spectrum of two fluoroquinolone-cephalosporin, dual-action compounds with carboxamido bonds

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1 ORIGINAL ARTICLE and : antimicrobial activity and spectrum of two fluoroquinolone-cephalosporin, dual-action compounds with carboxamido bonds David M.Johnson and Ronald N. Jones Medical Microbiology Division, Department of Pathology, University of Iowa College of Medicine, Iowa City, Iowa, USA Objective: To evaluate the potential spectrum of activity of two novel dual-action compounds vvith carboxamido bonds ( and ; Laboratorios Aranda, San Rafael, Mexico) against human pathogens. Methods: Approximately Gram-positive and Gram-negative aerobic clinical bacteria were tested in vitro using the Mueller-Hinton broth microdilution method of the National Committee of Clinical Laboratory Standards. Results: (cefamandole+enrofloxacin) and (cefamandole+norfloxacin) were equally potent against Enterobacteriaceae (MICgo range, - pg/ml and - ug/ml, respectively). Citrobacter freundii (MIC9, pg/ml) and frovidencia spp. (MIGO, >3 pg/ml) exhibited elevated study drug MICs. Enterobacteriaceae resistant to fluoroquinolones generally remained resistant. and were active against Stenotrophomonas maltophilia (MIGO, pg/ml) and oxacillin-susceptible staphylococci (MI&, pg/ml), but not oxacillin-resistant Staphylococcus aureus (MIC9, >3 pg/ml), Staphylococcus epidermidis (MIC9, pg/ml), and enterococci (MICgos, to >3 pg/ml). There was no difference in the dual-action drug activity (MIC9, pg/ml) between penicillin-susceptible and -resistant pneumococci. Haemophilus influenzae and Moraxella catarrhalis were very susceptible (MIC range, r.5- pg/ml) to both compounds. Conclusions: The activity of these novel dual-action compounds, formed from the bonding of older antimicrobials, warrants further investigation for potential human and/or animal health use, including toxicology and pharmacokinetics. Key words: Dual-action compounds, older cephalosporins, enrofloxacin, norfloxacin I NTROD UCTl ON and CQ-3 are synthetic broad-spectrum, dual-action combination (LIAC) antimicrobials coiisistiiig of a cephalosporin (cefaiiiandole-like) linked at the C-7 position by a carboxaniido bond to the fluoroyuinolones enrofloxacin and iiodoxacin, respectively. Aiiti~iiicrobial drugs having a dual mode of Corresponding author and reprint requests: D. M. Johnson, Medical Microbiology Division, Department of Pathology, 5 MRC, University of Iowa College of Medicine, Iowa City, Iowa 5, USA Tel: (39) Accepted 3 January 997 Fax: (39) action were first described two decades 'igo but, unfortunately, toxic side-effects limited their ther'ipeutic use [I]. However, further development of ])A<: agents led to cornpounds that icoinbined the cell wall synthesis-iiihibitory action of a cephalosporin with the DNA gyrase-inhibiting activity of a quinolone. For example, Ro3-9 (an t:stei--linked co-drug of desacetylcefotaxiiiie aiid fleroxacin) has demonstrated potent activity against Enterobacteriaceae, staphylococci, Stveprococc~rs spp, Haernophilus ir!fhrrznc~, Movaxella catnvdinlis, Nrisscria spp. aiid ~iuiiiero~~s strailis resistant to fluoroquinolones or third-generation cephalosporin? [3,]. Another IIAC, <5-53 (a combination of a catechol cephalosporin and ciprofloxacin) was generally less potent than Ro3-9, although it possessed superior activity against Strnotvophornonas maltopldia and Pseirdomonas spp. IS]. 335

2 336 Clinical Microbiology and Infection, Volume 3 Number 3, June 997 conh I;Ilj CH, ch-s I R COOH N- N R Compound R R CH5 Cyclopropyl H CH5 Figure Chemical structures of and and are structurally similar to the previously studied DAC K3-9, and have a fluoroquinolone moiety that is linked at its 3' carboxyl group to the cephalosporin component. However, this structure could limit the candidate DAC to cephalosporin-like activity until the fluoroquinolone is released []. Unique to the new Aranda compounds is the carboxyamido bond at the C-7 position, in contrast to the 3'-position bonds utilized in previous DAC drugs. In this study, we evaluated the in vitro potency and spectrum of activity of and (Figure ) compared with cefamandole, enrofloxacin and norfloxacin. In addition, ceftazidime was added as a third-generation comparator cephalosporin because of its broader and more potent spectrum of activity with respect to cefamandole. MATERIALS AND METHODS, and enrofloxacin were provided by Laboratories Aranda, S.A. (San Kafael, Mexico). and cefiazidime were obtained from Eli Lilly and Co. (Indianapolis, IN), and norfloxacin from Merck, Inc. (Kahway, NJ). Nearly Gram-positive and Gram-negative aerobic bacteria were tested (Tables -3). The organisms were recent clinical isolates ( 993-) cultured from blood and other sterile body fluids (not including urine) in patients at the University of Iowa Hospitals and Clinics (Iowa City, IA). The strains were stored at -7 C and subcultured twice prior to being tested. Minimum inhibitory concentrations (MICs) were determined in cation-adjusted Mueller-Hinton broth microdilution trays (Prepared Media Microbiologics, Tualatin, OR). Fastidious organisms, such as Streptococcus spp., Corynebacteriurn jeikeiurn, Bacillus cereus and Haeniophilus influenzae, were grown in medium supplemented with 5% lysed horse blood. After overnight incubation, the trays were examined for growth and MICs were determined according to National Committee for Clinical Laboratory Standards (NCCLS) methods [6,7]. Interpretive breakpoint criteria for norfloxacin, cefamandole and ceftazidime followed NCCLS [6] guidelines, while enrofloxacin and the new DACs were assigned the susceptible breakpoints of 5 pg/ml and 5 pg/ml, respectively. RESULTS AND DISCUSSION Table lists the results of the study compounds tested against 5 strains of Enterobacteriaceae. and were equally active (MICgo range, - pg/ml and - yg/ml, respectively) against this family of bacteria. Citvobacterjeundii (MICgo, pg/ml) and the Pvovidencia spp. (MICeo, >3 pg/ml) demonstrated greater resistance to the DAC drugs; however, the comparison cephalosporins, cefamandole and ceftazidime, remained active against the Provideucia spp. strains (MICoo range, - pg/nil). Several species (Entevohacter aerogenes, Enterohacter cloacae, Klehsiella oxytoca, Mouganella rnouganii, Panteoa aggloinerans, Proteus vulgaris and Servatia rnarcescens) that exhibited resistantrange MIC results (MICgo, >3 pg/ml) to cefamandole were more susceptible to both IIAC drugs (MIC9o range, - pg/ml). In general, when the

3 Johnson and Jones: In vitro activity of and 337 Table and antimicrobial activity and spectrum tested against 5 Enterobacteriaceae clinical icolates by NCCLS dilution methods MIC (pg/tnl) Organism (no. tested) Antimicrobial agent 5% 9% Range u/;i Susceptihlea Gram-negative bacilli Citrobacterjeundii () > 3 >6 > 3 > 3.5 to >3 5.5 to >3 5. to > 6.5 to > 6 to >3:! to >lh 9 (5) 9 (Xi) vo 35 5 Cifrobacter koseri ( ) O.OO I () () Enterobacter aeropzer () EIirofloxaciIi > 3 > 6 to >:5 to >.5. 5 to >. 6 to > 6 to >?I:! to > 6 (YO) (9) En teru barter cloacae () NorAoxacind Ceftazidinie. 5 > 3 > to >,3 to >3. to N 6 to > 6 to 3 to > I6 9 (9) YO (9) Ercherichia c~li () to > Lb to >3 5 to > 6 9 (9) YO (YO) 9 I Klebstella oxytora () Nodloxacin io.6 > to >3 (.6- () () 7 Cefamaiidole Ceftazidiine.5 to > 3 to > 3.5 to >>6 to > 6 to > (9) 9 (9) 9 VO I i Cefamaridole 6 > O ,6 to >3 5 to >6 () () 3 YO

4 33 Clinical Microbiology and Infection, Volume 3 Number 3, June 997 Table -continued MIC (pg/ml) Organism (no. tested) Antimicrobial agent 5% 9% Range % Susceptible Gram-negative bacilli Pantoea agriomerans ().5.5 >3 > to >3 to 3 () () 7 7 Proteus mirabilis () Nodloxacin to >3 5- () () Proteus vulpris () > 3 > to 3 5- () () Providencia rettgeri () Enro floxacin 5 5 >3 3 to >3 to >3 to >6 to >6 5 to >3 5- (9) () 9 Provideitcia stuartii ( ) 3 >3 to >3 to >3 to >6 to (7) (7) 6 7 Salmonella enteritidis () () () Serratia marcescens () Norfloxacm > 3 > 3 to 3 to >3-6 to >6 to >3 - () () 5 Shigella spp. () () ()

5 Johnson and Jones: In vitro activity of and 339 Table -continued Organism (no. tested) Antimicrobial agent 5% 9% Range % Suweptihle' MIC Gram-negative bacilli Ersinia entcromlitica () ( ) () Other Eiiterobacteriaceae (lqh Norlloxacin.5 > 3 > to >:5 to >'I6 ( ) () SO "Breakpoint criteria per National Committee for Clinical Laboratory Standards [6] for norfloxacin, cefamandole and ceftazidime. The enrofloxacin-susceptible breakpoint was assigned at 5 yg/rnl (wne as ciprofloxacin) and the commonly used cephalosporin breakpoint of 5 pg/nil was applied to and. The O/u susceptible values parentheses indicate the 5 pg/ml t'reakpoint. bincludes six species: Enterobacfer rakazakii (two strains), Enterobacfer taylorae (two strains), Hafnia aluei (one strain), Klebsiella ozueiiae (one strain), Salmonella typhi (two strains) and Serratia liquefciens (two strains), Table and antimicrobial activity and spectrum tested against various non-enteric Gram-negative clinical isolates by NCCLS dilution methods MIC (pg/nl) Organism (no. tested) Antimicrobial agent 5% 9% Range 96 Susceptiblea Gram-negative bacilli Acinetobacter spp. () -. () - ().5-.: -6 9 >3 > 3 3 to >3 - Psrudornona, aerrqinosa (3) (67) (67) > 3 > 3 > 3 to >6 Stenotrophomonas maltophilia () >6 > 3 > to >I6 6 to >3-6 ( ) () 9 Moraxella catarrhah p-lactamase-positive BKO-I ().5 Norlloxacin I5-5 5 () ()

6 3 Clinical Microbiology and Infection, Volume 3 Number 3, June 997 Table -coontinued MIC (pg/ml) Organism (no. tested) Antimicrobial agent 5% 9% Range % Susceptible" Gram-negative bacilli Moraxrlla catarrhatis P-lactamase-positive BRO- () ~ () () P-lactamase-negative () cq () () Haernophilus inpuenrae P-lactamase-positive () Noriloxacin () () P-lactaniase-negative, Ampicillin-susceptible () () () Ampicillin-resistant () to >3 to >6 () () 7 "Breakpoint criteria per NCCLS 6 for norfioxacin, cefanlatldole and ceftazidime. The enrofloxacin susceptible breakpoint was assigned at Sl pg/ml (same as ciprofloxacin) and the commonly used cephalosporin breakpoint of 5 pg/ml was applied to and. If a fluoroquinolone breakpoint (5 pg/nil) was used for or, the spectrum is indicated in parentheses. activities of and are compared with those of their component fluoroquinolones (enrofloxacin and norfloxacin), the best component drug was two- to four-fold more active than and CQ-. The spectrum and potency of the 'thirdgeneration' cephalosporin comparator were superior to those of both DAC drugs. Table contains the activity results for the study drugs tested against non-enteric Gram-negative organisms. The species causing respiratory tract infection, Haernophilus ivlfluenzae and Moraxella catarvhalis, were usually susceptible to all tested antimicrobial agents. had slightly elevated MICs for the P-lactamase-negative ampicillin-resistant (BLNAR) Haernophilus injuenzae strains, but none of the remaining compounds was significantly affected by strains possessing enzymatic or penicillin-bindingprotein-mediated resistances. (MICgo, pg/ml) was the most active compound against Pseudornonas aerqinosa. The DACs (MIC9o range, - pg/ml) were more potent than the other core structure compound cefaniandole (MICyo, > 3 pg/ ml) against Acinetobacter spp. and Stenotrophotnonas rnaltophilia.

7 Johnson and Jones: In vitro activity of and 3 Table 3 and antimicrobial activity and spectruni tested against Gram-positive clinical isolates by NCCLS dilution methods MIC (pg/nil) Organisin (no. tested) Antimicrobial agent 5% 9% Range o/o SusceptibleA Staphylococcus aureus Oxacillin-susceptible ( ) Ceftazidme.s O.O- to > (9) 9 (9) Oxacilln-resistant (5) 3 3 >6 >I6 > 3 > 3 > 6 > 6 6 >6 to >3 to >3 to > 6 to >6-3 to >6 36 (3) (3) Staphylococcus epidermidi3 Oxacillin-susceptible (3) Oil ( ) () 96 Oxacillin-resistant (7) Ceftazidinie > 6 6 >I6 to >.3 to >3 - to >6 - to 6 96 (9) 93 (9) Staphylococcus haeniolyticus Oxacillin-susceptible () Ceftazidiine O () ( ) 6 Oxacillin-resistant (3) >3 >3 6 >I6 >3 > 6 > 3 > 3 > 6 > 6 > 3 > 6 to > to.3 to >6 to >I6 to >3 >I6 () (() 3 Coagulase-negative staphylococci () 6 to > to >.5 to :~6 to >6 - to >I6 Oil (5) () 6 Streptococcus Group A () Ceftazidinie 5 zzo to >I6 5 - () (9i)

8 3 Clinical Microbiology and Infection, Volume 3 Number 3, June 997 Organism (no. tested) Antimicrobial agent 5% 9% Range % Susceptiblea Streptococcus Group B () () () 5 Group C () () () Group G () () () Streptococcus pneumoriiue Penicillin-susceptible (7) Ceftazidinie (9) (9) 9 76 Penicillin-intermediate and -resistant (3) to >6 () () 9 77 Enterococcus spp. Vanconiycin-susceptible (5) > 3 > 6 y3 >3 > 6 > 6 >3 >6 to >3 to >3 to >6 to 6 6 to >3 to >I6 56 () 56 () 5 56 Vancomycin-resistant vuna (9) 3 3 > 6 > 6 > 3 > 6 >3 > 3 >6 > 6 > 3 > 6 to >3 to >3 to >6 to >6 3 to 3 >6 () () vunb () >3 >3 > 6 > 6 3 > 6 > 3 > 3 > 6 >I6 >3 > 6 >6 to >3 to 3 to >6 to >6 > 3 () ()

9 Johnson and Jones: In vitro activity of and 33 Table 3-continued MIC (pg/ml) Organism (no. tested) Antimicrobial agent 5% 9% Range 96 Susceptible, Entrvocorrur spp. Vancomycin-resistant vanc (5) 6 Ceftazidiine > 6 3 >lh - (73) - (hij) to >3 7 >6 Covynebactevum jcikeium () > 6 Cefainandole > 3 > 6 > 3 to >.3 () > 3 to >3 () 6 to > 6 6 to >6 to > 3 to >3 >6 to >6 - - () - - () Cefainandolr >6-6 Breakpoint criteria per NCCLS [6] for norfloxacin, crfamandole and ceftazidime The enrofloxacin-susceptible breakpoint wa5 assigned at pg/ml (same a\ ciprofloxacm) and the commonly used cephalosporin breakpoint of 5 pg/inl was applied to CQ 397 and If a fluoroquinolone breakpoint (5 pg/ml) was used for or CQ, the Fpectruin is indicated in parentheses The activities of DAC drugs and the comparison compounds tested against Gram-positive organisms are listed in Table 3. Oxacillin-susceptible staphylococci were susceptible to and (MIC9 range, - pg/ml); however, oxacillin-resistant Staphylococcus haemolyticus and Staphylococcus aureus strains had very elevated MIC results (56- and - fold, respectively). Although oxacillin-resistant Staphylococcus epidermidis MICs were generally 6-fold greater than those of oxacillin-susceptible isolates, the vast majority (9%) still had DAC MICs of Spg/mL. was less active (MICgo, pg/ml) than the component drugs enrofloxacin (MIC9o, pg/ml) and cefamandole (MIC9, pg/ml) against oxacillinresistant Staphylococcus epidermidis, indicating potential antagonism. Only cefamandole had any measurable activity against oxacillin-resistant Staphylococcus haemolyticus. The remaining coagulase-negative Staphylococcus spp. were less susceptible to cefiazidime (% resistant) than to the remaining studied antimicrobials. There was no difference in the and CQ- potencies (MICso, pg/ml) between penicillinsusceptible and -resistant Streptococcus pneumoniae strains. had a slightly higher MIC (two- to 6-fold) than the component drugs against some P-haemolytic streptococci, again suggesting possible antagonism. DAC potency against Enterococcus spp. (% susceptible at 5 pg/ml) carrying the vanc gene was greater than that against vancomycin-susceptible strains (56% susceptible at 5 pg/ml). Both DACs were more active (MIC~O, pg/ml) than cefamandole (MICW, 3 pg/ml) and ceftazidime (MIC9o, > 6 pg/ml) against Bacillus cereus. All of the study conipounds (MIC9, 6 pg/ml) were inactive against Covynebacterium jeikeium. DAC antimicrobials continue to be promising agents with potential clinical use in humans and/or animals [l]. A wide variety of candidate cephemfluoroquinolone combinations have been studied using various linking bonds. The use of older fused agents (norfloxacin+a second-generation cephalosporin) indicates that activity can be enhanced by this process and applications to human practice could include DACs with known safety and efficacy (component drug history). We anxiously await the results of expanded in vitro trials of and, or similar compounds. Acknowledgments This study was supported by research funds from the Department of Pathology, University of Iowa College of Medicine. The authors thank: Ms Kay Meyer for her

10 3 Clinical Microbiology and Infection, Volume 3 Number 3, June 997 assistance in the preparation of this manuscript and the staff of the Anti-Infectives Research Center for technical contributions. References. Hamilton-Miller JMT. Dual-action antibiotic hybrids. J Antimicrob Chemother 99; 33: Georgopapadakou NH, Bertasso A, Chan KK, et al. Mode of action of the dual-action cephalosporin Ro3-9. Antimicrob Agents Chemother 99; 33: Jones RN, Barry AL, Thornsberry C. Antimicrobial activity of Ro3-9, a novel ester-linked co-drug of fleroxacin and desacetylcefotaxiine. Antimicrob Agents Chemother 99; 33: Beskid G, Fallat V, Lipschitz ER, et al. In vitro activities of a dual-action antibacterial agent Ro3-9, and comparative agents. Antimicrob Agents Chemother 99; 33: Jones RN, Sanchez ML. Antimicrobial activity of a new antipseudomonal dual-action drug, Ro5-53. Diagn Microbiol Infect Dis 99; : National Committee of Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing. Supplemental tables, M-S6. Villanova, PA: NCCLS, National Committee for Clinical Laboratory Standards. Approved standard M7-A3: standard methods for hlution antimicrobial susceptibility tests for bacteria that grow aerobically. Vdlanova, PA: NCCLS, 993.