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Antimicrobial Agents and Chemotherapy, November 2001, p. 3113-3121, Vol. 45, No. 11
Laboratory of Microbiology, Department of
Sanitary Microbiology and Parasitology, Division of Health Sciences,
Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain
Received 14 December 2000/Returned for modification 4 June
2001/Accepted 12 August 2001
E-4767 {( Since the discovery of nalidixic and
oxolinic acids (10, 13), many modifications have
been introduced in the chemical structure of the quinolonic
ring, yielding new drugs with improved antibacterial efficacy against
gram-positive and anaerobic bacteria and with enhanced bioavailability,
such as norfloxacin (11), ofloxacin (21),
ciprofloxacin (25), and tosufloxacin (23). In
a recent review, Domagala (4) established a model in which every junction of a substituent has a specific property in addition to
that of the substituent itself. Substituents at position 7 influence
antimicrobial potency, the spectrum of activity, and the
pharmacokinetic characteristics. Pyrrolidines at this position offer
great potency against gram-positive bacteria, and piperazines introduce
excellent activity against gram-negative bacteria. Furthermore, among
the variations described at position 7 (1, 2, 7, 12, 16),
it has been proven that the presence of an azetidine moiety (3,
5, 6, 8, 9) yields a broader spectrum and enhanced activity
against gram-positive bacteria. On the other hand, the substituent at
position 8 controls in vivo efficacy and is largely responsible for the
activity against anaerobic bacteria, with halogens being one of the
optimal substitution groups at this site. Among the quinolones with a
Cl at position 8, DU-6859 (20), BAY-Y-3118
(24), and clinafloxacin (19) have been the
most active.
E-4767 and E-5065 (Fig. 1) are
structurally characterized by the presence of an azetidine ring with
different C-2' and C-3' substitutions at position 7 of the molecule.
E-4767 has a 2-methyl-3-aminoazetidin-1-yl substituent
at position 7, while E-5065 is characterized by a 3-aminoazetidin-1-yl
group in the same position. Both compounds possess a Cl as
R8.
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.11.3113-3121.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Antibacterial Activities and Pharmacokinetics of
E-4767 and E-5065, Two New 8-Chlorofluoroquinolones with a
7-Azetidin Ring Substituent
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
)-7-[3-(R)-amino-2-(S)-methyl-1-azetidinyl]-8-chloro-1-cyclopropyl-1,4-dihydro-6-fluoro-4- oxo-3-quinolinecarboxylic acid} and E-5065
[()-7-(3-amino-1-azetidinyl)-8-chloro-1-cyclopropyl-1,4-dihydro-6-fluoro-4-oxo-3-quinolinecarboxylic acid] are two new chlorofluoroquinolones with an azetidine
moiety at position 7. Their in vitro activities were evaluated in
comparison with those of ciprofloxacin, ofloxacin, fleroxacin, and
tosufloxacin, while ciprofloxacin was used as a reference for in vivo
studies. Against gram-positive organisms, E-4767 and E-5065 were,
in general, eight- and fourfold more active than tosufloxacin, which is
the most potent of the reference compounds. E-4767 and E-5065 were also
more potent than the reference compounds against all species of enteric
bacteria tested. The MICs of E-4767 and E-5065 at which 90% of the
isolates tested were inhibited (MIC90s) were 0.007 to 0.5 µg/ml and 0.03 to 2 µg/ml, respectively, for gram-positive organisms and 
0.003 to 0.06 µg/ml and 0.007 to 0.12 µg/ml,
respectively, for members of the family
Enterobacteriaceae except Serratia
marcescens and Providencia spp.
(MIC90s of E-4767 and E-5065 for these species were 0.5
µg/ml and 2 µg/ml, respectively). For Pseudomonas
aeruginosa both compounds had a MIC90 of 0.5 µg/ml. E-4767 and E-5065 were 356- and 32-fold more potent than
ciprofloxacin against Bacteroides spp., and their
MIC90s for Clostridium spp. were 0.25 and
0.5 µg/ml, respectively. Both products showed a remarkable reduction of activity when the pH was below 4.8 and, in general, were less active
in the presence of 5 or 10 mM Mg2+. The presence of horse
serum or human urine (pH 7.2) decreased the activity of E-4767 and
E-5065 only two- to fourfold more than the activity observed in broth.
After an oral dose of 50 mg/kg of body weight, the maximum levels in
serum (the maximum concentration of drug in serum was reached 30 min
postadministration) of E-4767 and E-5065 were approximately threefold
higher than that of ciprofloxacin. The area under the
concentration-time curve from 0 to 4 h for ciprofloxacin was about
two- and fourfold lower than that for E-4767 and E-5065, respectively.
These two new chlorofluoroquinolones were as effective as or more
effective than ciprofloxacin against all experimental infections
evaluated, not only against gram-negative bacteria, such as
Escherichia coli or P.
aeruginosa, but also against gram-positive pathogens,
such as Staphylococcus aureus or Streptococcus
pneumoniae. E-4767 was the most effective compound, with a 50%
effective dose (ED50) of 17 mg/kg for all strains tested
except ciprofloxacin-resistant S. aureus
strains. The ED50 of E-4767 for these strains was 47.5
mg/kg. Against gram-positive experimental infections, the
ED50 values of E-4767 were 3- to 14-fold lower than those
of E-5065 and up to 25 times lower than those of ciprofloxacin.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (9K):
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FIG. 1.
Chemical structures of E-4767 and
E-5065.
In this work, the in vitro activities of E-4767 and E-5065 are compared with those of ciprofloxacin, fleroxacin, ofloxacin, and tosufloxacin against groups of pathogenic bacterial clinical isolates. The effects of various assay conditions on in vitro activity, such as pH, magnesium ion concentration, and the presence of serum and urine, were investigated. We also studied the pharmacokinetic properties and the in vivo protective efficacy of these compounds against lethal systemic infections in mice. Ciprofloxacin was selected as the reference compound for in vivo studies.
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MATERIALS AND METHODS |
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Introduction
Materials and Methods
Results
Discussion
References
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Antibacterial agents. E-4767, E-5065, ofloxacin, and tosufloxacin were synthesized at Laboratorios Esteve S.A. (Barcelona, Spain). Ciprofloxacin (Bayer A.G., Wuppertal, Germany) and fleroxacin (Roche S.A., Madrid, Spain) were obtained as standard powders of known potency. Solutions were prepared immediately before use. For determination of MICs, a stock solution was prepared in 0.1 N NaOH and diluted in broth medium to the appropriate concentration. For in vivo tests, all antibacterial agents were dissolved in 0.1 N NaOH, appropriately diluted with sterile water, and finally mixed in 0.1% carboxymethyl cellulose.
Organisms. Clinical isolates for in vitro activity evaluation were randomly collected from various hospitals in Spain. Selected strains (Citrobacter freundii HSP-73, Enterobacter aerogenes HSP-145, Escherichia coli HM-42, Klebsiella pneumoniae HSP-30, Proteus vulgaris HSP-99, Salmonella enterica serovar Enteritidis HSP-928/F, Pseudomonas aeruginosa HSP-116, Staphylococcus aureus HS-93, coagulase-negative Staphylococcus sp. HSP-10, and Enterococcus faecalis HSP-30) were used to evaluate the effects of different conditions upon the in vitro activity of the new chlorofluoroquinolones, and additional strains (ciprofloxacin-resistant [Cipr] P. aeruginosa B-464, Cipr S. aureus 73777, Cipr S. aureus 73940, Streptococcus pneumoniae 1625, S. pneumoniae 84551, and S. pneumoniae 1752) were used in previous studies for in vivo efficacy evaluation (6, 8, 9).
Five additional strains (S. aureus ATCC 25923, E. coli ATCC 25922, P. aeruginosa ATCC 27853, Clostridium perfringens ATCC 13124, and Bacteroides fragilis ATCC 25285) obtained from the American Type Culture Collection (Rockville, Md.) were used as susceptibility test controls for in vitro studies (data not shown). Bacillus subtilis ATCC 6633 was used as indicator strain in pharmacokinetic bioassay studies. All strains were stored frozen at70°C in our laboratories until used.
Determination of MICs and MBCs.
For aerobic and
facultatively anaerobic organisms, MICs were determined in
Mueller-Hinton (MH) broth (Oxoid Ltd., Basingstoke, England) by a
microtiter twofold dilution method using a Quick Spense II system
(Dynatech AG, Denkendorf, Germany) as recommended by the National
Committee for Clinical Laboratory Standards (17). For
Streptococcus species, brain heart infusion broth medium
(Oxoid Ltd.) was used. The inoculum was approximately 5 × 105 CFU/ml. MICs, defined as the lowest
concentrations of antibacterial agent that inhibited development of
growth, were recorded after 18 h of incubation at 37°C. Minimum
bactericidal concentrations (MBCs), defined as the lowest compound
concentrations that killed 
99.9% of the initial inoculum, were in
turn determined by subculturing 10 µl of broth from the drug-free
control well, the first well containing growth, and each clear well on
MH agar plates.
Factors affecting in vitro activity. The effects of medium pH, magnesium ion concentration, and the presence of serum and urine on the in vitro activities of E-4767 and E-5065 against C. freundii HSP-73, E. aerogenes HSP-145, E. coli HM-42, K. pneumoniae HSP-30, P. vulgaris HSP-99, S. enterica serovar Enteritidis HSP-928/F, P. aeruginosa HSP-116, S. aureus HS-93, coagulase-negative S. aureus HSP-10, and E. faecalis HSP-30 were determined as described above for aerobic and facultatively anaerobic organisms in MH broth supplemented with the different tested factors. Unsupplemented MH broth, MH broth adjusted to different pH values, and magnesium-, serum-, or urine-supplemented wells without antibacterial agents were used as control media.
(i) Medium pH. The effects of pH were determined in MH broth adjusted to pH 4.8, 5.8, 6.8, 7.8, and 8.8.
(ii) Mg2+ concentration. Magnesium (as MgCl2 · 6H2O) was added to MH broth at 1, 5, and 10 mM.
(iii) Serum. Horse serum inactivated at 56°C for 30 min was added to MH broth to a final concentration of 20 or 70% (vol/vol) with the pH adjusted to 7.2.
(iv) Urine. The urine samples used were early morning pooled and obtained from healthy human male volunteers. The pH was adjusted to 5.5 or 7.2, followed by sterilization by filtering through a 0.22-µm-pore-size membrane filter (Millipore Corp., Bedford, Mass.).
Pharmacokinetics in mice. Pharmacokinetic assays were performed using male Swiss mice (Charles River, Cleon, France) weighing approximately 30 g. Each compound (E-4767, E-5065, and ciprofloxacin) was administered once by gavage at a dose of 50 mg/kg of body weight after fasting for 4 h. Samples of blood were collected from groups of six mice at 30, 60, 120, and 240 min postadministration. The blood samples were immediately chilled and centrifuged at 1,000 × g for 15 min. Prior to analysis, the standards (initially solubilized in 0.1 N NaOH) and the plasma specimens obtained were suitably diluted with 0.07 M phosphate buffer (pH 6.0).
Concentrations of E-4767, E-5065, or ciprofloxacin in plasma were determined by the agar diffusion method under standard conditions using 7-mm-diameter Oxford cylinders. B. subtilis ATCC 6633 was used as an indicator organism. Plates were incubated at 37°C for approximately 18 h. The lower limit of detection was <0.05 µg/ml. The areas under the concentration-time curve from 0 to 4 h (AUC0-4) were calculated from the mean concentrations by the trapezoidal method. Results obtained with the agar diffusion method were validated by high-pressure liquid chromatography analysis.Mouse protection tests on experimental systemic infections. Male HC:CFLP mice weighing approximately 25 g (Interfauna U.K. Ltd., Huntingdon, England) were used in protection tests on systemic infections. Mouse protection tests were performed against the strains E. coli HM-42, P. aeruginosa HSP-116, Cipr P. aeruginosa B-464, S. aureus HS-93, Cipr S. aureus 73777, Cipr S. aureus 73940, S. pneumoniae 1625, S. pneumoniae 84551, and S. pneumoniae 1752.
Test organisms were grown overnight on MH agar plates, except S. pneumoniae strains, which were grown on MH agar supplemented with 5% horse blood and incubated in a candle jar. Mice were inoculated intraperitoneally with 0.5 ml of a bacterial suspension adjusted to the appropriate concentration (five times the minimum lethal dose) with physiological saline solution, with the exception of the Cipr strains, which were adjusted with 5% hog gastric mucin (ICN Biomedicals, Columbus, Ohio). The challenge inoculum was sufficient to kill 100% of untreated control mice within 2 days postinfection, with the exception of mice experimentally infected with S. aureus and S. pneumoniae strains, some of which died within 4 days after the challenge. Immediately after the challenge, the mice were subjected to a single oral administration of the test compound, with the exception of those experimentally infected with Cipr strains (which received an additional dose at 6 h postinfection) and those infected with S. pneumoniae strains (which received additional doses at 6, 12, and 24 h postinfection). Four groups of 10 mice each were treated with different doses of each antibacterial agent. ED50 (50% effective dose) and 95% confidence intervals were calculated by probit analysis (15) and the method of Litchfield and Wilcoxon (14), respectively, 7 days after infection. The ED50 values for Cipr and S. pneumoniae strains were determined for the total drug dose given.| |
RESULTS |
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Materials and Methods
Results
Discussion
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Susceptibilities in vitro.
The susceptibilities of clinical
isolates to E-4767 and E-5065 were compared with their susceptibilities
to ciprofloxacin, fleroxacin, ofloxacin, and tosufloxacin (Table
1). The two new compounds
were highly active against gram-positive cocci, including Staphylococcus spp. (methicillin-resistant
[Metr] S. aureus, Cipr S. aureus, coagulase-negative methicillin-susceptible
[Mets] Staphylococcus spp.,
coagulase-negative Metr Staphylococcus
spp., and coagulase-negative Cipr
Staphylococcus spp.), S. pneumoniae, Streptococcus viridans, and
E. faecalis. E-4767 and E-5065 were the most
effective compounds against all species of gram-positive
organisms tested. The MIC90s (MICs at which
90% of the strains tested were inhibited) of E-4767 and E-5065
for Mets Staphylococcus spp., including
S. aureus and coagulase-negative Staphylococcus
spp., were 0.007 and 0.06 µg/ml, respectively. Against
these groups of isolates, E-4767 and E-5065 were 16- and 2-fold
more potent than tosufloxacin, which was the most active of the
reference compounds tested. Against MetR Staphylococcus spp.
isolates (including S. aureus and coagulase-negative
Staphylococcus spp.), E-4767 and E-5065 were four- to
eightfold more active than any standard reference compound tested. In
addition, E-4767 and E-5065 exhibited significant activity
against Cipr Staphylococcus sp. strains. For
Cipr S. aureus isolates, the
MIC90s of E-4767 and E-5065 were 0.25 and 2 µg/ml, respectively, versus 0.25 and 1 µg/ml, respectively, for
coagulase-negative Cipr Staphylococcus
sp. organisms. In turn, the MIC90s of the four antibacterial agents used as reference controls against
Cipr Staphylococcus spp. isolates were higher
than 4 µg/ml in all cases. The MIC90 of E-4767
for S. pneumoniae isolates was 0.03 µg/ml, i.e., 8 times lower than that of tosufloxacin and 64 times lower than that of either ciprofloxacin or ofloxacin. Against S. pneumoniae isolates, E-5065 was eightfold more
active than ciprofloxacin and ofloxacin. For S. viridans and E. faecalis, the MIC90s of E-4767 and E-5065 were 0.5 and
1 µg/ml, respectively. Against these organisms, the activity of
all marketed compounds evaluated was, in general, eight times lower
than that of E-4767, and four times lower than that of E-5065.
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0.06 µg/ml versus a MIC90 of
0.12 µg/ml for E-5065, with the exception of Serratia
marcescens and Providencia spp. In all cases,
MIC90s of the two new chlorofluoroquinolones were
lower than those of standard reference compounds.
Regarding activity against S. marcescens
isolates, E-4767 and E-5065 (MIC90, 0.25 µg/ml)
were fourfold more potent than ciprofloxacin and eightfold more active
than fleroxacin, ofloxacin, or tosufloxacin. The
MIC90s of E-4767 and E-5065 for
Providencia spp., including Providencia
rettgeri and Providencia stuartii, were 0.5 and 2 µg/ml, respectively. In this case, the MIC90s
of ciprofloxacin, fleroxacin, ofloxacin, and tosufloxacin for
P. rettgeri were 0.5, 2, 2, and 1 µg/ml,
respectively. On the other hand, the activities of marketed compounds
against P. stuartii were even lower, with MIC90s of >4 µg/ml in all cases.
E-4767 and E-5065 showed potent activities against P. aeruginosa. The antibacterial activities of the
two new chlorofluoroquinolones (MIC90, 0.5 µg/ml) were comparable to that of ciprofloxacin
(MIC90, 0.5 µg/ml), and both compounds were
more potent than fleroxacin, ofloxacin, and tosufloxacin
(MIC90s of 4, 4, and 2 µg/ml, respectively).
Finally, the activities of E-4767 and E-5065 against anaerobic
organisms, such as Clostridium spp. and
Bacteroides spp., were compared with that of ciprofloxacin.
For Clostridium spp., the MIC90s of
E-4767 and E-5065 were 0.25 and 0.5 µg/ml, respectively. These activities were twofold higher than (for E-4767) and
equivalent to (for E-5065) that of ciprofloxacin
(MIC90, 0.5 µg/ml). However, against
Bacteroides spp., the activity of ciprofloxacin
(MIC90, 32 µg/ml) was as much as 256 times
lower than that of E-4767 and 32 times lower than the activity of
E-5065.
Factors affecting in vitro activities. Tables 2 to 4 show the effects of medium pH, increasing magnesium concentration, and the presence of serum or urine at different pH values on the MICs of E-4767 and E-5065.
(i) Effects of medium pH.
The effects of pH on the activities
of E-4767 and E-5065 are shown in Table
2. At pH 4.8, the MICs of E-4767 for the
isolates tested were 4- to 16-fold higher than at pH 6.8, with the
exception of the MIC for P. aeruginosa HS-116,
which increased only 2-fold. At pH 5.8, 7.8, and 8.8, the MICs of
E-4767 were in general two- to fourfold higher than at pH 6.8. Compared
with the MIC of E-4767 at pH 6.8, the MICs at pH 5.8, 7.8, and 8.8 for
P. vulgaris HSP-99 and the MIC at pH 5.8 for
E. coli HM-42 increased eightfold. The MICs of
E-5065 for the strains tested were generally between two- and eightfold
higher at pH 5.8 and two- to fourfold higher at pH 7.8 and 8.8 than the
MICs at pH 6.8. At pH 4.8, the MICs of E-5065 increased 8- to 16-fold,
with the exception of coagulase-negative Staphylococcus sp.
HSP-10, for which the increase was only 2-fold, and K. pneumoniae HSP-30, for which the MIC at pH 5.8 was more than
128-fold higher than at pH 6.8.
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(ii) Effects of magnesium ion concentration.
Table
3 shows the effects of increasing the
concentration of Mg2+ upon the MICs of E-4767 and
E-5065. MICs of both compounds in MH broth supplemented with 1 mM
Mg2+ remained unchanged, with the exception of
the E-4767 MICs for P. vulgaris HSP-99 and
P. aeruginosa HS-116 and the E-5065 MICs for
C. freundii HSP-73 and coagulase-negative
Staphylococcus sp. HSP-10. In the case of medium
supplemented with 5 or 10 mM Mg2+, MICs increased
two- to eightfold. In general, there was no difference between MICs and
MBCs.
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(iii) Effects of serum.
In medium containing 20 or 70% horse
serum, E-4767 and E-5065 were two to four times less active than in
broth against most species tested (Table
4). However, for P. vulgaris HSP-99, the MIC of E-4767 increased eightfold in
the presence of 20% serum. On the other hand, for K. pneumoniae HSP-30, P. aeruginosa
HS-116, and E. faecalis HSP-30, the MICs of
E-4767 were not affected by the presence of 20% serum. Finally, the
MICs of E-5065 for E. coli HM-42 and
E. faecalis HSP-30 were likewise not
affected by the presence of 20% serum.
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(iv) Effects of urine. The effects of urine on the in vitro activity of E-4767 and E-5065 are shown in Table 4. In general, the activity of E-4767 decreased two- to eightfold when tested in fresh urine at pH 7.2, with the exception of E. faecalis HSP-30, for which the MIC of E-5065 was more than 16-fold higher than in MH broth. Against P. vulgaris HSP-99, E-4767 and E-5065 were not active in fresh urine at pH 7.2. At pH 5.5, E-4767 and E-5065 were 8 to 32 times less active than in broth, except against P. vulgaris HSP-99 and coagulase-negative Staphylococcus sp. HSP-10. The MIC of E-4767 for E. faecalis HSP-30 showed a twofold increase compared to that obtained in urine with a pH of 7.2.
Pharmacokinetics in mice.
The time courses of drug levels in
serum for E-4767, E-5065, and ciprofloxacin in mice after oral
administration (dose, 50 mg/kg) are shown in Table
5.
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Antibacterial efficacy in vivo.
Table
6 shows the therapeutic efficacies of
E-4767 and E-5065 compared with that of ciprofloxacin against lethal
systemic infections in mice caused by selected gram-positive and
-negative pathogens, such as E. coli HM-42,
P. aeruginosa HSP-116, Cipr
P. aeruginosa B-464, S. aureus HS-93, Cipr S. aureus 73777, Cipr S. aureus 73940, S. pneumoniae 1625, S. pneumoniae 84551, and S. pneumoniae 1752. As can be seen, several Cipr
strains have been included in the in vivo evaluation panel to assess
the ability of the two new chlorofluoroquinolones to overcome resistance mechanisms.
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DISCUSSION |
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Materials and Methods
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Discussion
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Fluoroquinolones have proven to be a very useful therapeutic class of agents for the treatment of infectious diseases, including those of the respiratory tract, urinary tract, skin and soft tissues, and even those that are transmitted sexually. Marketed compounds, such as ciprofloxacin, ofloxacin, levofloxacin, and others, have been extensively used because of their potent activity against gram-negative organisms. However, their activities are not optimal against certain gram-positive pathogens, such as staphylococci and streptococci, or against anaerobes. This situation has pointed out the need to develop new compounds capable of overcoming these drawbacks.
In general, fluoroquinolones with C-7 azetidine-1-yl substituents seem to improve potency against gram-positive and anaerobic organisms (6, 8, 9). On the other hand, C-8 fluoro- or chloro- derivatives are more effective in vivo than their quinolone counterparts with no substitution at the C-8 position (4).
E-4767 and E-5065 are two new 8-chlorofluoroquinolones with a 7-azetidin ring substituent, which show increased in vitro activity and in vivo efficacy not only against gram-positive but also against gram-negative organisms.
Against staphylococci, streptococci, and enterococci, including
coagulase-negative and Metr Staphylococcus spp.
strains, E-4767 was 2- to 8-fold more potent in vitro than E-5065 but
4- to 32-fold and 8- to 64-fold more potent than tosufloxacin and
ciprofloxacin, respectively. E-4767 and E-5065 were active against
Cipr S. aureus strains, with
MIC90s of 0.25 and 2 µg/ml, respectively. The
MIC50s of ciprofloxacin, fleroxacin, ofloxacin,
and tosufloxacin for resistant strains were all >4 µg/ml. In
addition, the two new 8-chlorofluoroquinolones demonstrated excellent
activity against anaerobes. E-4767 showed twofold-greater potency than
E-5065 and ciprofloxacin against isolates of Clostridium
spp. Against Bacteroides spp., E-5065 was 32-fold more
potent than ciprofloxacin but 8-fold less potent than E-4767. Both
E-4767 and E-5065 have demonstrated activity against all members of the
Enterobacteriaceae family tested, including S. marcescens and P. rettgeri, with
MIC90s of 0.25 and 0.5 µg/ml. Against enteric
bacteria, E-4767 was about twice as potent as E-5065. Furthermore,
against this group of organisms, E-4767 showed 2- to 16-fold-greater in
vitro activity than ciprofloxacin and 4- to 32-fold-greater activity
than tosufloxacin. Against P. aeruginosa, the
activities of E-4767 and E-5065 were comparable to that of
ciprofloxacin, fourfold higher than that of tosufloxacin, and eightfold
higher than those of ofloxacin and fleroxacin.
As has been reported for most quinolones, E-4767 and E-5065 exhibited substantial reductions in activity when the medium pH decreased to under 4.8. The documented antagonistic effect of magnesium ions on quinolone antimicrobial activity was also observed with E-4767 and E-5065 in the presence of 5 or 10 mM Mg2+. The in vitro activities of E-4767 and E-5065 were lowered by the presence of horse serum or human urine at pH 7.2, although the activities were only reduced two- to fourfold compared to those observed in broth. Bacteriostatic and bactericidal activities of both drugs were usually achieved at similar compound concentrations.
In general, E-4767 and E-5065 have demonstrated excellent in vitro properties against a broad range of pathogenic bacteria. However, it is well known that the final outcome of any anti-infective treatment is a consequence of the in vitro activity and pharmacokinetic properties. This is the reason why recent efforts have focused on the development of new compounds not only with improved in vitro activities against gram-positive and anaerobic organisms but also with improved pharmacokinetic performance. Preliminary pharmacokinetic studies with E-4767 and E-5065 showed that after a single oral administration of 50 mg/kg, both compounds reached significantly higher concentrations in mouse serum than did ciprofloxacin. The latter was selected as the reference compound for in vivo studies since it is the most widely used of the marketed quinolones. The pharmacokinetic results showed E-4767 and E-5065 to be rapidly absorbed, reaching concentrations in serum of 5.7 and 6.2 µg/ml, respectively, within 30 min, compared to 2.3 µg/ml of ciprofloxacin in this same amount of time. The AUC was also greater than that obtained with ciprofloxacin. It is well known that compounds having a C-7 azetidine-1-yl substituent achieve significantly higher concentrations in serum and consequently show increased therapeutic efficacy in protection tests (6, 14).
E-4767 and E-5065 were effective in the treatment of a variety of experimental infections in mice, including those produced by gram-positive and -negative pathogens. The two new chlorofluoroquinolones were similar to or more effective than ciprofloxacin against all experimental infections studied. Against infections produced by gram-positive pathogens such as S. aureus and S. pneumoniae, E-4767 was between 3 and 14 times more effective than E-5065 and up to 25-fold more effective than ciprofloxacin. E-4767 and E-5065 were also effective in protecting mice infected with E. coli or P. aeruginosa strains. The protective effects shown by E-4767 and E-5065 against gram-positive cocci may be related to their higher in vitro activities and perhaps better pharmacokinetic properties.
In conclusion, E-4767 and E-5065 are new broad-spectrum compounds with potent in vitro activities against gram-negative organisms, gram-positive cocci, and anaerobes. Both drugs exhibit greater therapeutic efficacies in murine experimental infection models involving gram-positive and -negative organisms than ciprofloxacin, possibly because of their better in vitro activities against gram-positive cocci linked to their improved pharmacokinetic properties, these being key factors for enhancing in vivo drug efficacy.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratory of Microbiology, Department of Sanitary Microbiology and Parasitology, Division of Health Sciences, Faculty of Pharmacy, University of Barcelona, C/Joan XXIII s/n, 08028 Barcelona, Spain. Phone: 34 93 4024496. Fax: 34 93 4021896. E-mail: jguinea{at}farmacia.far.ub.es.
Present address: Research Department, GlaxoSmithKline, Parque
Tecnológico de Madrid, 28760 Tres Cantos, Madrid, Spain.
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REFERENCES |
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Abstract
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Materials and Methods
Results
Discussion
References
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Antimicrobial Agents and Chemotherapy, November 2001, p. 3113-3121, Vol. 45, No. 11
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.11.3113-3121.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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