Penaldic Acid

Figure 8.3 • Degradation of penicillins.

Penicilloaldehyde bacilli, however, may result from other poorly characterized "resistance factors" or constitutive j-lactamase elaboration. S-Lactamases produced by Gram-negative bacilli appear to be cytoplasmic enzymes that remain in the bacterial cell, whereas those elaborated by S. aureus are synthesized in the cell wall and released extracellularly. J-Lactamases from different bacterial species may be classified24-26 by their structure, their substrate and inhibitor specificities, their physical properties (e.g., pH optimum, isoelectric point, molecular weight), and their immunological properties.

Specific acylases (enzymes that can hydrolyze the acyl-amino side chain of penicillins) have been obtained from several species of Gram-negative bacteria, but their possi ble role in bacterial resistance has not been well defined. These enzymes find some commercial use in the preparation of 6-APA for the preparation of semisynthetic penicillins. 6-APA is less active and hydrolyzed more rapidly (enzymatically and nonenzymatically) than penicillin.

Another important resistance mechanism, especially in Gram-negative bacteria, is decreased permeability to penicillins. The cell envelope in most Gram-negative bacteria is more complex than in Gram-positive bacteria. It contains an outer membrane (linked by lipoprotein bridges to the pepti-doglycan cell wall) not present in Gram-positive bacteria, which creates a physical barrier to the penetration of antibiotics, especially those that are hydrophobic.27 Small hydrophilic molecules, however, can traverse the outer membrane through pores formed by proteins called porins.28 Alteration of the number or nature of porins in the cell enve-lope28 also could be an important mechanism of antibiotic resistance. Bacterial resistance can result from changes in the affinity of PBPs for penicillins.29 Altered PBP binding has been demonstrated in non—S-lactamase-producing strains of penicillin-resistant Neisseria gonorrhoeae30 and methicillin-resistant S. aureus (MRSA).31

Certain strains of bacteria are resistant to the lytic properties of penicillins but remain susceptible to their growth-inhibiting effects. Thus, the action of the antibiotic has been converted from bactericidal to bacteriostatic. This mechanism of resistance is termed tolerance and apparently results from impaired autolysin activity in the bacterium.

Penicillinase-Resistant Penicillins

The availability of 6-APA on a commercial scale made possible the synthesis of numerous semisynthetic penicillins modified at the acylamino side chain. Much of the early work done in the 1960s was directed toward the preparation of derivatives that would resist destruction by 3-lactamases, particularly those produced by penicillin-resistant strains of S. aureus, which constituted a very serious health problem at that time. In general, increasing the steric hindrance at the acarbon of the acyl group increased resistance to staphylococ-cal 3-lactamase, with maximal resistance being observed with quaternary substitution.32 More fruitful from the standpoint of antibacterial potency, however, was the observation that the a-acyl carbon could be part of an aromatic (e.g., phenyl or naphthyl) or heteroaromatic (e.g., 4-isoxazoyl) system.33 Substitutions at the ortho positions of a phenyl ring (e.g., 2,6-dimethoxy [methicillin]) or the 2-position of a 1-naphthyl system (e.g., 2-ethoxyl [nafcillin]) increase the steric hindrance of the acyl group and confer more 3-lactamase resistance than shown by the unsubstituted compounds or those substituted at positions more distant from the a-carbon. Bulkier substituents are required to confer effective 3-lactamase resistance among five-membered-ring heterocyclic derivatives.34 Thus, members of the 4-isoxa-zoyl penicillin family (e.g., oxacillin, cloxacillin, and di-cloxacillin) require both the 3-aryl and 5-methyl (3-methyl and 5-aryl) substituents for effectiveness against ¡-lactamase-producing S. aureus.

Increasing the bulkiness of the acyl group is not without its price, however, because all of the clinically available penicillinase-resistant penicillins are significantly less active than either penicillin G or penicillin V against most non-3-lactamase-producing bacteria normally sensitive to the penicillins. The 3-lactamase-resistant penicillins tend to be comparatively lipophilic molecules that do not penetrate well into Gram-negative bacteria. The isoxazoyl penicillins, particularly those with an electronegative substituent in the 3-phenyl group (cloxacillin, dicloxacillin, and floxacillin), are also resistant to acid-catalyzed hydrolysis of the 3-lactam, for the reasons described previously. Steric factors that confer 3-lactamase resistance, however, do not necessarily also confer stability to acid. Accordingly, methi-cillin, which has electron-donating groups (by resonance) ortho to the carbonyl carbon, is even more labile to acid-catalyzed hydrolysis than is penicillin G because of the more rapid formation of the penicillenic acid derivative.

Extended-Spectrum Penicillins

Another highly significant advance arising from the preparation of semisynthetic penicillins was the discovery that the introduction of an ionized or polar group into the a-position of the side chain benzyl carbon atom of penicillin G confers activity against Gram-negative bacilli. Hence, derivatives with an ionized a-amino group, such as ampicillin and amoxicillin, are generally effective against such Gram-negative genera as Escherichia, Klebsiella, Haemophilus, Salmonella, Shigella, and non-indole-producing Proteus. Furthermore, activity against penicillin G-sensitive, Gram-positive species is largely retained. The introduction of an a-amino group in ampicillin (or amoxicillin) creates an additional chiral center. Extension of the antibacterial spectrum brought about by the substituent applies only to the D-isomer, which is 2 to 8 times more active than either the L-isomer or benzylpenicillin (which are equiactive) against various species of the aforementioned genera of Gram-negative bacilli.

The basis for the expanded spectrum of activity associated with the ampicillin group is not related to ¡-lactamase inhibition, as ampicillin and amoxicillin are even more labile than penicillin G to the action of ¡-lactamases elaborated by both S. aureus and various species of Gram-negative bacilli, including strains among the ampicillin-sensitive group. Hydrophilic penicillins, such as ampicillin, penetrate Gramnegative bacteria more readily than penicillin G, penicillin V, or methicillin. This selective penetration is believed to take place through the porin channels of the cell membrane.35

a-Hydroxy substitution also yields "expanded-spectrum" penicillins with activity and stereoselectivity similar to that of the ampicillin group. The a-hydroxybenzylpenicillins are, however, about 2 to 5 times less active than their corresponding a-aminobenzyl counterparts and, unlike the latter, not very stable under acidic conditions.

Incorporation of an acidic substituent at the a-benzyl carbon atom of penicillin G also imparts clinical effectiveness against Gram-negative bacilli and, furthermore, extends the spectrum of activity to include organisms resistant to ampi-cillin. Thus, a-carboxybenzylpenicillin (carbenicillin) is active against ampicillin-sensitive, Gram-negative species and additional Gram-negative bacilli of the genera Pseudomonas, Klebsiella, Enterobacter, indole-producing Proteus, Serratia, and Providencia. The potency of carbenicillin against most species of penicillin G-sensitive, Gram-positive bacteria is several orders of magnitude lower than that of either penicillin G or ampicillin, presumably because of poorer penetration of a more highly ionized molecule into these bacteria. (Note that a-aminobenzylpenicillins exist as zwitterions over a broad pH range and, as such, are considerably less polar than carbenicillin.) This increased polarity is apparently an advantage for the penetration of carbenicillin through the cell envelope of Gram-negative bacteria via porin channels.35

Carbenicillin is active against both ¡-lactamase-producing and non-¡-lactamase-producing strains of Gramnegative bacteria. It is somewhat resistant to a few of the ¡-lactamases produced by Gram-negative bacteria, especially members of the Enterobacteriaceae family.36 Resistance to ¡-lactamases elaborated by Gram-negative bacteria, therefore, may be an important component of carbenicillin's activity against some ampicillin-resistant organisms. ¡-Lactamases produced by Pseudomonas spp., however, readily hydrolyze carbenicillin. Although carbenicillin is also somewhat resistant to staphylococcal 3-lactamase, it is considerably less so than methicillin or the isoxazoyl penicillins, and its inherent antistaphylococcal activity is less impressive than that of the penicillinase-resistant penicillins. The penicilli-nase-resistant penicillins, despite their resistance to most 3-lactamases, however, share the lack of activity of penicillin G against Gram-negative bacilli, primarily because of an inability to penetrate the bacterial cell envelope.

Compared with the aminoglycoside antibiotics, the potency of carbenicillin against such Gram-negative bacilli as Pseudomonas aeruginosa, Proteus vulgaris, and Klebsiella pneumoniae is much less impressive. Large parenteral doses are required to achieve bactericidal concentrations in plasma and tissues. The low toxicity of carbenicillin (and the penicillins in general), however, usually permits (in the absence of allergy) the use of such high doses without untoward effects. Furthermore, carbenicillin (and other penicillins), when combined with aminoglycosides, exerts a synergistic bactericidal action against bacterial species sensitive to both agents, frequently allowing the use of a lower dose of the more toxic aminoglycoside than is normally required for treatment of a life-threatening infection. The chemical incompatibility of penicillins and aminoglycosides requires that the two antibiotics be administered separately; otherwise, both are inactivated. Iyengar et al.37 showed that acylation of amino groups in the aminoglycoside by the 3-lactam of the penicillin occurs.

Unlike the situation with ampicillin, the introduction of asymmetry at the a-benzyl carbon in carbenicillin imparts little or no stereoselectivity of antibacterial action; the individual enantiomers are nearly equally active and readily epimerized to the racemate in aqueous solution. Because it is a derivative of phenylmalonic acid, carbenicillin readily decarboxylates to benzylpenicillin in the presence of acid; therefore, it is not active (as carbenicillin) orally and must be administered parenterally. Esterification of the a-carboxyl group (e.g., as the 5-indanyl ester) partially protects the compound from acid-catalyzed destruction and provides an orally active derivative that is hydrolyzed to carbenicillin in the plasma. The plasma levels of free carbenicillin achieved with oral administration of such esters, however, may not suffice for effective treatment of serious infections caused by some species of Gram-negative bacilli, such as P. aeruginosa.

A series of a-acylureido-substituted penicillins, exemplified by azlocillin, mezlocillin, and piperacillin, exhibit greater activity against certain Gram-negative bacilli than carbenicillin. Although the acylureidopenicillins are acyl-ated derivatives of ampicillin, the antibacterial spectrum of activity of the group is more like that of carbenicillin. The acylureidopenicillins are, however, superior to carbenicillin against Klebsiella spp., Enterobacter spp., and P. aeruginosa. This enhanced activity is apparently not because of 3-lactamase resistance, in that both inducible and plasmid-mediated 3-lactamases hydrolyze these penicillins. More facile penetration through the cell envelope of these particular bacterial species is the most likely explanation for the greater potency. The acylureidopenicillins, unlike ampi-cillin, are unstable under acidic conditions; therefore, they are not available for oral administration.

Protein Binding

The nature of the acylamino side chain also determines the extent to which penicillins are plasma protein bound.

Quantitative structure-activity relationship (QSAR) studies of the binding of penicillins to human serum38'39 indicate that hydrophobic groups (positive w dependence) in the side chain appear to be largely responsible for increased binding to serum proteins. Penicillins with polar or ionized sub-stituents in the side chain exhibit low-to-intermediate fractions of protein binding. Accordingly, ampicillin, amoxicillin, and cyclacillin experience 25% to 30% protein binding, and carbenicillin and ticarcillin show 45% to 55% protein binding. Those with nonpolar, lipophilic substituents (nafcillin and isoxazoyl penicillins) are more than 90% protein bound. The penicillins with less complex acyl groups (benzylpenicillin, phenoxymethylpenicillin, and methi-cillin) fall in the range of 35% to 80%. Protein binding is thought to restrict the tissue availability of drugs if the fraction bound is sufficiently high; thus, the tissue distribution of the penicillins in the highly bound group may be inferior to that of other penicillins. The similarity of biological half-lives for various penicillins, however, indicates that plasma protein binding has little effect on duration of action. All of the commercially available penicillins are secreted actively by the renal active transport system for anions. The reversible nature of protein binding does not compete effectively with the active tubular secretion process.

Allergy to Penicillins

Allergic reactions to various penicillins, ranging in severity from a variety of skin and mucous membrane rashes to drug fever and anaphylaxis, constitute the major problem associated with the use of this class of antibiotics. Estimates place the prevalence of hypersensitivity to penicillin G throughout the world between 1% and 10% of the population. In the United States and other industrialized countries, it is nearer the higher figure, ranking penicillin the most common cause of drug-induced allergy. The penicillins that are most frequently implicated in allergic reactions are penicillin G and ampicillin. Virtually all commercially available penicillins, however, have been reported to cause such reactions; in fact, cross-sensitivity among most chemical classes of 6-acylaminopenicillanic acid derivatives has been demonstrated.40

The chemical mechanisms by which penicillin preparations become antigenic have been studied extensively.20 Evidence suggests that penicillins or their rearrangement products formed in vivo (e.g., penicillenic acids)41 react with lysine e-amino groups of proteins to form penicilloyl proteins, which are major antigenic determinants.42,43 Early clinical observations with the biosynthetic penicillins G and V indicated a higher incidence of allergic reactions with un-purified, amorphous preparations than with highly purified, crystalline forms, suggesting that small amounts of highly antigenic penicilloyl proteins present in unpurified samples were a cause. Polymeric impurities in ampicillin dosage forms have been implicated as possible antigenic determinants and a possible explanation for the high frequency of allergic reactions with this particular semisynthetic penicillin. Ampicillin is known to undergo pH-dependent polymerization reactions (especially in concentrated solutions) that involve nucleophilic attack of the side chain amino group of one molecule on the ^-lactam carbonyl carbon atom of a second molecule, and so on.44 The high frequency of antigenicity shown by ampicillin polymers, together with

TABLE 8.3 Classification and Properties of Penicillins


Acid Absorption

Penicillin Source Resistance (%)

their isolation and characterization in some ampicillin preparations, supports the theory that they can contribute to ampicillin-induced allergy.45


Various designations have been used to classify penicillins, based on their sources, chemistry, pharmacokinetic properties, resistance to enzymatic spectrum of activity, and clinical uses (Table 8.3). Thus, penicillins may be biosynthetic, semisynthetic, or (potentially) synthetic; acid-resistant or not; orally or (only) parenterally active; and resistant to fS-lactamases (penicillinases) or not. They may have a narrow, intermediate, broad, or extended spectrum of antibacterial activity and may be intended for multipurpose or limited clinical use. Designations of the activity spectrum as narrow, intermediate, broad, or extended are relative and do not necessarily imply the breadth of therapeutic application. Indeed, the classification of penicillin G as a "narrow-spectrum" antibiotic has meaning only relative to other penicillins. Although the jS-lactamase-resistant penicillins have a spectrum of activity similar to that of penicillin G, they generally are reserved for the treatment of infections caused by penicillin G-resistant, jS-lactamase-producing S. aureus because their activity against most penicillin G-sensitive bacteria is significantly inferior. Similarly, carbenicillin and ticarcillin usually are reserved for the treatment of infections caused by ampicillin-resistant, Gram-negative bacilli because they offer no advantage (and have some disadvantages) over ampicillin or penicillin G in infections sensitive to them.


Penicillin G

For years, the most popular penicillin has been penicillin G, or benzylpenicillin. In fact, with the exception of patients allergic to it, penicillin G remains the agent of choice for the treatment of more different kinds of bacterial infection than any other antibiotic. It was first made available as the water-soluble salts of potassium, sodium, and calcium. These salts of penicillin are inactivated by the gastric juice and are not effective when administered orally unless antacids, such as calcium carbonate, aluminum hydroxide, and magnesium trisilicate; or a strong buffer, such as sodium citrate, is added. Also, because penicillin is absorbed poorly from the

Plasma j3-Lactamase

Protein Resistance Spectrum

Binding (%) (S. aureus) of Activity Clinical Use intestinal tract, oral doses must be very large, about five times the amount necessary with parenteral administration. Only after the production of penicillin had increased enough to make low-priced penicillin available did the oral dosage forms become popular. The water-soluble potassium and sodium salts are used orally and parenterally to achieve high plasma concentrations of penicillin G rapidly. The more water-soluble potassium salt usually is preferred when large doses are required. Situations in which hyperkalemia is a danger, however, as in renal failure, require use of the sodium salt; the potassium salt is preferred for patients on salt-free diets or with congestive heart conditions.

The rapid elimination of penicillin from the bloodstream through the kidneys by active tubular secretion and the need to maintain an effective concentration in blood have led to the development of "repository" forms of this drug. Suspensions of penicillin in peanut oil or sesame oil with white beeswax added were first used to prolong the duration of injected forms of penicillin. This dosage form was replaced by a suspension in vegetable oil, to which aluminum monostearate or aluminum distearate was added. Today, most repository forms are suspensions of high-molecular weight amine salts of penicillin in a similar base.

Penicillin G Procaine

The first widely used amine salt of penicillin G was made with procaine. Penicillin G procaine (Crysticillin, Duracillin, Wycillin) can be made readily from penicillin G sodium by treatment with procaine hydrochloride. This salt is considerably less soluble in water than the alkali metal salts, requiring about 250 mL to dissolve 1 g. Free penicillin is released only as the compound dissolves and dissociates. It has an activity of 1,009 units/mg. A large number of preparations for injection of penicillin G procaine are commercially available. Most of these are either suspensions in water to which a suitable dispersing or suspending agent, a

TABLE 8.3 Classification and Properties of Penicillins


Acid Absorption

Penicillin Source Resistance (%)

Plasma j3-Lactamase

Protein Resistance Spectrum

Binding (%) (S. aureus) of Activity Clinical Use




Poor (20)

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