The Cephalosporin Group of Antibiotics

The Cephalosporin Group of Antibiotics D . R. OWENS.D. K . LUSCOMBE. A . D . RUSSELL.AND P . J . NICHOLLS Department of Medicine. Welsh National School of Medicine. Cardiff and Welsh School of Pharmacy. University of Wales Institute of Science and Technology Cardifj Great Britain

I . Introduction . I1 . Chemical Aspects A . Production

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B . Structure-Activity

Relationships

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I11. Antibacterial Activity . . . . . . . . . . . . . . . . . . A . Spectrum of Activity . . . . . . . . . . . . . . . . . B . Bacterial Resistance

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C . Mode of Action . . . . . . . . . . . . . . . . . . .

IV. Pharmacology and Toxicology

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VI .

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A . Cephaloridine . . . . . B. Cephalothin . . . . . . C . Cephalexin . . . . . . D . Cephaloglycin . . . . . E . Other Cephalosporins . . . Clinical Aspects . . . . . . A . Urinary Tract Infections . . B. Respiratory Tract Infections . C . Venereal Disease . . . . D. Obstetrics and Gynecology . E . Pediatrics . . . . . . . F. Dermatology . . . . . . G . Ophthalmology . . . . . H . Meningitis . . . . . . I . Endocarditis . . . . . . J . Bones and Joints . . . . Hypersensitivity and Allergenicity A . General Considerations . . B. Conclusions . . . . . . References . . . . . . . . Addendum . . . . . . . . References to Addendum . . .

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I . Introduction In 1945. an antibiotic-producing organism was isolated (Brotzu. 1948) from the sea near a sewage outfall off the Sardinian coast . This was 83

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H$Jc(,.HA

H P p ?

0

COOH

0

’

CGOCOCH,

COOH (1)

FIG. 1. 6-Aminopenicillanic acid (I) and 7-aminocephalosporanic acid (11).

later identified as a species of Cephalosporium, which secreted material that inhibited the growth of several gram-positive bacteria but not of yeasts and molds. Subsequent studies on the Cephalosporium sp. were carried out at Oxford and at Clevedon, Somerset, England (see Abraham, 1962). An acidic antibiotic, termed cephalosporin P because it was active mainly against gram-positive bacteria, was isolated from culture fluids of this mold; however, this antibiotic differed from that described by Brotzu (Burton and Abraham, 1951), and a second, chemically unrelated acidic antibiotic was found in culture fluids from which cephalosporin P had been extracted (Crawford et a l . , 1952). This compound showed activity against gram-negative as well as against gram-positive bacteria and was thus named cephalosporin N. It was, however, subsequently found to be a new type of penicillin, since its structure was based on 6-aminopenicillanic acid (6-APA) [Fig. 1 (I)] and not on 7-aminocephalosporanic acid (7-ACA) [Fig. 1 (II)]. Cephalosporin N (penicillin N) was identical with synnematin B (Abraham et a l . , 1955), which was produced by Cephalosporium charticola and by a member of the genus Tilachlidium, the latter species subsequently appearing to be a new species of Cephalosporium salmosynnematum (Gottshall et a l . , 1951; Roberts, 1952; Olson et al., 1953). A third antibiotic, cephalosporin C (Fig. 2) was obtained during the purification of cephalosporin N (Newton and Abraham, 1955, 1956). Cephalosporin C is a true cephalosporin; its antibacterial activity was low, but it was found to be resistant to the enzyme P-lactamase (penicillinase; see Section II1,B) produced by some strains of Staphylococcus aureus, and hence to be of potential clinical importance. In addition, 7-ACA was obtained in low yield by mild acid hydrolysis of cephalosporin C, and this has been an important starting point in the production of new cephalosporins (Hale et a l . , 1961; Loder et a l . , 1961). Further chemical details are provided in Section 11. Extensive investigations by leading pharmaceutical companies in the United States and in Great Britain have resulted in the marketing of some clinically important cephalosporins; many other cephalosporins have been isolated, some of which may be added to this list. The more important cephalosporins are

T H E C E P H A L O S P O R I N GROUP OF A N T I B I O T I C S

Cephalosporin Cephalosporin C

R, HO,CCH(NH,)(CH,),C~ -

R, - OCOCH,

-&El

Cephaloridine QCHzCOCephalothin

- OCOCH,

Cephalexin

-H

Cephaloglycin

- OCOCH,

Cephacet rile

NGC-CH2CO-

Cefazolin

-OCOCH, -H

Cephradine

Cephapirin

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N>

S-CH2CO-

I

NzN h-CH2CON-=j

-OCOCH,

-S

l7ICH3 N-N

-s:

Cefamandole

N-N

II

H3C

FIG. 2. C h e m i c a l structures of t h e m o s t important cephalosporins.

considered in this paper, which deals with the chemical, microbiological, pharmacokinetic, pharmacodynamic, and clinical aspects of these drugs.

II. Chemical Aspects A. PRODUCTION The cephalosporin nucleus [7-ACA; Fig. 1 (11)] shows a close relationship to the penicillin nucleus [6-APA; Fig. 1 (I)], the main

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difference being the six-membered dihydrothiazine ring of the former in place of the five-membered thiazolidine ring of the latter, although both possess the characteristic p-lactam ring. Loder et al. (1961) succeeded in producing 7-ACA in small amounts by removing a-aminoadipic acid from the cephalosporin C molecule (Fig. 2). Later, Morin et al. (1962) described a method of obtaining better yields of 7-ACA from cephalosporin C; their procedure involved the formation of a readily hydrolyzed iminolactone by an intramolecular cyclization of cephalosporin C. Countless cephalosporins have been prepared from 7-ACA and examined for antibacterial activity. Details of the chemical evolution of the cephalosporin antibiotics are well documentated in the papers of O’Callaghan and Kirby (1970) and Flynn (1971). The first significant finding was the introduction of cephalothin (Fig. 2) (Boniece et al., 1962; Chauvette et al., 1963; Godzeski et al., 1963), which is produced by reaction of thiophene-2-acetic acid with 7-ACA. The second derivative of clinical importance, cephaloridine (Fig. 2) (Muggleton et al., 1964; Murdoch et al., 1964) is produced chemically from cephalosporin C by replacing the a-aminoadipic side-chain at position 7 by 2-thienylacetic acid, and the acetoxy group at position 3 by pyridine. Two other cephalosporins which are being used clinically are cephaloglycin and cephalexin (Fig. 2). Cephaloglycin (Wick and Boniece, 1965; Wick et al., 1971) is a synthetic analog of cephalosporin C which is well absorbed after oral administration. Cephalexin (Wick, 1967; Muggleton et al., 1969) is a semisynthetic analog of cephalosporin C in which the a-aminoadipic acid is replaced by phenylglycine, and esterlinked acetic acid is condensed to a simple methyl group. The chemical structure of cephalexin resembles that of ampicillin. More recently, other new cephalosporins have been described: cefazolin (Fig. 2) (Kariyone et al., 1970; Nishida et al., 1970a,b,c; Mine et a l . , 1970a,b; Wick and Preston, 1972) and other heterocyclic cephalosporins (Wick and Preston, 1972) are prepared from 7-ACA by substitution of heterocyclic groups; cephacetrile (CIBA 36,278-Ba), the sodium salt of 7-cyanacetamidocephalosporanic acid, is prepared from 7-ACA and the mixed anhydride of cyanacetic acid and trichloroacetic acid (Knusel et al., 1971). Further details of the chemistry of cephalosporin antibiotics may be found in the reviews of Abraham (1967), Van Heyningen (1967), Manhas and Bose (1969), Barton and Sammes (1971), and Sassiver and Lewis (1970). Enzymic synthesis of cephalexin and cephaloglycin from their corre-

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sponding organic acid esters and 7-aminocephem compounds in a single step by means of bacteria of the family Pseudomonadaceae has recently been demonstrated (Takahashi et al., 1972). The preparation of 7-ACA is described by Chauvette et al. (1972).

B. STRUCTURE-ACTIVITY RELATIONSHIPS Space does not permit a comprehensive review of the research carried out on this aspect of the cephalosporins, and what follows summarizes the excellent accounts of structure-activity relationships in the cephalosporins group provided by Abraham (1967), Van Heyningen (1967), and Sassiver and Lewis (1970). Two sites in the cephalosporin molecule [Fig. 1 (11)] have attracted particular interest among chemists. These are (1) the 7-acyl side chain, leading to the production of 7-acylaminocephalosporanic acids, e.g., ring-substituted phenylacetylcephalosporanic acids (analogous to benzylpenicillin) and ring-substituted phenoxyacetylcephalosporanic acids, members of both types being active mainly against gram-positive bacteria; and (2) the 3-acetoxymethyl side chain leading to the production of, e.g., deacetylcephalosporins and deacetoxylcephalosporins. The former have about half the activity of the parent compounds against gram-positive bacteria, such as staphylococci, but much reduced activity against gram-negative bacteria. Removal of the acetoxy group gives 3-hydroxyl derivatives that are less antibacterial than the parent acetoxy compounds (O’Callaghan and Muggleton, 1963). Cephalosporin C, cephalothin, and cephaloglycin (see Fig. 2) all lose antibacterial activity rapidly when incubated with rat liver homogenate, because of removal of the acetoxy group; however, cephaloridine and cephalexin do not possess these labile ester linkages and are, thus, not inactivated (O’Callaghan and Kirby, 1970). Flynn (1971) discusses those portions of the 7-ACA molecule [Fig. 1 (11)] which, when changed, will modify antibacterial activity. Some examples are listed in the following. a. The double bond of the dihydrothiazine ring: reduction of this bond destroys activity. Similarly, activity is lost if there is a migration of the double bond from the 3 4 position from normal A3-cephalosponns to the 2-3 position in A’-cephalosporins. b. C-7: simple epimerization of the hydrogen atom at C-7 destroys activity. c. S atom: oxidation of this, to give a sulfoxide or sulfone, destroys activity.

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d. The dihydrothiazine ring: opening of this gives an inactive molecule. At least three major components of the 7-ACA molecule [Fig. 1 (II)] are necessary for antibacterial activity (Benner, 1971; Flynn, 1971): (I) the p-lactam ring must remain chemically reactive [Van Heyningen and Aherne (1968) have ascribed the lack of antibacterial activity of A'cephalosporins to their much greater stability toward basic hydrolysis than A3-cephalosporins, since it is likely (see Fig. 3 in Section III,c1) that a covalent bond is formed by p-lactam acylation of an active site in the bacterial cell wall]; (2) definite chemical binding sites on the molecule are required; (3) the three-dimensional shape of the ring system must be maintained intact. A fourth factor, the transport (permeability ?) of a cephalosporin antibiotic to its active site in the cell is also mentioned by Flynn and Benner; this will be considered in Section III,B and C). The above has dealt very briefly with some of the structural changes that influence antibacterial activity in the cephalosporin group. The cephalosporins are not normally considered a s possessing antifungal or antimycoplasmal activity, because their mode of action is believed to involve an inhibition of bacterial cell wall synthesis (Section 111,C). There have, nevertheless, been some recent reports indicating that modification of the molecule may result in a cephalosporin with antifungal and/or antimycoplasmal activity. The introduction of a dimethyldithiocarbamate grouping at position 3 gives a cephalosporin [7(S-benzylthioacetamido)cephem-3-ylmethyl-N-dimethyldithiocarbamate-4carboxylic acid] with both activities i n vitro (Fallon, 1970; Russell and Fountain, 1971), and another cephalosporin with marked antifungal activity [sodium (N-benzyldithiocarbamoylacetamido)cephalosporanate] has been described recently by Gottstein et al. (1971); the minimum inhibitory concentration of this drug necessary against a strain of Cryptococcus neoformans was 8 &ml. The in vivo significance of these findings is difficult to assess; it is, however, a logical conclusion that, although at present such compounds may not give sufficiently high in vivo levels for systemic use, further studies should be made along these lines. Resistance to the various types of p-lactamases (Section II1,B) can be associated with the cephalosporin molecule. The resistance of cephalosporin C to staphylococcal f?-lactamase is associated with the ring system of the molecule and not with the nature of the N-acyl side chain, because cephalosporin N (penicillin N) is rapidly inactivated by the enzyme althoug!i also containing the 6-(D-a-aminoadipoyl) side chain

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(Abraham, 1967). Cephalosporins with N-acyl side chains have a high activity against most gram-positive bacteria including p-lactamaseproducing staphylococci. Cephalosporins with a 2,6-dimethoxybenzoyl side chain have a high affinity for, and are resistant to hydrolysis by, plactamases from some gram-negative bacteria, but have only a low order of activity against these organisms (Hamilton-Miller et a l . , 1965).

Ill. Antibacterial Activity A. SPECTRUM OF ACTIVITY 1. Cephaloridine Cephaloridine { 7-[(2-thienyl)acetamido1-3-(l-pyridylmethyl)-3-cephem4-carboxylic acid betaine} has a broad antibacterial spectrum, is bactericidal, and is highly active against a and P-hemolytic streptococci, pneumococci, Corynebacterium sp., penicillin-sensitive strains of Staphylococcus aureus, and various Clostridia sp. and Neisseria sp. (although N . gonorrhoeae is less sensitive than N. catarrhalis and N . menigitidis) (Barber and Waterworth, 1964; Murdoch et al., 1964; Muggleton et al., 1964; Stewart and Holt, 1964; Benner et al., 1965a; Vymola and Hejzlar, 1966; Muggleton and O’Callaghan, 1967; Newton and Hamilton-Miller, 1967; Gonnella et al., 1967; Perkins et a l . , 1967b; Turck et al., 1967; Hewitt and Parker, 1968; Stratford and Dixson, 1968; Acar, 1971; Cox and Montgomery, 1971; Kayser, 1971). Enterococci show a low degree of sensitivity (Kislak et a l . , 1966; Naumann, 1967), and penicillinase (p1actamase)-producing strains of S . aureus are less sensitive to cephaloridine than are non-p-lactamase producers; much higher concentrations of the drug are needed to inhibit the growth of large inocula of the p lactamase producers although small inocula are very sensitive (Benner et al., 1965a; Ridley and Phillips, 1965; Kislak et al., 1966; Editorial, 1967; Hewitt and Parker, 1968; Eykyn, 1971; Kayser, 1971; Russell, 1972a,b,). Cephaloridine and cephalothin are some 2 4 times more active than cloxacillin against Actinomyces israelii strains, but Actinomyces bovis is much less sensitive (Lerner, 1968). Methicillin-resistant strains of staphylococci frequently pose a serious clinical problem in hospitals (Finland, 1972; Kayser et al., 1972). The sensitivity of methicillin-resistant S. aureus to p-lactam antibiotics depends markedly on the test conditions, e.g., incubation at 30°, or the

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presence of 5% w/v sodium chloride in the culture medium (see Russell et a l . , 1973). Methicillin-resistant staphylococci are resistant to cephalosporins (Chabbert, 1967a,b), although cephaloridine is considered to be more active than other cephalosporins against such strains (Hallander and Laurell, 1972). It is, however, highly unlikely that cephaloridine is a practical in vivo proposition because of the high blood levels which would have to be attained. Activity of cephaloridine against some strains of Mycoplasma has also been described (Fallon and Hutchinson, 1967; Stewart et a l . , 1969) although generally concentrations of at least 100 pg/ml may be needed to achieve an inhibitory effect. Cephaloridine also shows a high degree of activity against many different species of gram-negative bacteria, and bactericidal levels are generally not much higher than minimum inhibitory concentrations (MIC’s) (Murdoch et a l . , 1964; Muggleton et a l . , 19M; Thornton and Andriole, 1966; Muggleton and O’Callaghan, 1967; Perkins et a l . , 1967b; Turck et a l . , 1967) although minimum bactericidal concentrations (MBC’s) of 16 to 64 times the MIC’s have been recorded in some cases (Gonnella et a l . , 1967). Indole-positive Proteus sp. are resistant to cephaloridine and to cephalexin and cephalothin, whereas the majority of Proteus mirabilis strains are sensitive. Pseudomonas aeruginosa is highly resistant to all cephalosporins, but Escherichia coli, Salmonella sp. (Adams and Nelson, 1968), and Shigella sp. (Nelson and Haltalin, 1972) are sensitive to cephaloridine, and Neisseria meningitidis and Neisseria catarrhalis are highly sensitive (Muggleton and O’Callaghan, 1967; Eykyn, 1971). P-Lactamase-producing gram-negative bacteria may inactivate cephaloridine (Muggleton et a l . , 1964; Hamilton-Miller et a l . , 1965; O’Callaghan and Muggleton, 1967) and this aspect is considered later in this section.

2 . Cephalothin The sodium salt of 7-(2-thienylacetamido)cephalosporanic acid, cephalothin is an important member of the cephalosporin group of antibiotics, with a fairly broad spectrum of activity (Godzeski et al., 1963). It was used clinically before cephaloridine had been discovered, but its relatively greater resistance to staphylococcal p-lactamase was not observed until some time later. It is less active than cephaloridine but rather more active than cephaloglycin or cephalexin against non+ -

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lactamase-producing Staphylococcus aureus strains, pneumococci, and a- and P-hemolytic streptococci (Kayser, 1971); cephalothin is bactericidal in its action against gram-positive bacteria, with the MBC generally being about twice the MIC. Low concentrations of cephalothin are inhibitory to both p-lactamase and non-p-lactamase-producing strains of S. aureus (Boniece et al., 1962). Cephaloridine is more active than cephalothin against penicillin-sensitive S. aureus strains and against small inocula of p-lactamase-producing S. aureus and has a greater bactericidal effect (Perkins et al., 1967b), but the MIC’s of cephalothin are less affected by changes in inoculum size of the latter organisms than are ampicillin (Sherris et a l . , 1967) or cephaloridine (Benner et a l . , 1965a; Hewitt and Parker, 1968; Russell, 1972a,b). However, inoculum size has a very marked effect on the MBC’s of cephalothin (Turck et a l . , 1965). Enterococci are resistant to cephalothin (Walters et al., 1964; Naumann, 1967; Eykyn, 1971). A close association between methicillin resistance and resistance to various cephalosporins. including cephalothin, with heteroresistance (Sutherland and Rolinson, 1964) a typical trait, has recently been observed (Hallander and Laurell, 1972). Methicillin-resistant strains are less sensitive to cephalothin than to cephaloridine (Barber and Waterworth, 1964; Benner and Morthland, 1967). Cephalothin is inhibitory and bactericidal to many types of gramnegative bacteria, but these are less sensitive and more variable than gram-positive bacteria (Walters et al., 1964; Turck et al., 1965). Barber and Waterworth (1964) suggested that there was little difference between cephalothin and cephaloridine in their activity against coliforms, but cephaloridine is rather more inhibitory against Escherichia coli and P . mirabilis (Turck et a l . , 1965, 1967; Perkins et al., 196713). Pseudomonas aeruginosa and indole-positive Proteus strains are highly resistant to cephalothin (Turck et al., 1965; Naumann, 1967). Some authorities consider cephalothin as having a low degree of activity against Klebsiella-Aerobacter strains (Turck et al., 1965; Steigbeigel et al., 1967); however, there is evidence that nearly all the motile strains (Aerobacter) in this group are highly resistant to cephalothin and cephaloridine, whereas nonmotile strains (Klebsiella) are sensitive to cephalothin and cephaloridine especially the former (Benner et ul., 1965b; Lerner and Weinstein, 1967; Kunin and Brandt, 1968). Cephalothin is inhibitory to Shigella strains (Nelson and Haltalin, 1972). The effects of some types of p-lactamases from gram-negative bacteria on cephalothin are considered in Section II1,B.

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3. Cephalexin The monohydrate of 7-(Diu-aminophenylacetamido)-3-methyl-3-~ephem-4-carboxylic acid, cephalexin is one of the newer group of cephalosporin antibiotics, and it is of particular interest because it is given orally. Details of its absorption, excretion, and tissue distribution and of its clinical efficacy are described later (Sections IV, V, and VI). Cephalexin is considered to be a broad-spectrum antibiotic (Griffith and Black, 1968), although its in vitro activity may be less than that of cephaloglycin (Wick, 1967; Braun et al., 1968; Kayser, 1971); however, cephaloglycin is incompletely absorbed after oral administration (Applestein et al., 1968) and higher blood and urine levels are obtained after orally given cephalexin (Wick, 1967). The majority of S. aureus strains are considered by Leigh et al. (1970) to be highly sensitive to cephalexin, and Thornhill et al. (1969) found that 88% of such strains were inhibited by 6.3 pg/ml and 100% inhibited by 12.5 pg/ml of cephalexin. Strains of a- and P-hemolytic streptococci, Streptococcus pneumoniae, gonococci, and meningococci are moderately to highly susceptible to cephalexin (Braun et al., 1968; Muggleton et al., 1969; Kayser, 1971). Despite being less active than cephaloridine against most bacteria (Hoeprich, 1968; Levison et al., 1969; Muggleton et al., 1969; Bond et a l . , 1970; Eykyn, 1971; James and Walker, 1971), cephalexin is more active than cephaloridine against N. gonorrhoeae (Muggleton et al., 1969). Cephalexin has a bactericidal action, and is considered to be equally effective against penicillin-sensitive and p-lactamase-producing strains of S. aureus (Wick, 1967). However, although it is more active than ampicillin against the latter strains and although it is more resistant than cephaloridine to destruction by p-lactamase-producing staphylococci and gram-negative bacteria, Muggleton et al. (1969) do not consider this is of a sufficient extent to make cephalexin effective against these gram-negative organisms. Some inoculum effect occurs with p-lactamase-producing staphylococci but to a much smaller extent than with cephaloridine (Eykyn, 1971). Methicillin-resistant strains of staphylococci are resistant to cephalexin (Kayser, 1971; Russell, 1972a,b). Gram-negative bacteria are considerably less sensitive to cephalexin than is S. aureus (Leigh et al., 1970), and strains of Haemophilus influenzae and many common gram-negative bacilli are moderately to highly resistant (Braun et a l . , 1968). Of Proteus strains, P . mirabilis is the most sensitive (Levison et al., 1969; Thornhill et al., 1969; Leigh et al., 1970); Thornhill et al. (1969) reported that in their studies, 56% of P . mirabilis strains, 80% of E. coli strains, and 72% of Aerobacter-

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Klebsiella strains were inhibited by 12.5 pglml of cephalexin. Pseudomonas aeruginosa and indole-positive Proteus strains are resistant (Thornhill et a l . , 1969; Russell, 1972a,b). A marked inoculum effect is noted with the activity of cephalexin against various types of gramnegative bacteria (Fountain and Russell, 1969; Muggleton et a l . , 1969; Bond et al., 1970; Russell, 1972a,b) but this does not appear to be associated with /I-lactamase production and drug destruction. Subinhibitory concentrations of cephalexin induce very long filamentous forms in various types of gram-negative bacteria (Fujii et al., 1969; Muggleton et a l . , 1969; Nakazawa et a l . , 1969; Russell and Fountain, 1970).

4. Cephaloglycin The first oral cephalosporin, cephaloglycin [7-(DaYaminopheny1acetamido)-3-acetoxymethylcephem-4-carboxylicacid] was described by Wick and Boniece (1965). It is a bactericidal antibiotic, the MBC generally being about twice the MIC for most groups of gram-positive and gram-negative bacteria (Johnson et a l . , 1968; Kayser, 1971). It is about twice as active against penicillin-sensitive staphylococci as against p-lactamase producers (Kayser, 1971; however, cf. Pitt et al., 1968). Cephaloglycin is inhibitory to a- and P-hemolytic streptococci, staphylococci, and pneumococci (Applestein et al., 1968; Johnson et a l . , 1968; Kayser, 1971). Cephaloglycin is also inhibitory and bactericidal to E. coli and P . mirabilis, with rather higher drug concentrations necessary for activity against the Klebsiella-Aerobacter group (Applestein et al., 1968; Pitt et al., 1968). Johnson et al. (1968) found cephaloglycin to be more active than tetracycline, ampicillin, and cephalothin, and of equal activity to chloramphenicol and colistin, against E. coli, whereas it was less active than ampicillin, cephalothin, and kanamycin against P . mirabilis. Waterworth (1971) showed that cephaloglycin was more active than other cephalosporins against some types of gram-negative bacteria. Pseudomonas aeruginosa is resistant to cephaloglycin (Pitt et al.,, 1968). It must be added that the activity of cephaloglycin is markedly pHdependent, with a much greater bactericidal effect at acid pH (Kunin and Brandt, 1968). It is unstable at alkaline pH (Wick and Boniece, 1965), and the rates of degradation in trypticase soy broth and in nutrient broth have been stated to be 14% per hour and 3.5% per hour, respectively (Applestein et al., 1968). Differences in MlC ranging from two- to eightfold occur when incubation at 37" is extended from 12 to 18 hours (Pitt et al., 1968) and this fact must be taken into account in considering MIC values. After oral administration, cephaloglycin is

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partially converted in man to a biologically active metabolite, deacetylcephaloglycin, which is as active as cephaloglycin against gram-positive bacteria but less active against gram-negative bacteria (Wick et al., 1971).

5 . Other Cephalosporins New cephalosporins continue to be developed. Of these, the following are worthy of comment. a . Cephacetrile (CIBA 36,278-Ba; CAA). The sodium salt of 7cyanacetamidocephalosporanic acid, this cephalosporin derivative is less active than cephaloridine against benzylpenicillin-sensitive staphylococci, but is more active than cephaloridine against p-lactamaseproducing strains; it is less active than cephaloridine and ampicillin against non+-lactamase-producing strains of E. coli, but is more active against E. coli R+ TEM (Kniisel et al., 1971; Russell, 1972a,b). Gramnegative p-lactamase-producing strains produce a red color when incubated in broth containing cephacetrile and this has been suggested as being of possible use in the detection of such organisms (Russell, 1972~).Cephacetrile is without effect on P. aeruginosa. b. Cephradine [Velosef (Squibb)].* There is, as yet, insufficient evidence on which to base an assessment of the usefulness of this new oral antibiotic. Limson et al. (1972) have listed the MIC values of cephradine against various gram-positive and gram-negative bacteria; the MIC against a penicillin-sensitive strain of S. aureus (inoculum size lo6 viable cellslml) was 3.1 and 18.7 p d m l against a comparable inoculum of a @-lactamase producer. Low inocula (lo3 viable cells/ml) of some gram-negative strains were sensitive to about 10 pg/ml or less of cephradine, but P . aeruginosa was resistant. According to Klastersky et al. (1973), cephradine inhibits most strains of Pneumococcus and Staphylococcus, as well as S. pyogenes. Landa (1972) has shown that oral administration to patients with infections caused by salmonellas and shigallae gave an 82% cure rate with no side effects. c. Cephapirin. A new cephalosporin antibiotic, cephapirin is administered parenterally (Gordon et al., 1971; McCloskey et al., 1972). As is usual with cephalosporins, it is more active against gram-positive bacteria than gram-negative bacteria (Wiesner et al., 1972), with an activity equivalent to that of cephalothin (Bran et al., 1972). Wiesner et al. (1972) found that all of their test strains of penicillin-sensitive and p -

* Further details are listed in the technical information published by E. R. Squibb & Sons, Ltd.

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g lactamase-producing staphylococci were inhibited and killed by 5 ~ r or less of cephapiridml against an inoculum of l o 4 or lo6 viable organisms/ ml, although an inoculum effect with S. aureus and various strains of gram-negative bacteria was also observed. Cephapirin is bactericidal to E. coli, pneumococci, and P . mirabilis but has negligible activity against P . aeruginosa and indole-positive Proteus strains. d . Cefazolin. Also a new cephalosporin antibiotic (Kariyone et al., 1970; Mine et al., 1970a,b; Nishida et a l . , 1970a,b), cefazolin shows promise clinically (Shibata and Fujii, 1971). It is more active against gram-positive than gram-negative bacteria, and its antibacterial spectrum includes @-lactamase-producing strains of S. aureus, although Group D streptococci are resistant, as are P . aeruginosa, indole-positive Proteus strains, and nonpigmented Serratia strains (Wick and Preston, 1972). Cefazolin is inhibitory to N . gonorrhoeae and N . meningitidis, but methicillin-resistant strains of S. aureus require high concentrations of cefazolin for inhibition, especially when sodium chloride is included in the medium. Cefazolin is less active than ampicillin or cefamandole (see below) against strains of Haemophilus influenzae. Overall, cefazolin could well prove to be a useful addition to the cephalosporin group of antibiotics. e . Cefamandole. This antibiotic was described by Wick and Preston (1972) as CMT. It shows activity against S. aurem, streptococci, Neisseria, Clostridia, and Corynebacterium and is more active than cefazolin against gram-negative bacteria including E. coli, Enterobacter sp., indole-positive Proteus, Salmonella sp., and Shigella sp. although P . aeruginosa and Serratia marcescens strains are resistant. The antiProteus activity is particularly interesting, and evidence has been presented to show that cefamandole is considerably more stable to cephalosporinase than is cephalothin. Cefamandole is very active against H . influenzae. f. Comments. Continuing research in the cephalosporin field has led to the development of some new and interesting antibiotics. Provided that it is shown to be clinically effective (see Wick and Preston, 1972), and if the preliminary findings are confirmed, cefamandole may prove to be a valuable addition to the cephalosporin range. Further research is, however, needed to produce a cephalosporin that combines high activity against gram-positive bacteria with potent bactericidal action against gram-negative bacteria, including P . aeruginosa and S . marcescens. It must also be pointed out that the type of assay medium used in determining the sensitivity of bacteria to cephalosporins may greatly influence the observed results (Pursiano et a l . , 1973). This finding could

96

D. R. OWENS ET AL.

be one reason in explaining the variable results which may occur from laboratory to laboratory.

B. BACTERIAL RESISTANCE 1. Development of Resistance When bacteria are, repeatedly subcultured in media containing gradually increasing concentrations of a cephalosporin, they frequently show an increase in resistance to that particular drug and to other p lactam antibiotics. Penicillin-resistant staphylococci may be selected in this manner in the laboratory, but the lack of emergence of such strains (as opposed to p-lactamase-producing ones) may be the result of the relatively low mutation rates in such organisms, because the levels of penicillin in the body will greatly exceed the sensitivity of both the original bacteria and first-step mutants as they arise (Rolinson, 1971). Generally, the type of resistance acquired to cephalosporins is of a similar, stepwise type (Jago, 1964; Bernard and Lambin, 1965; Ott and Godzeski, 1967; Fountain and Russell, 1970). Godzeski et al. (1963) found that gram-negative bacteria developed a stepwise resistance to cephalothin, whereas there was no in vitro development of resistance with S. aureus; gram-negative bacteria passaged in cephaloridine or benzylpenicillin have been reported to have a considerable increase in resistance to the antibiotic through which they were passaged and a moderate increase to the other antibiotic (Barber and Waterworth, 1964). Emergence of cephalexin-resistant S. aureus by repeated subculture has been reported (Bond et al., 1970) with as few as three steps giving variants resistant to high cephalexin concentrations (Kayser et al., 1970; Kayser, 1971), although Muggleton et a l . (1969) have proposed that there is no rapid emergence of organisms resistant to this drug. The manner in which these organisms with acquired resistance differ from the original bacterial population is by no means clear, although there are two distinct possibilities. (1) Alterations in the amount of p-lactamase produced. This could be applicable to a strain that originally produces no p-lactamase but does so when resistance has developed, or to a strain that produces an increased amount of fI-lactamase when resistance has been acquired. There is little evidence in support of this hypothesis (also see below) since cultures that have developed resistance to a penicillin or cephalosporin may not inactivate that drug (Ott and Godzeski, 1967; Park et a l . , 1971). Barber and Waterworth (1964) found that E. coli and

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97

P . mirabilis passaged in cephaloridine or benzylpenicillin showed no

change in B-lactamase activity, whereas Klebsiella aerogenes, passaged similarly, possessed an increased p-lactamase activity. (2) Changes in cell wall composition. There is, at present, insufficient information available on this topic for sound conclusions to be made. However, it has been found (Fountain and Russell, 1970) that E . coli made resistant to cephaloridine showed cross-resistance to other plactam antibiotics, that the resistant strain produced no P-lactamase, but that, significantly, ethylenediaminetetraacetic acid (EDTA) potentiated the effect of cephaloridine against this, but not the parent, strain. These findings suggest that changes in outer lipid layers might have occurred, a conclusion similar to that reached with carbenicillinresistant strains of P . aeruginosa (Thomas and Broadridge, 1972). Inaccessibility of the total murein transpeptidase to penicillin is one possible reason for the development of penicillin-resistant mutants of aureus strain H (Park et al., 1971).

s.

2. Cross-resistance An organism that has acquired resistance to one cephalosporin may be expected also to show cross-resistance to other members of this group. This need not be, necessarily so, however. S. aureus strains (Blactamase producers or not) made resistant to cephalexin also showed some cross-resistance with methicillin, whereas their susceptibility to cephalothin and cephaloridine was only decreased slightly (Kind et al., 1969a,b). In contrast, Kayser et al. (1970) showed that S. aureus could be induced to acquire intrinsic resistance to cephalexin with a concomitant increase in resistance to cephalothin and to the two Blactamase-stable penicillins, oxacillin and dicloxacillin. A marked crossresistance between cephalexin and methicillin in some naturally occurring methicillin-resistant strains has been observed (Kind et al., 1969b). Ott and Godzeski (1967) found extensive cross-resistance when S. aureus was cultured to higher levels of resistance with oxacillin, cephalothin, or cephaloglycin, but there was a marked lack of consistency among the various cultures in their response to the related antibiotics; the least cross-resistance was shown to cephaloridine, which was also the antibiotic with the least ability to give an increase in resistance level by serial passage. Staphylococci that had acquired in vitro resistance to one derivative of 7-ACA also showed strong cross-resistance to the other derivatives (Jago, 1964). In the case of gram-negative bacteria, some degree of cross-resistance is shown in a culture made resistant to one p-lactam antibiotic (Barber

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and Waterworth, 1964; Ott and Godzeski, 1967; Fountain and Russell, 1970; Russell and Fountain, 1970). 3. Effect of P-Lactamases on Cephalosporins P-Lactamase (penicillinase, cephalosporinase, penicillin amidoj3-lactam hydrolase; E.C.3.5.2.6.) is widely distributed among bacteria. It is extracellular with gram-positive bacteria and it is inducible in grampositive, but not gram-negative, bacteria (an exception occurs with P. aeruginosa, as described later; see also Mildvan et a l . , 1973). In gramnegative bacteria, p-lactamase is cell-bound, and these organisms may possess an “accessibility barrier” to many, but not all, p-lactam drugs so that /3 -1actamase activities in disrupted cell preparations may be several times greater than in intact cells (Hamilton-Miller et a l . , 1965; Smith et a l . , 1969). Most varieties of p-lactamases will hydrolyze most p-lactam antibiotics, but this hydrolysis proceeds at widely divergent rates, depending on the enzyme and the drug. p-Lactamase production may be either constitutive or it may be induced by the presence of a p-lactam antibiotic, and the enzyme activity may be transduced by phage in S. aureus (Richmond, 1965). Sabath et al. (1965), Hamilton-Miller et al. (1970a,b), and O’Callaghan et a l . (1972a) have shown that enzymic hydrolysis of the cephalosporins results in an opening of the p-lactam ring accompanied by expulsion of R’ (Fig. 2) as acetate (from cephalothin) or pyridine (from cephaloridine) with a concomitant disappearance of the characteristic UV absorption. This is replaced with an absorption band (A,, 230 nm) which itself disappears in turn after several hours (Sabath et al., 1965). However, opening of the p-lactam ring without these other changes occurs with deacetylcephalosporin C lactone, and the resulting product has a A,,, of 265 nm. In contrast to the relatively stable D-a-penicilloates produced by the action of p lactamases on penicillins, cleavage of the p-lactam rings of cephalosporins thus involves rapid initial changes in UV absorption spectra with further extensive degradation of cephaloridine and cephalosporin C (Newton et al., 1968). Hamilton-Miller et a l . (1970b) used proton magnetic resonance to study the nature of the compound with ,,,A of 230 nm and showed that the same compound with this Amax was formed from both cephalosporin C and deacetylcephalosporin C on enzyme hydrolysis. The /3 -1actamases from gram-positive and gram-negative bacteria differ to a considerable extent (Pollock, 1965, 1967, 1971; Citri and

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99

Pollock, 1966; Citri, 1971), and their effects on cephalosporins will thus be considered separately. a. p-Lactamases from Gram-Positive Bacteria. (i) Staphylococcal plactamases. In one of the original papers on cephaloridine, Murdoch et a l . (1964) reported that this compound was 100-1000 times more resistant than phenoxymethylpenicillin (penicillin V) to staphylococcal plactamase and, thus, that, for practical purposes, cephaloridine was unaffected by this enzyme. However, as has been pointed out earlier, cephaloridine is more sensitive than cephalothin or cephalexin to changes in inoculum size of P-lactamase-producing strains of S. aureus (Barber and Waterworth, 1964; Benner et a l . , 1965a; Ridley and Phillips, 1965; Kislak et al., 1966; Seligman and Hewitt, 1966; Newton and Hamilton-Miller, 1967; Thompson et al., 1967; Hewitt and Parker, 1968; Knusel et al., 1971; Russell, 1972b). Benner et a l . (1965a) found that many p-lactamase S. aureus producers required much larger amounts of cephaloridine than cephalothin for inhibition when large inocula were used; this has been claimed to be the most important reason for the failure of the former antibiotic in some staphylococcal infections (Seligman and Hewitt, 1966; Editorial, 1967; Hewitt and Parker, 1968). However, an alternative reason could be an increase in intrinsic resistance of the cells (Hamilton-Miller, 1967a,b). In support of this is the finding that, although cephaloridine is much more rapidly hydrolyzed by staphylococcal p-lactamase than cephalothin (HamiltonMiller and Ramsey, 1967; Muggleton and O’Callaghan, 1967) or cephalexin (Eykyn, 1971), it is, in fact, hydrolyzed at a rate of only 0.10.2% of that of benzylpenicillin (Hamilton-Miller, 1967a,b; Naumann, 1967; Knusel et al., 1971) (Table I). Cefazolin appears to be rather more, and cefamandole (CMT) less, susceptible than cephaloridine to staphylococcal P-lactamase (Wick and Preston, 1972). Several derivatives of 7-ACA behave as competitive inhibitors of the action of staphylococcal p-lactamase on penicillins (Crompton et al., 1962) although this does not apply to cephalosporin C and deacetylcephalosporin C (K,IK, values being >lo0 and >50, respectively); however, N-phenylacetyl-7-ACA, with a K J K , value of 1.7 x lo-’, is a powerful inhibitor (Abraham and Newton, 1956, 1961; Crompton et a l . , 1962). Methicillin ( 0 . 5 2 pglml) is a good inducer of staphylococcal p lactamase; negligible induction occurs with cephalosporin C at concentrations of 1 d m l although considerable induction occurs with higher concentrations (5 pg/ml or more) (Swallow and Sneath, 1962). All compounds tested that induce p-lactamase formation in Bacillus cereus

c,

0 0

TABLE I SUBSTRATE PROFILES OF SOME ~ L A C T A M A S E S " . ~ PLactamase source

Pen

Amp

Meth

Clox

Carb

Cer

Cet

Cex

CAA

Staphylococcus aureus 8325 i+p+

100 100

313 220 134 128 120 150

0.7 0.23 3.8 1 1

-

-

0.4 0.22 4.6 158 150 175

0.1 0.1 3.3 15.6

-

0.3 0.26 4.4 25

S.aureus 2999 i+p+ B. cereus 569 i+p+

Escherichia coli R+ TEM E. coli 53 Salmonella typhi 152, E. coli 1 E M or E . coli K-12 RTEMR-factor E. coli K-12 R,,,,R-factor E . coli K-12 R,,,,R-factor Enterobacter cloacae 214 (P99) Pseudomonas aeruginosa Enzyme Type I (inducible) Enzyme Type I1 (R-factor) Enzyme Type I11 (Dalgleish) Proteus morganii (2 strains)

+

+ +

100

100 100 100 100 100 100 100 100 100 100

91 100 1

10 160 100

-

2

-

21 0.1 1

126 1.5 -

-

-

-

-

-

0 10 150 -

-

-

86 145 8600 400 125 40 c.325

-

15 46 17 1250

-

-

c.220

-

-

-

-

780 140 0 0

-

-

-

' I Data of Datta and Kontomichalou (1%5), Hamilton-Miller (1967a,b), Hennessey and Richmond (1%8), Jack and Richmond (19701, newsom et al. (1970),Sykes and Richmond (1971),Kniisel et a l . (1971). " Abbreviations: Pen, benzylpenicillin; Amp, ampicillin; Meth, methicillin; Clox, cloxacillin; Carb, carbenicillin; Cer, cephaloridine; Cet, cephalothin; Cex, cephalexin; CAA, 7-cyanacetamidocephalosporanic acid.

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101

(Pollock, 1957; see below) will induce p-lactamase production in S. aureus. Nevertheless, there are striking differences in that the relative efficiencies of the inducer vary greatly from one organism to the other (Crompton et al., 1962). (ii) Bacillus cereus p-lactamase. Abraham and Newton (1956) showed that a crude p-lactamase preparation from B. cereus contained only 1/ 100 as much cephalosporinase activity as penicillinase activity; crude supernatant fluids from cultures of this organism, strain 569, had only about 1/20 as much activity against cephalosporin C as against benzylpenicillin, and Zn was found to be a cofactor for the cephalosporinase, but not for the penicillinase, activity of crude p-lactamase from B . cereus 569 and 569H (Sabath and Abraham, 1966; Sabath and Finland, 1968). The maximal rate of hydrolysis of several derivatives of 7-ACA by B. cereus p-lactamase is very low, with the exception of deacetylcephalosporin C lactone for which the rate of hydrolysis is of the same order as for benzylpenicillin (Crompton et al., 1962). The y-type p-lactamase from B. cerem 569H has been extracted and purified by Citri and Kalkstein (1967), who compared the substrate profiles of the y - and atype p-lactamases. The relative rates of hydrolysis of cephaloridine and methicillin by the y-type enzyme were very high, but were much lower with the y-type. Cephalothin and cephalosporin C were not hydrolyzed to any significant extent by either enzyme. Pollock (1956) had earlier shown that two types of cell-bound p-lactamases, the p - and y-types, were produced by this organism. The p-kype is thought to be identical with the a-type and with the p-lactamase Type I of Kuwabara and Abraham (1967, 1969); p-lactamase Type I1 of these authors is a Znrequiring enzyme that is active against a variety of cephalosporins and penicillins. Cephaloridine is more susceptible to B. cereus p-lactamase than cefazolin, cephalothin, and cephamandole (Wick and Preston,

1972). Unlike the case with the crude extracellular S. aureus enzyme described in the foregoing, cephalosporin C is a competitive inhibitor of B. cereus on benzylpenicillin (Abraham and Newton, 1956, 1961), and the general pattern of values for KiIK, with this enzyme and cephalosporin C, deacetylcephalosporin C, cephalosporin CA (pyridine), and N phenylacetyl 7-ACA falls within the range of 1 to 5. Cephalosporin C in optimal concentration is a better inducer of plactamase in B. cereus than is benzylpenicillin, although a higher concentration of the former compound is needed to produce a maximal effect (Pollock, 1967). Deacetylcephalosporin C behaves similarly to

102

D. R. OWENS ET AL.

cephalosporin C, but cephalosporin C A (pyridine) has a higher maximum inducing activity than cephalosporin C (Crompton et al., 1962). Some practical laboratory uses of these p -1actamases have recently been described; various types of p-lactamases may be used for inactivating cephaloridine and cephalothin, e.g., in assays of cephalosporin-noncephalosporin mixtures (Newson and Walsingham, 1973), and a highly purified p-lactamase (Neutrapen) from B. cereus may be employed (1) as an inactivating agent for cephaloridine in carrying out viable counts of bacteria treated with this antibiotic and (2) a s a means of carrying out sterility tests on cephaloridine and possibly cephalothin (Russell and Furr, 1973). Cephalexin is highly resistant to inactivation by this enzyme (Russell and Furr, 1973). b. p-Lactamases from Gram-Negative Bacteria. The situation with p lactamases from gram-negative bacteria is exceedingly complex (Richmond and Sykes, 1973) and at least eight distinct types of @-lactamase from gram-negative bacteria, apart from P. aeruginosa, are known (Jack and Richmond, 1970). High concentrations (5-10 mg/ml) of benzylpenicillin or cephalosporin C are needed to induce the enzyme in P. aeruginosa (Sabath et al., 1965; Sykes and Richmond, 1971). The best inducer of p-lactamase in strains of this organism appears to be 6-APA, and cephalothin the worst (Garber and Friedman, 1970). Significantly, carbenicillin appears to be resistant to the inducible p-lactamase enzyme in these organisms and, in fact, inhibits their activity against cephalosporins (Bobrowski and Borowski, 1971). A small number of P. aeruginosa strains produce a constitutive p lactamase, i.e., in the absence of inducer, and three types of plactamase have been identified (Sykes and Richmond, 1971) in these organisms . Type I: the p-lactamase produced is inducible and is common among p . aeruginosa strains. It is predominantly a cephalosporinase and rapidly hydrolyzes cephaloridine and cephalexin, whereas carbenicillin is resistant (also see above). Type 11: a constitutive p-lactamase of the R-factor-mediated type found in enteric bacteria. Cephalexin is not inactivated and carbenicillin only slightly so. Strains of P. aeruginosa producing this enzyme also produce Type I enzyme. Type 111: one strain (Dalgleish) produces a p-lactamase that is constitutive, but which is quite distinct from Type I1 and markedly inactivates carbenicillin. Cephaloridine is less labile, and cephalexin is resistant. This strain also produces an inducible Type I p-lactamase. The substrate profiles of these enzymes against various p-lactam

103

T H E CEPHALOSPORIN G R O U P OF ANTIBIOTICS

TABLE I1 SENSITIVITY TO SOME8-LACTAM ANTIBIOTICS OF Escherichia coli K-12 INFECTED WITH DIFFERENTR-FACTORS~ MIC (pglml) of

Escherichia coli

K-12 + K-12 + K-12 + K-12 + K-12 +

TEM

1818 1818 (cured) 7268 7268 (cured) Original K-12

+ R-factor

Pen

Amp

Cer

4000 250 200 1000 1000 30

4000 250 200 1000 1000 2

60 4 4 8 8 1

Data of Datta and Kontomichalou (1965). Inoculum size throughout, 10’ viable c e l l s h l . MIC, minimum inhibitory concentration; Pen, benzylpenicillin; Amp, ampicillin: Cer, cephaloridine. a

drugs, relative to benzylpenicillin, are shown in Table 11. The plactamase activity against cephaloridine of sonically disrupted P . aeruginosa cells is the same as whole cells, suggesting that this antibiotic readily penetrates all strains (Bobrowski and Borowski, 1971). The b-lactamases of the Enterobacteriaceae are not inducible but present an even more complex picture (Fleming et al., 1963, 1970; Jack and Richmond, 1970; Richmond, 1972; Richmond and Sykes, 1973). Extensive studies of the effects of these p-lactamases on penicillins and cephalosporins have been made (Jago et al., 1963; Ayliffe, 1964, 1965; Hamilton-Miller and Smith, 1964; Hamilton-Miller et al., 1965; Datta and Kontomichalou, 1965; Datta and Richmond, 1966; Hamilton-Miller, 1967a,b; Muggleton and O’Callaghan, 1967; O’Callaghan and Muggleton, 1967; O’Callaghan et al., 1967, 1968, 1969, 1972a,b; Hennessey and Richmond, 1968; Sykes and Richmond, 1970; Kniisel et al., 1971; Marshall et al., 1972; O’Callaghan and Morris, 1972; Wick and Preston, 1972; Russell, 1972c; Sykes and Nordstrom, 1972; Anderson and Sykes, 1973). Examples of the substrate profiles of the various types of p lactamases on cephalosporins and penicillins, relative to benzylpenicillin, are provided in Table I. The p-lactamase from Enterobacter cloacae strain 214 (believed to be identical with strain P99) has been isolated and purified; this enzyme hydrolyzes cephaloridine, cephalothin, and cephalosporin C at rates 80, 10, and 70 times, respectively, as rapidly as

104

D. R. OWENS ET AL.

benzylpenicillin, but ampicillin is not inactivated (Hennessey and Richmond, 1968). Nevertheless, this strain is highly resistant to ampicillin in terms of MIC values (Russell, 1974), although changes in morphology are induced over a short incubation period (Russell, 1973). There is no transfer of p-lactamase in this organism, and the gene specifying this type of enzyme is chromosomal (Jack and Richmond, 1970), in contrast to E. coli R+ TEM (Datta and Kontomichalou, 1965; Datta and Richmond, 1966; Jack and Richmond, 1970; see also Matsuhashi, 1971). Enterobacter cloacae P99 and Klebsiella aerogenes strain 1082E (also known as K1) are considered to represent extremes of cephalosporinase and penicillinase activity, respectively (Marshall et a l . , 1972). E . coli strain K-12 is sensitive to ampicillin and cephaloridine, and fairly sensitive to benzylpenicillin. However, when R-factors from 3 donors are transferred to K-12, a different sensitivity pattern emerges (Table 11) (Datta and Kontomichalou, 1965). The MIC values of cephaloridine in this table for K-12 infected with R-factors 1818 and 7268 are not very high, however, being some 4-8 times than for the original K-12 strain, whereas the substrate profiles in Table I indicate that this antibiotic is hydrolyzed at least as rapidly as ampicillin or benzylpenicillin. Medeiros and OBrien (1968) have shown that R-factors from different isolates mediate different levels of enzymic activity in the same recipient strains. Interestingly, their experimental findings also illustrate that increases in resistance of recipients to cephalosporins (and especially cephaloridine) are not necessarily high. Cephaloridine is believed to penetrate gram-negative bacteria readily, the p-lactamase activity of disrupted cell penetrations against this antibiotic being the same as that of whole cell suspensions (HamiltonMiller et a l . , 1965). This ratio, termed the permeability factor by Hamilton-Miller et a l . (1%5), is thus 1 for cephaloridine, in contrast to values of 1-18, 1-14, and 1-3 for benzylpenicillin, ampicillin, and 6APA, respectively, as found by these authors. Staphylococcal p-lactamase-stable penicillins, such as methicillin, cloxacillin, quinacillin, and nafcillin, may act as competitive inhibitors of p-lactamases produced by gram-negative bacteria on readily hydrolyzable substrates (Sabath et at!., 1965; Hamilton-Miller et a l . , 1965; O’Callaghan and Muggleton, 1967; O’Callaghan and Morns, 1972). Cloxacillin, methicillin, and nafcillin inhibit a cell-free P99 enzyme against cephaloridine, whereas only the first two penicillins “protect” cephaloridine from a cell-free R+ TEM P-lactamase, with only nafcillin inhibitory to a cell-free K. aerogenes K-1 enzyme (O’Callaghan and

T H E CEPHALOSPORIN GROUP OF ANTIBIOTICS

105

Morris, 1972). The situation may, nevertheless, be rather different with whole cells, the degree of synergism being less than might be expected (O’Callaghan and Morris, 1972) and varying with difference in inoculum size (O’Callaghan and Morris, 1972; Russell, 1974). Destruction by plactamase, although an important factor in the resistance of gramnegative bacteria to cephalosporins, is most certainly not the only reason for their resistance (Sabath and Finland, 1967; Madeiros and O’Brien, 1968; Garber and Friedman, 1970) and accessibility of the sensitive transpeptidase is possibly of equal importance (as discussed in Section 111,C).

4. Antibiotic Combinations a. Cephalosporin + P-Lactam Antibiotic. In an attempt to increase the sensitivity of a strain (NCTC 8203) of P. aeruginosa to cephalosporins, Sabath and Abraham (1964) used a combination of a p-lactamaseresistant penicillin (methicillin or cloxacillin) with low intrinsic activity against this organism, with cephalosporin C, cephalothin, or cephaloridine and found that the synergistic response noted appeared to be due to the delay in the destruction of the hydrolyzable cephalosporin. Methicillin and, especially, cloxacillin considerably increase the activity of cephaloridine against several types of p-lactamase-producing gramnegative bacteria by preventing its destruction in vitro and in experimental animals (O’Callaghan and Muggleton, 1967; O’Callaghan et a l . , 1967). Cephalosporins that are p-lactamase inhibitors still protect plactamase-sensitive cephalosporins from inactivation (O’Callaghan et a l . , 1968, 1969).

For synergism to occur between two p-lactam compounds against a certain bacterial strain, the following general criteria must be obeyed (Sabath, 1968; Hamilton-Miller, 1971a,b): (a) the strain must produce a p-lactamase; (b) the first p-lactam antibiotic must be hydrolyzable (hydrolyzable compound); (c) the second p-lactam compound (inhibitor) must be poorly, or not, hydrolyzed; ( d j the inhibitor must have a markedly greater affinity for the active center of the enzyme than the hydrolyzable antibiotic being protected and must be a stable competitive inhibitor of the p-lactamase in the presence of whole bacterial cells; and (ej the inhibitor must show no, or little, antibacterial activity at the concentration used. The p-lactamases from gram-negative bacteria may be distinguishable on the basis of their susceptibility to inhibition, because none of the potential inhibitors tested by O’Callaghan and Morns (1972) was a

106

D. R. OWENS ET AL.

potent inhibitor of all three enzymes tested, although nafcillin possessed the broadest activity. In addition, it must be added that an inhibitor may potentiate the activity of one p-lactam hydrolyzable antibiotic against a particular gram-negative strain but not of another p-lactam hydrolyzable compound against the same strain (Russell, 1974). Obviously, against intact cells, intrinsic resistance to a particular compound as well as drug inactivation must play a role here. The clinical significance of antibiotic combinations, including plactam combinations, has been discussed by Jawetz (1968). b. Cephalosporin Non- p-lactam Antibiotic. Inevitably, cephalosporins have been tested in the presence of other, no@-lactam antibiotics, kanamyin attempts to obtain a synergistic response. A cephalothin cin combination has been found to be synergistic against methicillinresistant, multiple antibiotic-resistant strains of S. aureus (Bulger, 1967a), Klebsiella strains (Bulger, 1967b), and against strains of E. coli (Kaplan and Koch, 1968), with bactericidal effects noted. Unfortunately, the reasons for this synergism remain unexplored. Enterococci are resistant to cephalosporins (see earlier); however, synergism is detectable i n vitro over a wide range of antibiotic concentrations with mixtures of streptomycin and benzylpenicillin, cephaloridine, cephalothin, or ampicillin. As a result of these findings, Fekety and Weiss (1967) have proposed that treatment with streptomycin plus one of these cephalosporins is worthy of consideration in patients with enterococcal endocarditis who are allergic to penicillin. The mechanism of synergism is again unknown, although the authors state that penicillin (and presumably, therefore, a cephalosporin also) rapidly enhances the intracellular uptake of streptomycin. Other examples of antibiotic combinations are provided later (see Section V).

+

+

c. MODEOF ACTION 1. Inhibition of Cell Wall Synthesis Cephalothin inhibits the incorporation of l y ~ i n e - ' ~ into C the cell wall, but not the cell protein fraction of S. aureus (Chang and Weinstein, 1964a). Cephalothin, cephaloridine, and cephalexin induce a considerable increase in the intracellular accumulation of N-acetylglucosamine in this organism (Chang and Weinstein, 1964a; Russell and Fountain, 1971). Both these findings suggest that the cephalosporins have a similar mode of action as the penicillins, involving an inhibition of cell wall

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synthesis. In brief, mucopeptide (murein, peptidoglycan) synthesis involves the formation and utilization of two uridine mucleotides, uridine diphosphate (UDP) N-acetylmuramic acid (MurNAc) pentapeptide and UDP N-acetylglucosamine (GlcNAc) to give a linear polymer, involving the participation of a membrane-bound phospholipid ca.l-rier; a complete disaccharide pentapeptide subunit is formed, which is detached from the carrier. In S . aureus there is the addition of ammonia to the acarboxyl group of glutamic acid to form isoglutamine, and five glycine residues are added to form a chain substituted on the €-amino group of lysine. A transpeptidation reaction then takes place, whereby cross-links are formed. The entire process is summarized in Fig. 3, which also shows the proposed site of action of cephalosporins (Strominger et al., 1967). However, Hartmann et al. (1972) have proposed that penicillin blocks the hydrolysis of two murein hydrolases from E. coli, i.e., ( a ) an endopeptidase, which has the properties of a transpeptidase and which may be predominantly involved in cell division, and (b) a glycosidase, which acts on the polysaccharide chains of murein, and which may be predominantly involved in cell elongation. Interestingly, it was found that penicillin concentrations affecting cell elongation and glycosidase activity were of the same order of magnitude, and likewise that there was a close similarity between penicillin concentrations stopping cell division and those blocking endopeptidase activity. In this context, it is thus important to note that a new penicillin, FL 1060, appears to have novel properties in that it does not inhibit D-Ala carboxypeptidase, transpeptidase or endopeptidase (Park and Burman, 1973). Although FL 1060 induces spherical forms, it does not induce filaments at any concentration (Greenwood and O’Grady, 1973a). By contrast, cephalexin induces filaments over a wide range of concentrations, and it has thus been proposed (Greenwood and O’Grady, 1973b) that, whereas FL 1060 might act by inhibiting the glycosidase, the filament formation, i.e., an inhibition of septum formation, induced by cephalexin results from an inhibition of endopeptidase.

2 . Uptake and Cellular Permeability The binding of radioactive penicillin to bacterial cells (S. aureus) has been extensively studied (see the review by Russell, 1969). Radioactive cephalosporins are not often available, and thus their binding to bacteria has to be measured by an indirect method, which involves preexposure of the cells to ordinary e2C) cephalosporin drugs, followed by exposure to benzylpenici1lin-l4C. If the cephalosporin is irreversibly bound to the

1. F o rm a t i on of Linear Po ly m e r UDP- MurNAc

I

pentapeptide

MurNAc-P-P-lipid

attachment to membranebound phospholipid carrier

I

Pentapeptide z-GlcNAc

Lipid P

LLipid-p-p GlcNAc-MurNAc- P- P-lipid Pentakptide

(In S .aureus, stepwise addition of G5)

GlcNAc-MurNAc-acceptor (in wall)

I

Pentapeptide (+G 5 in S. aureus )

2. Cross-LWring (a) Staphylococcus aureus

Glyco ptide

Glycopeptide

MurNAc

M ~ N A

'I"

L-Ala

L-Ala

D - G ~

-. '.

~-Lys-G5 D-Ala

Cephalosporin---

I

\,

tTranspeptidase o-; Glycopeptide

MurNAc

o-Ala

--=La Gly copeptide MU~NAC

L-Ala

L-Ala

D-G~u

D-G~u

I

--- D - A l a - ~ - L y s - G 5(b) Escherichia coli

D-G~u ~-Lys-G5

D-

Na-L-hys-G 5

+ Po- A la

I

GlcNAc-MurNAc-L- Ala-o-Glu- meso-DAP-o- Ala-o- Ala

/

/

-

+

t t

GLcNAC- MurNAc- L- Ma-D -Glu- m es o - DAP-0- Ala- D- Ala

/

Cephalosporin---

/

- - transpeptidase

GlcNAc-MurNAc-L-Ala-o-Glu-meso -DAP-D-Ala

/ I / GLcNAc-MurNAc-L-Ala-o-Glu-meso-DAP-o-Ala-D-Ala / Cephalosparin---

/

-- D-Ala

+ D- Ala

carboxypeptidase

GlcNAc-MurNAc-L- Ala-o-Glu-weso -DAP-D- Ala

I / / GlcNAc-MurNAc-L-Ala-o-Glu-meso-DAP-o-Ala+ o-Ala /

FIG. 3. Peptidoglycan (murein, mucopeptide) synthesis in Staphylococcus aureus and Escherichia coli and sites of inhibition by cephalosporins. Abbreviations: UDP, uridine diphosphate; UMP, uridine monophosphate; GlcNAc, N-acetylglucosamine; MurNAc, N acetylmuramic acid; D-G~u, D-glutamic acid; L-LYS, L-lysine, D-Ala, D-alanine; G5, pentaglycine chain; meso-DAP, meso-diaminopimelic acid.

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binding site, the subsequent uptake of penicillin-14C will be prevented. Edwards and Park (1969) have shown that the concentration of a test cephalosporin which caused a 50% inhibition of penicillin-14C uptake correlated very closely with the MIC of that cephalosporin. A similar technique has been used by Retsena and Ray (1972), who found that the extent of penicillin-14C binding by a competing cephalosporin-12C (or other penicillin) was a function of pH and that it could be correlated both with the net charge of the competing lZC molecule and the net charge of the S. aureus cell at a given pH. The ability of a cephalosporin or penicillin to compete for ben~ylpenicillin-'~Cbinding sites on the bacterial cell appears to be correlated with the hydrophobic nature of the molecule (Retsena and Ray, 1972), the more lipophilic penicillins exerting the greatest effect against S. aureus (Biagi et a l . , 1970). Hamilton-Miller et al. (1965) proposed the existence of permeability (accessibility) barriers in gram-negative bacteria to various p-lactam antibiotics except cephaloridine, and in this context it is of interest to note that the MIC of cephaloridine against a strain of E. coli was virtually the same in the presence and absence of EDTA (Fountain and Russell, 1970), a substance that is believed to increase the permeability of these cells (Russell, 1971).

3. Induction of Morphological Variants Penicillins have, for some considerable time, been known to induce morphological variants in gram-negative bacteria (for a review, see Russell, 1969). Likewise, long filaments, spheroplasts and L-forms are produced on exposure of gram-negative bacteria to cephalosporin C, cephalexin, cephalothin, cephaloridine, and cephaloglycin (Bond et d . , 1962; Chang and Weinstein, 1964b, 1966; Kagan et al., 1964; Kagan, 1965; Burdash et a l . , 1968; Russell, 1968; Fountain and Russell, 1969; Muggleton et al., 1969; D. G. Smith, 1969; Lorian and Sabath, 1972). It must also be noted, however, that studies with the scanning electron microscope have indicated a spectrum of morphological changes induced by inhibitors of protein synthesis similar to those induced by cephalothin (Klainer and Perkins, 1970, 1972; Fass et a l . , 1970a,b; Perkins and Miller, 1973). Naturally resistant organisms are unaffected, and organisms made cephalosporin-resistant may undergo changes in cellular form which are qualitatively the same but less intense than those induced in the parent cells (Chang and Weinstein, 1964b). Certainly, much higher concentrations of a cephalosporin are needed to induce spheroplasts in laboratory-induced cephalosporin-resistant cells (Fountain and Russell, 1970).

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Electron micrographs of cephalothin-induced long forms of P. vulgaris indicate that the only major difference between them and normal cells is in the length (Burdash et al., 1968), although Muggleton et al. (1969) consider that cephalexin modifies the cell surface of E. coli and inhibits the formation of fimbriae. Electron micrographs reveal that the cephalosporins inhibit cell division and the formation of transverse cell walls (Burdash et al., 1968; Muggleton et al., 1969; Russell and Fountain,

1970). Gram-negative bacteria that produce large amounts of p-lactamase would, by the nature of the high MIC values of ampicillin and some cephalosporins, be expected to be resistant to these p-lactam drugs; nevertheless, depending on the organism and the p-lactam drug under test, morphological variants similar to those described above may be produced (Russell, 1973a). Cephalosporins are not inhibitory to the growth of methicillin-induced or lysostaphin-induced spheroplasts of S. aureus (Watanakunakorn et al., 1969), of L-forms of staphylococci (Kagan et al., 1964; Kagan, 1965) or of gonococci (Roberts, 1966), or of protoplasts of B. megaterium (Fountain and Russell, 1969). 4. Lysis

Concentrations of a cephalosporin above its MIC will induce lysis of actively growing gram-negative bacteria. This has been observed with cephaloridine for E. coli by Lambin and Bernard (1967) and with cephaloridine, cephalexin, and cephaloglycin for E. coli and other Enterobacteriaceae by Fountain and Russell (1969) and Russell and (1970). The presence of a sufficient concentration of a Fountain stabilizing agent, such as sucrose, prevents lysis leading to the formation of osmotically sensitive spheroplasts, as described above.

5. Conclusions Current theories on the mode of action of penicillin consider that this antibiotic binds to, and inactivates, cell wall murein transpeptidase (Park et al., 1971). The findings described in the foregoing suggest that cephalosporins have a similar mechanism of action. Nevertheless, differences in morphological response in ten strains of P. mirabilis exposed to benzylpenicillin (very long forms up to 93 pm) as compared to cephaloridine, cephalothin, and cephaloglycin (maximum long form, 14 pm) have been reported by Lorian and Sabath (1972), who suggest

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that long filamentous forms could be a consequence of the selective inhibition of D-Ala-carboxypeptidase, the cephalosporin then specifically inhibiting cell septation at concentrations well below the level needed to inhibit transpeptidase.

IV. Pharmacology and Toxicology The biological activity of an antibiotic is characterized by selectivity; ideally such a compound has no effect on the host but exerts a profoundly toxic effect upon the foreign organism. It is not surprising, therefore, that a successful antibiotic possesses few pharmacological (pharmacodynamic) and toxicological properties in animals and man and that these are observed normally only at high doses or concentrations. An important aspect of the pharmacology of antibiotics is their fate in the body. In this, a correlation of the rate and extent of absorption, distribution, metabolism, and excretion processes (pharmacokinetics) to the therapeutic utility of the antibiotics in animals and man is attempted. Such knowledge allows for the intelligent planning of suitable dosage regimens and routes of administration. As well as the antibacterial spectrum, the characteristics of distribution and excretion are important for the selection of an antibiotic for a particular therapeutic purpose. Thus in cases of biliary tract infection, it would be logical to employ, ceteris paribus, an antibiotic that is extensively excreted by this route in an active form. The binding of a drug to plasma protein, in addition to being a factor potentially affecting the availability of the drug to its site of action, may also be a site of interaction with other drugs by competition or displacement from the binding. Information on tissue localization is useful in alerting investigators during the preliminary evaluation of a new antibiotic to possible therapeutic applications or to potential sites of toxicity, e . g . , penetration into the eye and the fetus. Knowledge of the penetration of an antibiotic into soft and dense tissue is of obvious importance for the successful treatment of infection at these locations. Whereas a complete study of drug distribution can be readily made in animals, this is not so for man. However, a useful general guide for the distribution of a drug in the various body compartments is given by the apparent volume of distribution. It is to be expected that greater use of this and other pharmacokinetic parameters will be made in clinical studies of the newer antibiotics.

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A. CEPHALORIDINE 1. Absorption, Distribution, and Excretion Although cephaloridine is better absorbed than cephalothin from the gastrointestinal tract, only low levels (about 0.9 &ml) of this antibiotic are found in the plasma after a 500-mg dose (Griffith and Black, 1971). Because of this a n d . the existence of orally active cephalosporins, cephaloridine is administered only parenterally. Following intramuscular injection of 500 mg cephaloridine, peak plasma levels of 15 &ml are obtained (Kislak et al., 1966). After 1 gm intramuscularly, peak cephaloridine concentrations in plasma of 10-30 &ml occur in 0.5 to 1 hour (Apicella et al., 1966b) and significant amounts of the antibiotic can be detected for up to 6 hours. Peak concentrations of the drug in the plasma of pregnant women appear to be reached slightly more slowly (Barr and Graham, 1967b). After equivalent doses, plasma levels of cephaloridine are about twice those of cephalothin measured at several times (Benner et al., 1966) and are more sustained. When administered by intravenous infusion at 0.5 gm/hour, the plasma concentration of cephaloridine continues to rise beyond 3 hours. However, a constant plasma level of 24.7 pg/ml is obtained when an initial loading dose (0.35 gm) is followed by an infusion rate of 0.25 gm/ hour (Kirby et al., 1971). Plasma levels of cephaloridine are slightly increased and antibacterial activity persists much longer when probenecid is administered concurrently with the drug (Kaplan et al., 1967; Tuano et al., 1967). In patients with chronic uremia, plasma levels are elevated above the normal range and persist at higher levels for a prolonged time following cephaloridine therapy (Perkins et al., 1969b). In normal volunteers, the plasma half-life of cephaloridine, after a single intramuscular dose, is 1-1.5 hours (Kunin and Atuk, 1966; Pryor et al., 1967). It is possible that the half-life of this antibiotic may be longer in patients over the age of 50 years than in the under-50 age group (Apicella et al., 1966b). When determined following a steady state achieved by continuous intravenous infusion of cephaloridine, the halflife in plasma has been found to be 1.12 hours (Kirby et al., 1971). Early investigators (Barber and Waterworth, 1964; Muggleton et al., 1964; Kunin and Atuk, 1966) concluded that the binding of cephaloridine to serum protein was very low because the i n vitro activity of this antibiotic was not affected by serum. However, more recently, using ultrafiltration techniques it has been shown that cephaloridine is 20% bound to serum protein (Kind et al., 1969a).

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From the limited studies made, it appears that cephaloridine is fairly widely distributed in human body tissues and fluid compartments. Substantial concentrations of the drug have been found in liver, spleen, stomach wall, and lung (Apicella et al., 1966b). A smaller amount was detected in the cerebral cortex. Cephaloridine is detected in pleural effusions in fairly high concentrations (Murdoch et al., 1964). After a dose of 1 gm cephaloridine, no antibacterial activity is detected in the aqueous humor. However, up to 28 d r n l is found in the secondary aqueous humor that refills the eye following aspiration of primary fluid (Records, 1969). Cephaloridine is capable of penetrating fibrin clots in higher concentrations than cephalothin because of the higher plasma concentrations and longer half-life of cephaloridine (O’Connell, 1971). Penetration of cephaloridine into the cerebrospinal fluid (CSF), in the absence of meningeal inflammation, is negligible (Brayton et al., 1967; Steigbeigel et al., 1968). In the presence of severe renal impairment and, therefore, of high plasma levels, cephaloridine is found in concentrations of 6 to 12% of the serum levels in the CSF of patients with normal meninges (Kabins and Cohen, 1966; Gonnella et al., 1967; Gabriel et al., 1970); in patients with meningeal inflammation, there is a greater penetration of the drug into the CSF (Lerner, 1971). However, penetration of cephaloridine into CSF is not so consistently related to CSF protein content as it is with cephalothin. When cephaloridine is given to pregnant women in active labor, the drug crosses the placenta and is found in the serum of the newborn at concentrations up to 54% of the level in the maternal serum (Barr and Graham, 1967b; Arthur and Burland, 1969). Levels are measurable in neonatal serum up to 22 hours postpartum. Following a 1-gm intramuscular dose to pregnant women, a peak of cephaloridine in cord blood occurs at about 4 hours. The concentration of cephaloridine in amniotic fluid also rises slowly, taking about 3 hours to reach an effective antibacterial concentration after this dose (Barr and Graham, 196733). Cephaloridine is widely distributed in various tissues of the rat and rabbit including brain and spinal fluid, aqueous humor, fetus, and milk (Welles et al., 1966). The highest concentrations occur in the kidney and 4 hours after administration the kidney/serum concentration ratio for cephaloridine is 30. In the rabbit, cephaloridine penetrates into the cornea, aqueous and vitreous humors, and optic nerve following its subconjunctival injection (Moll, 1970). Following an intramuscular injection of cephaloridine, 40-100% of the dose can be recovered in human urine (Benner et al., 1966; Kislak et al., 1966; Kaplan et al., 1967; Griffith and Black, 1971). The major

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portion of this is excreted in 6 hours (Benner et al., 1966) and, after a 1gm dose, urine concentrations of this cephalosporin are 286-1024 &ml in this time period (Kaplan et al., 1967). Excretion occurs primarily through the glomeruli, and renal clearance is approximately the same as the creatinine clearance (Kirby et a l . , 1971) but some renal tubular secretion is also present because probenecid slightly increased plasma levels and slightly decreased renal clearance of this antibiotic (Kaplan et al., 1967; Tuano et a l . , 1967). Normal kidney can excrete 4-6 gm total dose of cephaloridine daily, but this amount decreases over the age of 50 years (Apicella et a l . , 196613). Elimination of cephaloridine is very dependent on renal function, the plasma half-life becoming more and more prolonged as creatinine clearance decreases below 20 &minute. Thus, in patients with little or no kidney function, the half-life is 20-25 hours (Kabins and Cohen, 1966; Kunin and Atuk, 1966; Pryor et a l . , 1967). The bile is a minor pathway of excretion for this cephalosporin, concentrations of cephaloridine in bile being at most 50% of serum levels (Apicella et a l . , 1966b; Acocella et al., 1968; Brogard et a l . ,

1972).

In animals, renal excretion of cephaloridine is solely by glomerular filtration, except in the hen where a small but significant tubular secretion occurs (Child and Dodds, 1966; Welles et a l . , 1966). In perfused rabbit liver, the concentrations of cephaloridine in the bile are less than the concurrent serum levels (Brogard et al., 1972).

2. Metabolism The greatly increased plasma half-life of cephaloridine in patients with renal failure (Kabins and Cohen, 1966) suggests that this antibiotic is stable in the body. In fact, no degradation products of the drug have, as yet, been identified in human urine. This apparent resistance to metabolic transformation may be related to the replacement of acetyl (as in cephalothin) by pyridyl on C-3 of the cephalosporin nucleus. However, Kunin and Atuk (1966) observe that, although cephaloridine is retained in the presence of renal failure, this is not for nearly a s long a period of time as are drugs that depend almost entirely on the kidney for excretion, e.g., streptomycin and kanamycin. These authors suggest that a significant proportion of cephaloridine is inactivated by nonrenal mechanisms. Although results from perfusion experiments with rabbit liver suggest that cephaloridine may be fixed and inactivated in this organ (Brogard et

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al., 1972), it is possible to recover 92% of an intravenous dose as unchanged drug in dog urine (Welles et al., 1966).

3. Toxicology Cephaloridine is painless when administered intramuscularly, and it may be given by this route over long periods of time thus avoiding the general hazards of continuous therapy (Lerner, 1971). This may be related, in part, to the greater solubility of cephaloridine allowing for a small injection volume (Turck et al., 1967). This cephalosporin is an internal salt, a betaine, containing neither Na+ nor K+. Thus it has the advantage of not overloading patients with cations if this is contraindicated clinically. Most patients receiving cephaloridine experience no adverse effects (Murdoch et al., 1964; Stewart and Holt, 1964; Benner et al., 1966; Cohen et al., 1966; Kislak et al., 1966) and the side effects noted are usually quite minor. One case of the appearance of an extrapyramidal syndrome after cephaloridine has been reported (Mintz et al., 1971); this is not considered to be an allergic reaction. In this connection it is interesting to note that intrathecal administration of cephaloridine can give rise to hallucination, confusion, and nystagmus (Murdoch et al., 1964). Cephaloridine is well tolerated when given to mothers prior to amniotomy (Barr and Graham, 1971). A transient rise of SGOT has been observed in some patients receiving this antibiotic (Dennis et al., 1966; Hermans et al., 1966). Cephaloridine may also elevate prothrombin time, but this effect is readily reversible and does not lead to hemorrhage (Hermans et al., 1966). In cases of reduced renal function, it has been observed that treatment with cephaloridine is followed by further deterioration of kidney function in a small proportion of patients (Cohen et al., 1966; Holloway and Scott, 1966; Kislak et al., 1966). It was not possible to determine whether these changes were due to the antibiotic or to the progression of the disease. However, there is accumulating evidence that cephaloridine has a nephrotoxic action particularly when the drug is given in doses producing high plasma levels, i.e., 15(1-200 &ml (Lawson et al., 1970) or where kidney function is already impaired (Benner, 1970; Foord, 1971). In elderly patients with an apparently normal renal function, there is an increase in hyaline casts in urine when large daily doses (6 gm) of cephaloridine are administered (Linsell et al., 1967). The production of casts returns to normal on withdrawal of the drug. Fleming and Jaffe (1967) have also reported the presence of hyaline casts without accompanying proteinuria in children receiving

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high doses of cephaloridine. These authors find that cephaloridine (200 mg/kg) administered to rabbits produces proximal tubular necrosis and shedding of casts into the tubules. The nephrotoxic activity of cephaloridine is well-documented in animals (Atkinson et al., 1966a; Child and Dodds, 1966, 1967; Perkins et al., 1968), but the dose required to induce proximal tubular necrosis of the kidney varies with species, the rabbit being the most sensitive. From the results of a detailed study in rabbits by light and electron microscopy (Silverblatt et al., 1970), it appears that the first changes to be seen in the kidney are in the brush border of the outermost proximal tubules In a review of cases of renal failure associated with administration of cephaloridine (Foord, 1969), it was observed that in several patients there had been coincidental administration of frusemide, and it was suggested that this diuretic might enhance the nephrotoxicity of the antibiotic. Studies in rats and rabbits have confirmed that such an interaction occurs (Dodds and Foord, 1970; Lawson et al., 1970, 1972). In these experiments, nephrotoxicity developed at plasma levels of cephaloridine previously regarded as nontoxic. The mechanism of this interaction has not yet been elucidated, although involvement of the renin-angiotensin system has been suggested (Lawson et al., 1972). It has also been proposed that frusemide may aggravate the precipitation of cephaloridine from concentrated solution and the polymerization of the drug in the renal tubules, both of which events could be responsible for damage to the brush borders (Boyd et al., 1971). In the light of these findings it is now the clinical practice to reduce the dosage of cephaloridine adequately in the presence of preexisting renal impairment and also in the elderly and to avoid concurrent administration of frusemide. As determined in animals, the pharmacodynamic effects of cephaloridine are negligible. At high doses there is a transient fall of blood pressure related to a ganglion blocking action (Atkinson et al., 1966b).

.

B. CEPHALOTHIN 1. Absorption, Distribution, and Excretion

Cephalothin is available as a water-soluble sodium salt for parenteral administration. It is poorly absorbed by mouth and, after a dose of 500 mg, no or just assayable levels of the drug are detected in the serum

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(Griffith and Black, 1971). When given intramuscularly, 500 mg of cephalothin is rapidly absorbed, peak concentrations of 7.6 to 10 &ml of serum being obtained in 0.5 hour (Griffith and Black, 1964, 1971). A doubling of the dose increases the peak concentration by the same factor, and measurable serum levels of the drug persist for 4 hours after this higher dose. When infused intravenously at 0.5 gm/hour, constant plasma levels of 10 to 30 pg/ml are reached after 0.5-2.5 hours (Tuano et al., 1967; Griffith and Black, 1971). Plasma concentrations of cephalothin are elevated above the normal range when probenecid is also administered (Griffith and Black, 1968) and in the presence of renal insufficiency (Kabins and Cohen, 1965). After a dose of 12.5 mg/kg, distinctly higher and more sustained blood levels of cephalothin are observed in full-term newborn and premature infants than in older infants and children (Sheng et al., 1965). In the healthy adult, after a single intramuscular or intravenous dose, the plasma half-life of cephalothin vanes between 0.5 and 0.85 hour (Kabins and Cohen, 1965; Kunin and Atuk, 1966; Naumann, 1971). The half-life is somewhat shorter (0.47 hour) when determined after a steadystate plasma level has been reached by means of a constant intravenous infusion (Kirby et al., 1971). As determined by ultrafiltration, cephalothin is 65% bound to plasma protein (Kirby et al., 1971). The apparent volume of distribution appears to be slightly larger for cephalothin than for either cephaloridine or cephalexin (Kirby et al., 1971). The differences in plasma level between these three antibiotics is greater than can be accounted for by the differences in apparent volume of distribution and probably relate to differences in renal and nonrenal clearance rates. Cephalothin is widely distributed in the body, high concentrations being found in renal cortex, pleural fluid, skin, striated muscle, myocardium, and stomach wall (Perkins and Saslaw, 1966). A lower concentration was detected in the liver. Bactericidal amounts of the drug are found within a short time of administration in ascitic (Kabins and Cohen, 1965), pericardial, and synovial fluids (Gump and Lipson, 1968). The drug does not cross the normal blood-brain barrier as no cephalothin is detectable in the CSF of patients without meningitis. However, where the meninges are inflammed, cephalothin is present in the CSF at concentrations of 0.16 to 0.31 pg/ml with concomitant plasma levels of 10-80 &ml (Vianna and Kaye, 1967). Drug concentrations in CSF up to 5 &ml have been observed in patients with CSF protein in excess of 50 mg/100 ml (Lerner, 1969). Cephalothin traverses the placenta, and significant bactericidal concentrations are found in cord serum and amniotic fluid within 15

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minutes of administration (MacAulay and Charles, 1968; Paterson et al., 1970; Hirsch, 1971). The peak concentration of cephalothin in cord blood occurs later than in the maternal blood (Sheng et al., 1965). At the time of peak concentration in cord blood the levels are 2550% of those in the maternal circulation. Cephalothin has been found in fetal urine in concentrations up to 100 d m l (Morrow et al., 1968). A similar pattern of distribution of cephalothin has been found in animals (Lee and Anderson, 1963; Perkins et al., 1968). In addition, this antibiotic has been shown to penetrate into the aqueous humor in low concentration (Uwaydah and Faris, 1970). In rats, using 14C-labeled cephalothin, radioactivity was found in bone soon after subcutaneous administration (Kanyuck et al., 1971); this had a similar half-life to the cephalothin in serum. Cephalothin is rapidly cleared by the human kidney (Griffith and Black, 1971), and there is no evidence of accumulation when intravenous doses of 4 gm are given every 8 hours for 32 hours. Even in cases of severe impairment of creatinine clearance, there is little accumulation of the drug (Kabins and Cohen, 1965). However, in patients with serious impairment of the renal clearance mechanism, Benner (1970) has found that high serum concentrations of cephalothin occur when large doses of the antibiotic are administered for several days. In the newborn infant, cephalothin is quickly excreted (Sheng et al., 1965). In adults, following 500 mg intramuscularly, 60-90% of the administered dose appears in the urine within 6 hours (Griffith and Black, 1971). A mean recovery of 75.6% in 24-hour urine has been reported (Naumann, 1971). The secretion of this antibiotic involves a significant renal tubular component as may be seen from the clearance rate which is over twice that of creatinine (Kirby et al., 1971) and from the marked reduction of clearance when probenecid is administered (Tuano et al., 1967). Although the bile is only a minor excretory pathway for this cephalosporin, cephalothin is concentrated in gall bladder bile to the extent of 5 to 100 times the serum concentration (Rain, 1971; Brogard et al., 1973b). A pattern and mode of urinary excretion similar to that in man has been shown for the drug in animals (Lee et al., 1963).

2 . Metabolism The major route of metabolism following parenteral administration of cephalothin to man is by hydrolysis to 0-deacetylcephalothin (Lee et al., 1963). This metabolite is considerably less active in vitro than cephalothin, particularly against gram-negative bacteria (Kirby et al., 1971).

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The esterase capable of hydrolyzing cephalothin is present in human tissues, principally the liver, but insignificant amounts occur in the serum (Lee et al., 1963). The metabolite is excreted into urine in varying proportions together with unchanged cephalothin (Lee et al., 1963). In the 6-hour urine, after a single intramuscular dose of 1 gm cephalothin, approximately 65% of the excreted cephalosporin is as the parent compound and 35% as deacetylcephalothin (Griffith and Black, 1971). In uremic patients receiving. cephalothin, the antibacterial activity of the serum is characterized by two half-lives (Kabins and Cohen, 1965)-a rapid early decline in serum levels with a half-life of about 3 hours followed after 8 hours by a slower decline with a half-life of 12 to 18 hours. One explanation of this is that the first phase represents the clearance of the parent compound, whereas the second phase represents chiefly the deacetyl metabolite. Recently, the plasma levels of cephalothin and deacetylcephalothin were measured in an uremic patient receiving 1 gm of the drug intravenously (Kirby et al., 1971). While cephalothin levels fell rapidly with a half-life of 2 hours, concentrations of deacetylcephalothin increased over the first 12 hours and then declined slowly with a half-life of approximately 8 hours. It has also been observed that repeated dosage with cephalothin leads to an accumulation of deacetylcephalothin in the blood of an uremic patient (Kirby et al., 1971). In rabbits with reduced renal function, 45-90% of the antibacterial activity of plasma is in the deacetylated form 1 hour after cephalothin administration (Venuto et al., 1972). The metabolism of 14C-labeled cephalothin has been studied in the rat. After oral administration, the drug is degraded in the gut and the products are absorbed (Sullivan and McMahon, 1967). The urine contains only a trace of deacetylcephalothin and no parent compound. However, 32% of urinary radioactivity is present as thienylacetylglycine and 13% as thienylacetamidoethanol. By contrast, when cephalothin-14C is given intraperitoneally the major urinary metabolite is the deacetylated compound (Sullivan et al., 1971). Only a small proportion of urinary radioactivity is present as thienylacetylglycine, a metabolite that is not found in human urine. A long-lived minor metabolite occurs in rat blood and appears to be an albumin-cephalothin complex.

3. Toxicology Because of poor oral absorption, cephalothin must be given parenterally. However, intramuscular administration is painful and in some

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cases the local irritation may progress to induration or, rarely, sterile abcess or slough (Weinstein and Kaplan, 1970). Hence, the intravenous route is often preferred for large doses. By this latter route, thrombophlebitis may occur with continuous infusions of doses greater than 6 gm daily (Perkins and Saslaw, 1966). Cephalothin is administered as its sodium salt which contains 55 mg Na+/gm. Thus, if large doses are given to patients with severe renal impairment, there is the possibility of overloading the patient with this cation. The incidence of adverse reactions in patients treated with cephalothin is low. These appear to result largely from mild-to-moderate hypersensitization (Davis et al., 1964; Heitler et al., 1964; Weinstein et al., 1964; Kabins and Cohen, 1965; Molthan et al., 1967). Cephalothin has been extensively investigated clinically since 1963, and it is generally concluded that this antibiotic does not possess a nephrotoxic action in the doses employed therapeutically (Maynard, 1969; Benner, 1970) or at least that the risk of renal injury is very much less likely than it is from cephaloridine therapy (Perkins et al., 1968; Rahal et al., 1968; Turcotte et al., 1970). It has been observed that, in certain patients with impaired renal function and with high serum concentrations of cephalothin, deterioration of renal function occurs (Maynard, 1969; Benner, 1970 ). However, the renal necrosis in these patients could well occur from other factors such as congestive heart failure, low cardiac output, or unrecognized hypertension. There is as yet, no evidence suggesting an interaction between cephalothin and diuretics such as frusemide (Lawson et al., 1970). However, cases of deterioration in renal function after treatment with cephalothin in combination with other potentially nephrotoxic antibiotics has been observed (Thoburn and Fekety, 1970). Rabbits receiving large daily doses (500 mg/kg) of cephalothin for 3 weeks showed only slight histological changes in the kidney (Perkins et al., 1968). These consisted of minimal swelling or hydropic changes of the tubular epithelium. No renal histological changes were seen in monkeys similarly treated. Even in rabbits with an extensive reduction in glomerular filtration rate, subacute administration of cephalothin (300 mg/kg, daily) produced no further deterioration in renal function (Venuto et al., 1972). In rats with mild transient renal impairment induced by glycerol, cephalothin, at concentrations in plasma of 113 pg/ml, produced extensive acute tubular necrosis when combined with frusemide (Lawson et al., 1972). Until further evidence is produced it would appear to be prudent to regard cephalothn as being potentially nephrotoxic in

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man when at high plasma concentrations andfor in combination with diuretics. In animals cephalothin has minimal pharmacodynamic properties. Perrelli and Tempesta (1968) have demonstrated a slight transient decrease in diastolic and systolic pressures after injection of the drug.

C. CEPHALEXIN 1. Absorption, Distribution, and Excretion In cephalexin the substituent at C-7 of the cephalosporin nucleus is the same as that at C-6 in ampicillin. Cephalexin is a zwitterion and at pH 3.8 exists as an inner salt. It is used clinically as the monohydrate. The drug is stable in gastric acid, and, following oral administration to normal fasting subjects, absorption is rapid (O’Callaghan et al., 1971), peak plasma levels being obtained at about 1 hour. The magnitude of the peak is dose-related, and, after 500 mg cephalexin, peak levels of 13 to 18 pg/ml are reached (Braun et al., 1968; Griffith and Black, 1968, 1971; Levison et al., 1969; Meyers et al., 1969). Measurable concentrations persist in plasma for 4 to 6 hours. Absorption takes place primarily from the upper portions of the gastrointestinal tract (Griffith and Black, 1970). However, in animals, absorption from the rectum is also possible (Welles et al., 1969). The drug is well absorbed from the gastrointestinal tract following partial gastrectomy, but poor absorption may occur in patients with obstructive jaundice and pernicious anemia (Davies et al., 1970). Absorption of cephalexin is delayed when the drug is given with or shortly after food (Griffith and Black, 1968, 1971; Gower and Dash, 1969; O’Callaghan et al., 1971). The peak level, which is about 50% of that observed in the fasting state, is reached after 2 hours. However, there is a prolongation of plasma levels, and total absorption of cephalexin is not decreased. The presence of very small amounts of food does not interfere with the absorption of this cephalosporin (Thornhill et al., 1969). Delayed absorption of cephalexin is also observed in some anephric patients (Reisberg and Mandelbaum, 1971). Following a constant intravenous infusion at a rate of 0.5 gmthour, a steady-state cephalexin concentration of 27 pg/ml in plasma is reached in 2 hours (De Maine and Kirby, 1971). There is no accumulation of cephalexin on repeated oral administration (Griffith and Black, 1968, 1971) unless renal insufficiency is present (Levison et a l . , 1969). Administration of probenecid increases the height and duration of antibacterial activity in the plasma after a dose of cephalexin (Braun et al. 1968; Griffith and Black, 1968; Gower and Dash, 1969).

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In newborns and infants up to the age of 6 months, absorption of cephalexin is delayed, and peak plasma levels are lower compared with older children (Marget and Daschner, 1969; Marget, 1971). When administered after bottle feeding, cephalexin is more slowly absorbed by infants compared with fasting infants (Fujii et al., 1969). In subjects with normal renal function, the plasma half-life of cephalexin is 0.9-1.2 hours after oral administration (Bergan et al., 1970; Kabins et al., 1970; Kunin and Finkelberg, 1970). When given by intravenous infusion to achieve a steady state, the half-life is 0.61 hour (Kirby et al., 1971). The longer half-life after oral administration is probably related to absorption of the drug still occurring while the halflife is being determined. The half-life of orally administered cephalexin in neonates and infants up to 12 months of age is prolonged in patients with impaired renal function (Clark and Turck, 1969; Kabins et al., 1970; Kirby et al., 1971; Regamey and Humair, 1971). In patients with a creatinine clearance below 2-5 muminute, the half-life of cephalexin is about 22 hours (Kabins et al., 1970). This may be reduced considerably by either hemodialysis (Linquist et al., 1970; Kirby et al., 1971) or peritoneal dialysis (Yamasaku et al., 1970). Cephalexin is bound to plasma protein to the extent of 15% (Kirby et al., 1971). The value for the volume of distribution of cephalexin in man suggests that the drug is well distributed in total body water (Orsolini, 1970) and, considered with the low plasma binding, it indicates a good accessibility to tissues of the body. This is confirmed by results of tissue assays, the drug being found in lung, spleen, liver, adrenal,pancreas, kidney, myocardium, stomach, appendix, omentum, vein, genital organs, and tumor tissue (Mizuno et a l , 1969; Orsolini, 1970; Griffith and Black, 1971). Concentrations of cephalexin in these tissues are lower than the plasma level, except for the kidney which is higher. Cephalexin is well distributed in the tissues of children (Simon et al., 1970). The drug is also found in breast milk (Mizuno et al., 1969), aqueous humor (Gager et al., 1969; Boyle et al., 1970), and pus (Shibata and Kato, 1969). In the latter, relatively high peak concentrations of the antibiotic are reached more slowly than those in plasma. No (or very small amounts of) cephalexin has been found in the CSF of patients with normal meninges receiving this cephalosporin (Bergan et al., 1970; Davies et al., 1970). The passage of cephalexin into the CSF of patients with meningeal inflammation has not, as yet, been reported. Cephalexin traverses the placenta rapidly to pass into the fetus (Mizuno et al., 1969; Paterson et al., 1972). Five hours after

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administration the antibiotic concentration in cord blood is higher than in the maternal circulation. The level of cephalexin in amniotic fluid rises gradually up to 7 hours. A similar pattern of distribution to that in man has been found in animals (Welles et al., 1969, 1970; Kanyuck et al., 1971; Berte et al., 1972). A detailed review of the absorption and distribution of cephalexin has recently been published (Speight et al., 1972). From the plasma profile of cephalexin levels in patients with renal failure, it is evident that the antibiotic depends on the kidney for excretion. After oral doses, 75-100% of the drug is excreted in the urine within 8 hours (Braun et al., 1968; Clark and Turck, 1969; Kind et al., 1969a; Meyers et al., 1969). This high recovery is evidence of the efficiency of absorption of orally administered cephalexin. Thus, the drug is excreted in high concentration; after an oral dose of 250 mg, mean urine concentrations are 830 &ml over 6 hours (Thornhill et al., 1969). In patients with poor renal function, excretion is reduced to 3.6% in 8 hours (Meyers et al., 1969), but even in such patients the urinary concentrations of cephalexin are adequate for the treatment of most urinary tract infections (Reisberg and Mandelbaum, 1971). After intravenous administration to patients with normal renal function, 80% of the dose is excreted in urine within 2 hours (Davies and Holt, 1972). In normal subjects the renal clearance ratio of cephalexin to creatinine is 1.7 (Kirby et al., 1971). The urinary excretion of cephalexin occurs partly through glomerular filtration and partly by tubular secretion (Foord et al., 1969b). Concurrent administration of probenecid delays the renal tubular secretion of cephalexin (Braun et al., 1968; Thornhill et al., 1969). The renal clearance of this antibiotic is increased by 15% when urinary pH is reduced to 4.8 (Asscher et al., 1970). However, this is considered to have no therapeutic importance. After doses of 500 mg and 1 gm cephalexin, antibacterial activity is detected in the bile at 2 and 4 hours in concentrations that are lower than the concurrent plasma levels, except in a case of obstructive jaundice where the biliary concentration was significantly higher (Sales et al., 1969). In animal experiments (Welles et al., 1969, 1970), it is found that recovery of cephalexin in urine is slightly less complete than in man. In the species studied renal clearance is both by glomerular filtration and tubular secretion. In the dog and rabbit, a significant tubular reabsorption of antibiotic occurs. Biliary excretion is low in the dog. Up to 15% of the dose of cephalexin administered is found in rat feces in 24 hours and this is largely the result of biliary excretion (Sullivan et al., 1969b).

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2. Metabolism The antibacterial activity in human urine after administration of cephalexin is unchanged drug (Chou, 1969), and it is assumed that this cephalosporin is not metabolized. However, for similar reasoning applied to cephaloridine, because in cases of renal failure cephalexin is not retained in the body for nearly as long as antibiotics such as kanamycin, it is possible that metabolic inactivation of the compound may occur to some degree when raised plasma levels of the drug are prolonged. No evidence for the metabolism of cephalexin has been obtained from animal experiments (Welles et a l . , 1969; Sullivan et a l . , 1969b). 3. Toxicology The most frequent side effects (up to 2%) reported during cephalexin therapy are gastrointestinal disturbances such as diarrhea, nausea, vomiting, and abdominal cramp (Braun et a l . , 1968; Griffith and Black, 1968; Kind et a l . , 1969a; Levison et a l . , 1969; Page et a l . , 1970; Fass et al., 1970b). The incidence of allergic reactions, mainly rash or urticaria, is low (Griffith and Black, 1970; Landes et a l . , 1972) Although no nephrotoxicity from cephalexin has- been reported clinically, a slight vacuolar nephrosis has been found in some rabbits receiving a high dose of the antibiotic (4 gm/kg) (Welles et a l . , 1969, 1970). In 1 of 5 patients receiving a 1-week course of cephalexin, there was a fall in the creatinine clearance 2 weeks after the last dose of the antibiotic. However, renal function improved and the effect cannot be proved to be drug-related (Kunin and Finkelberg, 1970).

D. CEPHALOGLYCIN 1. Absorption, Distribution, and Excretion

An orally active cephalosporin, cephaloglycin (see Fig. 2) has a spectrum of antibacterial activity comparable to that of cephalothin and cephaloridine (Wick and Boniece, 1965). Unfortunately, cephaloglycin is not extensively absorbed from the gastrointestinal tract, only about onequarter of an orally administered dose being absorbed. This is in contrast to the nearly complete absorption of cephalexin (Griffith and Black, 1971) which is the deacetoxy analog of cephaloglycin. After an oral dose of 500 mg cephaloglycin, maximal concentrations of antibacterial activity in the blood occur at 2 hours, but these levels are

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extremely low and variable. For example, levels that range from 0.2-0.9 p d m l (Perkins et al., 1969a) to 1.0-3.0 pg/ml have been reported (Applestein et al., 1968; Boyer and Andriole, 1968; Braun et al., 1968; Johnson et al., 1968; Pitt et al., 1968; Griffith and Black, 1971). In some cases no antimicrobial activity has been detected in the blood (Kunin and Brandt, 1968). Serum concentrations have been shown by Braun et al. (1968) to be significantly elevated and prolonged when probenecid is concurrently administered with cephaloglycin. However, other workers using probenecid have failed to observe any increase in the serum levels, although antimicrobial activity in the serum was prolonged (Pitt et al., 1968). Because of the low blood levels achieved with oral cephaloglycin, many workers have indicated that this drug should not be administered to patients with suspected bacteremia or with infections located outside the urinary tract (Ronald and Turck, 1967; Boyer and Andriole, 1968; Johnson et al., 1968; Kunin and Brandt, 1968; Ronald et al., 1968). Little is known about the distribution of cephaloglycin in man. However, in studies carried out in mice, following an oral dose of 200 mdkg, the concentrations of antibacterial activity are highest in the liver and kidney, being 1.9 and 3.2 times, respectively, greater than in the serum. Other tissues such as spleen, heart, lung, and skeletal muscle possess approximately one-half the activity of the serum (Welles, 1972). Cephaloglycin is bound to human plasma to the extent of 67% (Pruitt and Dayton, 1971). Urine collected from human volunteers over the first 8 hours after a single oral dose of 500 mg cephaloglycin shows concentrations of antibacterial activity ranging from 76 to 1330 pg/ml. Between one-fifth and one-third of the administered dose is accounted for in the urine in 8 hours (Braun et al., 1968; Pitt et al., 1968; Griffith and Black, 1971). 2. Metabolism The metabolism of 14C-labeled cephaloglycin has been studied extensively in rats by Sullivan et al., (1969a). Following oral administration of labeled material, the total recovery of radioactivity in 24 hours was 90%, of which 20% was in the urine and 70% in the feces. The urine contained one microbiologically active substance, identified as deacetylcephaloglycin. The biologically inactive compound D-2-phenylglycine was also present. Thus, cephaloglycin is metabolized in the rat by two pathways; deacetylation to form deacetylcephaloglycin and hydrolysis of the amide linkage to form D-2-phenylglycine. When administered in large doses, some unchanged material is also recovered in the urine.

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These results, which indicate that cephaloglycin is substantially metabolized, contrast with results obtained for cephalexin which is not metabolized but is eliminated by the kidney in an unchanged form (Sullivan et al., 1969b).

3. Toxicology Toxicity does not constitute a major problem in therapy with cephaloglycin. No consistent alterations in blood cell counts, urinalysis, blood urea nitrogen, SGOT, and SGPT have been attributed to the drug (Ronald et al., 1968; Perkins et al., 1969a). However, Boyer and Andriole (1968) recommend that cephaloglycin should be used cautiously in patients with renal insufficiency. The most frequent adverse effects reported during cephaloglycin therapy have been gastrointestinal disturbances such as diarrhea, nausea, and vomiting (Ronald and Turck, 1967; Boyer and Andriole, 1968; Johnson et al., 1968; Ronald et al., 1968). A comparatively high incidence (in the order of 30%) of gastrointestinal tract reactions have been recorded when patients were administered oral cephaloglycin in daily doses of 2 gm for 10 days. Less common side effects associated with oral cephaloglycin therapy include skin rashes and eosinophilia (Boyer and Andriole, 1968; Johnson et al., 1968; Ronald et al., 1968).

E. OTHERCEPHALOSPORINS 1. Cephacetrile Like many other cephalosporin derivatives, cephacetrile (see Fig. 2) is poorly absorbed from the gastrointestinal tract. Therefore, to be therapeutically effective, the drug must be administered parenterally. In a study carried out in human volunteers by Luscombe et a l . (1972, 1973), a single intravenous dose of 500 mg cephacetrile produced mean serum levels of 30.7 &ml, 15 minutes after administration. Antibacterial activity rapidly disappeared from the blood and the serum half-life was calculated to be 33.2 2 4.2 minutes which is comparable with the half-life of cephalothin. A similar value (33.0 minutes) has been determined by other workers using intravenous doses of 1 gm cephacetrile (Brogard et al., 1973~).When determined following a steady state achieved with a continuous intravenous infusion of the drug, the serum half-life has been found to be 1.3 f 0.3 hours (Nissenson et a l . , 1972). Cephacetrile is readily absorbed from the intramuscular site, a peak serum concentration of 14.5 Fg/ml being observed 30 minutes after a 1-

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gm dose. The serum half-life by the intramuscular route has been calculated to be 1.5 hours (Brogard et al., 1973~).Blood levels can be slightly increased and prolonged by administering probenecid concurrently with cephacetrile. In animal studies, Luscombe et al. (1972) showed that in rabbits receiving probenecid (30 mg/kg intravenously) immediately prior to the antibiotic, the serum half-life of antibacterial activity is increased by 79% over the half-life achieved with the same dose of cephacetrile but without probenecid (i.e., an increase from 22.1 to 38.6 minutes). Cephacetrile is 33-36% bound to human serum proteins (Luscombe et al., 1973) compared with 15-20% for cephalexin and cephaloridine and with 65% for cephalothin (Kind et al., 1969a; Kirby et al., 1971). Following intravenous and intramuscular injections of cephacetrile, between 70 and 85% of the dose is recovered in the urine in 10 hours (Luscombe et al., 1972; Brogard et al., 1973~).The phase of maximum urinary excretion of antibacterial activity occurs in the first 2 hours of the drug being administered, but small amounts of the active substance continue to be excreted for up to 10 hours. From renal and creatinine clearance studies performed by Nissenson et al. (1972), cephacetrile appears to be excreted by both glomerular filtration and tubular secretion in normal subjects, On the other hand, in patients with renal failure, there is little evidence of tubular excretion. Animal experiments using probenecid-treated rabbits confirm that under normal conditions, renal excretion of cephacetrile is accomplished by both glomerular filtration and active tubular excretion (Luscombe et al., 1972). Antibacterial activity in human urine has been determined to be due largely to unchanged material (Luscombe et al., 1973). Studies in normal subjects and rabbits show that the excretion of cephacetrile in bile is low (Luscombe et al., 1972; Brogard et al., 1973a,c). Nevertheless, in patients with renal failure, biliary excretion of antibacterial activity is considerably increased (Brogard et ul., 1973a). Following intravenous and intramuscular injections of 25 mdkg cephacetrile in rabbits, antibacterial activity can be detected in the CSF. However, the ratio of antibacterial concentration in the CSF to the concurrent concentration unbound in the serum is less than unity. Thus, cephacetrile does not appear to penetrate readily the blood-brain barrier (Luscombe et al., 1973). In toxicological studies in laboratory animals carried out by Kradolfer et al. (1971), cephacetrile exhibited a low degree of toxicity. The acute intravenous LD,, in mice was 4.5 gm/kg although in dogs a 6-gm/kg dose by the same route was lethal. In rabbits, intravenous doses of up to 6

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g d k g had no detectable effects on renal function or kidney structure. When compared with cephaloridine and cephalothin, cephacetrile lacked nephrotoxicity in a similar manner to cephalothin, whereas cephaloridine even at 0.1 gm/kg was found to produce both functional and structural impairment of the proximal tubules of the kidney. The concurrent administration of frusemide and either cephalothin or cephaloridine (Dodds and Foord, 1970; Lawson et al., 1972; Linton et al., 1972) has been shown in animal studies to increase the incidence and extent of nephrotoxicity produced by large doses of cephalothin and cephaloridine. However, in a recent investigation carried out by Luscombe and Nicholls (1975), no biochemical or histological evidence of increased nephrotoxicity was observed when frusemide was administered concurrently with 500-mg and 1-gm doses of cephacetrile in rats and rabbits. In a similar study carried out in dogs, no adverse acute effects of cephacetrile on glomerular filtration rate and effective renal plasma flow was found either in the presence or absence of concurrently administered doses of frusemide (Naber and Madsen, 1973). This evidence, therefore, suggests that cephacetrile, unlike cephaloridine, is relatively free from any nephrotoxicity. Little is known about the possible side effects of continuous cephacetrile therapy. However, in volunteer studies the drug is well tolerated after intravenous injection, no discomfort being experienced either locally or systemically after dosing. In addition, no changes in hematological parameters and hepatic or renal function have been observed (Luscombe et al., 1972).

2. Cephradine Chemically, cephradine is 7-[D-%amino-%(1,4-cyclohexadien-l-yl)acetamido]-3-methyl-3-cephen-4 carboxylic acid (Fig. 2). It is of particular interest since it is rapidly absorbed from the gastrointestinal tract and can, therefore, be administered orally. In a recent human volunteer study carried out in Belgium (Harvengt et al., 1973), a single oral dose of 500 mg in fasted individuals was rapidly absorbed giving peak serum levels of 18.3 ? 2 d m l 1 hour after ingestion. In nonfasted persons, cephradine was less rapidly absorbed although peak serum concentrations also occurred at 1 hour and were similar (19.2 4.1 pg/ml) to those observed in fasted volunteers. Antibacterial activity is rapidly excreted in the urine in high concentrations, more than 85% of the administered dose being recovered in the initial 6-hour period following ingestion. This is a further

*

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indication of the high degree of gastrointestinal absorption following oral doses of cephradine. At present it is not known whether the antibacterial activity in urine samples is due to unchanged cephradine or to a metabolite. Limson et al. (1972) have recently carried out a trial demonstrating that cephradine is highly effective in patients suffering from moderateto-severe, acute infective disease. In addition, these workers found that orally administered cephradine is relatively safe since no toxic effects were observed on the renal, hepatic, or hemopoietic systems although mild adverse reactions involving the gastrointestinal tract were noted in 2 of the treated patients. Landa (1972) has also demonstrated the absence of side effects attributable to the drug in a trial in which the usefulness of cephradine in the treatment of intestinal infections caused by Shigella or Salmonella organisms was examined.

3. Cephapirin Chemically, cephapirin is the sodium salt of 7-[~-(4-pyridylthio)acetamido]cephalosporanic acid (Fig. 2). It is effective only when administered parenterally and possesses antibacterial and pharmacological properties similar to those of cephalothin (Chisholm et a l . , 1970; Gordon et a l . , 1971; Axelrod et al., 1972; Bodner and Koenig, 1972). In pharmacological studies carried out in human volunteers by Axelrod et a l . (1972), 1-gm doses of cephapirin administered intravenously and intramuscularly produced mean serum levels after 1 hour of 6.1 and 18.6 pg/ml, respectively. These levels are similar to those occurring in patients with bacterial infections after the same dose regimen (Bran et al., 1972). Following intramuscular injection, Axelrod et a l . (1972) found a peak serum level after 30 minutes of 24.2 CLglml. Serum half-lives were calculated to be 21 and 47 minutes for the intravenous and intramuscular routes, respectively. Within 6 hours of administration, antibacterial activity in the urine represented 72% of the intravenous and 53% of the intramuscular dose of cephapirin. Gordon et al. (1971) examined the serum levels of cephapirin in pediatric patients. After a 20-mg/kg intramuscular dose, mean peak serum concentrations of 14.5 &ml occurred at 30 minutes. Following rapid intravenous administration of the same dose, levels as high as 54 pg/ml were recorded at 15 minutes. However, antibacterial activity was absent from the serum 4 hours after both intravenous and intramuscular administration. The recovery in the urine of children was comparable to that found in adults (Khan and Pryles, 1973).

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With regards to protein binding, cephapirin is bound to human serum proteins to the extent of 44 to 50% (Chisholm et al., 1970). Cephapirin is fairly well tolerated although pain is produced at the site of intramuscular injection (Bran et al., 1972; Wiesner et al., 1972), and some workers have even advocated that the drug should not be given by this route for this reason (Axelrod et al., 1972). Phlebitis is an additional problem with cephapirin following repeated intravenous administration (Jameson et al., 1971; Bodner and Koenig, 1972; Wiesner et al., 1972) although many workers have stated that the incidence and severity of phlebitis is milder than that which occurs with cephalothin (Lane et al., 1972; Carrizosa et al., 1973; Inagaki and Bodey, 1973). There is no evidence of renal or hepatic toxicity in animals or humans (Wiesner et al., 1972) even after prolonged therapy at high dosage (Bodner and Koenig, 1972). However, eosinophilia is a side effect that has been definitely attributed to cephapirin (Gordon et al., 1971; Bodner and Koenig, 1972; Khan and Pryles, 1973). Bone marrow depression with leukopenia, neutropenia, and anemia has also been observed in isolated cases (Bran et al., 1972).

4. Cefazolin Cefazolin, 7-[ 1-(lH)-tetrazolylacetamido]-3-[2-(5-methyl-1,3,4thiadiazoly1)-thiomethyll-A3-cephem-4-carboxylicacid (Fig. 2), was first described by Kariyone et al. (1970). It is readily absorbed from the intramuscular site, a 500-mg dose in human volunteers giving mean peak serum concentrations ranging from 34.9 (De Schepper et al., 1973) to 44.6 &ml (Nishida et a l . , 1970a). This is 2-3 times as high as serum levels obtained after an equivalent dose of cephaloridine. Serum levels of cefazolin steadily decline after 1 hour, but therapeutically effective concentrations are maintained for as long as 6-10 hours after intramuscular administration (Nishida et al., 1970a,c; Shibata and Fugii, 1971; Phair et al., 1972). Although there are no data available for the serum half-life of cefazolin in humans, in the mouse the half-life after subcutaneous injection of a 20-mg/kg dose has been calculated to be 36.2 k 3.5 minutes which is similar to that found with cephaloridine (Wick and Preston, 1972). Nishida et al. (1970a) examined the degree of binding of cefazolin to human serum protein and found that the drug is bound to the extent of 74%. However, more recently Wick and Preston (1972) have arrived at the somewhat lower figure of 48%. The distribution of I4C-labeled cefazolin has been extensively studied

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in rats and mice by Kozatani et a l . (197233) using whole-body radioautography and liquid scintillation counting. Following intravenous and intramuscular injections of 20-mglkg doses of labeled drug, radioactivity was rapidly distributed throughout the whole body except for the brain. Its absence from the brain indicates that cefazolin does not cross the blood-brain barrier. Radioactivity was almost completely removed from all organs and tissues at 4 hours except from the kidney, liver, and gastrointestinal tract which are responsible for the elimination of the drug. In pregnant mice after intravenous administration, the placenta showed a considerable amount of radioactivity but the fetuses showed a much lower activity than that occurring in the maternal circulation. Placental transfer of cefazolin, therefore, appears to be of a low order. Antibacterial activity is rapidly excreted in the urine following an intramuscular dose of 500 mg and maximal concentrations in excess of 1 mg/ml have been recorded in an initial 3-hour sample (Nishida et a l . , 1970a). Therapeutically effective urine levels of antibacterial activity are maintained for at least 8 to 10 hours after dosing. A total of about 80% of the dose is recovered in a 24-hour urine sample which is a little greater than the recovery of cephaloridine after a comparable dose (Nishida et a l . , 1970a; De Schepper et a l . , 1973). From human (Nishida et a l . , 1970a) and animal studies (Nishida et a l . , 1970c; Ishiyama et a l . , 1971; Kozatani et al., 1972a) it appears that cefazolin is metabolically stable when administered parenterally and is excreted in the urine as unchanged compound. In rats and dogs, cefazolin is excreted in the bile in levels higher than observed with other cephalosporins such as cephaloridine and cephalothin (Nishida et a l . , 1970~).For example, after an intramuscular dose of 20 mg/kg, the 24-hour biliary recovery of cefazolin in rats was 17% compared with 0.6% for cephaloridine and 1.0% for cephalothin. In the dog, 3.3% of cefazolin was recovered in 24 hours but only 0.1 and 0.2% of cephaloridine and cephalothin, respectively. In metabolism and excretion studies using 14C-labeled cefazolin in the rat, Kozatani et al. (1972a) demonstrated that antibacterial activity in the bile is due almost entirely to unchanged drug, providing further evidence that cefazolin is a metabolically stable compound. 5. Cephanone

Chemically, cephanone is 3-(5-methyl-l, 3,4-thiadiazol-2-yl-thiomethyl)-7-[2-(3-sydnone) acetamido]-3-cephem-4-carboxylic acid. It is not absorbed after oral administration and must, therefore, be administered

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parenterally. In human volunteer studies carried out by Meyers et al. (1972b), 1-gm doses of cephanone produced peak serum concentrations of 38.0 and 81.2 d m l following intramuscular and intravenous administration, respectively. Cephanone is only slowly removed from the serum and even at 6 hours the serum concentration of antibacterial activity was found to be in excess of the MIC’s for most of the gram-positive organisms which these authors investigated. A recovery of 96 and 50% of antibacterial activity was found in the urine after intravenous and intramuscular doses, respectively, during an initial 8-hour period following drug administration. The total 24-hour recovery was 100% for the intravenous and 60% for the intramuscular route of administration. Cephanone was well tolerated in these volunteer studies, no untoward side effects or changes in hematological parameters, nor hepatic and renal function being noted. In animal studies, Wick and Preston (1972) administered 20 mg/kg cephanone subcutaneously to mice and found peak serum concentrations of 10.9 d m l at 30 minutes. The calculated serum half-life (29.5 & 2.2 minutes) was similar to that of cephaloridine (33.8 2 4.1 minutes) and cefazolin (36.2 f 3.5 minutes). Serum protein binding was found to be concentration-dependent, increasing from 32 to 50% as the serum concentration of cephanone decreased from 10 to 2.5 ,ug/ml. High concentrations of antibacterial activity were found in the urine and this was shown to be due largely to unchanged cephanone.

V. Clinical Aspects A. URINARY TRACTINFECTIONS The complexity of urinary tract infection (Kass and Zinner, 1969; Cox and Montgomery, 1971) is such, that the determination of efficacy and comparative efficacy of antimicrobial agents is very difficult. The most commonly encountered organisms include E. coli, P. mirabilis, Klebsiella, and S . aureus which cause between 60 and 85% of all urinary tract infections (Cox and Montgomery, 1971). Of the cephalosporins, cephalothin appears to be slightly more effective against P. mirabilis isolates, cephaloglycin against the Enterobacter species, and cephaloridine against enterococci, cephalexin being generally slightly less active than the others (Cox and Montgomery, 1971); all are ineffective against the pseudomonads (see Section II1,A). The success or failure of antimicrobial therapy in urinary tract

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infections is dependent on many factors (Hodson, 1959; McCabe and Jackson, 1965; Condie et al., 1968; Norden and Kass, 1968; Smellie and Normand, 1968; Brumfitt et a l . , 1970), not solely the antimicrobial potency of the compound.

1. Cephalothin Cephalothin has been shown to be valuable in the treatment of patients with urinary tract infection both in adults (Anderson and Petersdorf, 1963; Philson et a l . , 1964; Walters et a l . , 1964; Turck et al., 1965; Wilson et al., 1966; Cox and Montgomery, 1971) and in children (Heitler et a l . , 1964; Hallberg and Svenningsen, 1970). It is well tolerated in patients with penicillin allergy (Griffith and Black, 1964) and those with renal insufficency (Holloway and Scott, 1965; Bulger and Petersdorf, 1970; Lang and Levin, 1971). Urologists have used cephalothin prophylactically in irrigation solutions of the bladder during cytoscopy and retrograde pyelography (Seneca and Lattimer, 1965) and in urological surgery (Wilson et a l . , 1971). By combining cephalothin with other antibacterial agents, its success rate in patients with resistant urinary tract infection is increased (Thoburn and Fekety, 1970; Weissbach et a l . , 1971).

2 . Cephaloridine The value of cephaloridine in patients with urinary tract infections is well documented (Murdoch et a l . , 1964; Stewart and Holt, 1964; Gherardi et a l . , 1965; Ishigami et a l . , 1965; Lenti et a l . , 1965; Apicella et a l . , 1966b; Dennis et al., 1966; MacLean et al., 1967; Foord, 1967; McAllister and Mack, 1967), a very high success rate being recorded in acute urinary tract infections (Rocca-Rossetti et a l . , 1965; Foord, 1967; Landes et al., 1967), but a slightly less impressive result being observed in those cases with acute on chronic infections. In resistant gram-negative infections, cloxacillin and methicillin (Ohkoshi et a l . , 1967) have been added to cephaloridine with encouraging results. Chronic urinary tract infections are invariably difficult to treat irrespective of antimicrobial agent employed (Garrod et a l . , 1955). Cephaloridine is no exception, reinfection occurring in the majority of patients after an initial favorable response (Murdock et a l . , 1964; McAllister and Mack, 1967). A high failure rate with respect to cephaloridine may be related to the development of L-forms of E . coli in patients with chronic urinary tract infection. Better results are obtained

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by increasing the duration of treatment (Seneca et al., 1967; Stratford and Dixson, 1967; Ueda et al., 1967; Burgess et al., 1968). Cephaloridine has also been used prophylactically in patients undergoing gynecological operations (Matthews et al., 1967). It is also considered of value in patients undergoing prostatectomy (Lacy et al., 1971). Drach et al. (1971) showed that the addition of a closed neomycin-polyrnixin-irrigated catheter system, in addition to systemic cephaloridine, further reduced the rate of acquired bacteriuria. In very severe urinary tract infections, cephaloridine alone in a high dose or in combination with gentamicin, or a combination of polymixin B or colistimethate and kanamycin may be necessary (Cox and Montgomery, 1971).

3 . Cephalexin Acute urinary tract infections respond extremely well to cephalexin (Griffith, 1969; S. Ishigami et al., 1969; Brumfitt et al., 1970; Daikos et al., 1970; Erikssen et al., 1970; Kawamura, 1970; Leigh et al., 1970; Wise and Schwartz, 1971; Seneca et al., 1972). Long-term results in patients with chronic urinary tract infections are poor and essentially similar to all the other antimicrobial agents in this indication (Griffiths, 1969; Kind et al., 196913; Levison et a l . , 1969; Ohkoshi et al., 1969; Richards, 1969; Spiers et al., 1969; Fass et al., 1970b; Rohner, 1970; Mohring et al., 1971; Speight et al., 1972). However, good results have been recorded by Kaye et al. (1970) and Smith and Williams (1970). Eyckmans (1970), Fairley (1970), Ohkoshi et a l . (1970), and Acar (1971) found that the rate of eradication of bacteriuria is dependent on .the site of the infection. Gruneberg and Brumfitt (1967) and Davies et al. (1971) found cephalexin and ampicillin equally effective in the treatment of urinary tract infections. In bacteriuria of pregnancy, Brumfitt and Pursell (1972) found ampicillin and cephalexin equally effective in their hospital patients, whereas ampicillin was clearly superior in general practice. Acar (1971) compared cephalothin, cephaloridine, cephaloglycin, and cephalexin, and found no difference in the rate of cure, but failures were more common with cephaloglycin. 4. Cephaloglycin A variable response has been seen with cephaloglycin in urinary tract infections (Eyckmans et al., 1968; Hogan et al., 1968; Cox and Montgomery, 1971; Wick et al., 1971). It is also more effective in

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patients with lower urinary tract infections (Ronald et al., 1968), and its effectiveness is likewise dependent on certain host factors (Boyer and Andriole, 1968; Johnson et al., 1968). The treatment of chronic urinary tract infections has been essentially unsatisfactory (Seneca et al., 1970). Cephaloglycin sometimes is poorly tolerated (Eyckmans et al., 1968; Hogan et al., 1968; Ronald et al., 1968), but others have found the drug well tolerated even in patients with chronic urinary tract infections on long-term therapy, with reduced renal function (Landes et al., 1969; Lowentritt et al., 1971).

5. Others Cephapirin (Bodner and Koenig, 1972), cephradine (Elkins et al., 1972), cephacetrile (Benner, 1972; Opitz et al., 1972; Maurice et al., 1973), and cefazolin (Benner, 1972; Cox et al., 1972) are also valuable in

the treatment of urinary tract infections.

B. RESPIRATORYTRACTINFECTIONS 1. Cephaloridine This drug has been used successfully in the treatment of various lower respiratory tract infections (Verney, 1967; Eyckmans, 1970). Numerous authors have noted the effectiveness of cephaloridine in pneumococcal pneumonia (Katsu et al., 1965; Foord, 1967; Galbraith, 1967; MacLean et al., 1967; Brayton et al., 1967). In such infections, cephaloridine compares favorably with penicillin G (Cohen et al., 1966; Thornton and Andriole, 1966; Tempest and Austrian, 1967) and ampicillin (Matts, 1967), although it is inferior to erythromycin in cases with Mycoplasma infections (Sterner et al., 1967). The response in staphylococcal infections has also been most encouraging (Holloway and Scott, 1965; Apicella et al., 1966b; Foord, 1967; Galbraith, 1967; Merchant, 1967). Cephaloridine has a similar high clinical and bacteriological rate of success (Galbraith, 1967) in streptococcal infections. However, it is not as effective against Haemophilus influenzae respiratory infections-not an unexpected finding (May, 1967). Also, certain Klebsiella organisms are equally resistant (Hinman and Wolinsky, 1967). However, H . influenzae and Klebsiella pneumoniae lung infections cleared in 68 and 60% of cases, respectively, in a multicenter trial with cephaloridine (Foord, 1967). Even better clinical results have been achieved by

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Galbraith (1967) and Eyckmans (1970). Coliform pneumonia or bronchopneumonia, on the other hand, responds very poorly to cephaloridine (Foord, 1967). Superinfection by P. aeruginosa has been occasionally reported in patients with pneumonia who were treated with cephaloridine (Apicella et a l . , 1966b; Dennis et al., 1966). This is not a problem specific to cephaloridine. Cephaloridine compares well with ampicillin (Matts, 1967; Bailey, 1970; Pines, 1970), penicillin, and streptomycin (Pines et al., 1967; Pines and Raafat, 1967; Citron et a l . , 1968) in acute and acute on chronic bronchitis. Pines et al. (1971) also have shown cephaloridine to be effective in very troublesome purulent bronchitis. Cephaloridine has also been administered by intravenous infusion (Matsumoto et al., 1970) and as inhalations (Kennedy, 1967). In the treatment of lung abscess, cephaloridine compares favorably with the combination of penicillin and streptomycin (Seftel et a l . , 1970). Also, Le Roux (1970) has demonstrated its value in chronic suppurative cavitated lung lesions. Merchant (1967) also successfully treated children with lung abscesses.

2. Cephalothin

This drug has been used with success in patients with respiratory tract infections (Griffith and Black, 1964; Klein et al., 1964; Weinstein et al., 1964). Cephalothin and cephaloridine are equally effective in respiratory tract infections (Smith, 1971), cephalothin being usually restricted to the patient with severe life-threatening infections (Merrill et al., 1966). 3 . Cephalexin This antibiotic has been found valuable in various infections of the upper respiratory tract (Busca, 1969; Seftel et a l . , 1969; Fass et al., 1970a; Marks and Garrett, 1970). A large number of studies indicate its value in acute pneumonia or bronchopneumonia (Bieder, 1969; Daikos, 1969; Fujii et a l . , 1969; Hedlund, 1969; Jacquot, 1969; Lidman et al., 1969; Stratford, 1969; Bailey et a l . , 1970; Daikos et al., 1970; Macquet and Lafitte, 1970; Poggiolini, 1970; Rohner, 1970; Solberg et a l . , 1972). Gould (1970) showed cephalexin to be slightly inferior to doxycycline in acute Haemophilus and S. pyogenes respiratory tract infections in general practice. Better results are obtained in acute bronchitis (Seftel et a l . , 1969; Bailey et al., 1970; Fass et a l . , 1970b) than in chronic

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bronchitis (Bailey et al., 1970; Eyckmans, 1970; Macquet and Lafitte, 1970; Pines, 1970; Rohner, 1970) or bronchiectasis (Bieder, 1970; Marks and Garrett, 1970). Increasing the dosage and duration of treatment improves the response (Pines, 1972). A varying response has been observed with cephalexin in the treatment of lung abscess (Seftel et al., 1969; Daikos et al., 1970). Oral plus intrapleural injections have been employed with success by Macquet and Lafitte (1970) in subjects with empyema.

4. Cephaloglycin

This drug has been used with success in upper respiratory tract infections (Leiderman et al., 1970; Stillerman, 1970; Matsen, 1971). A poorer response is seen in cases with lower respiratory tract infections (Rohner, 1970), although Hogan et al. (1968) have obtained a satisfactory response. 5. Others Cephapirin (Benner, 1972; Bran et al., 1972; Quintiliani et al., 1972) and cephacetrile are effective in cases with respiratory tract infection (Hodson and Holloway, 1973; Maurice et al., 1973), the latter having comparable efficacy to ampicillin in acute exacerbations of chronic bronchitis (Eckolt et al., 1972).

C. VENEREALDISEASE New chemotherapeutic agents have to be compared to penicillin with respect to efficacy and tolerability in venereal disease (Fiumara, 1972).

1. Gonorrhea Penicillin remains the drug of choice in the treatment of gonorrhea (Meyer-Rhon, 1972). The cephalosporin analogs have an advantage in penicillin-sensitive patients although cross-sensitization has been occasionally reported (Meyler and Herxheimer, 1968) (see Section VI). a. Cephaloridine. This antibiotic has been successfully employed in the treatment of gonorrhea (Seftel et al., 1966), following encouraging in vitro sensitivity of Neisseria gonorrhoeae to cephaloridine (Barber and Waterworth, 1964; Muggleton et al., 1964). A single 1-gm dose has been found to be inadequate (Marshall and Curtis, 1967; Oller, 1967a), better results being obtained with higher doses (Oller, 1967b; Jouhar and Fowler, 1968; Haberman et al., 1969; Shapiro and Lentz, 1970). Despite

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higher doses the failure rate may still be high (Lucas et a l . , 1966; McClone et a l . , 1968; Keyes and Halverson, 1969; Molin and Nystrom, 1970; Pariser and Marino, 1970), penicillin-resistant strains of gonococci being an ever increasing problem (Fowler, 1969). b. Cephalexin. This drug is more active against N. gonorrhoeae than cephaloridine (Muggleton et al., 1969). Other advantages are ease of administration and high urinary excretion (Foord et al., 1969b). The sensitivity of gonococci to cephalexin runs parallel to their penicillin sensitivity (Oller and Smith, 1969). The cure rate is seen to be related to total dosage and frequency of administration (Oller et a l . , 1970; Csonka, 1971; Taylor and Holloway, 1972). Unfortunately, patient default rate with repeated administrations is high (Willcox and Woodcock, 1970) as seen previously with kanamycin (Wilkinson et al., 1967). The addition of probenecid further improves the response rate (Fowler, 1970; Landes et

al., 1972). Wilcox (1971) compared cephalexin with demethylchlortetracycline and found the latter achieved a lower failure rate. 2. Nongonococcal Urethritis (N.G.U.)

This is probably among the commonest of the sexually communicable diseases (Csonka, 1971; King, 1971). The treatment of nongonococcal urethritis with cephaloridine has been unimpressive (Csonka and Murray, 1967); tetracyclines remain the treatment of choice (Csonka,

1967).

3. Ophthalmia Neonatorum and Lymphogranuloma Venerum Cephaloridine has been successful in the treatment of these two

conditions (Vegas, 1965; Oller et al., 1970).

4. Syphilis Penicillin is still the most valuable treatment of syphilis, since its first application to man by Mahoney, Arnold, and Harris in 1943. Success can be expected in more than 95% of cases (Jefferiss and Willcox, 1963; King, 1971). No evidence of true resistance of Treponema pallidum to penicillin has been yet demonstrated (Guthe, 1965). Penicillin hypersensitivity reactions present a real problem (Frank et a l . , 1965; Minkin and Lynch, 1968), especially in pregnant patients when alternative treatments have also well-recognized hazards (Kline et a l . , 1964; South et a l . , 1964; Davis and Kaufman, 1966). Also Hardy (Hardy et a l . , 1970)

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reported the failure of penicillin to prevent congenital syphilis in an adequately treated mother with syphilis. a . Cephaloridine. The effectiveness of cephaloridine against syphilis was initially demonstrated by Galla et a l . (1965), although the antisyphilitic activity of cephaloridine was shown to be less than that of penicillin G (Flarer, 1967). Several encouraging reports have been published on its use in early syphilis (Flarer et al., 1965; Ochoa and Cravioto, 1965; Seftel et a l . , 1966; Oller, 1967b; Glicksman et a l . , 1968; Duncan and Knox, 1971). Pregnant patients with syphilis respond very satisfactorily to cephaloridine (Flarer, 1967; Oller, 1967a). b. Cephalexin. Unimpressive results have been seen with cephalexin in the treatment of syphilis (Duncan and Knox, 1971). AND GYNECOLOGY D. OBSTETRICS

The cephalosporins are readily transferred across the placenta to the fetal blood and later into the amniotic fluid, as evidenced by the following reports: cephalothin (Lee and Anderson, 1963; Reichelderfer and Reichelderfer, 19M; Sheng et a l . , 1965; MacAulay and Charles, 1968; Morrow et a l . , 1968; Hirsch, 1970; Paterson et a l . , 1970; Stephen and Bolognese, 1970; cephaloridine (Barr and Graham, 1967a; Arthur and Burland, 1969a; cephalexin (Mizuno et a l . , 1969; Bert6 et a l . , 1972; Paterson et al., 1972.) If they are to be of value in the treatment of infections in the pregnant patient, these drugs should satisfy certain basic criteria (Atkinson et a l . , 1966a; Hirsch, 1970, 1971). The antibiotic to be employed must be capable of attaining therapeutically effective concentration both in the mother, fetus, and amniotic fluid, while at the same time being free of any unwanted effects.

1. Cephalothin

This drug has been used with success in obstetric practice (Holloway and Scott, 1965; Hirsch, 1968; Paterson et al., 1970; Soto et al., 1970). Urinary tract infections associated with pregnancy respond well to cephalothin therapy (Conti and Iurlaro, 1967). Allen et a l . (1972) showed quite clearly that, in gynecological use, cephalothin reduced morbidity following hysterectomy.

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2. Cephaloridine This antibiotic is valuable in a variety of obstetric problems (Josey and Farrar, 1970; Ostergard, 1970; Soto et a l . , 1970), amniotitis and postpartum endometritis (Holloway and Scott, 1965; Conti and Iurlaro, 1967), various pelvic infections, postabortal sepsis (Tully and Smith, 1968), and mastitis. Cephaloridine has also been used successfully in pregnant women with urinary tract infections (Landes et a l . , 1967; Brumfitt et a l . , 1970). It has also been used prophylactically in patients with premature rupture of the membranes, but Barr and Graham (1967a) and Charles and MacAulay (1970) feel that neither cephaloridine or any other antibiotic (Davies, 1972) should be given in all cases with premature rupture of the membranes. Cephaloridine used prophylactically in patients undergoing gynecological operations reduced significantly the incidence of postoperative urinary tract infection (Matthews et a l . , 1967).

3. Cephalexin This drug has been successfully used in treatment of pelvic infections, puerperal mastitis, bartholinitis (Mizuno et a l . , 1969; Poggiolini, 1970), and in pregnant patients with associated urinary .tract infections (Hirsch, 1971). Good results have also been obtained with cephalexin in cases of gynecological infections (Daikos et a l . , 1970).

E. PEDIATRICS It is difficult to evaluate new antibiotic agents in newborn (Butler, 1967), infection being a serious problem (Butler and Bonham, 1963) yet amenable to prevention and treatment (Keay et a l . , 1967). A significant change has also been noticed in the type of organism involved (Nyham and Fousek, 1958; Forfar et al., 1966; Burland and Simpson, 1967; Keay et a l . , 1967).

1. Cephalothin This is a valuable drug for a variety of infections in infants and children (Riley et a l . , 1963; Flux et al., 1964; Hallberg and Svenningsen, 1970). Davies (1970) regards it as the treatment of choice in penicillinallergic children with serious infections.

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2. Cephaloridine This drug has been used successfully in pediatric practice (Flux et a l . , 1966; Sweetman et a l . , 1966; Albores et a l . , 1967). It has also been used effectively in combination with streptomycin (Fine et a l . , 1966; Fekety and Weiss, 1967; Burland et a l . , 1970; Gould, 1970; Keay and Fleming, 1970) and polymixin B or E (Marget, 1967). Cephaloridine is of undoubted value in children with septicemia, meningitis, and respiratory and urinary tract infections (Corda and Scane, 1965; Flux et a l . , 1966; Merchant, 1967). It has been shown to have little affinity for albumin binding, as apposed to cephalothin, which may be of importance in the treatment of premature infants (Malaka-Zafiriu and Strates, 1969).

3. Cephalexin This is also a valuable antibiotic in the treatment of the severe infections of the newborn (MacMillan, 1971). Respiratory tract infections in older infants and children respond well to cephalexin (Bamatter and Hazeghi, 1969; Marget and Daschner, 1969; Donnison and Davison, 1970; Grislain, 1970; Leiderman et a l . , 1970; Stillerman and Isenburg, 1971; Azimi et a l . , 1972; Disney et a l . , 1971; Gau et a l . , 1972; Paschetta and De Biasi, 1971; Riley, 1971).

4. Cephaloglycin This drug is also effective in children with mild infections (Leiderman et a l . , 1970; Matsen, 1971). F. DERMATOLOGY The cephalosporins, cephalothin (Limson and Santos, 1968; Water-

mann et a l . , 1968; Bernard et a l . , 1969; Smith, 1971; Tong, 1972),

cephaloridine (Dennis et a l . , 1966; Holloway and Scott, 1965; Thornton and Andriole, 1966; Foord, 1967; Ishiyama et a l . , 1967; Da Silva et a l . , 1967; Smith, 1971; Kaplan et a l . , 1968a; Brown et a l . , 1970; Polk and Lopez-Mayor, 1969; Dillon et a l . , 1972), cephalexin (Goto, 1969; Hedlund, 1969; Kienitz and Mann, 1969; Lidman et a l . , 1969; Shibata and Kato, 1969; Page et a l . , 1970; Grislain, 1971; Lyons and Andriole, 1971; Lautre and Baker, 1972), cephapirin (Bodner and Koenig, l972), and cephacetrile (Benner, 1972; Hodges et a l . , 1973; Maurice et a l . , 1973), have all been shown to be most valuable in the treatment of skin and soft tissue infections.

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G. OPHTHALMOLOGY Excellent intraocular penetration of cephaloridine occurs following both i.m. and i.v. administration in man (Records, 1968a,b; Riley et a l . , 1968; Richards et a l . , 1972) and is superior to that following cephalothin (Mizukawa et a l . , 1965; Hatano et a l . , 1966; Records, 1969a). Even higher concentrations are achieved following local subconjunctival injection (Records and Ellis, 1969; Moll, 1970; Moll et a l . , 1971). Cephaloridine has been used for severe occular infections (Rosa, 1967; Richards et al., 1972) and prophylactically in patients undergoing intraocular surgery (Ikui et a l . , 1966; Records and Ellis, 1969; Moll,

1971). Cephalexin penetrates the eye both in animal and in man (Gager et a l . , 1969; Boyle et a l . , 1970) and has been used with success in the treatment of various eye infections and also as an antibiotic cover during surgery (Constantindes, 1971). Cephalosporins may also be found to be useful in eradicating Treponema pallidum from the eyes of syphilitic patients (J. L. Smith, 1969).

H. MENINGITIS Cephalothin and cephaloridine are able to reach adequate concentrations in the CSF (Klein et a l . , 1964; Perkins and Saslaw, 1966; Ruedy, 1967; Vianna and Kaye, 1967; Lerner, 1969; Oppenheimer and Beaty, 1969; Walker and Gonzales, 1969; Brown et a l . , 1970), both in experimental meningitis and true clinical meningitis in man.

1. Cephalothin Satisfactory response has been obtained in the treatment of pneumococcal meningitis (Griffith and Black, 1964; Perkins and Saslaw, 1966; Herrell, 1968) and staphylococcal meningitis (Weinstein et al., 1964); a less favorable and variable response is observed in meningococcal and H . influenzae meningitis (Binns and Pankey, 1966; Almond, 1969; Southern and Sanford, 1969; Brown et a l . , 1970). The combination of cephalothin and gentamicin may be necessary in gram-negative meningitis (Rahal, 1972).

2. Cephaloridine Murdock et a l . (1964), Katsu et a l . 1965), Apicella et a l . (1966b), McKenzie et a l . (1967), Love et a l . (1970), and Lerner (1971) have all

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shown cephaloridine to be valuable for the treatment of pneumococcal meningitis. Impressive results have also been seen in staphylococcal (Toscano et a l . , 1967; Walker and Gonzales, 1969), streptococcal, and H. influenzae meningitis (McKenzie et a l . , 1967; Lerner, 1971). Meningococcal meningitis is a more serious infection with the increasing occurrence of sulfonamide-resistant strains (Eickhoff and Finland, 1965), and cephaloridine may or may not (Walker and Gonzales, 1969) prove to be effective.

I. ENDOCARDITIS Treatment with the cephalosporin analogs should be considered particularly in patients allergic to penicillin (Koya and Misawa, 1970) (see Section VI).

1. Cephalothin The value of cephalothin in endocarditis is well documented (Flux et al., 1964; Walters et al., 1964; Merrill et al., 1966; Siguier et al., 1966; Tuano et a l . , 1967; Rahal et al., 1968). Both streptococcal and staphylococcal endocarditis respond well to cephalothin (Saslaw and Perkins, 1966; Limson and Santos, 1968; Weinstein, 1971; Meyers et al., 1972a). Also aerobic diphtheroid endocarditis (Kaplan and Weinstein, 1969; Goldsweig et a l . , 1972) responds well to this drug. In the treatment of severe gram-positive and gram-negative endocarditis, combination with methicillin, oxacillin, streptomycin (Herrell et al., 1965, 1967), polymixin B (Pressman et al., 1966), or kanamycin (Foster, 1969) may be necessary. Cephalothin has also been used prophylactically in patients undergoing cardiac surgery (Okies, Vitoslav and Williams, 1971). 2 . Cephaloridine

This drug has also been used with success in cases ol' endocarditis (Ueda et al., 1965; Apicella et al., 1966a; Parker et al., 1968; Kump et al., 1969). The response of staphylococcal endocarditis has been less encouraging (Rowntree and Bullen, 1967), despite adequate serum concentrations (Burgess and Evans, 1966). Therapy may need to be precise and prolonged (Foord and Snell, 1966; Vacek and MolikoviWagnerova, 1967) to achieve better results. Similar combinations to those mentioned for cephalothin may be valuable (Ehrenkranz, 1971). Cephaloridine may be employed prophylactically in patients at risk

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undergoing surgery, as it can kill strains of Streptococcus viridans more rapidly than erythromycin or vancomycin (Tozer et al., 1966).

3. Cephalexin The value of cephalexin in the treatment of endocarditis remains unclear (Zabransky et al., 1969; Stratford, 1970). J. BONESAND JOINTS The main causative organism in acute osteomyelitis is S. aureus; less frequent offenders include streptococci, E. coli, Proteus sp., and H. influenzae (Smith, 1958; Blockey and Watson, 1970). The expected effectiveness of the cephalosporin analogs is in the region of 80 to 90% (Smith, 1971), equivalent to the combination of dicloxacillin, ampicillin, and benzylpenicillin (Kienitz, 1971). Treatment should be commenced with a high dosage of the antibiotic, in order to prevent a recurrence rate of up to 30% observed in chronic osteomyelitis by Waldvogel et a l . (1970). 1. Cephalothin Success with cephalothin in bone infection has been well-documented (Griffith and Black, 1964; Barrett and Ehrenkranz, 1965; Hess and Martin, 1971). Norden (1971) found that cephalothin penetrates well into the diseased bone, and parenteral administration, therefore, should be adequate (Argen et al., 1966; Nelson, 1971).

2 . Cephaloridine It has been shown that cephaloridine is of value in treating both acute and chronic osteomyelitis and septic arthritis (Cabitza, 1965; Taylor and Fallon, 1966; Lai, 1967; Azimi and Cramblett, 1968; Kaplan et a l . , 1968b; Steigbeigel et al., 1968; Fleming et al., 1970; Hermans, 1971; Kienitz, 1971).

3 . Cephalexin Cephalexin has also been used successfully in patients with acute osteomyelitis (Daikos et a l . , 1969, 1970; Hedlund, 1969, 1970), due mainly to staphylococcal infection. In chronic osteomyelitis, results have been encouraging (Cheng et al., 1970; Herrell, 1971), oral medication having undoubted value in the long-term management of chronic bone infection.

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4. Other Cephalosporins Benner (1972) has shown cephacetrile and cefazolin valuable in osteomyelitis.

VI. Hypersensitivity and Allergenicity A. GENERALCONSIDERATIONS It is a well-known fact that, although they are not toxic in the pharmacological sense, the penicillins may elicit allergic responses in a small percentage of patients. The immunological properties of penicillins are discussed in detail by Shaltiel et al. (1971), who point out that commercially available benzylpenicillin and 6-APA (the latter being implicated as a major antigenic determinant in penicillin allergy) may contain trace amounts of penicilloylated protein impurities and that the removal of these impurities markedly reduces the immunological manifestations induced by these antibiotics. The position with cephalosporins is, in many respects, less clear-cut. The Coombs test is widely used to detect the presence of blood-group antigens absorbed on the surface of red blood corpuscles. Coombspositive hemolytic anemia is known to occur in patients receiving large doses of intravenous benzylpenicillin, cephaloridine, cephalothin, or cefazolin (Gralnick et al., 1967; Molthan et al., 1967; Perkins et al., 1967a; Kaplan et al., 1968a,b; York and Landes, 1968; Kosakai and Miyakawa, 1970; Mine et al., 1970a; Gralnick, 1971; Petz, 1971). Much higher concentrations of cefazolin are needed to produce a positive direct Coombs reaction than penicillin or other cephalosporins, and the intensity of the positive reaction appears to be related to the lytic action of the antibiotics on the red blood cells (Mine et al., 1970a). However, the minimum concentration of a cephalosporin needed for a positive reaction varies markedly with the antiglobulin serum used (Gralnick, 1971). Stewart (1962) found that cephalosporin C was not cross-allergenic with the penicillin series in skin tests in human volunteers. However, subsequent studies have indicated that the cephalosporins have crossreactivity with benzylpenicillin (Brandriss et al., 1964; Batchelor et al., 1966; Shibata et at!., 1966; Mine et at!., 1970b; Mashimo, 1971). Brandiss et al. (1964) proposed that the benzylpenicilloyl group was an antigenic determinant in benzylpenicillin hypersensitivity and that it was formed by conjugation of the highly reactive intermediate, benzylpenicillenic

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acid, to protein. However, cephaloridine was also antigenic in rabbits despite its inability to undergo a penicillanic acid-type rearrangement in vitro ; in addition, both the hemagglutinating and passive cutaneous anaphylaxis reacting antibodies formed in response to injection with this antigen cross-reacted with the benzylpenicillin antigen. Batchelor et al. (1966) showed that cephaloridine and cephalothin formed protein conjugates that stimulated the production of hemagglutinating, precipitating, and guinea pig-sensitizing antibodies in the rabbit and that there was cross-reaction between benzylpenicillin and cephalosporins; they suggested that this cross-reaction was caused by an immunologically similar side chain. Cephaloridine and cephalothin were found by Mashimo (1971) to have a higher affinity to antibenzylpenicilloyl antibody than cephalexin, ampicillin, or oxacillin; it was proposed that similarities in the stereochemical structure of the side chain of cephaloridine, cephalothin, and benzylpenicillin are associated with the degree of hapten inhibition. Ky et al. (1970) described a very sensitive lymphoblastic transformation test for studying cross-reactions between benzylpenicillin and cephaloridine and found that such reactions were rare (circa 12%); they suggested that the p-lactam nucleus is, in some patients, a common hapten responsible for cross-reactions. Cefazolin gives a minimal cross-reactivity with benzylpenicillin, ampicillin, and cephaloridine (Mine et al., 1970b), and Mine et al. (1970b) agreed with other authors cited above that the cross-reactivity between cephalosporins and related penicillins against benzylpenicillin appears to be mediated mainly by the acyl side chain. Reaction of cephalosporins with ammonia, amino acids, and other simple amino compounds in weakly alkaline solutions gives labile compounds with a A,,, of 230 nm (Hamilton-Miller et al., 1970a,b). Such compounds obtained from cephalosporin C are less stable in concentrated than in dilute solution, and their breakdown is followed by the appearance of new chromophores with A,,, 270 and 278 nm. These are attributable to the fragmentation of the molecule with the formation of penaldates and penamaldates from the side chain and the carbon atoms of the p-lactam ring. Derivatives similar to those obtained with simple amino compounds, but which are virtually unaffected by dilution, may be formed when cephalosporins react with lysine polymers such as serum). Interestingly, Hamilton-Miller et al. polylysine or poly (lysine (1970a,b) concluded that “haptens resulting from the primary reaction of cephalosporins, deacetylcephalosporins and deacetoxycephalosporins with the ammonia groups of proteins will differ from each other and from the penicilloyl haptens derived from the penicillins in several respects other than in the nature of their N-acyl side-chains.’’

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Traces of macromolecular protein or peptide complexes that possess immunogenic and allergenic properties are found in natural penicillins and cephalosporins (Stewart, 1968). Cross-immunization and crosssensitization between the two types of p-lactam antibiotics occurs especially when macromolecular conjugates are used (Stewart et al.,

1970).

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Addendum Since the main body of this article was written, several important papers on cephalosporins have been published. This brief Addendum will thus consider these papers in order to bring this chapter as up-todate as possible. The works of Flynn (1972) and Garrod et al. (1973)

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should also be consulted (as should the following brief review articles: Hewitt, 1973; Levine, 1973; Mandell, 1973; Anon, 1974; Garrod, 1974; Sanders et al., 1974).

A. ANTIBACTERIALASPECTS The new cephalosporin cefamandole, has been shown (Eykyn et al., 1973; Williams and Geddes, 1973; Williams and Andrews, 1974) to be more active than cephaloridine, cephalothin, cephradine, cephalexin, or cephacetrile against H. influenzae and against gram-negative bacilli susceptible to cephalosporins. Cefamandole has also been found to be active against many strains resistant to other cephalosporins, such as indole-positive Proteus sp., but minimum bactericidal concentrations are considerably higher than minimum inhibitory concentrations (MICs) (Eykyn et al., 1973; Russell, 1974), and there is a marked inoculum effect (Eykyn et al., 1973). Isenberg et al. (1973) have observed a great similarity in the response of gram-negative rods and gram-positive cocci (other than enterococci) to cephalothin and cephacetrile, with the latter antibiotic more active than cephalothin against enterococci. Misiek et a l . (1973) have described the structure-activity relationship of more than 600 cephalosporins against M. tuberculosis H 37Rv, and have shown that among the most active derivatives of cephalosporin C were those in which a pyridyl or an aminomethylphenyl moiety was present in the side chain. The cephamycins are not cephalosporins, but are closely related to this antibiotic group, and as such are worthy of mention here, especially as a new cephamycin, cefoxitin, has shown considerable promise as an antibacterial agent. The cephamycins are produced by various strains of Actinomycetes (Stapley et al., 1972) and their isolation and chemical characteristics have been described (Miller et al., 1972). Cephamycins have a broad antibacterial spectrum, including many strains resistant to cephalosporins and penicillins, and induce spheroplast formation in gram-negative bacteria (Stapley et al., 1972) suggesting a similarity in mode of action. Cefoxitin is more active than cephalothin against indolepositive Proteus sp. and is not destroyed by the p-lactamases produced by gram-negative bacteria; it is, however, considerably less active than cephalothin against gram-positive bacteria (Kosmidis et al., 1973). Further studies (Wallick and Hendlin, 1974; Miller et a l . , 1974) have confirmed these findings. Dusart et al. (1973) have presented evidence that the same enzyme performs DD-carboxypeptidase and transpeptidase activities in the

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Streptomyces strains they examined and that this enzyme is the killing site of penicillins and cephalosporins. Nishino and Nakazawa (1973a) have used the scanning electron microscope to investigate the surface changes in cephalexin-treated S . aureus and E . coli and by means of viable counting and ultrathin sections in the electron microscope they have demonstrated an antagonistic effect between cephalexin and an inhibitor of protein synthesis, erythromycin, in S. aureus (Nishino and Nakazawa, 1973b). Topp and Christensen (1974) have considered, from a theoretical viewpoint, the structure-activity relationship of fi -1actam antibiotics, based upon a two-step mechanism of inhibition of bacterial growth: (a) reversible binding of drug to enzyme, followed by (b) irreversible acylation of transpeptidase enzyme via thiol attack at the p lactam carbonyl carbon. A rapid assay method for cephalosporins, based on their inhibition of glucose or inositol fermentation by a strain of gentamycin-resistant Providencia (group A) has been described by Noone (1973). Reller et al. (1973) have made an in vitro and in vivo evaluation of cefazolin, and Saslaw and Carlisle (1973) have compared the use of various cephalosporins in the therapy of staphylococcal infections in monkeys.

B. RESISTANCE Jackson et al. (1973) have compared the ability of p-lactamases from staphylococci, Enterobacteriaceae, and two types of P. aeruginosa to hydrolyze benzylpenicillin and five different cephalosporins. Medeiros and O’Brien (1973) tested the sensitivity of ampicillin-resistant strains of E. coli to cephaloridine, cephalothin, cephalexin, and cefazolin, and found three distinct classes of ampicillin-resistant strains possessing different P-lactamases. These classes were as follows: Class I. Hydrolysis of different cephalosporins occurred at widely differing rates. The MICs against large inocula correlated with the rates of hydrolysis, except for cephalexin; against small inocula, the MICs were low for all four cephalosporins. Class ZZ. Much more “cephalosporinase” than “ampicillinase” activity occurred in these strains. There was resistance to the four cephalosporins even when tested with small inocula. Cephalexin was again the most resistant cephalosporin, but ampicillin was destroyed even less. Class ZZZ. The p-lactamase of these strains had low levels of specific activity, and the strains were only slightly more resistant to cephalosporins than were ampicillin-sensitive E. coli strains. Hedges et al. (1974) examined the molecular specifities of R-factor-

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determined p-lactamases, and showed that the p-lactamases determined by 29 resistance plasmids could be divided into two groups: (a) TEM-type (20 out of 29 cases). These were very uniform with respect to substrate specificity, e.g., taking the rate of hydrolysis of benzylpenicillin as 100 (see Table I in main chapter), the relative rates of hydrolysis of oxacillin, ampicillin, and cephaloridine were, respectively, low (about 5), 100-110, about 75. (b) Oxacillin-hydrolyzing type. These were less common (9 out of 29 cases), showed lower absolute levels of activity, but were heterogenous as regards substrate specificities, e.g., with benzylpenicillin as 100, rate for oxacillin was 184-646, ampicillin 133-408, and cephaloridine 30-62. Hewitt (1973) has stated that there is a high correlation between p lactamase production and bacterial resistance to cephalosporins, but points out (see the main part of the present article) that (a) intrinsic resistance and (b) cellular permeability are also important in this context. Del Bene and Farrar (1973) studied p-lactamase activity in 10 strains of Bacteroides fragilis and found that these caused low rates of hydrolysis of cephalosporins, whereas penicillin substrates appeared to be undetected. In two strains, this “cephalosporinase” activity was increased some 40 to 80-fold by growing the cells in the presence of penicillin. Of the four cephalosporins used as substrates, cephaloridine was the most, and cephalexin the least, labile. Farrar and Newsome (1973) present evidence in support of the hypothesis that the synergistic antibacterial effects of a combination of p-lactam antibiotics on gram-negative p-lactamase-producing bacteria is due to the inhibition of p-lactamase by one of the antibiotics and the lethal effect of the second, “protected” drug. The cephamycin, cefoxitin, is highly resistant to inactivation by several p-lactamases (Daoust et al., 1973; Onishi et al., 1974), although the drug is degraded by some strains, and this enzymatic destruction could be an important factor in their resistance (Onishi et al., 1974). Hou and Poole (1973) have studied the effect of competitive inhibitors on the p-lactamase activity of S. aureus and B . cereus, and showed that the degree of inhibition depended on (i) the structural characteristics of the inhibitor, (ii) the time of exposure of enzyme to inhibitor, and (iii) the ratio of amount of inhibitor to amount of substrate. Haque and Russell (1974) have studied the effect of some chelating agents on the subsequent susceptibility of some gram-negative plactamase and non- @-lactamase producers to some @-lactam antibiotics and other antibacterial agents. Transferable resistance associated with cellular impermeability to penicillins and cephalosporins, rather than

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inactivation by a P-lactamase, has been described by Richmond and his colleagues (Curtis et al., 1973). With P. aeruginosa, a major factor conferring resistance appears to be impermeability rather than drug destruction (Mills and Russell, 1974). Richmond and Curtis (1973) have considered the resistance of gram-negative bacteria in relation to plactamase production and intrinsic factors. Vernon and Russell (1974) have described the effects of methicillin, cephaloridine, and cephalothin on the growth, lysis, and viability of methicillin-resistant strains of S. aureus, and have shown that it is the treatment temperature, rather than the pretreatment growth temperature, which is of importance in determining resistance to these p-lactam antibiotics. Further studies on the combined action of p-lactam antibiotics against gram-negative bacteria have been described (Bobrowski et al., 1973). C. PHARMACOLOGICAL AND CLINICAL ASPECTS The absorption, distribution, and excretion of cephradine has recently been investigated in mice, rats, and dogs by Weliky et al. (1974). The antibiotic was well absorbed after oral and subcutaneous administration and its plasma half-life was about 1.0 hour. Cephradine was widely distributed throughout the body tissues with the greatest concentrations being in the liver and kidneys. Virtually all of an administered dose was recovered during a 24-hour urine collection period, cephradine being excreted in an unchanged form (Miraglia et al., 1973; Weliky et al., 1974). Cephradine has been reported to be well tolerated in laboratory animals (Gadebusch et al., 1972) and has a low order of acute and chronic toxicity after oral and parenteral administration to rats, dogs, and monkeys (Hassert et al., 1973). In a clinical study conducted by Zaki et al. (1974), cephradine was observed to be rapidly absorbed after oral administration, peak serum concentrations being reached in 1.0 hour. After single doses, nearly all the antibiotic was excreted in the urine as unmetabolized material within 6.0 hours of dosing. Trujillo et al. (1974) have described the usefulness of parenterally administered cephapirin in acute lung diseases in children. Evidence suggests, however, that the continuous intravenous administration of large doses of cephapirin produces a high incidence of side reactions. For example, in a study conducted by Sanders et al. (1974), most subjects experienced profound malaise, weakness, fever, lymphadenopathy, and a pruritic skin rash following administration of a 2.0 gm dose of

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cephapirin by rapid intravenous injection, four times a day for 2 to 4 weeks. The pharmacokinetic properties of cefazolin have recently been compared with other cephalosporins by a number of workers (Bergeron et al., 1973; Kirby and Regamey, 1973; Cahn et al., 1974). After intravenous and intramuscular administration, serum levels of cefazolin have been found to be about twice those following equal doses of cephaloridine and three to four times those after cephalothin. Peak serum levels of 3 2 4 2 pg/ml have been reached after an intramuscular injection of 500 mg cefazolin. Following this dose, the half-life of cefazolin in the serum of normal persons was approximately 2.0 hours, although this has been observed to be extended up to 12.0 hours in patients with severely compromised renal function (Levison et al., 1973) and up to 35.0 hours in severely uremic patients (Craig et al., 1973). The long half-life (2.0 hours) in serum compared with those of cephaloridine (1.1 hour) and cephalothin (0.5 hour) appears to be primarily due to the low serum and renal clearance of cefazolin (Regamey and Kirby, 1973). These factors, together with an apparent small volume of distribution, no doubt contribute to the high peak levels of the antibiotic in the blood after dosing. Cefazolin is bound to human plasma protein to the extent of 7686% (Nishida et al., 1970a; Kirby and Regamey, 1973). This is relatively high when compared with the values for cephaloridine (20%), cephalexin (15%), and cephalothin (65%), although the recently introduced cephanone is also highly bound (88%). In toxicological studies in laboratory animals carried out by Birkhead et al. (1973), cefazolin exhibited low acute toxicity in mice and rats and was not teratogenic for mice or rabbits. In subacute and chronic toxicity experiments in rats and dogs, the major finding was damage to muscles following intramuscular injection. In nephrotoxic studies, cefazolin was less nephrotoxic than cephaloridine (Birkhead et al., 1973; Silverblatt et al., 1973).

Madhaven et al. (1973) have compared the clinical usefulness of cefazolin with other cephalosporins. It has been found to be effective in the treatment of bacterial pneumonia (Turck et al., 1973), bronchitis (Gold et al., 1973), bacterial endocarditis (Quinn et al., 1973; Reinarz et al., 1973), soft tissue infections (Gold et al., 1973), urinary tract infections (Cox, 1973), and in a preliminary report by Pickering et al. (1973) cefazolin has been observed to be effective in a number of childhood infections. On the other hand, cefazolin has only a limited

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usefulness in uncomplicated gonorrhea (Karney et al., 1973; Nelson, 1973). The only adverse clinical effect so far reported with cefazolin is pain at the site of injection following both intravenous and intramuscular administration. The incidence is greater with cefazolin than with cephaloridine (Cahn et al., 1974), however, like cephaloridine, cefazolin appears to cause less ,pain on parenteral injection than cephalothin. Furthermore, cefazolin does not cause thrombophlebitis despite extended intravenous administration (Reller et al., 1973). In a study carried out by Regamey and Kirby (1973), cephanone administered intramuscularly at a dose level of 7 mg/kg (approx. 500 mg) to five normal volunteers gave peak serum concentrations (36 pg/ml) approximately four times as high as with the same dose of cephalothin, twice as high as with cephaloridine, but slightly lower than with cefazolin. The serum half-life of cephanone was about 2.5 hours and over 90% of the dose administered was recovered in the urine within 24 hours of dosing. Cephanone has a small apparent volume of distribution, probably related to its high serum protein binding (88%). Jackson et al. (1974) have made a double-blind comparison of cephacetrile with cephalothin or cephaloridine and concluded that cephacetrile can be considered as being comparable to cephalothin in antimicrobial treatment and overall reactions. The possibility that the cause of cephalothin-induced phlebitis may be associated with the acid pH of cephalothin when in solution (pH 4.8-5.0) has recently been investigated by Carrizosa et al. (1974). These authors carried out a double-blind comparison of phlebitis produced by intravenous infusions of cephalothin at acid and neutral pH values (7.2-7.4). However, they found that neither the incidence, degree, nor time of onset of phlebitis was altered by a change in pH of the cephalothin infusion.

REFERENCES TO ADDENDUM Anon. (1974).Pharmaceut. J. 212, 181. Bergeron, M. G., Brusch, J. L., Barza, M., and Weinstein, L. (1973). Antimicrob. Ag. Chemother. 4, 396. Birkhead, H. A., Briggs, G. B., and Saunders, L. Z. (1973). J. Infec. Dis. Suppl. 128, s379. Bobrowski, M. M., Gbbicka, K., and Borowski, J. (1973). J. Hyg. Epidemiol. Microbiol. Immunol. 17, 129. Brogard, J. M., Kuntzmann, J. and Lavillaureix, J. (1973). Schweiz. Med. Wochenschr. 103, 110. Cahn, M. M., Levy, E. J., Actor, P. and P a d s , J. F. (1974).J. Clin. Pharmacol. 14, 61. Carrizosa, J., Levison, M. E., and Kaye, D. (1974). Antimicrob. Ag. Chemother. 5 , 192.

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Cox, C. E. (1973). J. Znfec. Dis. Suppl. 128, S397. Craig, W. A., Welling, P. G., Jackson, T. C., and Kunin, C. M. (1973). J. Znfec. Dis. Suppl. 128, S347. Curtis, N. A. C., Richmond, M. H., and Stanisich, V. (1973). J. Gen. Microbiol. 79, 163. Daoust, D. R., Onishi, H. R., Wallick, H., Hendlin, D., and Stapley, E. 0. (1973). Antimicrob. Ag. Chemother. 3 , 254. Del Bene, V. E., and Farrar, W. E. (1973). Antimicrob. Ag. Chemother. 3 , 369. Dusart, J., Marquet, A., Ghuysen, J. M., Frkre, J. M., Morens, R., Leyh-Bouille, M., Johnson, M., Lucchi, C., Perkins, H. R., and Nieto, M. (1973). Antimicrob. Ag. Chemother. 3 , 181. Eykyn, S., Jenkins, C., King, A., and Phillips, I. (1973). Antimicrob. Ag. Chemother. 3 , 657. Farrar, W. E., and Newsome, J. K. (1973). Antimicrob. Ag. Chemother. 4, 109. Flynn, E. H. (1972). Ed., “Cephalosporins and Penicillins. Chemistry and Biology.” Academic Press, New York and London. Gadebusch, H. H., Miraglia, G. J., Basch, H., Goodwin, C., Pan, S., and Renz, K. J. (1972). Aduan. Antimicrob. Antineoplastic Chemother. 1, 1059. Garrod, L. P. (1974). Brit. Med. J. 3 , 96. Garrod, L. P., Lambert, H. P.. and O’Grady, F. (1973). “Antibiotic & Chemotherapy,” 3rd. Ed. Churchill Livingstone. Edinburgh and London. Gold, J. A., McKee, J. P., and Ziv, D. S.(1973). J. Znfec. Dis. Suppl. 128, S415. Haque, H., and Russell, A. D. (1974). Antimicrob. Ag. Chemother. 6, 200. Hassert, G. L., DeBaecke, P. J., Kulesza, J. S., Traina, V. M., Sinha, D. P., and Bernal, E. (1973). Antirnicrob. Ag. Chemother. 3 , 682. Hedges, G. R., Scholand, J. F., and Perkins, R. L. (1973). Antirnicrob. Ag. Chemother. 3 , 228. Hedges, R. W., Datta, N., Kontomichalou, P., and Smith, J. T. (1974). J. Bacteriol. 117, 56. Hewitt, W. L. (1973). J. Znfec. Dis. Suppl. 128, S312. Hou, J. P., and Poole, J. W. (1973). Chemotherapy 19, 129. Hughes, S. P. F., Hughes, L., and Dash, C. H. (1974). Brit. J. Clin. Pruct. 28, 51. Isenberg, H. D., Painter, B. G., Sampson-Scherer, J., and Siegel, M. (1973). Amer. J. Clin. Pathol. 59, 700. Jackson, G. G., Lolans, V. T., and Gallegos, B. G. (1973). J. Znfec. Dis. Suppl. 128, S327. Jackson, G. G . , Riff, L. J., Zimelis, V. M., Daood, M., and Youssuf, M. (1974). Antimicrob. A g . Chemother. 5 , 247. Karney, W. W., Turck, M., and Holmes, K. K. (1973). J. Znfec. Dis. Suppl. 128, S399. Kirby, M. M., and Regamey, C. (1973). J. Znfec. Dis. Suppl. 128, S341. Kosmidis, J., Hamilton-Miller, J. M. T., Gilchrist, J. N. G., Kerry, D. W., and Brumfitt, W. (1973). Brit. Med. J. 4, 653. Levine, B. B. (1973). J. Znfec. Dis. Suppl. 128, S364. Levison, M. E., Levison, S. P., Ries, K., and Kaye, D. (1973). J. Infec. Dis. Suppl. 128, s354. Madhaven, T.. Quinn, E. L., Freimer, E., Fisher, E. J., Cox, F., Burch, K., and Pohlod, D. (1973). Antirnicrob. Ag. Chemother. 4, 525. Mandell, G. L. (1973). Ann. Intern. Med. 79, 561. Medeiros, A. A. and O’Brien, T. F. (1973). J. Znfec. Dis.Suppl. 128, S335. Miller, A. K., Celozzi, E., Kong, Y., Pelak, B. A., Hendlin, D., and Stapley, E. 0. (1974). Antimicrob. Ag. Chemother. 5, 33.

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Miller, T. W., Goegelman, R. T., Weston, R. G., Putter, I., and Wolf, F. J. (1972). Antimicrob. Ag. Chemother. 2, 132. Mills, A. P., and Russell, A. D. (1974). J . Pharm. Pharmacol. 26, 102p. Miraglia, G. J., Renz, K. J. and Gadebusch, H. H. (1973). Antimicrob. Ag. Chemother. 3 , 270. Misiek, M., Moses, A. J., Pursiano, T. A., Leitner, F., and Price, K. E. (1973). J. Antibiot. 26, 737. Nelson, M. (1973). J. Znfec. Dis. Suppl. 128, S404. Nishino, T., and Nakazawa (1973a). J a p . J. Microbiol. 17, 383. Nishino, T., and Nakazawa (197333). J. Antibiot. 26, 362. Noone, P. (1973). J. Clin. Pathol. 2 6 , 5 0 6 . Onishi, H. R., Daoust, D. R., Zimmerman, S. B., Hendlin, D., and Stapley, E. 0. (1974). Antimicrob. Ag. Chemother. 5 , 38. Pickering, L. K., O’Connor, D. M., Anderson, D., Bairan. A. C., Feigin, R. D., and Cherry, J. D. (1973). J. Znfec. Dis. Suppl. 128, S407. Quinn, E. L., Pohlod, D., Madhavan, T., Burch, K., Fisher, E., and Cox, F. (1973). J. Infec. Dis. Suppl. 128, S386. Regamey, C., and Kirby, W. M. M. (1973). Antimicrob. Ag. Chemother. 4, 589. Reinarz, J. A., Kier, C. M., and Guckian, J. C. (1973). J. Znfec. Dis. Suppl. 128, S392. Reller, L. B., Karney, W. W., Beaty, H. N., Holmes, K. K., and Turck, M. (1973). Antimicrob. Ag. Chemother. 5 , 488. Richmond, M. H., and Curtis, N. A. C. (1974). Ann. N.Y. Acad. Sci. 255, 553. Russell, A. D. (1974). Unpublished data. Sanders, W. E., Johnson, J. E., and Taggart, J. G. (1974). New Engl. J . Med. 2 9 8 , 4 2 4 . Saslow, S., and Carlisle, H. N. (1973). J . Znfec. Dis. Suppl. 128, S373. Silverblatt, F., Harrison, W. O., and Turck, M. (1973). J . Znfec. Dis. Suppl. 128, S367. Stapley, E. O., Jackson, M., Hernandez, S., Zimmerman, S. B., Currie, S. A., Mochales, S., Mata, J. M., Woodruff, H. B., and Hendlin, D. (1972). Antimicrob. Ag. Chemother. 2, 122. Topp, W. C., and Christensen, B. G. (1974). J. Med. Chem. 17, 342. Trujillo, H., Manotas, R., Salazar, C., Rodriguez, A., Uribe, A., Agudelo, N., and de Vidal, E. L. (1974). J . Intern. Med. Res. 2, 125. Turck, M., Clark, R. A., Beaty, H. N., Holmes, K. K., Karney, W. W., and Reller, L. B. (1973). J. Infec. Dis. Suppl. 128, S382. Vernon, G. N., and Russell, A. D. (1974). J. Pharm. Pharmacol. 2 6 , 102p. Wallick, H., and Hendlin, D. (1974). Antimicrob. Ag. Chemother. 5 , 25. Weliky, I . , Gadebusch, H. H., Kripalani, K., Arnow, P., and Schreiber, E. C. (1974). Antimicrob. Ag. Chemother. 5 , 49. Westenfelder, S. R., Naber, K. G., andMadsen, P. 0. (1973). Infection 1, 157. Williams, J. D., and Andrews, J. (1974). Brit. Med. J . 1, 134. Williams, J. D., and Geddes, A. M. (1973). Brit. Med. J . 2, 613. Zaki, A., Schreiber, E. C., Weliky, I., Knill, J. R., and Hubscher, J. A. (1974). J. Clin. Pharmacol. 14, 118.