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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1990, p. 326-331 Vol. 34, No.

2
0066-4804/90/020326-06$02.00/0
Copyright © 1990, American Society for Microbiology

Application of Mathematical Model to Experimental Chemotherapy


of Fatal Murine Pneumonia
TAMOTSU HISHIKAWA,1* TADASHI KUSUNOKI,2 KANJI TSUCHIYA,3 YOSHIO UZUKA,4
TASUKU SAKAMOTO,5 TSUYOSHI NAGATAKE,5 AND KEIZO MATSUMOTO5
New Product Planning and Development Division,' Management and Planning Division,2 and Central Research
Division,3 Takeda Chemical Industries, Ltd., Osaka, Third Department of Internal Medicine, Teikyo University,
Ichihara,4 and Department of Internal Medicine, Institute of Tropical Medicine, Nagasaki University, Nagasaki,5 Japan
Received 7 April 1989/Accepted 6 November 1989

Two beta-lactam antibiotics, cefazolin and cefmenoxime, were administered in an experimental model of
murine pneumonia caused by Kkebsiella pneumonuae in a way which enabled us to approximate the serum
antibiotic concentration time course in humans. Bacterial counts during the experiments were subjected to
nonlinear least-squares analyses by using a mathematical model that explained the bacterial killing by the
antibiotic concentration time course and other factors associated with antimicrobial potency and bacterial
growth. Cefazolin gave a killing curve that changed synchronously with the drug levels in serum; in contrast,
cefmenoxime gave a curve that was prolonged as compared with the change in the drug levels in serum.
Multiple correlation coefficients were about 0.9, and the model worked well for bacteriail count data.

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Parameters relating to antimicrobial potency of the drugs, bacterial growth rate, and drug distribution into the
tissue were estimated numerically.

Bacterial reduction is an essential measure in evaluating with the antibiotic concentration time course in serum. The
the effect of antimicrobial therapy in patients and in deter- purpose of this study is to evaluate the goodness of fit of the
mining the optimal dosage regimen for antibiotics. However, model and to quantify the contribution of the parameters
bacterial c6unts are not always ineasurable in clinical stud- referring to the above factors.
ies, which have other experimental and ethical limitations.
To avoid i'uch difulties we established a fatal murine MATERIALS AND METHODS
pneumonia model. It was used in the present study on Animal. A total of 100 3-week old male DDY mice were
experimental therapy with two beta-lactam antibiotics, ce- infected with aerosolized Klebsiella pneumoniae B-54 by
fazolin and cefmenoxime. using the exposure chamber of Matsumoto et al. (9), based
Since antibiotic therapy cannot be successful without a on the method of Nishi and Tsuchiya (13).
sufficient concentration of a drug in the infectious focus, Bacteria. K. pneumoniae B-54 was isolated from a patient
many pharmacokinetic studies have been carried out; e.g., with very severe pneumonia in 1978. The 50% lethal dose of
studies on antibiotic penetration into tissues by Grasso et al. this strain was 9.7 CFU per mouse in this experimental
(5), Ryan et al. (14), and Shibl et al. (15) and protein binding pneumonia model.
by Bergan et al. (1), Dudley et al. (3), and Wise et al. (19). Antibiotics. Cefazolin (Fujisawa Pharmaceutical Co., Ja-
Furthermore, Craig and Ebert (2) studied the correlation of pan) and cefmenoxime (Takeda Chemical Industries Ltd.,
in vivo bacterial reduction with the area under the time- Japan) were used. MICs of cefazolin and cefmenoxime
concentration curve (AUJC), peak level, and other pharma- against K. pneumoniae B-54 were measured by the agar well
cokinetic parameters to identify the major factors dominat- method. The MIC of cefazolin was 1.56 jig/ml and the MIC
ing efficacy. Grasso et al. (5) obtained in vitro bacterial of cefmenoxime was 0.013 p.g/ml.
killing curves by using an experimental apparatus that repro- Doses and dosage regimens,. The doses and the schedules of
duced the antibiotic concentration time course in human the four treatments, two dosage regimens each for both
serum; the study showed that the regrowth phase took place drugs (Table 1) were determined to approximate the concen-
when the drug concentration dropped below the MIC. tration time course in hutnan serum produced by usual
These studies are useful for better understanding of anti- clinical treatment: drip infusion over 1 h with doses of 20 to
microbial efficacy and to construct better clinical treatments. 40 mg/kg of body weight for both drugs. Since drip infusion
In the present paper we propose a mathematical model to was impossible in mice and the time course was much faster
obtain more quantitative informnation about the relationship in mice than in humans, frequent subcutaneous injections
between bacterial killing and antibiotic pharmacokinetics. were necessary.
Various pharmacokinetic and pharmacodynamic parameters Twenty-four hours after infection, the mice were divided
are included in the model to explain the bacterial count time into four groups, and each group of mice received one of four
course under the experimental conditions.
Of the few studies applying mathematical models, Garrett treatments (A, B, C, and D; Table 1).
and Won (4), Hamano et al. (6), Mattie and van der Voet Antibiotic assay. Cefazolin and cefmenoxime in plasma
(10), and Tsuji et al. (16) dealt with in vitro killing curves, but samples were assayed by the agar well method with Esche-
only the model of Kono and Asai (8), which is the origin of richia coli NIHJ as the test organism (7). The sensitivity was
the model proposed here, dealt with in vivo killing curves about 0.1 ,ug/ml, and the variation coefficient of replicate
analyses was ±7.0%.
Measurement of bacterial count. Immediately before and 2,
*
Corresponding author. 4, 8, 12, 16, 20, and 24 h after the initial dosing, three mice
326
VOL. 34, 1990 MATHEMATICAL MODEL FOR EXPERIMENTAL CHEMOTHERAPY 327

TABLE 1. Doses and dosing regimens for micea n attached to D has a specific value corresponding to the
Doses (mg/kg) administered at time tissue. Ai and Bi (i = 1, ..., p) are the pharmacokinetic
Total after initial dosing parameters representing serum concentration time courses
Treatment Drug dose and p is the number of exponential terms.
(mg/kg) 0 20 40 1 1.5 2 2.5 3 4 In equation 2, Cn") at time t is equal to serum level if n is
min min min h h h h h h
equal to 1 and less than the serum level if n is less than 1.
A Cefazolin 126 16 20 24 16 16 10 10 8 6 Since Cf") changes synchronously with the level in serum,
B Cefazolin 252 32 40 48 32 32 20 20 16 12 the change in bacterial count should also be synchronous
with the change in serum drug level and C(n). Some gap in
C Cefmenoxime 84 20 20 24 10 5 3 2 time can exist, however, between the serum and tissue
D Cefmenoxime 168 40 40 48 20 10 6 4 antibiotic concentration time courses, and between the bac-
a Both drugs were subcutaneously injected in infected mice. terial reduction and the antibiotic concentration time course
in serum.
To allow the possible gaps, we modified the model by
of each treatment group were sacrificed, and the lungs of introducing an additional parameter, E. Replacing the ex-
each mouse were aseptically removed. Each lung was ho- ponential term in the equation 2, Atexp(-B,t), with
mogenized, and the number of viable bacteria was measured Aiexp(-B1t/E)/E, and assuming that the AUC in the tissue
by the quantitative culture method. The detection limit was does not change from equation 2, cjn) can be expressed as
about 10 CFU per lung.
Mathematical model. The basic idea is to express the n') = DIP'Atexp(-B1tIE)IE. (3)
competition between bacterial growth and antimicrobial kill When E 1, equation 3 is the same as equation 2.
=
as rate of change in bacterial count (growth/kill) = growth Since E values of >1.0 prolong the time course of an) and
rate - kill rate. bacterial count, more or less lag time can exist before the

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Considering the logarithimic growth phase of the bacteria, onset of killing, and bacterial growth can be suppressed
and assuming that the growth rate is proportional to the continuously even after the drug concentration in serum
bacterial count at the moment and that the kill rate is drops below the effective level. E has thus a meaning of
proportional to the product of the bacterial count and the "delay factor" or "continuity parameter." The generalized
drug concentration, Kono and Asai (8) have indicated this equation 3 is applicable to cases such as that noted by Ryan
relationship in the form of a simple differential equation et al. (14), in which there was a marked delay of beta-lactam
dCJdt = KbCb- KCn )Cb (1) concentration time course in the tissue determined by the
surface/volume ratio of the infectious tissue.
where t denotes the time after administration, Cb and C(n) are If Cn) is directly measurable in tissues, pharmacokinetic
the bacterial count and the drug concentration in the infec- parameters for the tissue antibiotic concentration time
tious tissue at time t, and Kb and K are the growth rate and course should be used in equation 3; however, it is not the
kill rate, respectively. Garrett (4) used equation 1 a few years case in this study.
later for in vitro experiments. Kono and Asai have assumed Integration of equation 1 over time t yields
that on) is proportional to the level in serum, which is a
function of time t expressed by the usual multicompartment p
model, and described C(n) as ln (CbICbo) = Kbt - KDn >.{A.IBj
C(nl - DnlpA exp(-Bit), (2)
where D is the dose of the drug administered and the power [1 - exp(-Bit/E)]} (4)

C C
100- Cef azolin 100 Cefmenoxi me

80 C = 38.0(1-e ) + 168.5 (1_e-0 40t) 80


C= 34.2e +55.5e ,t' = t-10

co
_E 60 AUC = 207 60
._
C=39.4( 1-e )+28.9(1-e )
0 A'.
Man 2.58 t' -0. 69 t'
.a C = 36.3e +14.4 e 0 t'= t-1 .0
40 40 A
0
U C =68.3

se AIJC= 6 9.3
Q
20.
co

0 1 2 3 0 1 3 4 5 6
Time after administration (h) Time after administration(h)
FIG. 1. Antibiotic concentration time courses in serum with the dose of 20 mg/kg. Multiple subcutaneous injections of treatments A and
C (Table 1) were used for mice. Single-drip infusions over 1 h were used for humans. The doses of the drugs were both 20 mg/kg of body
weight.
328 HISHIKAWA ET AL. ANTIMICROB. AGENTS CHEMOTHER.

where CbO is the initial condition, bacterial count at time tional assumption (common CbO, and separate Kb, K, n, and
zero. E were estimated for each drug), and model 3 is the general
Estimation of pharmacokinetic parameters. Pharmacoki- model with common Cbo and Kb (common Cbo and Kb, and
netic parameters were calculated by applying a two-com- separate K, n, and E were estimated for each drug).
partment open model. Simulation curves for mice under Akaike's information criterion was computed for each
frequent dosings were provided, assuming that the concen- model and compared to select the optimal model with the
tration time course produced by the succeeding injections smallest value.
could be superimposed linearly, and were compared with
those for humans under single-drip infusion. RESULTS
Estimation of Cbo, Kb, K, n and E. Given the values of Pharmacokinetic parameters and simulation of antibiotic
pharmacokinetic parameters and D, the data of bacterial concentration time courses. The pharmacokinetic parameters
count in the mice lung were subjected to nonlinear least- and simulated serum antibiotic concentrations for both drugs
squares analyses to estimate the other unknown param- in human and mouse serum are shown in Fig. 1. Since the
eters, and the overall goodness-of-fit of the model was time course of serum drug concentration in mice is similar to
examined by multiple correlation coefficient (R). The pro- that in humans, the design of Table 1 is regarded as an
gram NONLIN (11) was used, taking Wi as equal to 1 and approximation of the time course in humans. By using the
1/Cb as the weighting factor, to estimate the unknown differences between the mice and human AUCs, the total
parameters. dose in each treatment for mice was converted to the
Since both antibiotics have two treatments with different corresponding dose for humans: to 17.0 and 34.0 mg of
doses, separate estimation of K and n is possible; however, cefazolin per kg in treatments A and B and to 20.3 and 40.6
it is impossible if only one dose is used, and the estimation of mg of cefmenoxime per kg in treatments C and D.
a compound parameter, KD', becomes inevitable. In the following analyses, these doses and pharmacoki-
The above procedures were applied to three models based netic parameters in human subjects were used to approxi-

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on equations 3 and 4: model 1 is the Kono model where E = mate the antibiotic concentration time course in the infected
1.0 (common Cbo, and separate Kb, K, and n were estimated mice, and the experimental therapy in mice is regarded as a
for each drug), model 2 is the general model without addi- simulation of clinical therapy of pneumonia in humans.

11 0

Cefazoli n Cefmenoxime
o 0
0
0 Treatment C 0
Q
-I' 0

0
0 '1."\ 0 D

A and B 8
0
0
a.
5
4)

C.) --- Model I ----Model I


CD
Model 2 - Model 2
0 *Treatment A 5
* Treatment C
oTteatment B O Treatment D

8 12 16 20 24 0 4 8 12 16 24

100 1 00 -
Cefazolin (Treatment A) Cefmenoxime (Treatment C )
0
80 80 -
._
a)
06
ce
4)
60 60
0 Model 1 Model 1
0 Model 2 Model 2
c;
40 40 - . .
.I|
co
c; 20 20 -
Eq- 0
0 4 8 12 16 20 24 0 4 8 12 16 20 24
Time after administration (h) Time after administration(h)
FIG. 2. Time courses of bacterial counts and tissue drug concentrations.
VOL. 34, 1990 MATHEMATICAL MODEL FOR EXPERIMENTAL CHEMOTHERAPY 329

TABLE 2. Estimates of parametersa


Multiple Residual
Model Drug ln(Cbo) Kb K E n correlation sum of AIC
coefficient squares
1 Cefazolin 15.95 ± 0.41 0.43 ± 0.04 0.35 ± 0.08 1.0 0.000 ± 0.000 0.886 191.8 250.6
Cefmenoxime 0.37 ± 0.03 0.27 ± 0.23 1.0 0.382 ± 0.219
2 Cefazolin 15.81 ± 0.43 0.42 ± 0.05 0.29 ± 0.08 0.73 ± 0.49 0.003 ± 0.004 0.909 154.8 244.9
Cefmenoxime 0.50 ± 0.06 0.79 ± 0.41 3.52 ± 0.71 0.240 ± 0.125
3 Cefazolin 15.97 ± 0.39 0.47 ± 0.04 0.40 + 0.08 1.08 ± 0.46 0.007 ± 0.007 0.906 159.7 244.3
Cefmenoxime 0.66 ± 0.33 3.09 ± 0.64 0.271 ± 0.130
a Values ofln(Cbo), Kb, K, E, and n are given as estimates ± standard errors. Model 1 is the Kono model, in which E is 1.0. Model 2 is the general model in
equation 4 without additional assumptions. In model 3, Kb is assumed to be common to cefazolin and cefmenoxime. AIC, Akaike's information criterion.

Bacterial count time courses. The log1o CFU of the three expected little effect of increasing the dose of cefazolin on
mice at time t and the simulated time courses with models 1 the in vivo antimicrobial activity in the tissue.
and 2 and the tissue antibiotic concentration time courses in The two killing curves with higher and lower doses of
treatments A and C are shown in Fig. 2. cefazolin overlapped because of the small n (Fig. 3). The
The initial mean log1o CFU was 6.94 for all treatments. prolonged simulation curves for cefmenoxime shifted to the
The lowest mean log1o CFU and the times they were right as compared with the curves for cefazolin. The mini-
observed were 6.26 (8 h), 6.12 (4 h), 6.35 (8 h), and 5.84 (8 h) mum bacterial counts on the curve are arranged in the order

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in treatments A, B, C, and D, respectively. The higher dose of D < C - B - A. The higher dose of cefmenoxime was the
of cefmenoxime (40.6 mg/kg) gave the lowest mean CFU most effective.
among the four treatments. When the minimum bacterial count is reached at time t,
Comparison between model 1 (Kono model) and model 2 just before regrowth takes place, bacterial growth and anti-
(general model). The estimates of the parameters and other microbial killing stop, and a relation dCbldt = 0 holds. If
results are summarized in Table 2. Since the choice of Co(') denotes the tissue antibiotic concentration at this
weighting factors (Wi = 1 or JICb) did not affect the results, moment, Co(n) is derived from equation 1 as Co(n) = Kb/K;
those from Wi = 1 were adopted here. Models 1 and 2 using the estimates of Kb and K in Table 2, Co0") is 1.18 ,ug/ml
showed high multiple correlation coefficients of 0.886 and for cefazolin and 0.71 ,ug/ml for cefmenoxime.
0.909. The residual sum of squares decreased from 191.8 in The length of time until dCbldt = 0 is estimated at 2.0 h for
model 1 to 154.8 in model 2, and the Akaike information cefazolin and 5.0 h for cefmenoxime. Killing activity of
criterion decreased from 250.6 in model 1 to 244.9 in model cefmenoxime continues longer than that of cefazolin.
2.
No appreciable gap was noted between the cefazolin DISCUSSION
concentration time course with models 1 and 2 and between The shape of the killing curves can be divided into depth
the corresponding time courses of bacterial counts (Fig. 2). and length of killing phase. In the model proposed here the
In contrast, the simulation for cefmenoxime, based on model
2 with E = 3.52, showed a better fit to the observed bacterial
counts versus time. The cefmenoxime concentration time 1 0-
course with E = 3.52 showed a marked delay compared with
that based on model 1 with E = 1.0. These results suggest //
that the E of cefazolin does not differ from 1.0 and that the E /l
le

of cefmenoxime is larger than 1.0. 9-


Optimal model. Models 2 and 3 had almost the same
optimality in terms of Akaike information criterion (244.9
and 244.3) and residual sum of squares (154.8 and 159.7) 8-
(Table 2). However, model 3 is considered to be optimal,
since it meets the requirement of parsimony with the mini- 7-77 I

mum number of parameters to be estimated. The assumption


of model 3 that Kb is common regardless of the drugs is also 7,I
preferred. Figure 3 shows the simulation based on model 3 -a,-

and the results of Table 2. The continuity parameter E was 6-


estimated in model 3 to be 1.08 and 3.09 for cefazolin and
cefmenoxime, respectively. Treatments A and B (Cefazolin )
Comparison between two drugs in parameter estimates
from model 3. The kill rate constant K was estimated at 0.66 5 --- TTreatmenit C' (Cefmenoxime)
for cefmenoxime. It was about 1.5 times larger than the K of -.Treat meidt D (Ce fmenoxime)
0.40 for cefazolin. These facts show that cefmenoxime has a
stronger in vivo potency than that of cefazolin against K. 1
pneumoniae B-54. 0 4 8 12 16 20 24
The estimate of n was less than 1.0 for both drugs. It was
especially small for cefazolin (n = 0.007), which was almost Time af ter administration (h)
1/40 of that for cefmenoxime (n = 0.271). Consequently, we FIG. 3. Simulated time courses of bacterial counts with model 3.
330 HISHIKAWA ET AL. ANTIMICROB. AGENTS CHEMOTHER.

length is determined by the antibiotic time course in serum


and the parameter E. In contrast to the fact that cefmenox-
-, 160- (A)
ime showed a prolonged killing curve associated with E =
3.09, no appreciable delay was noted in cefazolin with E = 3 120-
- E= 3.0
1.08. The Kono model, which is the origin of our model, was 0
-_-- E= 1.0
applicable to cefazolin. It.4
co 80- II
We have already reported similar preliminary results of E I'
= 1 for cefazolin and E = 3 for cefmenoxime with a lag
before onset and a delayed end of the killing phase (12). But ru 40 -
C)
0
separate estimation of the parameters K and n was impossi-
ble because only one dose level each for both drugs was used v
in that experiment. 0 4 8 12 16 20 24
Despite this, reproducibility problems will remain if de- Time after administration (h)
layed killing curves are attributed solely to the simultaneous -

2.0
delay of antibiotic concentration time course in tissues. The CB)
postantibiotic effect proposed by Vogelman and Craig (18) , 1.5-
and phenotypic tolerance in stationary phase proposed by
Tuomanen et al. (17) should also be considered. -. 1.0- A*
ol

The roles of E and other parameters in the model are I


I O-oo
illustrated in Fig. 4, which shows a simulation of treatment D AUC
I
0.5- II
of cefmenoxime based on model 3 and numerical estimates Q I

of the parameters other than E. The solid and dotted lines ¢ 0 I0 4 I 2 24


denote the simulations using E = 3 and E = 1. 0 4 8 12 16 20 24

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An E of 3 (>1) caused a delay of antibiotic concentration 0
time course in the tissue and a decrease of peak concentra- _-l

tion (Fig. 4A), and this delay produced a little lag time before a
the onset of kill and a prolonged killing phase (Fig. 4D).
Garrett and Won used lag time as another parameter to
explain this kind of phenomenon; however, the model in - 6
0C
equation 4 is correct if the true factor is the antibiotic time 0C;
course in the tissue. Additional experiments and careful Q 5-
explanations are necessary to identify the true factor and to Q._

select the correct model.


The AUC shown in Fig. 4B, dose level (D), kill rate (K), ~> A
and distribution into the tissue (n) cooperate each other in
the fashion of the second term of equation 4, KD'AUC, to
determine the depth (Fig. 4C). Larger values of K, D', and
AUC produce greater antimicrobial effect and greater depth.
Protein binding, studied by Dudley et al. (3), would be a real a
mechanism affecting n. Emphasis is necessary on the fact
that the strength of killing is dominated solely by the c;

magnitude of K when n approaches 0 (n << 1), as noted for C.


0
cefazolin.
:s
K is the direct expression of in vivo antimicrobial potency,
having specific values for the respective antibiotics. It might
be compared to the MIC, the representative parameter of in .t
vitro antimicrobial potency. A more appropriate parameter 0 4 8 12 16 20 24
for the in vivo situation, Co(n), was constructed from our FIG. 4. Relation between parameter E and antibiotic concentra-
model. Comparing the empirical COW with the MIC, COt" = tion time courses, AUC, and bacterial counts.
1.18 for cefazolin is comparable to the MIC of 1.56 ,ug/ml,
but Co(n) = 0.71 for cefmenoxime was larger than the MIC of
0.013 ,ug/ml. crobial kill (-KD'AUC) to form the overall bacterial counts
In Fig. 4C the difference between the solid and the dotted time course as Fig. 4D which was represented in equation 4.
lines (E = 1 and E = 3) is noted only during the killing phase, Since this model worked well to explain the observed
when the AUCs are expanding and two lines are getting bacterial counts by pharmacokinetic data with an apprecia-
together when the AUCs are reaching to the common limit, ble goodness of fit, it is a possible approach to quantitation of
AUC°. the in vivo relationship between antimicrobial effects, real-
The killing phase is the first 8 h, which include only three ized as bacterial killing curves, and factors inherent in
points of bacterial count measurements, 2, 4, and 8 h; the antibiotic-microorganism-animal tissue combinations. The
other four points are located on the regrowth phase (Fig. 4C results obtained here will be useful in considering optimal
and D). This makes the estimate of Kb reliable; however, it clinical treatment with cefazolin and cefmenoxime.
is unfavorable for accuracy of parameter estimates relating
to killing effects.
Despite the above problems in definition, interpretation, ACKNOWLEDGMENTS
and accuracy of various parameters, we combined the We are grateful to G. L. Drusano and two reviewers for their
competition between the bacterial growth (Kbt) and antimi- constructive comments and suggestions. We also thank Y. Kibune,
VOL. 34, 1990 MATHEMATICAL MODEL FOR EXPERIMENTAL CHEMOTHERAPY 331

T. Morikawa, and M. Yoshida for their kind advice and encourage- pneumonia due to gram negative bacilli by air-borne infection.
ment. Nippon Kyoubushikkan Gakkai Zasshi 16:581-588. (In Japa-
nese.)
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