In vitro activity of new macrolides against respiratory pathogens

~E FULL POTENTIAL OF THE MACROLIDES HAS BEEN LIMITED .l due to the incidence of side effects, variable oral absorption, short half-life necessitating qid dosing, and limited spectrum of activity against several pathogens, including Haemophilus injluenzae. In contrast to older macrolides, which often have been considered as second choice drugs with a very narrow spectrum of activity, clarithromycin and other newer macrolides, including azithromycin, roxithromycin, rokitamycin and dirithromycin, can now be considered as broad spectrum antimicrobial agents and will now be used as first choice drugs for the treatment of several infectious diseases. This review evaluates the comparative activity of clarithromycin with other macrolides, and several antimicrobial agents used for the treatment of respiratory pathogens.


STRUCTURE/FUNCTION ACTIVITY
Macrolides are natural or semi-synthetic agents characterized by a classic macrocyclic ring of lactone type composed of 12 t.o16 atoms from which the name is derived (Table 1).Erythromycin is a 14-ring macrolide (13 carbon and one oxygen).Introducing chemical modifications at.C6.Cs.Cg, C11 and C12 in the basic molecule has allowed for more stability in acid medium, better absorption and improved pharmacology.and better in vitro and in vivo antibacterial activity in infected.inflamed and purulent sites which are often acidic.
Clarithromycin differs from erythromycin by an 0methyl substitution at position 6 of the lactone ring.Clarithromycin is metabolized by the liver in vivo to an active metabolite.14-hydroxy clarithromycin (Figure 1).This metabolite is more active in vitro against several pathogens including beta-lactamase negative and positive H injluenzae strains.
All macrolides inhibit RNA-dependent protein synthesis.can block transpeptidation and/or translocation and bind reversibly to ribosomal 50s subunit, specifically the P site.In vitro lincomycin and chloramphenicol compete with erythromycin and can be antagonistic.

ANTIBACTERIAL ACTIVITY
The macrolides can be bactericidal or bacteriostatic.depending on concentration .microbial species, inoculum size and growth phase of bacteria.In general, their activity is increased by an alkaline environment.This can be explained by the fact.U1at.U1e penetration of weak bases like macrolides through the bacterial wall is better when they are ionized.Being acid-stable.the newer macrolides.like clarithromycin.h ave an improved in vitro activity over that of older macrolides in such an enviromnenl.In vitro activity of clarithromycin and its 14-hydroxy metabolite against H in.fluenzae: One of the major facto rs lin1iting the use of erythromycin in respiratory tract infections has been it.spoor in vitro aclivity against H iqjluenzae.In a recently publish ed study (1).we evaluated U1e in vitro activity of clarithromycin and its major 14-hydroxy metabolite, alone or in combination.against beta-lactamase positive and negative strains of H injluenzae. Two hundred and U1ree strains selected from our previous study on the epidemiology of H irifl.uenzae resistance in Canada (2) were evaluated.Characteristics of the strains studied are presented in Table 2.
Antibiotic susceptibility testing was done according to the National Committee for Clinical Laboratory Standards (NCCLS) (3) .In short.log-phase broth dilution susceptibility testing was performed with a microdilution method using Haemophilus Test Media (HTM).HTM was prepared as described by Jorgensen et al (4).The checkerboard technique was used to determine the activity of combinations of clarithromycin and 14hydroxy clarithromycin (5).The technique was performed in 96-well microplates containing HTM. Fractional inhibitory concentrations (FICs) were calculated for each agent by dividing the minimum inhibitory concentration (MIC) of the agent in combination by the MIC of the agent alone.
The FIC index was defined as the s u m of the individual FICs at the most effective concentrations.The com- bination was defined as synergistic if the FIC index was 0.5 or less, additive if it was 1, and antagonistic if the FIC index was 4.0 or greater.The MIC5o, MICgo and minimum bactericidal concentration (MBC) for clarithromycin and 14-hydroxy clarithromycin are presented in Table 3.There were no major differences in the susceptibility between B and non-B serotypes.Moreover, for the non-B serotypes or for the different biotypes, no special pattern of susceptibility was observed.
Results of the checkerboard tests for the combination of clarithromycin and 14-hydroxy clarithromycin are presented in Table 4. Overall, activity was additive in 92% (176 of 191) and synergistic in 8% (15 of 191).There was no difference in the activity of clarithromycin against beta-lactamase positive and negative strains.The site of isolation did not the susceptibility of H irifl.uenzae to the combination of clarithromycin and 14-hydroxy clarithromycin.
The effect of the combination clarithromycin and 14-hydroxy clarithromycin was also demonstrated using growth curves on strain 133, a strain against which the combination was shown to be synergistic (Figure 2).
Overall. when compared to macrolides available and under investigation, the 14-hydroxy clarithromycin was shown to be the most effective drug against H irifl.uenzae (Table 5) (1.6).Approximately 25 to 30% or more H injluenzae strains are developing resistance to ampicillin in many regions of the world (2,7,8); therefore, clarithromycin may prove to be a potential alternative to ampicillin.
Based on pharmacokinetic considerations, Hanson et al (9) and Hoover et al (10) have proposed interpretive susceptibility criteria for clarithromycin.Interpretive criteria for in vitro testing of clarithromycin have now been established by the NCCLS (11).Table 6 shows the interpretive criteria for all pathogens including H injiuenzae. Using these criteria, H influenzae is usually susceptible to clarithromycin on HTM incubated in normal atmosphere.As demonstrated in the study presented here, the anti-haemophilus activity of clarithromycin is significantly enhanced by the presence of its active major metabolite, 14-hydroxy clarithromycin.
In this study all of 203 clinically obtained H irifl.uenzae strains (including beta-lactamase positive strains) were susceptible to clarithromycin and to its major human metabolite.14-hydroxy clarithromycin.Clarithromycin and its metabolite were additive or even synergistic in killing these same H influenzae strains.Overall, the isolation site, the production or absence of beta-lactamase, the serotype and the biotype did not modify the susceptibility of either drug or their combination.
Other data suggest that these in vitro results with clarithromycin will correlate with favorable in vivo outcomes.When orally administered to gerbils with experimental H irifl.uenzae otitis media.clarithromycin and

MACROLIDES AND PNEUMOCOCCI
The pneumococci are still today one of the most virulent and most important pathogens responsible for severe pneumonia.This pathogen, which has been generally considered to be uniformly susceptible to penicillin and erythromycin, is now becoming resistant to both agents.7 reveals worldwide distribution of pneumococci resistant to either agent.While in Canada most S pneumoniae strains are still susceptible to both drugs, the percentage of resistance to penicillin has reached 36% in Spain while close to 30% are now resistant to erythromycin in France (15,16).While the penicillin-resistant strains are usually susceptible to erythromycins and vice versa, strains resistant to erythromycin are also resistant to all macrolides.Table 8 lists the MIC breakpoints for susceptibility testing of pneumococci.Clarithromycin has also been demonstrated to have the lowest MIC of the macrolides against pneumococci (Table 9) (17) .Other streptococci, including Streptococcus agalactiae and S pyogenes, are exclusively susceptible to all macrolides (6).

MACROLIDES AND M CATARRHALIS
M catarrhalis is now considered one of the most commonly recovered pathogens in upper (otitis) and lower respiratory tract infections.A recent report from England and Scotland shows that 91% of the isolates were resistant to ampicillin (18) .Most strains of 16A M catarrhalis were exclusively susceptible to amoxicillin-clavulanate, cefaclor, loracarbef, ceftxime, and tetracycline.The MICgo for old or new macrolides has been in the region of 0.25 mg/L.with the exception of roxithromycin where the MICgo has been around 1 mg/L, suggesting that macrolides may be used for the treatment of this infection.

MACROLIDES AGAINST BORDETELLA PERTUSSIS, ANAEROBES, MYCOPLASMA, CHLAMYDIA, LEGIONELLA AND MYCOBACTERIUM SPECIES
Bordetella pertussis is uniformly susceptible lo all macrolides.Gram-negative anaerobes including Bacteroides .fragilis,Bacteroides oralis and Fusobacterium species have variable susceptibility patterns to old and newer macrolides .MICs of these agents range between 0.25 and 30 mg/L or greater for most pathogens.
Macrolides have good in vitro activity against Mycoplasma pneumoniae (Table 10) and much lower MICs for this pathogen than tetracycline or quinolones (19) .Clarithromycin's MIC against Chlamydia pneumoniae is 0.03 mg/L and compares favorably with erythromycin (0.125 mg/L), roxithromycin (0.25 mg/L), azithromycin (0 .25 mg/L), doxycycline (0 .1 25 mg/L) and ofloxacin (0.5 mg/L) (20).Against Legionella pneumophila, all macrolides are effective.The MIC of clarithromycin for this organism was between 0 .25 and l mg/L.ln the pig model of L pneumophila, clarithromycin was shown to have better in vivo activity than erythromycin (14) .In the beige mouse infected with Mycobacterium avium in- tracellulare, clarith romycin was shown to be more active than ciprofloxacin, rifampin and amikacin.In the rabbit foot Mycobacte1ium leprae infection , clarithromycin was more effective than roxithromycin and azithromycin (14).Whether these animal experiments will be applicable in humans remain to be proven.Against other Mycobacterium species including Mycobacterium jortuitum and Mycobacterium chelonae, clarithromycin was also superior to azithromycin and roxithromycin (14) .
With their respective protein binding of 50 lo 70% for azithromycin.65 to 70% for clarithromycin and 90% or greater for roxithromycin , one may have to waH for further in vivo compa risons between these new generation macrolides to understand the real significance of the differen ces noted in th e in vitro activity of those compounds.Moreover.these agents have marked pharmacokinetic differences which may further modify their in vivo activity.Penetration of newer macrolides into polymorphonuclear leukocytes is excellent (21).Some investigations even s uggest that macrolides may have immunomodulatory functions (22).

SU MMARY
Clarithromycin and its microbiologically active 14hydroxy metabolite h ave additive and. at Urnes.syn ergistic , activity against H injluenzae. suggesting the potential clinical utility of clarithromycin for U1e treatment of H injluenzae infections.This increased antibacterial activity of the new macrolides and llieir potential use against a multitude of paU1ogens s uggest that clarithromycin and newer macrolides can now be called broad s pectrum antibiotics.

TABLE 1
Macrolides of today and tomorrow