In vitro antibacterial activities of the fluoroquinolones PD 117596, PD 124816, and PD 127391

In vitro antibacterial activities of the fluoroquinolones PD 117596, PD 124816, and PD 127391

DIAGN MICROBIOLINFECTDIS 1991;14:245-258 245 In vitro Antibacterial Activities of the Fluoroquinolones PD 117596, PD 124816, and PD 127391 Michael A...

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DIAGN MICROBIOLINFECTDIS 1991;14:245-258

245

In vitro Antibacterial Activities of the Fluoroquinolones PD 117596, PD 124816, and PD 127391 Michael A. Cohen, Michael D. Huband, Gail B. Mailloux, Steven L. Yoder, Gregory E. Roland, and Carl L. Heifetz

Three new aminopyrrolidine-substituted fluorocyclopropyl quinolones--PD 117596, PD 124816, and PD 127391-were tested for in vitro antibacterial activity against 349 bacterial strains, which are primarily clinical isolates. The minimum inhibitory concentrations (MIC) in ~g/ml required for ~90% of strains were 0.03-0.06 for staphylococci (26 strains); 0.06-0.25 for Streptococcus pyogenes, S. agalactiae, S. pneumoniae, and Enterococcus faecalis (80); 40.015 for BranhameUa catarrhalis, Haemophilus influenzae, and Neisseria gonorrhoeae (42); 0.06 for Enterobacteriaceae (97); 0.125-0.25 for Acinetobacter spp. (14); 0.5 for Pseudomonas aeruginosa (20); 0.125-1.0 for Bac-

teroides fragilis (13); and 0.25-0.5 for anaerobic cocci (11). These activities were generally superior to that of ciprofloxacin, imipenem, ampicillin, penicillin G, oxacillin, cefazolin, ceftazidime, cefoxitin, cefsulodin, aztreonam, piperacillin, amikacin, spectinomycin, doxycycline, erythomycin, clindamycin, metronidazole, and vancomycin. The activities of the new quinolones were generally unchanged with light, 50% human serum, aerobic/anaerobic atmosphere, 5% sodium cholate, cation supplementation, and lO0-fold increased or decreased inoculum; as with other quinolones, potency was measurably diminished with decreasing pH (pH ~ 6.0) and in 100% urine.

INTRODUCTION

1987; Heifetz, 1987; Hoban, 1989; Neuman and Esanu, 1988; Stahlman, 1988; Wood, 1988). The ParkeDavis Pharmaceutical Research Division of Warner-Lambert has already achieved successes in constructing quinolones without these deficits (Cohen et al., 1985 and 1986; Rolston et al., 1989; Sesnie et al. 1989). This report details the enhanced in vitro activities of PD 117596, PD 124816, and PD 127391 (Domagala et al., 1988; Sanchez et al., 1988; Shah, 1989), which are among the most potent fluoroquinolones in the latest generation of their drug class. The following data demonstrate activities of high order against both Gram-positive and -negative medically significant bacterial species.

Much interest has been focused on the quinolone antibacterial antiinfectives since nalidixic acid was introduced in 1964. This drug was limited to therapy of urinary tract infections caused by Gramnegative microorganisms. Since then, discovery and development of quinolones has reached a fever pitch similar to that of cephalosporin research in recent years. Newer quinolones are designed to expand the variety of infections treatable by members of this drug class. Until now, one obstacle has been the low level of activity against Gram-positive organisms, particularly streptococci, pneumococci, and enterococci (Andersen and Goldstein, From Parke-Davis PharmaceuticalResearch Division, Ann Arbor, Michigan, USA. Address reprint requests to Dr. M.A. Cohen, Parke-Davis PharmaceuticalResearch Division, 2800 PlymouthRoad, Ann Arbor, MI 48105, USA. Received 29 May 1990; revised and accepted 16 July 1990. ©1991 ElsevierScience Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/91/$3.50

MATERIALS A N D METHODS Antimicrobial Agents The compounds used in this study were obtained through the following sources: PD 117596, PD 124816, PD 127391 (Figure 1), ciprofloxacin, and ofloxacin

246

M.A. Cohen et al.

X

0

(Chemistry Department of Parke-Davis Pharmaceutical Research Division of Warner-Lambert, Ann Arbor, MI); amoxicillin and clavulanic acid (Beecham Laboratories, Bristol, TN); ceftazidime (Glaxo Pharmaceuticals, Greenford, Scotland); sulfamethoxazole (Hoffman-LaRoche, Nutley, NJ); cefaclor (Eli Lilly, Indianapolis, IN); imipenem (Merck Sharp and Dohme, Rahway, NJ); cefoperazone (Pfizer, New York, NY); amikacin, ampicillin, cefazolin, clindamycin, doxycycline, erythromycin, gentamicin, metronidazole, oxacillin, penicillin G, spectinomycin, and vancomycin, (Sigma Chemical Company, St. Louis, MO); aztreonam (E.R. Squibb and Sons, New Brunswick, NJ); cefsulodin (Takeda Chemical Industries, Osaka, Japan); and piperacillin (United States Pharmacopeial Convention, Rockville, MD).

bacterial drug susceptibility tests were performed in microbroth dilution trays prepared with 100 Ixl per well by using a Quick Spense II multichannel reagent dispenser (Sandy Spring Instrument Company/Dynatech Labs, Chantilly, VA) and inoculated with an initial concentration of 5 x 105 CFU/ml by means of an automatic MIC-2000 pin inoculator (Dynatech). Nonfastidious cultures were tested in MuellerHinton Broth (Difco Laboratories, Detroit MI) with cation-adjusted Mueller-Hinton broth (CAMHB), fastidious streptococci were tested in CAMHB-5% lysed horse blood, Haemophilus strains were tested in Haemophilus test medium, Neisseria strains were tested in gonococcal broth (Shapiro et al., 1984), and anaerobes were tested in anaerobe broth (Difco). All incubations were at 35°C. Neisseria strains were grown under 5% CO2. Anaerobic tests were performed in an anaerobic glove box (Forma Scientific, Marietta, OH). To study the effect of light on MICs during incubation, we topped two stacked trays with an empty tray and placed them 6 in under a fluorescent light (twin 15-W bulbs). Minimum bactericidal concentrations (MBCs) were determined in compliance with NCCLS (1987a and b) guidelines after MIC readings by transferring ->100Ixl aliquots to blood agar. Readings were taken at 24 and 48 hr of incubation. The MBC was defined as the lowest concentration of drug that yielded ->99.9% reduction of the original inoculum.

Bacterial Strains

RESULTS

A total of 350 medically significant bacterial strains are included in this study. Several reference organisms were from the American Type Culture Collection (Rockville, MD). All other isolates were from the Parke-Davis culture collection and were largely obtained from clinical sources.

In vitro Antibacterial Activity

F

/COzH

H2N

FIGURE 1 Chemical structures of PD 117596, PD 124816, and PD 127391: PD 117596, x = H and y = F; PD 124816, x = NH2 and y = F; and PD 127391, x = H and y = CI.

Antimicrobial Susceptibility Testing Minimum inhibitory concentrations (MICs) were determined by using methods prescribed by the National Committee for Clinical Laboratory Standards (NCCLS, 1990a and b) except where otherwise indicated. Studies of inoculum size effects were performed in tubes containing 1-ml volumes. Anaerobiosis effects were performed by agar dilution with 104 colony-forming units (CFU) per spot. All other

Details of the in vitro activities of PD 117596, PD 124816, and PD 127391 against 349 clinically significant Gram-positive and -negative bacterial isolates are shown in Tables 1 and 2, and compared against relevant antimicrobic agents. The MICranse, MIC for 50% of isolates (MICs0), and MIC for 90% of isolates (MIC9o) in Ixg/ml are shown in most cases. The data indicate the activities of the three quinolones to be generally comparable, and their MIC90s were almost always within twofold agreement against a particular bacterial group. It is also noteworthy that the MIC,angewas often narrow for each of these drugs against individual species, an important trait which makes the MIC9o value even more representative than usual in describing antimicrobic activity.

In vitro A c t i v i t y of PD 117596, PD 124816, a n d PD 127391

TABLE 1

247

A n t i b a c t e r i a l A c t i v i t y of PD 117596, PD 124816, a n d PD 127391 MIC (~g/ml)a

Organism Methicillin-resistant Staphylococcus spp. b

No. of Isolates 15

Antimicrobial Agent

Range

50%

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Oxacillin Amikacin Vancomycin

0.015-0.03 0.015-0.03 0.015-0.06 0.125-1 0.06-128 16->128 0.5-32 0.5-4

0.03 0.015 0.03 0.25 8 64 8 1

90% 0.03 0.03 0.06 0.5 128 >128 32 2

MethiciUin-susceptible Staphylococcus spp. c

11

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Cefazolin Oxacillin Amikacin Vancomycin

0.008-0.06 0.004-0.03 0.015-0.06 0.125-0.5 0.015-1 0.25-4 0.125-4 0.25-8 1-2

0.03 0.015 0.03 0.25 0.03 1 1 1 1

Streptococcus pneumoniae

20

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Penicillin G Cefazolin Doxycycline Erythromycin

0.06-0.25 0.015-0.06 0.03--0.125 0.5-2 0.008--1 0.015-16 0.125-64 0.06-32 0.03-> 128

0.125 0.03 0.06 1 0.125 1 2 4 0.06

0.125 0.06 0.125 1 0.5 8 16 16 > 128

Streptococcus pyogenes,

20

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Penicillin G Cefazolin Doxycycline Erythromycin

0.06--0.125 0.03-0.125 0.03-0.125 0.25-1 0.004-0.008 0.004-2 0.125-0.25 0.06-0.5 0.03- > 128

0.125 0.06 0.06 0.5 0.004 0.008 0.125 0.125 0.06

0.125 0.125 0.06 0.5 0.008 0.015 0.25 0.5 0.125

20

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Penicillin G Cefazolin Doxycycline Erythromycin

0.125-0.25 0.06-0.125 0.06-0.125 0.5-2 0.008-0.03 0.03-0.06 0.125-0.25 0.125-32 0.03--4

0.125 0.06 0.125

0.125 0.125 0.125 1 0.015 0.06 0.25 16 1

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Ampicillin Vancomycin

0.06-0.25 0.03-0.25 0.06-0.25 0.25-1 0.5-2 0.5-2 1-4

2

PD 117596 PD 124816

0.06-0.25 0.06--0.125

0.125 0.06

Gp. A

Streptococcus agalactiae, Gp. B

Enterococcus faecalis

Other streptococcia

20

6

1

0.015 0.06 0.125 16 0.06 0.125 0.125 0.125 1 1 1

0.06 0.03 0.06 0.5 0.5 4 4 8 2

0.25 0.125 0.125 1 1 2 4

248

M . A . C o h e n et al.

TABLE 1

Continued MIC (~,g/ml) a

Organism

No. of Isolates

Antimicrobial Agent

Range

PD 127391 Ciprofloxacin Imipenern Penicillin G Cefazolin Doxycycline Erythromycin

0.06-0.125 1-2 0.008-2 0.03-1 0.06-8 0.03-16 0.015-4

50%

90%

0.06 1 0.06 0.06 1 0.5 0.03

Branhamella catarrhalis

20

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Augmentin Cefaclor Doxycycline Erythromycin

0.008-0.03 0.004-0.015 0.008-0.03 ~<0.03 0.015-4 ~<0.002-0.03 0.03-2.5 0.008-0.03 ~<0.08-0.6

0.015 0.008 0.015 ~<0.03 0.03 0.008 0.6 0.015 0.3

0.015 0.008 0.015 ~<0.03 0.06 0.03 1.25 0.03 0.3

Haemophilus influenzae

10

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Ampicillin Cefazolin Ceftazidime Doxycycline

0.004-0.008 0.001-0.002 0. 002-0. 004 ~<0.03 0.03-2 0.125-128 4-16 0.015-0.125 0.5-1

0.004 0.001 0.002 ~<0.03 0.5 0.5 8 0.03 0.5

0.004 0.002

~<0.0005-0.015 ~<0.0005-0.015 ~<0.0005-0.008 ~<0.03-0.06 0.015-0.125 0.015->128 0.06-2 0.03-1 8->128

0.001 0.001 0.001 ~<0.03 0.03 0.125 0.25 0.06 16

0.004 0.002 0.002 ~<0.03 0.125 128 2 0.5 >128 0.25 0.125 0.25 1 0.5 128 32 8 32

Neisseria gonorrhoeae

12

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Penicillin G Doxycycline Erythromycin Spectinomycin

Acinetobacter spp.

14

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Ampicillin Aztreonam Ceftazidime Amikacin

0.015-0.25 0.004-0.125 0.015-0.25 0.06-2 0.06-0.5 1->128 2-32 1-8 0.125-64

0.03 0.015 0.03 0.25 0.25 16 32 4 2

Citrobacter spp. e

10

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Ampicillin Aztreonam Ceftazidime Amikacin

0.001-0.06 0. 004-0.125 O.008-0.06 ~<0.03-0.125 O. 125-1 8->128 0.03->128 0.06->128 1-4

0.008 0.008 0.008 40.03 0.25 64 0.125 0.25 2

Enterobacter spp.f

10

PD 117596

O.008-0.03

0.015

0.004

~<0.03 2 128 16 0.125 1

0.03 0.06 0.06 ~<0.03 1 >128 8 8 2 0.015

I n v i t r o A c t i v i t y of P D 117596, P D 124816, a n d P D 127391

TABLE 1

249

Continued MIC (~g/ml) a

Organism

No. of Isolates

Antimicrobial Agent

Range

50%

90% 0.03 0.03 ~<0.03 1 >128 32 64 2

PD 124816 PD 127391 Ciprofloxacln Imipenem Ampicillin Aztreonam Ceftazidirne Amikacin

0.008-0.03 0. 008--0. 03 ~<0.03-0.06 0.125-2 16->128 0.125->128 0.25-128 2-4

0.008 0.015 ~<0.03 0.5 >128 1 2 2

Escherichia coli

11

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Ampicillin Aztreonam Ceftazidime Amikacin

0.001-0.06 0.004-0.06 0.008-0.06 ~<0.03-0.125 O.125-1 4->128 0.06-64 O.125-64 1-8

0.004 0.004 0.008 ~<0.03 0.125 8 0.125 0.25 4

0.008 0.015 0.015 ~<0.03 0.25 32 0.25 0.25 4

KlebsieUa s p p d

11

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Ampicillin Aztreonam Ceftazidime Amikacin

0.008-4 0.015-2 0.015-2 ~<0.03-8 O.125-1 32->128 0.06-0.5 O.125-1 1-2

0.015 0.015 0.015 ~<0.03 0.5 128 0.125 0.25 2

0.03 0.03 0.03 0.125 0.5 128 0.25 0.5 2

6

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Ampicillin Aztreonam Ceftazidime Amikacin

~<0.0005-0.25 0.001-0.5 0.001-0.5 <~0.03-2 0.25-1 0.5->128 0.015-128 0.034 2-32

0.001 0.002 0.002 ~<0.03 1 16 0.03 0.125 4

12

PD 117596 PD 124816 PD 127391 Ciprofloxacln Imipenem Ampicillin Aztreonam Ceftazidime Amikacin

0.008-0.125 0. 008--0.125 0. 008-0.125 ~<0.03-0.25 1-16 4->128 0.004-1 0.03-128 0.5-32

0.015 0.015 0.015 <~0.03 4 >128 0.03 0.25 4

0.06 O.125 0.125 O.125 8 >128 0.25 2 16

10

PD 117596 PD124816 PD 127391 Ciprofloxacin Imipenem Amipicillin Aztreonam Ceftazidime Amikacin

0.008--0.03 0.015-0.06 0.008-0.03 ~<0.03 4-16 1->128 0.015 0.06 1-8

0.015 0.03 0.015 <~0.03 4 2 0.015 0.06 4

0.03 0.06 0.03 ~<0.03 8 2 0.015 0.06 8

Miscellaneous Gramnegative rods h

Indole-positive Proteeaei

Proteus mirabilis

250

M . A . C o h e n e t al.

TABLE 1

Continued MIC (~.g/ml)~ Antimicrobial Agent

Organism

No. of Isolates

Pseudomonas aeruginosa

20

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Cefsulodin Ceftazidime Piperacillin Amikacin

0.03--4 0.06-4 0.06-4 0.06-16 0.5-32 1->128 1-64 2->128 1-64

Pseudomonas cepacia

10

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem PiperaciUin Ceftazidime Cefsulodin Amikacin

0.5-8 0.25-8 0.5-8 1-16 8->128 8->128 2-32 >128 128->128

Salmonella and Shigella sppd

12

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Ampicillin Aztreonam Ceftazidime Amikacin

0.001-0.008 0.004-0.008 0.004-0.015 ~<0.03 0.125-2 0.5->128 0.03-1 0.125-1 0.5-16

Serratia marcescens

20

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Ampicillin Aztreonam Ceftazidime Amikacin

0.008-4 0.015-4 0.015-4 ~<0.03-16 0.5-4 8->128 0.06-32 0.125-8 1-32

0.03 0.06 0.06 0.06 1 >128 0.5 0.25 4

0.25 0.25 0.25 1 2 >128 4 1 32

Bacteroides spp. k

20

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Penicillin G Cefoxitin Clindamycin Metronidazole

0.125-1 0.25-2 0.03-0.25 2-32 0.03-1 2->128 2-64 0.03->128 0.254

0.5 0.5 0.06 4 0.06 8 8 0.25 1

0.5 1 0.25 16 0.5 >128 32 4 2

Anaerobic cocci t

11

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Penicillin G Cefoxitin Clindamycin Metronidazole

0.015-0.5 0.015-0.5 0.015-0.25 0.25-2 0.004-0.5 0.015-4 0.015-4 0.015->128 0.25-2

0.06 0.25 0.06 0.5 0.015 0.03 0.125 0.5 1

Range

50% 0.25 0.125 0.25 0.25 2 4 2 8 8 2 2 2 4 32 32 8 >128 >128 0.002 0.004 0.008 ~<0.03 0.25 4 0.25 0.25 4

90% 0.5 0.5 0.5 1 8 128 8 64 32 8 8 8 16 128 >128 32 >128 >128 0.008 0.008 0.015 <~0.03 2 8 0.5 0.5 8

0.5 0.5 0.25 2 0.03 1 1 1 2

In vitro Activity of PD 117596, PD 124816, a n d PD 127391

TABLE 1

251

Continued

a50% and 90% refer to MICs0 and MICg0, respectively. blncludes 11 Staphylococcus aureus strains, two S. epidermidis strains, and two S. haemolyticus strains. ~Includes five S. aureus strains, four S. epidermidis strains, one S. haemolyticus strain, and one S. saprophyticus strain. elncludes one Streptococcus group C strain, one S. bovis strain, and four viridans streptococcal strains. elncludes four Citrobacter diversus strains and six C. freundii strains. fIncludes three Enterobacter aerogenes strains, one E. agglomerans strain, five E. cloacae strains, and one E. sakazakii strain. ~Includes one Klebsiella oxytoca strain and 10 K. pneumoniae strains. hlncludes two Aeromonas hydrophila strains, one Alcaligenes odorans strain, one Pasteurella multocida strain, one Plesiomonas shigeUoides strain, and one Yersinia enterocolitica strain. ilncludes four Morganella morganii strains, two Proteus vulgaris strains, one Providencia alcalifaciens strain, two P. rettgeri strains, and three P. stuartii strains. JIncludes four Salmonella enteritidis strains, two S. typhi strains, two Shingella dysenteriae strains, two S. flexneri strains, and two S. sonnei strains. klncludes two Bacteroides distasonis strains, 13 B. fragilis strains, two B. thetaiotaomicron strains, and three B. vulgatus strains. lIncludes eight Peptococcus spp. strains and three Peptostreptococcus anaerobius strains.

PD 117596, PD 124816, a n d PD 127391 d i s p l a y e d high activity against G r a m - p o s i t i v e bacteria. The lowest MICs w e r e v e r s u s staphylococci (MICrange 0.004-0.06 p,g/ml), a n d g e n e r a t e d MIC90s of 0.030.06 g,g/ml regardless of species or methicillin susceptibility. The d r u g s w e r e at least eightfold m o r e active t h a n ciprofloxacin w a s against these 26 strains of staphylococci, a n d at least 32-fold m o r e active t h a n the n o n q u i n o l o n e antimicrobials i m i p e n e m , cefazolin, oxacillin, amikacin, a n d v a n c o m y c i n . W h e n the three n e w quinolones were tested against a c o m b i n e d total of 80 Streptococcus pyogenes, S. agalactiae, S. pneumoniae, a n d Enterococcus faecalis strains, no d r u g p r o d u c e d an MIC > 0.25 ~g/ml for a n y

TABLE 2

isolate; the s a m e p o t e n c y w a s o b t a i n e d against three Listeria monocytogenes isolates. The MIC90s ranged from 0.125 to 0.25 p,g/ml against E. faecalis a n d 0.06-0.125 p,g/ml against the a b o v e streptococci. Similar activities w e r e o b s e r v e d against a n a s s o r t m e n t of other streptococci (viridans g r o u p , S. bovis, a n d Lancefield g r o u p C). These activities w e r e at least fourfold superior to ciprofloxacin. A n u m b e r of G r a m - n e g a t i v e o r g a n i s m s w e r e exquisitely susceptible to PD 117596, PD 124816, a n d PD 127391. A collection of 42 strains of Branhamella catarrhalis, Haemophilus influenzae, a n d Neisseria gonorrhoeae generated MIC9os -<0.015 p,g/ml (MICs against s o m e N. gonorrhoeae isolates w e r e -<0.0005 p,g/ml).

Antibacterial Activity of PD 117596, PD 124816, a n d PD 127391 A g a i n s t Miscellaneous I n d i v i d u a l Strains a

Organism

No. of Isolates

Antimicrobial Agent

MIC of Individual Strains (~g/ml)

Enterococcus faecium

3

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Ampicillin Vancomycin

1, 1, 1 0.5, 0.5, 2 1, 1, 1 2, 2, 4 32, 32, 32 16, 16, 8 0.5, 0.5, 4

Listeria monocytogenes

3

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Penicillin G Cefazolin Doxycycline Erythromycin

0.25, 0.25, 0.25 0.125, 0.25, 0.25 0.125, 0.125, 0.125 1, 1, 1 0.125, 0.06, 0.25 0.25, 0.25, 0.5 1, 1, 2 0.25, 0.25, 0.25 0.125, 0.125, 0.25

Xanthomonas maltophilia

3

PD 117596 PD 124816 PD 127391

0.06, 0.5, 2 0.06, 0.25, 1 0.06, 0.5, 1

252

M.A. C o h e n et al.

TABLE 2

Continued

Organism

No. of Isolates

Antimicrobial Agent

MIC of Individual Strains (~g/ml)

Ciprofloxacin Imipenem Ampicillin Aztreonam Ceftazidime Amikacin

0.5, 4, 8 128, >128, >128 >128, >128, >128 >128, >128, >128 64, 64, 64 8, >128, 8

Clostridium perfringens

3

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Penicillin G Cefoxitin Clindamycin Metronidazole

0.06, 0.06, 0.125 0.015, 0.03, 0.06 0.06, 0.06, 0.06 0.5, 0.5, 1 0.06, 0.06, 0.06 0.015, 0.06, 0.25 0.5, 1, 1 0.008, 8, 4 1, 1, 2

Clostridium spp. b

2

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Penicillin G Cefoxitin Clindamycin Metronidazole

0.5, 1 4, 0.5 0.25, 1 8, 16 4, 0.25 0.5, 0.03 128, 4 4, 8 0.5, 2

Fusobacterium spp. C

2

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Penicillin G Cefoxitin Clindamycin Metronidazole

0.5, 1, 1, 1 0.5, 1 8, 16 1, 2 0.5, 0.5 4, 4 4, 32 0.5, 0.5

Propionibacterium acnes

2

PD 117596 PD 124816 PD 127391 Ciprofloxacin Imipenem Penicillin G Cefoxitin Clindamycin Metronidazole

0.125, 0.125 0.25, 0.125 0.06, 0.06 0.5, 0.5 0.008, 0.008 0.015, 0.03 0.125, 0.125 0.06, 0.06 >128, >128

qndividual MICs are shown for species represented by five strains or fewer. blndudes one Clostridium difficilestrain and one C. ramosum strain. qncludes one Fusobacteriummortiferum strain and one F. varium strain.

The three q u i n o l o n e s d i s p l a y e d MIC90s of 0.06 ~g/ml against 97 Enterobacteriaceae strains. O n l y o n e isolate each of Klebsiella pneumoniae (10 strains tested) a n d Serratia marcescens (20 tested) p r o d u c e d M I C s as high as 2 - 4 ~,g/ml. A g a i n s t these latter t w o isolates, ciprofloxacin M I C s w e r e 2 - 4 times higher. In short, 0.06 ~g/ml of PD 117596, PD 124816, or PD 127391

w a s sufficient to inhibit at least 90% of strains identified as Escherichia coli, Proteus mirabilis, or species of Citrobacter, Enterobacter, Klebsiella, Salmonella, a n d ShigeUa; this concentration also inhibited at least 70% of the Serratia marcescens a n d indole-positive Proteeae (Morganella morganii, Proteus vulgaris, a n d Providencia spp.) strains tested. All Acinetobacter spp. tested (14

253

In vitro Activity of PD 117596, PD 124816, and PD 127391

TABLE 3 Minimum Inhibitory Concentrations of PD 117596, PD 124816, and PD 127391 Against Reference Bacterial Strains a MIC (~g/ml) Organism

PD 117596

PD 124816

PD 127391

Staphylococcus aureus ATCC 29213 Enterococcus faecalis ATCC 29212 Escherichia coli ATCC 25922 Pseudomonas aeruginosa ATCC 27853 Bacteroides fragilis ATCC 25285 Bacteroides thetaiotaomicron ATCC 29741 Clostridium perfringens ATCC 13124

0.03 0.125 0.004 0.5 0.25 1 0.125

0.015 0.06 0.004 0.25 0.5 1 0.125

0.03 0.125 0.008 0.25 0.06 0.25 0.125

aTest results for one run in duplicate.

strains) were inhibited by the three new quinolones at a concentration range of 0.004-0.25 ~g/ml. Some representative strains of an assortment of Gram-negative rod species were highly susceptible to PD 117596, PD 124816, and PD 127391. Concentrations <--0.008 ~,g/ml inhibited Aeromonas hydrophila, Pasteurella multocida, and Plesimonas shigelloides; somewhat higher levels of 0.25-0.5 ~g/ml were required against an Alcaligenes odorans. Pseudomonads are somewhat less susceptible than the above organisms. The three new quinolones shared an MIC90 of 0.5 ~g/ml versus P. aeruginosa (20 strains) and 8 ~g/ml versus P. cepacia (10 strains); approximately twofold higher concentrations were needed for inhibition by ciprofloxacin. Testing against a limited number of Xanthomonas (formerly Pseudomonas) maltophilia strains revealed an MIC range of 0.06-2.0 ~g/ml for these new compounds; approximately eightfold higher concentrations were needed for inhibition by ciprofloxacin. Because anaerobic bacteria are generally insusceptible to quinolones (Andersen and Goldstein, 1987; Hoban, 1989; Neuman and Esanu, 1988; Stahlman, 1988; Wolfson and Hooper, 1985), potencies of these quinolones are noteworthy. The MIC90s versus 20 Bacteroides fragilis group isolates were 0.25, 0.5, and 1 p,g/ml for PD 127391, PD 117596, and PD 124816, respectively; ciprofloxacin was at least 16-fold less active. A potpourri of 16 anaerobic cocci, Propionibacterium acnes, and Clostridium perfringens isolates was inhibited by 0.015-0.5 ~g/ml of the three new compounds; PD 127391 was slightly more active than the other two, and ciprofloxacin activity was at least fourfold lower. Finally, Table 3 shows the PD 117596, PD 124816, and PD 127391 MICs obtained against ATCC cultures sanctioned as reference susceptibility test strains by the NCCLS.

Effectiveness Against Resistant Bacteria Strains possessing an assortment of resistance characters (methicillin-resistant Staphylococcus aureus, penicillin-resistant pneumococci, ampicillin-resistant Haemophilus influenzae, spectinomycin-resistant gonococci, Gram-negative rod [3-1actamase producers, clindamycin-resistant Bacteroides fragilis, and so on) are included in Tables 1 and 2. Some selected isolates are displayed in Table 4. Examination of the MICs of PD 117596, PD 124816, and PD 127391 clearly indicate that each drug has a broad spectrum of activity encompassing different types of resistant bacteria.

TABLE 4 Activity of PD 117596, PD 124816, and PD 127391 Against Representative Resistant Organisms Organism

Antimicrobial Agent

MIC (~g/ml)

Staphylococcus aureus SA-1010 (Methicillin resistant)

PD 117596 PD 124816 PD 127391 Cefazolin Oxacillin Amikacin Vancomycin

0.015 0.015 0.03 32 128 8 1

Streptococcus pneumoniae SP-4 (Penicillin resistant)

PD 117596 PD 124816 PD 127391 Penicillin G Cefazolin Doxycycline Erythromycin

0.06 0.03 0.06 4 8 4 0.03

254

M.A. Cohen et al.

TABLE 4

Continued

Organism Haemophitus influenzae HI-1 (B-lactamase positive)

Antimicrobial Agent PD 117596 PD 124816 PD 127391 Ampicillin Cefazolin Ceftazidime Doxycycline

Neisseria gonorrhoeae NG-1273 (~-lactamase positive) PD 117596 PD 124816 PD 127391 Penicillin G Doxycycline Erythromycin Spectinomycin Serratia marcescens SM-1178 (Multiresistant)

ShigeUa dysenteriae SD-5 (Multiresistant)

Bacteroides fragilis BF-1363 (Clindamycin resistant)

MIC (~g/ml) 0.004 0.002 0.004 64 8 0.03 0.5 0.004 0.002 0.002 >128 2 0.25 >128

PD 117596 PD 124816 PD 127391 Ampicillin Aztreonam Ceftazidime Amikacin

0.25 0.25 0.25 > 128 32 8 32

PD 117596 PD 124816 PD 127391 Ampicillin Aztreonam Ceftazidime Amikacin

0.001 0.004 0.004 > 128 0.125 0.125 2

PD 117596 PD 124816 PD 127391 Penicillin G Cefoxitin Clindamycin Metronidazole

0.5 0.5 0.06 8 8 > 128 0.5

Influence of Various Factors on in vitro Activity Increasing or decreasing the inoculum 100-fold from the 105 CFU/ml starting concentration changed the PD 117596, PD 124816, and PD 127391 MICs generally no more than twofold for the eight strains tested (Table 5); the substantial effect of inoculum size on ampicillin activity has been included to validate the test system. The quinolones were bactericidal, and there was also a lack of inoculum effect on the MBCs (data not shown). The effects of vary-

ing other test conditions on the three new quinolones versus standard strains are displayed in Tables 6-8. Diminished cation concentration in Mueller-Hinton broth had little influence on activity. An eightfold decrease in the gentamicin MIC when Pseudomonas aeruginosa was tested (data not shown) in Mueller-Hinton broth compared with that in CAMHB corroborates previous reports by other workers (Gilbert et al., 1971; Reller et al., 1974) and validates the methodology. Light also had minimal effects on PD 117596, PD 124816, and PD 127391 MICs, although nalidixic acid has been reported labile on exposure to light (Martindale, 1977). Little susceptibility change was measured in the presence of (1% or 5%) sodium cholate, an important component of bile. The addition of 50% human serum failed to change the MICs of the three new quinolones, whereas cefazolin, with high serumprotein-binding activity (Kirby and Regamey, 1973), displayed up to an eightfold loss of potency (data not shown). The quinolones' activities against the facultative anaerobes Staphylococcus aureus, Enterococcus faecalis, and E. coli was unaffected by anerobiosis, whereas gentamicin MICs increased to 16-fold; aminoglycoside resistance in bacteria is dependent on transport driven by oxidative metabolism (Bryan and Van Den Elzen, 1977). PD 117596, PD 124816, and PD 127391 activities generally decreased (up to 128-fold) with acidity and increased (up to eightfold) with alkalinity, when MICs across a p H range of 5-9 in CAMHB were compared to the MIC obtained at pH 7.4. Activity decreases of 4- to 32-fold also were revealed during testing in 100% human urine (pH 6.1).

DISCUSSION Efforts have been made to design quinolones with high potency and broad spectrum, especially versus Gram-positive bacteria (Domagala et al., 1988). Published data show that the new fluoroquinolones PD 117596, PD 124816, and PD 127391 are among the most active members of this drug class versus both Gram-positive and -negative bacterial species of medical significance (Auckenthaler et al., 1986; Espinoza et al., 1988; Stahlmann, 1988; Stature et al., 1986; Wolfson and Hooper, 1985). This article compares the side-by-side testing of these agents for the first time. There was close agreement between our data and those of earlier individual reports (King et al., 1988; Neu and Chin, 1989; Neu et al., 1989; Norrby and Jonsson, 1988; Smith et al., 1988; Wise et al., 1988). Small deviations, however, probably arising from strain differences, were evident in a few instances. These three compounds were generally

I n v i t r o A c t i v i t y of P D 117596, P D 124816, a n d P D 127391

TABLE 5

255

Effect of I n o c u l u m S i z e o n P D 117596, P D 124816, a n d P D 127391 Activities MIC (p,g/ml) LOgl0 CFU/ml

PD 117596

PD 124816

PD 127391

Staphylococcus aureus H228

3 5 7

0.06 0.03 0.06

0.015 0.03 0.03

0.03 0.06 0.06

Enterococcus faecalis

3 5 7

0.125 0.25 0.50

0.06 0.06 0.125

0.125 0.125 0.125

1 1 2

3 5 7

0.125 0.125 0.25

0.06 0.06 0.125

0.125 0.125 0.125

0.5 1 1

3 5 7

0.06 0.125 0.25

0.125 0.25 0.5

0.03 0.03 0.06

3 5 7

0.015 0.015 0.03

0.008 0.015 0.015

0.015 0.03 0.03

3 5 7

0.008 0.06 0.125

0.008 0.015 0.06

0.008 0.03 0.06

>128 >128 >128

3 5 7

0.002 0.004 0.008

0.001 0.002 0.004

0.002 0.004 0.008

1 8 >128

3 5 7

0.008 0.03 0.03

0.015 0.015 0.06

0.015 0.015 0.03

32 64 >128

Organism a

EF1-1729

Enterococcus faecium EF4-1419

Bacteroides fragilis ATCC 25285

Branhamella catarrhalis BC 1730

Enterobacter cloacae ECL-10

Haemophilus influenzae HI-1

Klebsiella pneumoniae MGH-2

Ampicillin 2 >128 >128

8 16 16 ~0.125 ~0.125 128

aAll organisms listed except Enterococcusfaecalis EF1-1729 and E. faecium EF4-1419 are/3-1actamase producers.

TABLE 6

Effect o f L i g h t , p H , a n d C a t i o n s o n i n v i t r o A c t i v i t i e s of P D 117596, P D 124816, a n d P D 127391 MIC (p,g/ml) in: CAMHB a pH

Agent a n d Organism

Light

5.0

6.0

7.4

8.0

9.0

MHB

0.06 0.125 0.008 0.5

0.25 1 0.25 4

0.06 0.25 0.03 1

0.06 0.125 0.008 0.25

0.03 0.125 0.004 0.25

0.03 0.06 0.015 0.06

0.03 0.06 0.004 0.125

0.015 0.125 0.004 0.5

0.125 0.5 0.25 32

0.03 0.125 0.03 1

0.015 0.06 0.004 0.25

0.03 0.06 0.002 0.25

0.008 0.03 0.001 0.03

0.008 0.03 0.001 0.06

PD 117596

S. E. E. P.

aureus b faecalis coli aeruginosa

PD 124816

S. E. E. P.

aureus faecalis coli aeruginosa

256

M.A. C o h e n et al.

TABLE 6

Continued MIC (~g/ml) in: CAMHBa pH

Agent and Organism

Light

5.0

6.0

7.4

8.0

9.0

MHB

PD 127391 S. aureus E. faecalis E. coli P. aeruginosa

0.03 0.125 0.008 0.25

0.06 0.5 0.25 2

0.03 0.125 0.03 0.5

0.03 0.125 0.008 0.25

0.03 0.06 0.008 0.25

0.03 0.06 0.004 0.125

0.03 0.06 0.004 0.125

aMicrobroth dilution performed in CAMHB at pH 7.4 in the dark unless otherwise indicated. bStrains used were Staphylococcus aureus ATCC 29213, Enterococcusfaecalis ATCC29212, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853.

comparable (within a twofold concentration) in their p o t e n c y against each particular bacterial group. PD 117596, PD 124816, a n d PD 127391 were fourto eightfold m o r e active versus aerobic a n d anerobic Gram-positive cocci than was ciprofloxacin; two- to eightfold more active versus Listeria monocytogenes and anerobic Gram-positive rods; as active or more active, versus Enterobacteriaceae; t w o - to eightfold more

TABLE 7

active versus Acinetobacter spp. a n d p s e u d o m o n a d s ; and 16- to 64-fold more active versus Bacteroides fragilis group. Generally, these n e w agents b e h a v e d similarly to other fluoroquinolones. There was no cross resistance b e t w e e n these n e w quinolones a n d m e m b e r s of other d r u g classes. PD 117596, PD 124816, a n d PD 127391 were generally unaffected by inoculum,

Effect of S o d i u m Cholate, Serum, a n d Urine on in vitro Activities of PD 117596, PD 124816, a n d PD 127391 MIC (p,g/ml) in: Sodium Cholate

Agent and Organism

CAMHBa

1%

5%

Serum b 50%

Urine c 100%

PD 117596 S. aureus E. faecalis E. coli P. aeruginosa

0.06 0.125 0.008 0.25

NG a 0.125 0.002 0.125

NG 0.25 0.008 0.25

0.06 0.125 0.008 0.25

0.25 1 0.06 2

PD 124816 S. aureus E. faecalis E. coli P. aeruginosa

0.015 0.06 0.004 0.25

NG 0.06 0.002 0.5

NG 0.25 0.004 0.25

0.03 0.06 0.004 0.25

0.125 1 0.06 8

PD 127391 S. aureus E. faecalis E. coli P. aeruginosa

0.03 0.125 0.008 0.25

NG 0.06 0.002 0.25

NG 0.25 0.008 0.5

0.03 0.125 0.008 0.25

0.25 0.5 0.06 2.

aMicrobroth dilution performed in CAMHB. bPooled human serum (from normal individuals) inactivated by heating at 56°C for 30 rain and diluted 1:1 with CAMHB. cPooled, filtered urine (pH 6.1) from normal individuals. aNG, no growth.

In vitro Activity of PD 117596, PD 124816, and PD 127391

TABLE 8

257

Influence of Anaerobiosis on in vitro Activities of PD 117596, PD 124816, a n d PD 127391 a MIC (~g/ml) Organism

Drug

Aerobic

Anaerobic

Staphylococcus aureus ATCC 29213

PD 117596 PD 124816 PD 127391 Gentamicin

0.03 0.015 0.03 0.5

0.03 0.015 0.015 8

Enterococcus faecalis ATCC 29212

PD 117596 PD 124816 PD 127391 Gentamicin

0.125 0.06 0.125 16

0.125 0.06 0.06 8

Escherichia coli ATCC 25922

PD 117596 PD 124816 PD 127391 Gentamicin

0.008 0.008 0.008 1

0.008 0.008 0.008 16

aAgar dilution in Mueller-Hinton agar. cations, light, s o d i u m cholate, h u m a n serum, and anaerobiosis; activities w e r e d i m i n i s h e d with decreasing p H a n d in urine (pH 6.1). These characteristics a n d the bactericidal nature of these c o m p o u n d s are consistent with the b e h a v i o r of other quinolones. O u r in vitro results describe the high potential of PD 117596, PD 124816, and PD 127391 as c h e m o -

therapeutic agents for infections caused b y a wide variety of frequently e n c o u n t e r e d bacterial species. These agents are comparable to each other in their high d e g r e e of in vitro p o t e n c y a n d spectrum. Extensive in vivo studies will be required to explore further the potential clinical utility of these compounds.

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