillins) as well as the nature of the C-3 substituent and its ability to serve as a leaving group. Considerable support for this hypothesis comes from the finding that isomerization of the olefinic linkage to C-3,4 leads to great losses in antibiotic activity. Most cephalosporins are comparatively unstable in aqueous solutions. 98 Metabolism. The cephalosporins that have an acetyl group in the side chain are subject to enzymatic hydrolysis in the body. This results in a molecule with a hydroxymethyl moiety at C-3. A hydroxy moiety is a poor leaving group, so this change is considerably deactivating with respect to breakage of the β-lactam bond. In addition, the particular geometry of this part of the molecule leads to facile lactonization with the carboxyl group attached to C-2 (see figure next slide). 99 3 2 Metabolism of C-3-acetyl–substituted cephalosporins. 100 This results is an inactivation of the drugs. The penicillin binding proteins have an absolute requirement for a free carboxyl group to mimic that of the terminal carboxyl of the d-alanyl-d-alanine moiety in their normal substrate. Lactonization masks this docking functional group and, as a result, blocks affinity of the inhibitor for the enzyme. 101 Structure-Activity Relationship. The addition of an amino and a H to the α and α′ positions, respectively, results in a basic compound that is protonated under the acidic conditions of the stomach. The ammonium ion improves the stability of the β-lactam of the cephalosporin, leading to orally active drugs. The 7β amino group is essential for antimicrobial activity (X = H), whereas replacement of the hydrogen at C-7 (X = H) with an alkoxy (X = OR) results in improvement of the antibacterial activity of the cephalosporin. 102 Within specific cephalosporin derivatives, the addition of a 7α CH3O also improves the drugs stability toward β-lactamase. The derivatives where Y = S exhibit greater antibacterial activity than if Y = O, but the reverse is true when stability toward β-lactamase is considered. The 6α H is essential for biologic activity. And finally, antibacterial activity is improved when Z is a five-membered heterocycle versus a six-membered heterocycle. 103 The following changes improved β-lactamase resistance: (1) the l-isomer of an α amino α′ hydrogen derivative of a cephalosporin was 30-40 times more stable than the d-isomer; (2) the addition of a methoxyoxime to the α and α′ positions increased stability nearly 100-fold; and (3) the Z-oxime was as much as 20,000-fold more stable than the E-oxime (see figure below). These changes have been incorporated into a number of marketed and experimental cephalosporins (e.g., cefuroxime, ceftizoxime, ceftazidime, cefixime). Z and E oxime configuration. 104 Mechanism of Action. The cephalosporins are believed to act in a manner analogous to that of the penicillins by binding to the penicillin-binding proteins followed by cell lysis (figure slide 73). Cephalosporins are bactericidal in clinical terms. Resistance. Susceptible cephalosporins can be hydrolyzed by βlactamases before they reach the penicillin-binding proteins. Certain β-lactamases are constitutive (chromosomally encoded) in certain strains of gram-negative bacteria (Citrobacter, Enterobacter, Pseudomonas, and Serratia) and are normally repressed. These can be induced by certain β-lactam antibiotics (e.g., imipenem, cefotetan, and cefoxitin). Specific examples will be seen in the next slides wherein resistance to β-lactamase hydrolysis is conveyed by strategic steric bulk near the side chain amide linkage. Penetration barriers to the cephalosporins are also known. 105 Allergenicity. Allergenicity is less commonly experienced and is less severe with cephalosporins than with penicillins. Cephalosporins are frequently administered to patients who have had a mild or delayed penicillin reaction; however, cross allergenicity is possible. Nomenclature and Classification. Most cephalosporins have generic names beginning with cef- or ceph-. The cephalosporins are classified by a trivial nomenclature system loosely derived from the chronology of their introduction but more closely related to their antimicrobial spectrum. 106 The first-generation cephalosporins are primarily active in vitro against gram-positive cocci (penicillinase-positive and -negative S. aureus and S. epidermis), group A β-hemolytic streptococci (S. pyogenes), group B streptococci (S. agalactiae), and S. pneumoniae. They are not effective against methicillin-resistant Staphylococcus aureus. They are not significantly active against gram-negative bacteria, although some strains of E. coli, K. pneumoniae, P. mirabilis, and Shigella sp. may be sensitive. 107 The second-generation cephalosporins generally retain the anti– gram-positive activity of the first-generation agents but include H. influenzae as well and add to this better anti–gram-negative activity, so that some strains of Acinetobacter, Citrobacter, Enterobacter, E. coli, Klebsiella, Neisseria, Proteus, Providencia, and Serratia are also sensitive. Cefotetan and cefoxitin also have antianaerobic activity as well. 108 The third-generation cephalosporins are less active against staphylococci than the first-generation agents but are much more active against gram-negative bacteria than either the first- or the second-generation drugs. They are frequently particularly useful against nosocomial multidrug-resistant hospital-acquired strains. Morganella sp. and P. aeruginosa can also be added to the list of species that are often sensitive. Newer third-generation agents have been combined with β- lactamase inhibitors to further improve the spectrum against βlactamase–producing strains. 109 The fourth-generation cephalosporins have an antibacterial spectrum like enterobacteria the third-generation drugs that are to resistant the but add some third-generation cephalosporins. They are also more active against some grampositive organisms. Adverse Effects. Aside from mild or severe allergic reaction, the most commonly experienced cephalosporin toxicities are mild and temporary nausea, vomiting, and diarrhea. 110 First-Generation Cephalosporins 111 At C-3 a thio-linked thiadiazole ring is in cefazolin. This group is an activating leaving group, the moiety is not subject to the inactivating host hydrolysis reaction. Cephalexin does not have an activating side chain at C-3, and as a consequence is somewhat less potent. It does not undergo metabolic deactivation and thus maintains potency. 112 Second-Generation Cephalosporins 113 The N-methyl-5-thiotetrazole (MTT) group enhances potency and prevents metabolism by deacetylation. Cefuroxime has a Z-oriented methoxyimino moiety as part of its C-7 side chain. This conveys considerable resistance to attack by many β-lactamases but not by all. This is believed to result from the steric demands of this group. Resistance by P. aeruginosa is attributed to lack of penetration of the drug rather than to enzymatic hydrolysis. The carbamoyl moiety at C-3 is intermediate in metabolic stability between the classic acetyl moieties and the thiotetrazoles. 114 Cefuroxime axetil is a prodrug. The ester bond is cleaved metabolically, and the resulting intermediate form loses acetaldehyde spontaneously to produce cefuroxime itself. 115 Cefoxitin. The most novel chemical feature of cefoxitin is the possession of an α-oriented methoxyl group in place of the normal H-atom at C-7. This increased steric bulk conveys very significant stability against β-lactamases. Agents that contain this 7α methoxy group are commonly referred to as cephamycins. Cefoxitin has useful activity against gonorrhea and against some anaerobic infections (including B. fragilis) as compared with its secondgeneration relatives. On the negative side, cefoxitin has the capacity to induce certain broad-spectrum β-lactamases. 116 Cefotetan. It is a cephamycin. Like cefoxitin, cefotetan : - has better activity against anaerobes than the rest of this group, - is stable to a wide range of β-lactamases but is also an inducer in some bacteria. Cefaclor. It is quite acid stable and also quite stable to metabolism. It is less active against gram-negative bacteria than the other second-generation cephalosporins but is more active against gram- negative bacteria than the first-generation drugs. Cefprozil. The C=C bond is present in its 2 geometric isomeric forms, both of which exhibit antibacterial activity. The predominant trans form (Table 29.8) is much more active against gram-negative organisms. Cefprozil most closely resembles cefaclor in its properties but is a little more potent. 117 Third-Generation Cephalosporins 118 119 Cefotaxime. The Z-methoxyimino moiety at C-7 conveys significant β-lactamase resistance. It has excellent anti–gram-negative activity. It has a metabolically vulnerable acetoxy group attached to C-3 and loses about 90% of its activity when this is hydrolyzed. Cefotaxime should be protected from heat and light and may color slightly without significant loss of potency. Cefotaxime has less activity against staphylococci but has greater activity against gram-negative organisms. Ceftizoxime. The whole C-3 side chain has been omitted to prevent deactivation by hydrolysis. It rather resembles cefotaxime in its properties; however, not being subject to metabolism, its pharmacokinetic properties are much less complex. 120 Ceftriaxone. The C-3 side chain consists of a metabolically stable and activating thiotriazinedione in place of the normal acetyl group. The C-3 side chain is sufficiently acidic that at normal pH, it forms an enolic sodium salt, and thus the commercial product is a disodium salt. It is useful for many severe infections and notably in the treatment of some meningitis infections caused by gram-negative bacteria. It is quite stable to many β-lactamases but is sensitive to some inducible chromosomal β-lactamases. 121 Ceftazidime with avibactam. Ceftazidime has more pronounced βlactamase stability, greater P. aeruginosa activity, and increased activity against gram-positive organisms. The C-3 side chain is charged pyridinium moiety which enhances water solubility and also highly activates the β-lactam bond toward cleavage. It is attacked readily in sodium bicarbonate solutions. Resistance is mediated by chromosomally mediated β-lactamases and also by lack of penetration into target bacteria. It has a very broad antibacterial spectrum. It has been combined with avibactam (broad-spectrum βlactamase inhibitor) to treat urinary tract infections caused by β- lactamase–producing organisms. 122 Cefixime. It has anti–gram-negative activity intermediate between that of the second-generation and third-generation agents. It is poorly active against staphylococci because it does not bind satisfactorily to a specific penicillin binding protein (PBP-2). Ceftibuten. It has an enhanced β-lactamase stability and may contribute to oral activity as well. Ceftibuten has no C-3 side chain and thus is not measurably metabolized. It is highly (75%-90%) absorbed on oral administration, but this is decreased significantly by food. Being lipophilic and acidic, it is significantly serum protein bound (65%). Some isomerization of the geometry of the olefinic linkage appears to take place in vivo before excretion. It is mainly used for respiratory tract infections, otitis media, and pharyngitis, as well as urinary tract infections by susceptible microorganisms. 123 Cefpodoxime Proxetil. It is a prodrug. It is cleaved enzymically to isopropanol, CO2, acetaldehyde, and cefpodoxime in the gut wall. It has better anti–Staphylococcus aureus activity than cefixime and is used to treat pharyngitis, urinary tract infections, upper and lower respiratory tract infections, otitis media, skin and soft tissue infections, and gonorrhea. Cefdinir. It has an enhanced anti–gram-positive activity. It has reasonable resistance to β-lactamases. 124 Cefditoren Pivoxil. It is an orally active prodrug. The pivoxil ester is hydrolyzed following intestinal absorption to release the active drug, cefditoren, along with formaldehyde and pivalic acid. The bioavailability of cefditoren pivoxil is increased if taken with food. It is indicated for mild to moderate infections in adults and adolescents with chronic bronchitis, pharyngitis/tonsillitis, and uncomplicated skin infections associated with gram-negative bacteria. 125 Fourth-Generation Cephalosporins Cefepime. It is a semisynthetic agent containing a Z-methoxyimine moiety and an aminothiazolyl group at C-7, broadening its spectrum, increasing its β-lactamase stability, and increasing its antistaphylococcal activity. The quaternary N-methylpyrrolidine group at C-3 seems to help penetration into gram-negative bacteria. The fourth-generation cephalosporins are characterized by enhanced antistaphylococcal activity and broader anti–gramnegative activity than the third-generation group. It is used against urinary tract infections, skin and skin structure infections, pneumonia, and intra-abdominal infections. 126 Ceftaroline Fosamil. It is bactericidal against methicillin-resistant Staphylococcus aureus due to its affinity for PBP-2a and against S. pneumoniae due to its affinity for PBP-2x. It can be used to treat methicillin-resistant Staphylococcus aureus infections of skin and soft tissues. It is administered as a water-soluble prodrug that is readily hydrolyzed to the active ceftaroline (loss of the phosphate ester). 127 Carbapenems. Thienamycin, the first of the carbapenems, was isolated from Streptomyces cattleya. It differs structurally from the penicillins and cephalosporins. The S atom is not part of the five-membered ring but rather has been replaced by a methylene moiety at that position. 128 The carbapenem ring system is highly strained and very susceptible to reactions cleaving the β-lactam bond (including reaction between 2 molecules resulting in inactivation). Inactivation of thienamycin via intermolecular reaction. 129 Carbapenems bind differently to the penicillin-binding proteins (especially strongly to PBP-2), but the result is very potent broad- spectrum activity. However, none of the current carbapenems have activity against methicillin-resistant Staphylococcus aureus. Imipenem (structure next slide), like thienamycin, penetrates very well through porins and is very stable, even inhibitory, to many βlactamases. Imipenem is not orally active. Renal dehydropeptidase-l metabolizes imipenem through hydrolysis of the β-lactam and deactivates it. An inhibitor for this enzyme, cilastatin, is coadministered with imipenem to protect it. The combination of imipenem and cilastatin is useful for treatment of serious infections by aerobic gram-negative bacilli, anaerobes, and S. aureus. 130 Carbapenem antibiotics and coadministered compounds. 131 Meropenem is a synthetic carbapenem. It has a chiral methyl group at C-4 which conveys intrinsic resistance to hydrolysis by dehydropeptidase-1. Doripenem contains the 4-β-methyl group, which confers stability toward dehydropeptidase-1. It is similar in spectrum to imipenem and meropenem but is considered more potent against Pseudomonas species. Ertapenem is another synthetic carbapenem. The 4-β-methyl group confers stability toward dehydropeptidase-1. It is not active against Pseudomonas or Acinetobacter and thus should not be substituted for other carbapenems for these organisms. 132 Monobactams. They are a class of monocyclic β-lactam antibiotics. Aztreonam is a totally synthetic antibiotic whose antimicrobial spectrum is devoted almost exclusively to gramnegative microorganisms, and it is capable of inactivating some βlactamases. Its molecular mode of action is closely similar to that of the penicillins, cephalosporins, and carbapenems, the action being characterized by strong affinity for PBP-3, producing filamentous cells as a consequence. 133 The monobactams demonstrate that a fused ring is not essential for antibiotic activity. The α-oriented methyl group at C-2 is associated with the stability of aztreonam toward β-lactamases. The protein binding is moderate (∼50%), and the drug is nearly unchanged by metabolism. Aztreonam is primarily excreted in the urine. Aztreonam is used against severe infections caused by gramnegative microorganisms (mainly urinary tract, upper respiratory tract, bone, cartilage, abdominal, obstetric, and gynecologic infections and septicemias). 134