Oj3Lactamase inhibitors

The strategy of using a ¡-lactamase inhibitor in combination with a ¡-lactamase-sensitive penicillin in the therapy for infections caused by ¡-lactamase-producing bacterial strains has, until relatively recently, failed to live up to its obvious promise. Early attempts to obtain synergy against such resistant strains, by using combinations consisting of a ¡-lactamase-resistant penicillin (e.g., methicillin or oxacillin) as a competitive inhibitor and a ¡-lactamase- sensitive penicillin (e.g., ampicillin or carbenicillin) to kill the organisms, met with limited success. Factors that may contribute to the failure of such combinations to achieve synergy include (a) the failure of most lipophilic penicillinase-resistant penicillins to penetrate the cell envelope of Gram-negative bacilli in effective concentrations, (b) the reversible binding of penicillinase-resistant penicillins to ¡-lactamase, requiring high concentrations to prevent substrate binding and hydrolysis, and (c) the induction of ¡-lactamases by some penicillinase-resistant penicillins.

The discovery of the naturally occurring, mechanism-based inhibitor clavulanic acid, which causes potent and progressive inactivation of ¡-lactamases (Fig. 8.4), has created renewed interest in ¡-lactam combination therapy. This interest has led to the design and synthesis of additional mechanism-based ¡-lactamase inhibitors, such as sulbactam and tazobactam, and the isolation of naturally occurring ¡-lactams, such as the thienamycins, which both inhibit ¡-lactamases and interact with PBPs.

The chemical events leading to the inactivation of ¡-lactamases by mechanism-based inhibitors are very complex. In a review of the chemistry of ¡-lactamase inhibition, Knowles53 has described two classes of ¡-lactamase inhibitors: class I inhibitors that have a heteroatom leaving group at position 1 (e.g., clavulanic acid and sulbactam) and class II inhibitors that do not (e.g., the carbapenems). Unlike competitive inhibitors, which bind reversibly to the enzyme they inhibit, mechanism-based inhibitors react with the enzyme in much the same way that the substrate does. With the ¡-lactamases, an acyl-enzyme intermediate is formed by reaction of the ¡-lactam with an active-site serine hydroxyl group of the enzyme. For normal substrates, the acyl-enzyme intermediate readily undergoes hydrolysis, destroying the substrate and freeing the enzyme to attack more substrate. The acyl-enzyme intermediate formed when a mechanism-based inhibitor is attacked by the enzyme is diverted by tautomerism to a more stable imine form that hydrolyzes more slowly to eventually free the enzyme (transient inhibition), or for a class I inhibitor, a second group on the enzyme may be attacked to inactivate it. Because these inhibitors are also substrates for the enzymes that they inactivate, they are sometimes referred to as "suicide substrates."

Because class I inhibitors cause prolonged inactivation of certain ¡-lactamases, they are particularly useful in combination with extended-spectrum, ¡-lactamase-sensitive penicillins to treat infections caused by ¡-lactamase-producing bacteria. Three such inhibitors, clavulanic acid, sulbactam, and tazobactam, are currently marketed in the United States for this purpose. A class II inhibitor, the carbapenem derivative imipenem, has potent antibacterial activity in addition to its ability to cause transient inhibition of some ¡-lactamases. Certain antibacterial cephalosporins with a leaving group at the C-3 position can cause transient inhibition of ¡-lactamases by forming stabilized acyl-enzyme intermediates. These are discussed more fully later in this chapter.

The relative susceptibilities of various ¡-lactamases to inactivation by class I inhibitors appear to be related to the molecular properties of the enzymes.25,54,55 ¡-Lactamases belonging to group A, a large and somewhat heterogenous group of serine enzymes, some with narrow (e.g., penicillin-ases or cephalosporinases) and some with broad (i.e., general ¡-lactamases) specificities, are generally inactivated by class I inhibitors. A large group of chromosomally encoded serine ¡-lactamases belonging to group C with specificity for cephalosporins are, however, resistant to inactivation by class I inhibitors. A small group of Zn2+-requiring metallo-¡-lactamases (group B) with broad substrate specificities56 are also not inactivated by class I inhibitors.

Figure 8.4

Transient Inhibition

Mechanism-based inhibition of ^-lactamases.

Products

Clavulanate Potassium

Clavulanic acid is an antibiotic isolated from Streptomyces clavuligeris. Structurally, it is a 1-oxopenam lacking the 6-acylamino side chain of penicillins but possessing a 2-hydroxyethylidene moiety at C-2. Clavulanic acid exhibits very weak antibacterial activity, comparable with that of 6-APA and, therefore, is not useful as an antibiotic. It is, however, a potent inhibitor of S. aureus ^-lactamase and plasmid-mediated ^-lactamases elaborated by Gramnegative bacilli.

Combinations of amoxicillin and the potassium salt of clavulanic acid are available (Augmentin) in various fixed-dose oral dosage forms intended for the treatment of skin, respiratory, ear, and urinary tract infections caused by ß-lactamase-producing bacterial strains. These combinations are effective against ß-lactamase-producing strains of S. aureus, E. coli, K. pneumoniae, Enterobacter, H. influenzae, Moraxella catarrhalis, and Haemophilus ducreyi, which are resistant to amoxicillin alone. The oral bioavailability of amoxicillin and potassium clavulanate is similar. Clavulanic acid is acid-stable. It cannot undergo peni-cillanic acid formation because it lacks an amide side chain.

Potassium clavulanate and the extended-spectrum penicillin ticarcillin have been combined in a fixed-dose, injectable form for the control of serious infections caused by ^-lactamase-producing bacterial strains. This combination has been recommended for septicemia, lower respiratory tract infections, and urinary tract infections caused by jS-lactamase-producing Klebsiella spp., E. coli, P. aeruginosa, and other Pseudomonas spp., Citrobacter spp., Enterobacter spp., S. marcescens, and S. aureus. It also is used in bone and joint infections caused by these organisms. The combination contains 3 g of ticarcillin disodium and 100 mg of potassium clavulanate in a sterile powder for injection (Timentin).

Sulbactam

Sulbactam is penicillanic acid sulfone or 1,1-dioxopenicillanic acid. This synthetic penicillin derivative is a potent inhibitor of S. aureus jS-lactamase as well as many j-lactamases elaborated by Gram-negative bacilli. Sulbactam has weak intrinsic antibacterial activity but potentiates the activity of ampicillin and carbenicillin against j-lactamase-producing S. aureus and members of the Enterobacteriaceae family. It does not, however, synergize with either carbenicillin or ticar-cillin against P. aeruginosa strains resistant to these agents. Failure of sulbactam to penetrate the cell envelope is a possible explanation for the lack of synergy.

Fixed-dose combinations of ampicillin sodium and sulbactam sodium, marketed under the trade name Unasyn as sterile powders for injection, have been approved for use in the United States. These combinations are recommended for the treatment of skin, tissue, intra-abdominal, and gynecological infections caused by jS-lactamase-producing strains of S. aureus, E. coli, Klebsiella spp., P. mirabilis, B. frag-ilis, and Enterobacter and Acinetobacter spp.

Tazobactam

Tazobactam is a penicillanic acid sulfone that is similar in structure to sulbactam. It is a more potent jS-lactamase inhibitor than sulbactam57 and has a slightly broader spectrum of activity than clavulanic acid. It has very weak antibacterial activity. Tazobactam is available in fixed-dose, injectable combinations with piperacillin, a broad-spectrum penicillin consisting of an 8:1 ratio of piperacillin sodium to tazobactam sodium by weight and marketed under the trade name Zosyn. The pharmacokinetics of the two drugs are very similar. Both have short half-lives (t1/2 ~1 hour), are minimally protein bound, experience very little metabolism, and are excreted in active forms in the urine in high concentrations.

Approved indications for the piperacillin-tazobactam combination include the treatment of appendicitis, postpartum endometritis, and pelvic inflammatory disease caused by j-lactamase-producing E. coli and Bacteroides spp., skin and skin structure infections caused by j-lactamase-producing S. aureus, and pneumonia caused by j-lactamase-producing strains of H. influenzae.

CARBAPENEMS Thienamycin

Thienamycin is a novel j-lactam antibiotic first isolated and identified by researchers at Merck58 from fermentation of cultures of Streptomyces cattleya. Its structure and absolute configuration were established both spectroscopically and by total synthesis.59,60 Two structural features of thienamycin are shared with the penicillins and cephalosporins: a fused bicyclic ring system containing a j-lactam and an equiva-lently attached 3-carboxyl group. In other respects, the thien-amycins represent a significant departure from the established j-lactam antibiotics. The bicyclic system consists of a car-bapenem containing a double bond between C-2 and C-3 (i.e., it is a 2-carbapenem, or A2-carbapenem, system). The double bond in the bicyclic structure creates considerable ring strain and increases the reactivity of the j-lactam to ring-opening reactions. The side chain is unique in two respects:

it is a simple 1-hydroxyethyl group instead of the familiar acylamino side chain, and it is oriented to the bicyclic ring system rather than having the usual S orientation of the penicillins and cephalosporins. The remaining feature is a 2-aminoethylthioether function at C-2. The absolute stereochemistry of thienamycin has been determined to be 5r:6s:8s. Several additional structurally related antibiotics have been isolated from various Streptomyces spp., including the four epithienamycins, which are isomeric to thienamycin at C-5, C-6, or C-8, and derivatives in which the 2-aminoethylthio side chain is modified.

Thienamycin displays outstanding broad-spectrum antibacterial properties in vitro.61 It is highly active against most aerobic and anaerobic Gram-positive and Gramnegative bacteria, including S. aureus, P. aeruginosa, and B. fragilis. Furthermore, it is resistant to inactivation by most j-lactamases elaborated by Gram-negative and Grampositive bacteria and, therefore, is effective against many strains resistant to penicillins and cephalosporins. Resistance to lactamases appears to be a function of the a-1-hydroxyethyl side chain because this property is lost in the 6-nor derivative and epithienamycins with S stereochemistry show variable resistance to the different j-lactamases.

An unfortunate property of thienamycin is its chemical instability in solution. It is more susceptible to hydrolysis in both acidic and alkaline solutions than most j-lactam antibiotics, because of the strained nature of its fused ring system containing an endocyclic double bond. Furthermore, at its optimally stable pH between 6 and 7, thienamycin undergoes concentration-dependent inactivation. This inactivation is believed to result from intermolecular aminolysis of the j-lactam by the cysteamine side chain of a second molecule. Another shortcoming is its susceptibility to hydrolytic inac-tivation by renal dehydropeptidase-I (DHP-I),62 which causes it to have an unacceptably short half-life in vivo.

Imipenem-Cilastatin

Imipenem is N-formimidoylthienamycin, the most successful of a series of chemically stable derivatives of thienamycin in which the primary amino group is converted to a nonnu-cleophilic basic function.63 Cilastatin is an inhibitor of DHP-I. The combination (Primaxin) provides a chemically and en-zymatically stable form of thienamycin that has clinically useful pharmacokinetic properties. The half-life of the drug is nonetheless short (t1/2 ~1 hour) because of renal tubular secretion of imipenem. Imipenem retains the extraordinary broad-spectrum antibacterial properties of thienamycin. Its bactericidal activity results from the inhibition of cell wall synthesis associated with bonding to PBPs 1b and 2. Imipenem is very stable to most j-lactamases. It is an inhibitor of S-lactamases from certain Gram-negative bacteria resistant to other j-lactam antibiotics (e.g., P. aeruginosa, S. marcescens, and Enterobacter spp.).

Imipenem is indicated for the treatment of a wide variety of bacterial infections of the skin and tissues, lower respiratory tract, bones and joints, and genitourinary tract, as well as of septicemia and endocarditis caused by S-lactamase-producing strains of susceptible bacteria. These include aerobic Grampositive organisms such as S. aureus, Staphylococcus epider-midis, enterococci, and viridans streptococci; aerobic Gramnegative bacteria such as E. coli, Klebsiella, Serratia, Providencia, Haemophilus, Citrobacter, and indole-positive Proteus spp., Morganella morganii, Acinetobacter and Enterobacter spp., and P. aeruginosa and anaerobes such as B. fragilis and Clostridium, Peptococcus, Peptidostreptococcus, Eubacterium, and Fusobacterium spp. Some Pseudomonas spp. are resistant, such as P. maltophilia and P. cepacia, as are some methicillin-resistant staphylococci. Imipenem is effective against non—S-lactamase-producing strains of these and additional bacterial species, but other less expensive and equally effective antibiotics are preferred for the treatment of infections caused by these organisms.

The imipenem-cilastatin combination is marketed as a sterile powder intended for the preparation of solutions for intravenous infusion. Such solutions are stable for 4 hours at 25°C and up to 24 hours when refrigerated. The concomitant administration of imipenem and an aminoglycoside antibiotic results in synergistic antibacterial activity in vivo. The two types of antibiotics are, however, chemically incompatible and should never be combined in the same intravenous bottle.

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