Streptococcus pneumoniae Pneumonia in Mice: Optimal Amoxicillin Dosing Predicted from a Pharmacokinetic-Pharmacodynamic Model
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0022-3565/99/2913-1086$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 291, No. 3
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A.
JPET 291:1086 –1092, 1999
Streptococcus pneumoniae Pneumonia in Mice: Optimal
Amoxicillin Dosing Predicted from a Pharmacokinetic-
Pharmacodynamic Model
PIERRE MOINE and JEAN XAVIER MAZOIT
Service et Laboratoire d’Anesthésie, Centre Hospitalier Universitaire de Bicêtre, Université Paris-Sud, Faculté de Médecine du Kremlin-Bicêtre.
Le Kremlin-Bicêtre, France (P.M., J.X.M.); and Contrat de Recherche Institut National de la Santé et de la Recherche Médicale CRI 4U 002 D
Pharmacologie de la résistance aux anti-infectieux, Centre Hospitalier Universitaire Bichat-Claude Bernard, Paris, France (P.M.)
Accepted for publication June 3, 1999 This paper is available online at http://www.jpet.org
Downloaded from jpet.aspetjournals.org at ASPET Journals on September 30, 2015
ABSTRACT
In an attempt to better understand the interaction of amoxicillin after a single dose of amoxicillin, bacterial counts in lung rapidly
with Streptococcus pneumoniae in the lung, and to determine decreased and the bacterial growth remained suppressed dur-
the parameters of therapeutic efficacy of the antimicrobial ing a long period of time after cessation of exposure of micro-
agent amoxicillin, we used a pharmacokinetic-pharmacody- organisms to amoxicillin; and 2) the duration of bacterial growth
namic model to describe the overall dose-effect relationship of suppression was related to the intrinsic properties of S. pneu-
amoxicillin against 12 strains of S. pneumoniae with penicillin moniae strains rather than to host environment because ke0
minimum inhibitory concentrations ranging from ,0.01 to 16 was significantly different between strains. These two premises
mg/ml in a neutropenic murine pneumonia model. We were able clearly demonstrate that bacterial growth suppression is related
to correlate amoxicillin dosing, pharmacokinetics, and the tem- to an in vivo postantibiotic effect. Furthermore, we have shown
poral changes in bacterial count in lung. Moreover, survival that the major determinant of amoxicillin in vivo bactericidal
rates measured in one strain at different dosing were signifi- activity and therapeutic efficacy appeared to be the dose of
cantly related to the number of bacteria in lung calculated from amoxicillin because amoxicillin exhibits a rapid dose-depen-
the pharmacokinetic-pharmacodynamic model. Disappearance dent killing regardless of the S. pneumoniae strain. Our findings
of amoxicillin from the effect compartment appeared to be very may have implications for the clinical use of amoxicillin. In view
slow and the rate constant (ke0) governing this process was of our results, the guidance to increase the amoxicillin-loading
significantly different between strains, ranging from 0.00131 to dose in pneumococcal pneumonia appears to be immediately
0.03945 h21. These findings have two major implications: 1) clinically relevant.
Infections caused by Streptococcus pneumoniae resistant to classes of antibiotics to predict bactericidal activity and ther-
b-lactam antimicrobials are an increasingly frequent problem apeutic efficacy in different animal models: the area under
in clinical practice but the optimal antibiotic therapy for peni- the inhibitory serum concentration-time curve, the time that
cillin-resistant S. pneumoniae pneumonia is not clear (Austri- the serum concentration exceeds MIC, and the ratio between
an, 1994; Boswell et al., 1994; Finch, 1995). In a murine S. peak serum concentration and MIC (Hyatt et al., 1995; Craig,
pneumoniae pneumonia model, with an Emax model, we have 1998). The duration of time that serum levels exceed MIC has
recently demonstrated that the standard in vitro minimum been shown to be the PK parameter most frequently corre-
inhibitory concentration (MIC) and minimum bactericidal con- lated with b-lactam efficacy in different animal models
centration (MBC) of amoxicillin were excellent predictors of the (Hyatt et al., 1995; Craig, 1998). Frimodt-Moller et al. (1986),
relative in vivo potency of amoxicillin against pneumococcal with a mouse model with i.p. inoculation of S. pneumoniae
species, including highly penicillin-resistant strains with peni- type 3 to compare in vivo effects of 14 cephalosporins, dem-
cillin MICs ranging from ,0.01 to 16 mg/ml (Moine et al., onstrated a significant correlation between the 50% effective
1997a). Nevertheless, we do not know which pharmacokinetic dose and the time the serum concentration remained above
(PK) and pharmacodynamic (PD) parameters correlated with the MIC for each drug. Moreover, in a neutropenic murine S.
efficacy in this S. pneumoniae pneumonia model. pneumoniae thigh infection model, Vogelman et al. (1988a)
Numerous PK parameters have been proposed for various demonstrated that maximum bactericidal activity was
achieved when serum amoxicillin levels were constantly
Received for publication August 20, 1998. maintained above the MIC.
ABBREVIATIONS: MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; PK, pharmacokinetic; PD, pharmacody-
namic; CFU, colony forming units; S, survival; MTD, minimal therapeutic dosage; PAE, postantibiotic effect.
10861999 Amoxicillin in a Mouse S. pneumoniae Pneumonia Model 1087
Nevertheless, all the above-mentioned PK parameters are Antibiotic Susceptibility Tests: Bactericidal Activity In Vivo
totally interdependent. b-Lactam antibiotics follow linear As stated above, we used published data for 12 different S. pneu-
PKs and because of the principle of superposition, all these moniae strains (Moine et al., 1997a). MICs and MBCs were deter-
parameters are always directly proportional to the dose. mined for each strain in Mueller-Hinton infusion broth supple-
They are also complex functions of the multicompartmental mented with 5% lysed horse blood by means of the tube dilution
characteristics of the drug and of the dosing interval. How- method (National Committee for Clinical Laboratory Standards,
ever, the dose remains the first determinant of kinetics. 1995). Bactericidal activity in vivo was determined in neutropenic
In an attempt to better understand the interaction of mice receiving various doses of amoxicillin according to the infective
amoxicillin with S. pneumoniae in the lung, and to tenta- strain as described in detail elsewhere (Moine et al., 1997a). Briefly,
the total CFU recovered from whole-lung homogenates was deter-
tively determine the parameters of therapeutic efficacy of the
mined 1, 3, 6, and 9 h after amoxicillin injection. The lower limit of
antimicrobial agent amoxicillin, we used a PK-PD model to detection was 4.6 ln CFU/lung (2 log10 CFU/lung), which corre-
describe the overall dose-effect relationship of amoxicillin sponded to the weakest dilution tissue homogenates (1021) that
against 12 strains of S. pneumoniae with penicillin MICs avoided significant drug carryover with control inocula. In control
ranging from ,0.01 to 16 mg/ml in a neutropenic murine animals mean bacterial count increased 2-fold (from 16.8 to 17.5 ln
pneumonia model. CFU/lung) during the time of observation (1–9 h after amoxicillin
injection).
Materials and Methods Survival Studies
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With the exception of serum and lung amoxicillin concentration- We have previously determined for each tested strain the minimal
time data, all other data used were obtained from previously pub- therapeutic dosage [MTD (mg/kg)] of amoxicillin (treatment sched-
lished studies (Azoulay-Dupuis et al., 1991; Moine et al., 1994, ule consisting of s.c. injections at 12-h intervals over 3 days) as the
1997a,b). dose required to achieve 75 to 85% survival (Moine et al., 1994,
1997a). For each strain, the survival rate was not significantly im-
Challenge Organisms and Experimental Pneumococcal proved with larger doses of amoxicillin than the respective MTD
(Moine et al., 1997a).
Pneumonia in Mice
Twelve S. pneumoniae clinical strains were used (Moine et al., Modeling Procedure
1997a): two strains were penicillin-susceptible (penicillin MIC ,
We separately fitted the PK, PD, and survival (S) data with a
0.012 mg/ml) PS [P52181 and P30923]; four were penicillin interme-
linear, stationary parametric approach (Fig. 1).
diate resistant (0.012 , penicillin MIC 5 1 mg/ml) PI [P31192,
Structural Model. PK. We made two assumptions. First, amoxi-
P30189, P40225, and P54B]; and six were penicillin resistant (pen-
cillin bioavailability was complete after s.c. injection because no
icillin MIC . 1 mg/ml) PR [P54988, P12698, P15986, P40422, P41375,
extrahepatic metabolism and/or excretion of amoxicillin has been
and P53681]. Pneumonia was induced in 20- to 24-g b.wt. female
described. Second, no flip-flop effect occurred after this injection.
Swiss mice rendered neutropenic by injecting cyclophosphamide (150
Serum concentration-time and lung tissue concentration-time data
mg/kg i.p. daily), starting 3 days before infection. Animals were
were simultaneously fitted to the following parametric models: one-,
infected by direct intratracheal bacterial suspension instillation [50
two-, and three-compartment open models (with the lung included in
ml of bacterial suspension, e.g., 107 colony forming units (CFU) per
the central compartment or constituting a peripheral compartment)
mouse] via the mouth as described elsewhere (Azoulay-Dupuis et al.,
with first-order absorption and linear first-order or Michaelis-Men-
1991; Moine et al., 1994, 1997a,b). Leukopenic mice developed acute
ten elimination from the central compartment. In the models with
bacteremic pneumonia and died within 2 to 3 days. Bacterial counts
the lung considered as part of the central compartment, lung tissue
exceeded 108 CFU/lungs at the time of death (106 CFU/ml blood).
concentration was related to the serum concentration by a partition
coefficient (K). Fitting was done with standard equations (Gibaldi
PKs and Assay and Perrier, 1982) with the procedures ADVAN 5 and ADVAN 6 from
In neutropenic Swiss mice, amoxicillin (Beecham Paris, France) the program NONMEM (Sheiner et al., 1979 –1984). The following
was determined in serum and lung after single s.c. injection of 2.5, 5, parameters were calculated: the volume of the central compartment
10, 25, 50, 100, 200, 300, and 400 mg/kg. At 0.5, 1, 2, 4, 6, 8, 12, and (Vc), the absorption rate constant (ka), and the individual compart-
24 h following drug administration, three to five animals per dose ment rate constants from compartment i to compartment j (kij). For
group were sacrificed with CO2, and exsanguinated by cardiac punc- the two- and three-compartment models, we also calculated the
ture. Blood samples were centrifuged to isolate serum, which was volume of distribution at steady state (Vss) and the total body clear-
frozen at 280°C until assay. Lungs were harvested from exsangi- ance (CL).
nated mice, washed in sterile saline solution, and frozen. On the day PD. The relationship between effect (E) (decrease in the natural
of assay, organs were weighed, pooled, and homogenized in phos- logarithm of bacterial count in lung [ln CFUzg21]) and drug concen-
phate buffer (pH 6.8). Homogenates were centrifuged and superna- tration at the (virtual) site of action (Ce) was modeled with the well
tants were used for assay. Amoxicillin concentrations were deter- known Hill equation (Holford and Sheiner, 1981; Verotta and Shei-
mined by the agar well diffusion method with Sarcina lutea ATCC ner, 1991):
9341 (American Type Culture Collection, Manassas, VA) as the bio-
assay organism and Antibiotic Medium 1 (Difco Laboratories, De-
troit, MI) as the growth medium. Standard curves for serum and
tissue concentrations were determined with solutions of amoxicillin
in phosphate buffer. Correlations between standard curves in buffer
and in serum or in lung homogenates were found to follow the line of
identity with a correlation coefficient between 0.96 and 0.94 for Fig. 1. PK and PD model. Conversion of dosing to plasma concentration
(Cp) and to virtual effect compartment concentration (Ce) is governed by
serum and lung, respectively. Standard curves were linear from
PKs. L represents the link between Cp and Ce. The observed effect
0.125 to 32 mg/ml. The lower limit of detection was 0.1 mg/ml, with a (decrease in bacterial count in lung) (E) is related to Ce by the classical
between- and within-day coefficient of variation of #7.5% at 0.5, 1, Hill equation and the resulting survival rate is related to E by a simple
7.5, and 20 mg/ml. exponential survival function (EXP).1088 Moine and Mazoit Vol. 291
Emax Ce to Emax after 72 h. A random interdose variability effect h also was
E 5 E0 2 added like in model S2.
Ce 50 1 Ce
where E0 is the initial bacterial count in lung, and Ce50 is the Results
effect-site concentration producing half Emax, which is the maximum
effect attainable. We did not include any factor of sigmoidicity. The
Fitting was adequate with the combined constant CV and
link between PKs and PDs was considered a linear first-order pro- additive error model (Fig. 2). The best PK model was found to
cess: Ce 5 Cp R L, where Cp is serum concentration, R is the be the two compartment open model with linear first-order
convolution operator, and L is a nonnegative, continuous, integrable elimination from the central compartment and the lung in-
function (Verotta and Sheiner, 1991) (Fig. 1). L has been taken to be cluded in the central compartment with a lung serum parti-
ke0 exp(2 ke0 t). It is then possible to express E as function of ke0, the tion ratio K 5 0.447 (Table 1). Neither Michaelis-Menten
rate constant of drug exit from effect compartment and Cpss50, the elimination nor interdose variability significantly improved
serum drug concentration corresponding to Ce50 at steady state the quality of fitting. Therefore, the kinetics of amoxicillin
(Sheiner et al., 1979). The PD values were comparatively fitted to may be considered linear in mice in the range of concentra-
models with Ce in the central compartment, in the peripheral com-
tions studied. PK parameters are displayed in Table 1.
partment, or in a special effect compartment. As usual, we consid-
PDs was best modeled with the effect in a special compart-
ered negligible mass transfer between Cp and Ce.
Survival analysis. Survival data with the same strain (P15986) ment linked to the central PK compartment rather than
directly in the central compartment or in the peripheral
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were obtained from previous studies (Moine et al., 1994, 1997a,b).
Briefly, amoxicillin 400, 300, 200, or 150 mg/kg was administered at compartment. The best PD model was the full model with ke0,
12-h intervals with a total of 6 injections, 100 mg/kg at 8-h intervals Emax, and Cpss50 relevant for each strain (P , .0001). Pa-
with a total of 9 injections, and 150 mg/kg at 6-h intervals with a rameters for each strain obtained by post hoc procedure are
total of 12 injections. In each experiment the animals were infected displayed in Table 2. Figure 3 displays experimental data
simultaneously. In two experiments, 15 animals per group were and fitted lines for three representative strains. The ke0,
used, and in a third experiment, 30 animals per group were used. which represents the temporal distance between central com-
The observation period was 14 days. Death rates were recorded daily
partment and effect compartment, was very low. T1/2ke0 the
and cumulative survival rates were compared. Control animals re-
elimination half-life of amoxicillin from the effect compart-
ceived identical treatment with saline.
Fifteen to 30 animals were used per experiment and the fractional ment (in fact after total disappearance of the drug from the
number of surviving animals (S) was recorded every day during 2 central compartment) ranged from 17.5 h (P40422) to 22 days
weeks. We fitted the raw survival data with the simplest exponential (P12698) (Fig. 4). For the 12 strains of S. pneumoniae, Cpss50
model: S(t) 5 exp(ut). u was first considered as a constant parameter, highly correlated with MIC (Spearman r 5 0.93, P , .0001)
thus allowing statistical comparisons between doses (see infra), and and MBC (Spearman r 5 0.90, P , .0001). In contrast, there
in a second step as a function of bacterial count in lung: u 5 a. E(t), was no significant correlation between ke0 and MIC or MBC
where a is a constant parameter (fixed effect) and E is the predicted nor between Emax and MIC or MBC. The MTD highly corre-
effect (ln CFU z g21) obtained at the PD step.
Statistical Model and Fitting Procedure. We used the pro-
gram NONMEM (version IV, level 2.1, Sheiner et al., 1979 –1994). It
uses extended least-squares as measure of goodness-of-fit (Sheiner
and Beal, 1985) and allows the fitting of mixed effects models by
using two levels of random errors (intra- and interindividual vari-
ability). The choice between the different PK and PD models was
made with the Akaike criterion (Yamaoka et al., 1978). The choice
between full models (i.e., with all interindividual variability param-
eters considered as relevant) and reduced models was made with the
log-likelihood ratio test (Eadie et al., 1982). Intraindividual variabil-
ity (assay error, model mispecification) was modeled with a combined
constant coefficient of variation and additive error. Interindividual
variability was modeled as uexp(h) (assuming a log-normal distribu-
tion), were u is the fixed effect parameter and h is the vector of
interindividual variability with mean zero and variance v2. We as-
sumed no covariance between the elements of h and between the
elements of e, the vector of residual error due to intraindividual and
measurement variability.
In addition to Michaelis-Menten analysis, we modeled linear PKs
with each dose considered as an “individual” with random interindi-
vidual variability. For PD analysis we modeled the entire data set as
a whole, with the kinetic parameters calculated at the PK step. Each
strain was treated as an individual subject. The different parameters
for each strain were obtained by post hoc procedure. Goodness-of-fit
was assessed for each strains with the root mean square error (Shei-
ner and Beal, 1981). S was fitted with u considered as a constant
parameter (fixed effect) (model S1), with interdose variability h with
mean zero and variance v2 (model S2). The effect of dose was tested
with the log-likelihood ratio test. In a second step, survival data were
modeled as S(t) 5 exp(u E(t) z t) (model S3), where E is as previously Fig. 2. Adequacy of fitting. The logarithm of the measured/predicted
defined, i.e., the effect calculated at the PD step and considered equal value ratio is displayed for PKs (top) and for PDs (bottom).1999 Amoxicillin in a Mouse S. pneumoniae Pneumonia Model 1089
TABLE 1
Pharmacokinetics
Models tested and parameters obtained with model 3. Parameter values are nominal with asymptotic coefficient of variation (CV%). Vss and CL have been derived a
posteriori. C, compartment.
Model No. of Objective Function
No. Parameters (22 Log Likelihood)
1 2 C lung in peripheral compartment 6 605.013
2 3 C lung in peripheral compartment 8 547.496
3 2 C lung in central compartment 6 543.561 P , .001 versus model 1
4 2 C Idem 1 interdose variability in k10 6 1 1a 540.786 P . .05 versus model 3
5 2 C lung in central compartment:Michaelis-Menten elimination 7 614.618
6 2 C lung in peripheral compartment:Michaelis-Menten elimination 7 615.316
Model 3 ka k10 k12 k21 Vc Vss CL K
h21 h21 h21 h21 l z kg21 l z kg21 l z h21 z kg21
Typical value 2.86 2.29 0.476 0.458 0.739 1.507 1.692 0.447
CV (%) 15 5.2 26 19 24 13
C, compartment; Idem, lung in central compartment.
a
Six structural parameters 1 one interdose variability parameter (h).
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TABLE 2
Pharmacodynamics
Individual parameters have been obtained by post hoc (Bayesian) procedure. Therefore, some shrinkage to the mean may exist, especially in strains with a small number
of observations. MIC and MTD (determined in previous studies) are reported in this table for the purpose of comparison between these conventional PD parameters and PD
parameters described in the present study (especially Cpss50). Objective function (22 log likelihood) for the full model, 1487.933; with no interstrain variability in ke0,
1508.931; in Emax, 1681.108; and in Cpss50, 1508.978. Differences between objective function for the full model and these reduced models are all significant at P , .0001.
Strain NObs ke0 Emax Cpss50 RMSE MIC MTD
h21 lnCFU z g21 mg z ml21 mg z ml21 mg z kg21
P30923 24 0.0026 13.77 0.00180 1.1778 0.1 5
P52181 52 0.0106 9.76 0.00488 2.1379 0.03 5
P31192 43 0.0015 8.24 0.00859 2.2389 0.5 25
P40225 35 0.0125 7.01 0.01139 1.1656 0.5 10
P30189 26 0.0373 9.55 0.02116 1.2212 0.5 10
P54B 76 0.0074 8.56 0.04851 1.2608 1 50
P12698 121 0.0013 16.76 0.86001 1.8943 2 100
P54988 97 0.0191 15.53 1.08820 1.6190 1 50
P15986 75 0.0100 3.83 3.02760 1.0599 2 300
P41375 61 0.0027 6.89 4.54140 0.7762 4
P53681 46 0.0031 5.58 8.67750 0.6882 8
P40422 57 0.0395 5.06 27.82700 1.0769 2 150
21
NObs, number of observations (lnCFU z g 2 time points for each strain); RMSE, root mean square error.
lated with MIC (Spearman r 5 0.98, P , .0001) and Cpss50 calculated from the PK-PD model (Table 3 and Fig. 5). The
(Spearman r 5 0.92, P , .0001). latter results fully confirmed the reliability of the model.
Survival analysis showed that the survival function was PK analysis showed that amoxicillin distributes in mice in
significantly different between doses (S1 versus S2, P , a central compartment probably including blood and the rich
.0001). Moreover, the best model used E(t), the effect modeled vascularized organs (lung was included in this central com-
by the PK-PD parameters as covariate to predict the propor- partment with a partition coefficient), and in a deeper pe-
tion of surviving mice (S3 versus S2, P , .0001) (Table 3 and ripheral compartment. However, effect (decrease in ln
Fig. 5). CFUzg21 in lung) was best modeled in a separate effect com-
partment linked to the central compartment. Under these
Discussion conditions, the peripheral compartment acts only as a reser-
voir buffering drug input and output.
The methodology of our study differs markedly from those
Disappearance of amoxicillin from the effect compartment
published previously in infectious disease. We used a PK-PD
appeared to be very slow and the rate constant (ke0) govern-
model to describe the overall dose-effect relationship of
amoxicillin against 12 strains of S. pneumoniae with penicil- ing this process was significantly different between strains,
lin MICs between ,0.01 and 16 mg/ml in a neutropenic mu- ranging from 0.00131 to 0.03945 h21 (Table 2). These find-
rine pneumonia model. We were able to accurately describe ings have two major implications: 1) after a single dose of
the complex relationship between antibiotic dosing, PKs, and amoxicillin, bacterial counts in lung rapidly decreased and
the temporal changes in bacterial count in lung. In a previous the bacterial growth remained suppressed during a long pe-
study (Moine et al., 1997a), we have shown that amoxicillin riod of time after cessation of exposure of microorganisms to
dose producing half-maximal decrease in bacterial count in amoxicillin (Fig. 4), and 2) the duration of bacterial growth
lung (ED50) closely correlated with the reciprocal of MICs suppression was related to the intrinsic properties of S. pneu-
measured in vitro. The present study permits to predict the moniae strains rather than to host environment becase ke0
decrease in bacterial count in lung from the dosing scheme. was significantly different between strains.
Moreover, we were able to show that the animal survival rate These two premise clearly demonstrate that bacterial
was significantly related to the number of bacteria in lung growth suppression is related to an in vivo postantibiotic1090 Moine and Mazoit Vol. 291
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Fig. 3. Effect of various doses of amoxicillin on bacterial count in lung (ln
CFUzg21). Symbols are experimental data (means 6 S.E.); lines are fitted
curves. At zero time, mice received either saline (control) or various doses
of amoxicillin. From top to bottom are represented strain P12698 (with
control, 25, 50, 100, 220, and 400 mg/kg amoxicillin); strain P15986
(control, 100, 200, and 400 mg/kg amoxicillin), and strain P40422 (con-
trol, 100, 200, and 400 mg/kg amoxicillin). Some shrinkage to the mean is
perceptible especially in strains with relatively few data, due to the
Bayesian procedure.
effect (PAE). The persisting suppression of bacterial growth
after a short exposure to antimicrobial agents is known as
the PAE (Zhanel and Craig, 1994). In vitro experiments with Fig. 4. Effect of amoxicillin on bacterial count in lung (ln CFUzg21).
Gram-positive cocci such as Staphylococcus aureus, S. pneu- Simulations have been performed with the parameters obtained at the
PD stage. The different curves represent the effect obtained after injec-
moniae, Enterococcus faecalis, and Streptococcus spp. consis- tion of the same daily dose (23 MTD) equally divided into 1, 2, 4, 8, and
tently demonstrated a PAE with different b-lactams (Eagle 12 injections. The curve corresponding to the first injection of one MTD
et al., 1950a; Sande et al., 1981). An in vivo PAE of penicillins dose (i.e., the twice-daily injection scheme) is extended after the 12th h to
show the rate of decay of effect with time (dashed line). From top to
on S. pneumoniae also has been demonstrated (Eagle et al., bottom are represented strain 12698 (ke0 5 0.0013 h21; MTD 5 100
1950a,b; Sande et al., 1981). However, studies with neutro- mgzkg21), strain 15986 (ke0 5 0.0100 h21; MTD 5 300 mgzkg21), and
penic animals and infection models in animals with impaired strain 40422 (ke0 5 0.0395 h21; MTD 5 150 mgzkg21).
host resistance provided sharply contrasting data and the in
vivo pertinence of PAE has been questionned (Vogelman et icantly different between strains, thus demonstrating that
al., 1988b). These authors among others suggested that the PAE is function of strain intrinsic properties (Table 2 and
observed PAE was due to residual antibiotic concentrations Fig. 4). Then, both postantibiotic leukocyte enhancement and
that were below the limit of detectability of the microbiolog- antimicrobial activity of residual amoxicillin concentrations
ical assay (Tauber et al., 1984; Vogelman et al., 1988b), or to cannot be incriminated in this neutropenic murine pneumo-
the postantibiotic leukocyte enhancement effect (McDonald nia model. These differences in strains remain to be charac-
et al., 1981; Vogelman et al., 1988b). Indeed, these two fac- terized and do not have something to do with inherent viru-
tors have been clearly demonstrated to be involved in the lence not captured in the PK-PD model because all these
duration and intensity of bacterial suppression in various strains belonged to serotypes 6, 19, and 23 (Moine et al.,
animal models (Tauber et al., 1984; Vogelman et al., 1988b). 1997a), which are naturally avirulent for mice independently
Nevertheless, our results clearly demonstrate the presence of their isolation sites in humans (Bédos et al., 1991; Briles et
of an in vivo PAE for amoxicillin with S. pneumoniae in this al., 1992).
pneumonia model. Duration of this PAE (T1/2ke0) was signif- Duration of PAE (ke0) was not correlated with in vitro1999 Amoxicillin in a Mouse S. pneumoniae Pneumonia Model 1091
TABLE 3
Survival data fitted with increasingly complex models
Parameters are given with their asymptotic coefficient of variation (CV%). v2 is the variance of (h), the interdose variability and LL is the log likelihood of the data with the
particular model.
Model Parameter CV% v2 22 LL
21
S1 5 exp(2ut) u 5 0.00491 h 20 2190.5
S2 5 exp(2ut) [u 5 f(Q)g(h)] u 5 0.00273 h21 40 1.00 2280.3*
S3 5 exp(2u z lnCFU(t) z t) [u 5 f(Q)g(h)] u 5 0.00112 h21 z ln CFU21 22 0.434 2308.4**
* P , .0001 versus S1; ** P , .0001 versus S2.
bacterial data in lung (Moine et al., 1997a). Indeed, with a
simpler dose-effect model, we showed that the dose producing
half-maximal effect (ED50) highly correlated with MIC and
MBC, indicating that the doses leading to in vitro and in vivo
bacterial growth inhibition (or killing) were correlated.
In fact, in our model, the two major determinants of amoxi-
cillin in vivo bactericidal activity and therapeutic efficacy
appeared to be the dose of amoxicillin because amoxicillin
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exhibits a rapid dose-dependent killing regardless of the S.
pneumoniae strain tested, and the PAE duration, which was
an intrinsic bacterial property. Indeed, for all strains, even
strains with short PAE (ke0 . 0.01 h21 in our model), the
amoxicillin-loading dose is the primary factor governing bac-
tericidal activity. This finding may have implications for the
clinical use of amoxicillin. Thus, increasing the amoxicillin-
loading dose appears to be immediately clinically relevant.
Furthermore, the “clinical” significance of the PAE lies in its
application to dosing regimens (Vogelman et al., 1988b;
Zhanel and Craig, 1994). For the strains with moderate-to-
long duration of PAE (i.e., with ke0 , 0.01 h21 in our model),
a persisting suppression of bacterial growth after the initial
bactericidal effect appears to be effective with only one or two
daily injections, and the extent of amoxicillin efficacy on
bacterial count is related to the dose (P12698 and P15986
strains (Fig. 4). We found no gain in bactericidal activity with
more frequent administration of the same daily amount of
amoxicillin against S. Pneumoniae P15986 strain. In fact,
dividing the same daily dose into multiple administrations
led to a slower decrease in ln CFU z g21 in animals infected
with P15986 or P12698 strain (Fig. 4) and to a lesser survival
rate in animals infected with P15986 strain (Fig. 5). In ani-
mals infected with P15986 strain, at 600 mg/kg daily dose,
Fig. 5. Survival rate of mice infected with strain P15986 and having amoxicillin 300 mg/kg administred at 12-h intervals resulted
received different dosing protocols. Raw data are represented by symbols in a better survival rate that amoxicillin 150 mg/kg admin-
and fitted curves by lines. Fitting has been performed only for the first
week. The top graph shows that the same daily dose (600 mg/kg/24 h, i.e., istred at 6-h intervals (Fig. 5). This better survival rate was
23 MTD) led to better survival rate when injected twice a day than when mainly due to the fact that the initial loading dose (300
divided in four injections. Increasing the dose from 300 mg/kg/12 h to 400 mg/kg) led more rapidly to an important bactericidal effect in
mg/kg/12 h did not improve the survival rate. The bottom graph displays
all various dosing patterns used showing that a simple exponential model the lungs (Fig. 4). However, the strains with the shorter
incorporating the result of PD modeling (ln CFUzg21) correctly predicts duration of PAE (ke0 . 0.01 h21 in our model) need a more
survival rate. f, 400 mg/kg/12 h; L, 300 mg/kg/12 h; Œ, 150 mg/kg/6 h; F, frequent dosing to achieve a constant decrease in ln CFUzg21
200 mg/kg/12 h; M, 100 mg/kg/8 h; ‚, 150 mg/kg/12 h.
(P40422 strain) (Fig. 4). Nevertheless, because amoxicillin
also exhibits a rapid dose-dependent killing with these
amoxicillin susceptibility tests, i.e., MIC and MBC. In fact, strains, increasing the amoxicillin-loading dose appears to be
ke0 is related to duration of effect, not to the magnitude of relevant.
this effect. The intrinsic activity of amoxicillin against bac- In conclusion, with a PK-PD model to describe the overall
terial growth in lung (Emax) was significantly different be- dose-effect relationship of amoxicillin against 12 strains of S.
tween strains. We did not show any relationship between pneumoniae with penicillin MICs between ,0.01 and 16
Emax and MIC or MBC for amoxicillin against S. Pneumoniae mg/ml in a neutropenic murine pneumonia model, we have
strains. However, there was a highly significant correlation shown that the major determinant of amoxicillin in vivo
between Cpss50, the serum drug concentration producing bactericidal activity and therapeutic efficacy appeared to be
half-maximal effect at steady state and MIC and MBC. The the dose of amoxicillin because amoxicillin exhibits a rapid
latter result confirms our previous findings with the same dose-dependent killing whatever the S. pneumoniae strain.1092 Moine and Mazoit Vol. 291
We also clearly demonstrated an in vivo PAE for amoxicillin Hyatt JM, McKinnon PS, Zimmer GS and Schentag JJ (1995) The importance of
pharmacokinetic/pharmacodynamic surrogate markers to outcome. Focus on an-
with S. pneumoniae, which was a nonpredictable intrinsic tibacterial agents. Clin Pharmacokinet 28:143–160.
bacterial property. Our findings may have implications for McDonald PJ, Wetherall BL and Pruul H (1981) Postantibiotic leukocyte enhance-
the clinical use of amoxicillin and could provide a theoritical ment: Increased susceptibility of bacteria pretreated with antibiotics to actvity of
leukocytes. Rev Infect Dis 3:38 – 44.
rationale in its application to dosing regimens. Determina- Moine P, Mazoit JX, Bédos JP, Vallée E and Azoulay-Dupuis E (1997a) Correlation
tion of the PAE does offer important information on the between in vitro and in vivo activity of amoxicillin against Streptococcus pneu-
moniae in a murine pneumonia model. J Pharmacol Exp Ther 280:310 –315.
interaction between antimicrobial agent and microorganism Moine P, Sauve C, Vallée E, Bédos JP, Pocidalo JJ and Azoulay-Dupuis E (1997b) In
that simple susceptibility testing and PK studies do not pro- vivo efficacy of cefotaxime, a broad-spectrum cephalosporin, against penicillin-
vide. This study gives a new approach on the PD properties of susceptible, penicillin-resistant and penicillin-cephalosporin-resistant strains of
Streptococcus pneumoniae in a mouse pneumonia model. Clin Microbiol Infect
antimicrobial agents and further studies, including search 3:608 – 615.
for rapid, predictive, and clinically accessible PAE quantifi- Moine P, Vallée E, Azoulay-Dupuis E, Bourget P, Bédos JP, Bauchet J and Pocidalo
cation tests, are needed before direct application of these JJ (1994) In vivo efficacy of a broad-spectrum cephalosporin, ceftriaxone, against
penicillin-susceptible and -resistant strains of Streptococcus pneumoniae in a
principles to patients. However, in view of our results, the mouse pneumonia model. Antimicrob Agents Chemother 38:1953–1958.
guidance to increase the amoxicillin-loading dose appears to National Committee for Clinical Laboratory Standards (1995) Performance Stan-
dards for Antimicrobial Susceptibility Testing: Document M100-56, National Com-
be immediately clinically relevant in pneumococcal pneumo- mittee for Clinical Laboratory Standards, Villanova, PA.
nia. Sande MA, Korzeniowski OM, Allegro GM, Brennan RO, Zak O and Scheld WM
(1981) Intermittent or continuous therapy of experimental meningitis due to
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