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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, July 2001, p. 2169-2172, Vol. 45, No. 7
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.7.2169-2172.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Activity of LY333328 in Experimental Meningitis
Caused by a Streptococcus pneumoniae Strain Susceptible
to Penicillin
Joachim
Gerber,1
Alexander
Smirnov,1
Andreas
Wellmer,1
Jasmin
Ragheb,1
Juliane
Prange,1
Eckhardt
Schütz,2
Klaus
Wettich,3
Siegfried
Kalich,3 and
Roland
Nau1,*
Departments of
Neurology1 and Laboratory
Medicine,2 University of Göttingen,
Göttingen, and Lilly Deutschland, Bad
Homburg,3 Germany
Received 5 September 2000/Returned for modification 13 January
2001/Accepted 15 April 2001
 |
ABSTRACT |
In a rabbit model of Streptococcus pneumoniae
meningitis single doses of 10 and 2.5 mg of the glycopeptide LY333328
per kg of body weight reduced bacterial titers in cerebrospinal fluid (CSF) almost as rapidly as ceftriaxone at 10 mg/kg/h (changes in log
CFU, 
0.29 ± 0.21 and 0.26 ± 0.22 versus 0.34 ± 0.15/ml/h). A dose of 1 mg/kg was bacteriostatic (change in log CFU,
0.01 ± 0.11/ml/h). In two animals receiving LY333328 at a dose of
40 mg/kg the bacterial titers were reduced by 0.54 and 0.51 log
CFU/ml/h. The penetration of CSF by LY333328 was 1 to 5%. The
concentrations of lipoteichoic and teichoic acids in CSF and neuronal
damage were similar in ceftriaxone- and LY333328-treated animals.
| |
TEXT |
Since the first description of a
penicillin-resistant pneumococcal strain in 1967 in Australia
(6), penicillin-resistant pneumococci have spread
worldwide (1). Furthermore, reduced sensitivity of
Streptococcus pneumoniae isolates to cephalosporins and
other 
-lactam antibiotics (7) resulting in clinical
failures of cefotaxime and ceftriaxone in the treatment of meningitis
has increased in frequency (2, 4).
Vancomycin is active against penicillin-resistant pneumococci. Although
bactericidal concentrations of vancomycin were achieved in
cerebrospinal fluid (CSF) in children with bacterial meningitis (9), clinical failures in adults have been reported
probably because of a diminished blood-brain barrier penetration during adjunctive steroid therapy (19). This emphasizes the need
for compounds either with a higher activity or with an improved CSF penetration compared to vancomycin for treatment of bacterial meningitis. LY333328 diphosphate is a semisynthetic glycopeptide inhibiting the synthesis of peptidoglycan in gram-positive bacteria. It
is highly active against many resistant organisms, including methicillin- and teicoplanin-resistant staphylococci,
vancomycin-resistant enterococci, and penicillin-resistant and
multiresistant pneumococci (3, 5, 13). In a search for new
therapeutic agents, we investigated its activity and its influence on
the release of proinflammatory bacterial compounds and on neuronal
damage in the rabbit model of S. pneumoniae meningitis with
a strain susceptible to penicillin.
In vitro antimicrobial activity.
S. pneumoniae type
3 (MIC and minimal bactericidal concentration [MBC] of LY333328,
0.015 and 0.03 µg/ml, respectively; MIC and MBC of ceftriaxone, 0.03 and 0.06 µg/ml, respectively; MIC and MBC of penicillin, <0.1
µg/ml) (gift of M. G. Täuber, Department of Medical
Microbiology, University of Bern, Bern, Switzerland) was grown,
centrifuged, and resuspended in fresh tryptic soy broth to a final
concentration of 6.83 ± 0.16 log CFU/ml (n = 5).
Control cultures were grown after resuspension without antibiotics.
Ceftriaxone and LY333328 (final concentration, 10 µg/ml) were added
to 15-ml aliquots, and bacterial counts were determined at 0, 1, 3, 6, 9, and 12 h.
Rabbit model.
Meningitis was induced by intracisternal
injection of approximately 106 CFU of S. pneumoniae. Blood (3 ml) and CSF (300 µl) were drawn before and
at 12, 14, 17, 20, and 24 h after infection. Twelve hours after
infection, therapy was initiated. Rabbits were treated with a single
dose of LY333328 (1 [n = 5], 2.5 [n = 5], 10 [n = 10], or 40 [n = 2] mg/kg of body weight). LY333328 was dissolved in 5% glucose
and infused over 30 min. Control animals received a bolus of 20 mg of
ceftriaxone (Rocephin; Hoffmann-La Roche, Grenzach-Wyhlen, Germany) per
kg followed by a continuous infusion of 10 mg/kg/h over 12 h
(n = 12). Ceftriaxone was administered continuously
because of its relatively short half-life in rabbits.
Sample processing.
CSF white blood cells were quantified in a
Fuchs-Rosenthal hemocytometer. Pneumococcal titers in CSF were counted
by plating 10-µl undiluted samples and serial 10-fold dilutions on
blood agar plates. To avoid carryover effects, 300-µl samples of
1:100 dilutions were plated on blood agar plates.
Bacterial titers at 12, 14, 17, 20, and 24 h after infection were
subjected to log-linear regression analysis. Neuron-specific enolase
concentrations were determined by an immunoluminometric method (LIA-mat
NSE Prolifigen; Byk-Sangtec, Dietzenbach, Germany). Lactate was
measured enzymatically (Greiner, Flacht, Germany), CSF protein
concentration was measured photometrically (BCA Protein Test; Pierce,
Rockford, Ill.), and pneumococcal lipoteichoic (LTA) and teichoic acid
(TA) levels were measured by enzyme immunoassay (16).
After acidification of serum and CSF and extraction on a C2
cartridge, concentrations of LY333328 were determined by liquid chromatography-mass spectrometry with positive-ion electrospray ionization using a Supelco Discovery C18 column (150 by 2.1 mm; inside diameter, 5 µm) as stationary phase and
methanol-water-formic acid (45, 55, and 0.1%, respectively) as mobile
phase. Peaks were quantitated by calculating area ratios with
calibration standards (BAS Analytics, West Lafayette, Ind.). The
detection limit was 0.15 µg/ml in serum and CSF.
In situ tailing of apoptotic neurons.
In deparaffinized and
hydrated 1-µm sections, DNA double-strand breaks were stained by in
situ tailing, and the density of apoptotic neurons in the granular cell
layer of the dentate gyrus in the hippocampal formation was determined
by planimetry (20).
Pharmacokinetics and statistics.
The areas under the
concentration-time curve from the start of the infusion (0 h = 12 h after infection) until the end of the experiment (12 h = 24 h after infection) in serum (AUCS 0-12h) and CSF
(AUCCSF 0-12h) were estimated by the linear trapezoidal rule. Noncompartmental methods were used to estimate clearance, the
elimination constant, and volume of distribution.
Leukocyte density, concentrations of protein, lactate, and LTA and TA
in CSF, and the density of apoptotic neurons in the dentate gyrus in
the groups receiving LY333328 were compared with those for
ceftriaxone-treated animals by analysis of variance with correction for
multiple testing by the Bonferroni method. The half-maximal dose and
the maximum bactericidal rate of LY333328 were estimated by a
Lineweaver-Burk plot assuming an increase in the CSF bacterial titers
of 0.15 log CFU/ml/h in untreated rabbits.
In vitro results.
At 10 µg/ml LY333328 decreased the
bacterial titers of S. pneumoniae rapidly to levels below
the detection limit of 102 CFU/ml within 1 h, whereas
cultures treated with 10 µg of ceftriaxone/ml were sterile after
12 h. The release of LTA and TA was lower after exposure to
LY333328 (141.1 ± 93.5 versus 491.4 ± 298.3 ng/ml at 3 h
[P = 0.020], 141.6 ± 89.9 versus 418.0 ± 293.3 ng/ml at 6 h [P = 0.042], 126.2 ± 92.1 versus 465.0 ± 295.8 ng/ml at 9 h [P = 0.035], and 157.8 ± 92.0 versus 425.6 ± 277.1 ng/ml
at 12 h [P = 0.051]).
Rabbit model.
Maximum concentration of LY333328 in
serum rose linearly with the dose administered. A plateauing effect,
however, appeared to occur at the dose of 40 mg/kg. A similar pattern
was observed in an earlier study with LY333328 at very high doses
(T. J. Brown, personal communication). Consistently, the apparent
volume of distribution (approximately 0.25 liter/kg) in the 1-, 2.5-, and 10-mg/kg-treated animals equalled the extracellular fluid space, whereas it increased to approximately 0.7 liter/kg at a dose of 40 mg/kg. The elimination half-life in serum generally ranged from 5 to
8 h (Table 1). Maximum
concentrations in CSF were reached several hours after intravenous
administration of LY333328 (8.1 ± 3.8 h for the 10-mg/kg
dose and 4.4 ± 1.3 h for the 2.5-mg/kg dose). For this
reason, half-life in CSF could not be estimated reliably. The entry
into CSF as estimated by AUCCSF
0-12h/AUCS 0-12h in most cases was between 1 and
5% (Table 1). A single 10-mg/kg dose of LY333328 (n = 10) reduced the bacterial titers in CSF almost as rapidly as
ceftriaxone (n = 12) (0.29 ± 0.21 versus 0.34 ± 0.15 log CFU/ml/h). The bactericidal effect was slightly lower for LY333328 at a dose of 2.5 mg/kg (n = 5)
(0.26 ± 0.22 log CFU/ml/h), whereas the bactericidal activities
at a dose of 40 mg/kg were 0.54 and 0.51 log CFU/ml/h (n = 2) (all groups statistically not significant versus
ceftriaxone). A 1-mg/kg dose of LY333328 (n = 5) was
only bacteriostatic (0.01 ± 0.11 CFU/ml/h [P < 0.05 versus ceftriaxone]) (Fig. 1).
Plotting 1/effect versus 1/dose and 1/effect versus 1/AUCCSF
0-12h (Lineweaver-Burk plot) yielded estimates of the
half-maximal dose (4.6 mg/kg), the half-maximal AUCCSF
0-12h (4.2 mg × h/liter), and the maximum bactericidal
rate (0.68 and 0.58 log CFU/ml/h) of LY333328.
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FIG. 1.
Bacterial titers in CSF. Antibiotic therapy was
initiated 12 h after infection. Only at a dose of 1 mg/kg did
LY333328 show significantly less activity than ceftriaxone (CRO)
(P < 0.05).
|
|
Leukocyte density and concentrations of protein and lactate in CSF at
24 h did not differ between LY333328- and ceftriaxone-treated animals (Table 1). The concentrations of LTA and TA showed no significant difference between ceftriaxone- and LY333328-treated animals at each time point except at 24 h, when animals treated with LY333328 at 1 mg/kg (log 3.99 ± 0.89 ng/ml) contained
significantly more LTA in CSF than rabbits treated with ceftriaxone 10 at mg/kg/h (log 2.81 ± 0.72 ng/ml [P < 0.05])
or LY333328 at 10 mg/kg (log 2.86 ± 0.62 [P < 0.05]). Parameters of neuronal damage (20) did not
differ significantly among the treatment groups (Table 1). In
uninfected animals (n = 4), the density of neuronal
apoptosis 24 h after saline injection was 12 ± 4/mm2,
and CSF protein and lactate concentrations were 1,021 ± 240 µg/ml and 1.9 ± 0.3 mmol/liter, respectively.
In vivo, a short-duration infusion of LY333328 at doses of 10 and 2.5 mg/kg was slightly less active than a continuous infusion of
ceftriaxone (total dose, 140 mg/kg over 12 h) (differences not
significant), whereas LY333328-treated cultures in vitro were sterile
after 1 h. Probable reasons for the discrepancy between in vitro
and in vivo activities are different growth rates and the high binding
of LY333328 to serum and CSF proteins. The bactericidal activity of
LY333328 in broth was reduced when it was tested in the presence of
90% rabbit serum (8). The binding to serum proteins and
the high molecular mass (1,989 Da) resulted in relatively low
concentrations in CSF after intravenous administration of LY333328.
Consequently, the AUCCSF 0-12h/AUCS 0-12h ratio was lower for LY333328 than for ceftriaxone (Table 1).
The dose and AUCCSF 0-12h of LY333328 estimated to produce
half-maximal killing were approximately 5 mg/kg and 4.2 mg × h/liter, respectively. This implies that half-maximal killing occurs at
a ratio of the CSF drug concentration to MIC of 20 to 30 and an
AUCCSF 0-24h/MIC ratio (10) of approximately 550. In the central compartment, with most quinolones and -lactam antibiotics the half-maximal effect occurs at lower ratios of the CSF
drug concentration and AUCCSF 0-24h to MIC (11, 17). In addition to the slow bacterial replication as a cause of
the reduced antibacterial activities of many compounds in the CSF
compartment, the relatively high ratios of the CSF drug concentration and AUCCSF 0-24h to MIC necessary to produce half-maximum killing by LY333328 may reflect substantial binding to CSF proteins in meningitis.
The 10-mg/kg dose of LY333328 was chosen for the present study to
produce concentrations in serum intended for human use; in healthy
volunteers, doses of up to 3 mg/kg producing concentrations in plasma
of approximately 50 µg/ml at the end of the infusion were tolerated
without serious side effects (Eli Lilly & Co., data on file). In the
present study, the dose of 40 mg/kg substantially increased the
bactericidal activity. The tolerability of such high doses in humans
remains to be determined.
Although LY333328 released smaller quantities of LTA and TA from
S. pneumoniae than those released by ceftriaxone in vitro, in vivo no significant differences were observed between animals treated with LY333328 at doses of 2.5, 10, and 40 mg/kg and those treated with ceftriaxone. This discrepancy probably originated from the
different velocities of bacterial killing by LY333328 in vitro and in
the animal model. Consequently, in the CSF compartment the inflammatory
reaction and parameters of neuronal damage during treatment with
LY333328 were comparable to those observed during ceftriaxone therapy.
This suggests that, unlike rifamycins and quinupristin-dalfopristin
(12, 14, 18), LY333328 probably does not alter the course
of the inflammatory reaction during treatment of S. pneumoniae meningitis. With the bacteriostatic 1-mg/kg dose of
LY333328, CSF LTA concentrations at 24 h were higher than those in
animals treated with LY333328 at 10 mg/kg or ceftriaxone. This agrees
well with previous reports of an increased liberation of
proinflammatory bacterial compounds at low antibiotic concentrations
both in broth and in CSF (15, 16).
In conclusion, despite low CSF penetration, the extremely low MICs and
MBCs against pneumococci and the high in vivo activity in the rabbit
model indicate that clinical studies with LY333328 for the treatment of
pneumococcal meningitis should be conducted. The selective activity of
LY333328 against gram-positive organisms, however, will favor use
against penicillin-resistant pneumococci but not in empiric therapy.
Since a reduced sensitivity to penicillin does not increase the MIC of
LY333328, the compound should be equally active against
penicillin-susceptible and -resistant strains, although the in vivo
activity of LY333328 against resistant strains in bacterial meningitis
remains to be determined.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Neurology, University of Göttingen, Robert-Koch-Str. 40, D-37075
Göttingen, Germany. Phone: 49-551-398455 or -396684. Fax:
49-551-398405. E-mail: rnau{at}gwdg.de.
| |
REFERENCES |
| 1.
|
Appelbaum, P. C.
1987.
World-wide development of antibiotic resistance in pneumococci.
Eur. J. Clin. Microbiol.
6:367-377[CrossRef][Medline].
|
| 2.
|
Bradley, J. S., and J. D. Connor.
1991.
Ceftriaxone failure in meningitis caused by Streptococcus pneumoniae with reduced susceptibility to beta-lactam antibiotics.
Pediatr. Infect. Dis. J.
10:871-873[Medline].
|
| 3.
|
Fasola, E.,
S. K. Spangler,
L. M. Ednie,
M. R. Jacobs,
S. Bajaksouzian, and P. C. Appelbaum.
1996.
Comparative activities of LY333328, a new glycopeptide, against penicillin-susceptible and -resistant pneumococci.
Antimicrob. Agents Chemother.
40:2661-2663[Abstract].
|
| 4.
|
Figueiredo, A. M. S.,
J. D. Connor,
A. Severin,
M. V. Vaz Pato, and A. Tomasz.
1992.
A pneumococcal clinical isolate with high-level resistance to cefotaxime and ceftriaxone.
Antimicrob. Agents Chemother.
36:886-889[Abstract/Free Full Text].
|
| 5.
|
Garcia-Garrote, F.,
E. Cercenado,
L. Alcalá, and E. Bouza.
1998.
In vitro activity of the new glycopeptide LY333328 against multiply resistant gram-positive clinical isolates.
Antimicrob. Agents Chemother.
42:2452-2455[Abstract/Free Full Text].
|
| 6.
|
Hansman, D., and M. M. Bullen.
1967.
A resistant pneumococcus.
Lancet
ii:264-265.
|
| 7.
|
Jetté, L. P.,
F. Lamothe, and The Pneumococcus Study Group.
1989.
Surveillance of invasive Streptococcus pneumoniae infection in Quebec, Canada, from 1984 to 1986: serotype distribution, antimicrobial susceptibility, and clinical characteristics.
J. Clin. Microbiol.
27:1-5[Abstract/Free Full Text].
|
| 8.
|
Kaatz, G. W.,
S. M. Seo,
J. R. Aeschlimann,
H. H. Houlihan,
R.-C. Mercier, and M. J. Rybak.
1998.
Efficacy of LY333328 against experimental methicillin-resistant Staphylococcus aureus endocarditis.
Antimicrob. Agents Chemother.
42:981-983[Abstract/Free Full Text].
|
| 9.
|
Klugman, K. P.,
I. R. Friedland, and J. S. Bradley.
1995.
Bactericidal activity against cephalosporin-resistant Streptococcus pneumoniae in cerebrospinal fluid of children with acute bacterial meningitis.
Antimicrob. Agents Chemother.
39:1988-1992[Abstract].
|
| 10.
|
Li, R. C.,
M. Zhu, and J. J. Schentag.
1999.
Achieving an optimal outcome in the treatment of infections. The role of clinical pharmacokinetics and pharmacodynamics of antimicrobials.
Clin. Pharmacokinet.
37:1-16[Medline].
|
| 11.
|
Nau, R.,
T. Schmidt,
K. Kaye,
J. L. Froula, and M. G. Täuber.
1995.
Quinolone antibiotics in therapy of experimental pneumococcal meningitis in rabbits.
Antimicrob. Agents Chemother.
39:593-597[Abstract].
|
| 12.
|
Nau, R.,
A. Wellmer,
A. Soto,
K. Koch,
O. Schneider,
H. Schmidt,
J. Gerber,
U. Michel, and W. Brück.
1999.
Rifampin reduces early mortality in experimental Streptococcus pneumoniae meningitis.
J. Infect. Dis.
179:1557-1560[CrossRef][Medline].
|
| 13.
|
Saleh-Mghir, A.,
A. Lefort,
Y. Petegnief,
S. Dautrey,
J. M. Vallois,
D. Le Guludec,
C. Carbon, and B. Fantin.
1999.
Activity and diffusion of LY333328 in experimental endocarditis due to vancomycin-resistant Enterococcus faecalis.
Antimicrob. Agents Chemother.
43:115-120[Abstract/Free Full Text].
|
| 14.
|
Schmidt, H.,
G. Zysk,
R. R. Reinert,
W. Brück,
A. Stringaris,
K. Stuertz,
R. Bartels,
K. Schaper,
S. Weinig, and R. Nau.
1997.
Rifabutin for experimental pneumococcal meningitis.
Chemotherapy
43:264-271[Medline].
|
| 15.
|
Stuertz, K.,
H. Schmidt,
H. Eiffert,
P. Schwartz,
M. Mäder, and R. Nau.
1998.
Differential release of lipoteichoic and teichoic acids from Streptococcus pneumoniae as a result of exposure to -lactam antibiotics, rifamycins, trovafloxacin, and quinupristin-dalfopristin.
Antimicrob. Agents Chemother.
42:277-281[Abstract/Free Full Text].
|
| 16.
|
Stuertz, K.,
H. Schmidt,
F. Trostdorf,
H. Eiffert,
M. Mäder, and R. Nau.
1999.
Lower lipoteichoic and teichoic acid CSF concentrations during treatment of pneumococcal meningitis with non-bacteriolytic antibiotics than with ceftriaxone.
Scand. J. Infect. Dis.
31:367-370[CrossRef][Medline].
|
| 17.
|
Täuber, M. G.,
C. A. Doroshow,
C. J. Hackbarth,
M. G. Rusnak,
T. A. Drake, and M. A. Sande.
1984.
Antibacterial activity of beta-lactam antibiotics in experimental meningitis due to Streptococcus pneumoniae.
J. Infect. Dis.
149:568-574[Medline].
|
| 18.
|
Trostdorf, F.,
R. R. Reinert,
H. Schmidt,
T. Nichterlein,
K. Stuertz,
M. Schmitz-Salue,
I. Sadowski,
W. Brück, and R. Nau.
1999.
Quinupristin/dalfopristin attenuates the inflammatory response and reduces the concentration of neuron-specific enolase in the cerebrospinal fluid of rabbits with experimental Streptococcus pneumoniae meningitis.
J. Antimicrob. Chemother.
43:87-94[Abstract/Free Full Text].
|
| 19.
|
Viladrich, P. F.,
F. Gudiol,
J. Linares,
R. Pallares,
I. Sabate,
G. Rufi, and J. Ariza.
1991.
Evaluation of vancomycin for therapy of adult pneumococcal meningitis.
Antimicrob. Agents Chemother.
35:2467-2472[Abstract/Free Full Text].
|
| 20.
|
Zysk, G.,
W. Brück,
J. Gerber,
Y. Brück,
H. W. Prange, and R. Nau.
1996.
Anti-inflammatory treatment influences neuronal apoptotic cell death in the dentate gyrus in experimental pneumococcal meningitis.
J. Neuropathol. Exp. Neurol.
55:722-728[Medline].
|
Antimicrobial Agents and Chemotherapy, July 2001, p. 2169-2172, Vol. 45, No. 7
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.7.2169-2172.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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