Pharmacokinetics and Pharmacodynamics in Antibiotic Dose Optimization

Expert Opinion on Drug Metabolism & Toxicology
ISSN: 1742-5255 (Print) 1744-7607 (Online) Journal homepage: http://www.tandfonline.com/loi/iemt20
Pharmacokinetics and Pharmacodynamics in

Antibiotic Dose Optimization
Sherwin K. B. Sy, Luning Zhuang & Hartmut Derendorf
To cite this article: Sherwin K. B. Sy, Luning Zhuang & Hartmut Derendorf (2015):
Pharmacokinetics and Pharmacodynamics in Antibiotic Dose Optimization, Expert Opinion on
Drug Metabolism & Toxicology, DOI: 10.1517/17425255.2016.1123250
To link to this article: http://dx.doi.org/10.1517/17425255.2016.1123250
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Dec 2015.
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Download by: [University of Nebraska, Lincoln] Date: 13 December 2015, At: 21:43
Publisher: Taylor & Francis
Journal: Expert Opinion on Drug Metabolism & Toxicology
DOI: 10.1517/17425255.2016.1123250
Pharmacokinetics and Pharmacodynamics in Antibiotic Dose
Optimization
Sherwin K. B. Sy1,2, Luning Zhuang1,3 and Hartmut Derendorf1‡

Downloaded by [University of Nebraska, Lincoln] at 21:43 13 December 2015
1
Department of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville,
Florida, USA
2
Post-Graduate Program in Biostatistics, Universidade Estadual de Maringá, Maringá,
PR, Brazil
3
Division of Pharmacometrics, Office of Clinical Pharmacology, Center for Drug
Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA
‡
Author of correspondence:
Hartmut Derendorf, PhD

Chair, Distinguished Professor of Pharmaceutics, V. Ravi Chandran Professor in
Pharmaceutical Sciences
Department of Pharmaceutics
1345 Center Drive, Room P3-20
PO Box 100494
Gainesville, FL 32610, USA
Tel: +1.352.273.7856
Fax: +1.352.273.7854
E-mail: hartmut@ufl.edu
1
Abstract
Introduction: Identifying the optimized dosing regimen and algorithm is critical in the
development of antibiotics. Suboptimal regimens and inappropriate choice of drug give
rise to drug-resistant bacteria which have limited the therapeutic utility of many
commercially available antimicrobial agents. Strategies to optimize therapy of
antimicrobial candidates to speed up the development process are urgently needed.

Areas covered: We examined pharmacokinetics and pharmacodynamics of
antimicrobial agents with modeling and simulation approaches. The approach that is
based on minimum inhibitory concentration to evaluate antimicrobial dosing strategy is
widely utilized in drug development. The modeling approach utilizing information from
time-kill kinetic studies is a tool that can provide more information on the time-course of
bacterial response to a particular dosing regimen. Animal studies of dosing regimens
that mimic human pharmacokinetics are another option to evaluate antimicrobial
efficacy. Empirical, semi-mechanistic and mechanistic models of bacterial dynamics and
development of drug resistance in response to drug therapy are discussed.
Expert opinion: Both theories and applications of these approaches provide an overall
understanding of how the tools can streamline drug development process and help
make crucial decisions. Many opportunities and potentials are presented to incorporate
more rigorous integration of PK-PD modeling approaches even at preclinical stage to
extrapolate to clinical settings, thus enabling successful trials and optimizing dosing
strategies in relevant populations where the drug is mostly used.
Keywords: PK-PD modeling, antibiotics, in vitro models, translational research
2
Article Highlights
 We identified and summarized PK-PD approaches that are routinely practiced in
drug development of antimicrobial agents
 The MIC-based approach is the most extensively used approach to identify PD
index of antibiotic that is predictive of microbiological efficacy and outcome
 The state-of-the-art modeling and simulation strategies are now available and
can be more rigorously applied in the evaluation of drug candidates
 The population PK-PD modeling and simulation approach can elucidate more
information related to the time-course of antibacterial activities as well as
development of drug resistance in bacteria
 PK-PD has been successfully applied in optimizing antimicrobial dosing regimens
in special population such as patients with renal disease and critically ill patients
with obesity
3
1. Introduction
The utility of pharmacokinetic-pharmacodynamic (PK-PD) approach in
antimicrobial drug development is very well established and now acknowledged by
regulatory authorities. Thus, we will discuss how PK-PD information can be used for
rational drug design, dose finding and optimization of dosing regimen from the context
of antibiotics. There are several articles that discussed optimizing treatment with
existing commercially available antimicrobial agents to combat global rise in drug-
resistant bacteria.1-3 In fact, the same PK-PD concepts can also be used for candidate
selection and dose finding. A survey from 2002 showed that for drugs in therapeutic
areas other antimicrobial drugs, the “defined daily dose” were reduced whereas the
dosage regimens for antimicrobial drugs were increased after the drugs were approved
during the period between 1988 and 2000.4 For antibiotics, the dosage increase was
due to change in susceptibility towards higher drug resistance in the target
microorganism.4 Therefore, it is important that drug developers determine the optimal
dosage regimen in the first place before the drug receives regulatory approval and
labeling. The dosage regimen refers to a dose with a dosage interval and duration of
treatment. The PK-PD approaches are useful in determination of the first two; the
duration of treatment is more of a clinical issue. In this chapter, tools to help drug
developers get the correct dose and dosing interval will be discussed in detail. These
concepts are broadly applicable to drug development in other therapeutic areas.
4
2. Concepts in Pharmacokinetics and Pharmacodynamics
Once an antimicrobial agent is administered to a patient, there are processes
that govern the drug presence as well as its removal in the systemic circulation and in
the tissues. These processes are collectively known as ADME (Absorption, Distribution,
Metabolism and Excretion). Pharmacokinetics, also referred to as “what the body does
to the drug”, studies these kinetic behavior or processes and the concentration-time
profile of the drug, by determining PK parameters such as total body clearance, volume
of distribution, bioavailability and protein binding. These parameters allow one to
characterize and predict the time-course of drug kinetics under either physiological and
pathological conditions.5
When the drug reaches its site of action with the appropriate concentration and
stays at this site for a sufficient duration of time, its interaction with the target ultimately
results in some pharmacological action and the study of which is commonly referred to
as pharmacodynamics, also known as “what the drug does to the body”. In the case of
antimicrobial therapy, the site of action for the drug is the bacterial pathogen. The PK-
PD approach is a concept that links the PK and PD of the drug, describing the time-
course of the pharmacological effect, which in turn is dependent on the time-course of
the PK behavior and the dosing regimen.
In antimicrobial therapy, the time-course of the pharmacological effect, which is
characterized by the change in bacterial density over time in the host, is very difficult to
obtain in the clinical setting. Instead, a “snapshot” view using minimum inhibitory
concentration (MIC) can provide valuable information on the susceptibility of the
5
pathogen toward a specific or combination of antimicrobial agent(s).6 The MIC, which is
the lowest antimicrobial concentration that inhibits the growth of the microorganism
usually over a 16-20 h incubation window, is simple and relatively easy to determine.7
However, this simplistic approach also has its shortcomings and does not represent the
complex interactions between the host, the pathogen and the drug itself. There are
complex interactions between these three players namely, host, pathogen and the drug
that have to be considered in the drug development process: role of the host immune
response against the infection; development of multi-drug resistant bacteria in response
to suboptimal drug dosing regimens; adverse effects of the drug to humans; and the
drug’s PK and PD properties.
2.1 MIC-Based Approach
The PD surrogate indices in antimicrobial treatment was first identified in the 40s
and 50s by Eagle, who noticed the time-dependent properties of penicillin bactericidal
activity in rodents and the concentration-dependent killing of streptomycin and
bacitracin and the mixed pattern in tetracyclines.8, 9 Knowing the implication of these
characteristic PD properties for clinical therapies, Eagle realized that penicillins are best
administered as continuous infusion while concentration-dependent agents are better
given as intravenous bolus to achieve high maximum concentrations.10, 11 In the 90s,
Craig expanded on the concept of PD indices through a number of rodent studies
correlating PK parameters with bacterial kill over 24 hours.12 This PK-PD information is
very useful in the selection of the dose and dosing regimen for antimicrobial candidates
entering the clinical stage of drug development, as well as for clinicians in deciding
6
whether a specific antibiotic is suitable for the patient’s infection and how the antibiotic
should be administered.
2.1.1 Pharmacodynamic Indices
The quantitative relationship between PK parameter and microbiological
outcome, the change in log10 colony forming unit (cfu) in post-dose 24-h sampling from
the starting bacterial density often obtained from dose fractionation in the rodent or the
time-kill kinetic studies, is referred to as a PD surrogate index. The characterization of
PD surrogate index is primarily based on the relationship between drug exposure and
MIC. The three common PD indices used to predict antimicrobial efficacy are time at
which the drug concentration is above MIC over the 24-h interval (T>MIC), the ratio of
peak drug concentration and MIC (Cmax/MIC), and the 24-h area under the
concentration-time curve over MIC (AUC/MIC) at steady state conditions. These indices
are often prefixed by an italic f, representing the free and unbound drug concentration,
as it is often assumed that only the unbound drug can exert its pharmacological effect.
In the literature, one often finds the percentage of time above MIC over the 24-h period
rather than the absolute time above MIC being reported.13 These patterns of
antimicrobial killing effect are summarily characterized as either time-dependent or
concentration-dependent killing effects. Some investigators included a third
classification as concentration-independent killing with prolonged post-antibiotic
effects.1 The post-antibiotic effect is the duration from the time after the microorganism
is exposed to the drug until the survivors start to multiply rapidly.14
7
Time-dependent killing with short or no post-antibiotic effects. Drugs exhibiting
this killing pattern is best described by the PD index fT>MIC. The dosing strategy is to
maintain the free drug concentration above the MIC value for an extended period of
time. The class of β-lactam antibiotics is commonly associated with the term
“concentration-independent” or “time-dependent” kill. That is because the efficacy of β-
lactams is enhanced with longer exposure times of the free drug concentration
maintained above MIC. β-lactams with the exception of carbapenems have limited
postantibiotic effect against gram-negative bacteria.14, 15 A classic illustration of β-lactam
exhibiting time-dependent killing effect is shown in Figure 1. In this example of
ceftazidime against Klebsiella pneumoniae ATCC 43816 in the lungs of neutropenic
mice, the time above MIC is best correlated with the 24-h cfu.16 The vertical line in
Figure 1 indicates that ceftazidime concentration above the MIC in at least 60% of the
dosing interval achieves the greatest bacterial kill.
Concentration-dependent killing with extended post-antibiotic effects. Drugs of
this killing trend tend to be best characterized by the fCmax/MIC ratio. The killing pattern
of aminoglycosides is best correlated with this ratio. Whether the concentrations of
these antimicrobial agents are maintained above the MIC for an extended period or not
seem to have only limited impact on their efficacy. This is due to their post-antibiotic
effect. Even when the active free drug concentration falls below the MIC, the extended
effects provide protection against the regrowth of bacteria. The magnitude of the peak
concentration is often associated with its bacterial killing efficiency. The initial kill rate of
aminoglycosides in the time-kill kinetic studies is proportional to the drug concentration
measured by the number of fold of MIC, as shown in Figure 2 for tobramycin.17 In
8
contrast, the initial kill rates do not change significantly with increasing concentration of
ticarcillin, a β-lactam which exhibits a time-dependent killing pattern. The shapes of the
initial kill from the static time-kill curves are not necessarily indicative that the drug is a
concentration-dependent agent. The presence and the duration of the post-antibiotic
effect are just as important.14
Concentration-independent killing with extended post-antibiotic effects. Drug

showing this type of killing pattern is often characterized by fAUC/MIC. The quinolones,
tetracyclines, oxazolidinones, vancomycin, collistin, as well as the macrolide antibiotics,
exhibit this type of killing pattern. The prolonged effect inhibits bacteria regrowth even
when the drug concentration is below MIC but their drug effect is not dependent on the
peak drug concentration. The example of linezolid in Figure 3 shows that the 24-h
AUC/MIC has the tightest correlation with the change in 24-h log10 cfu with a coefficient
of determination, R2 value, of 0.82.18 The criteria for deciding as to which of the PD
indices best characterizes the drug killing effect is based on fitting a sigmoidal Emax
model on the microbiological outcome such as the bacterial 24-h log cfu against the
three PD indices. The parameter that had the tightest correlation with the
microbiological outcome is selected as the PD index for the drug.
As a well-established approach to predict the success or failure of therapy, the
three empirical pharmacodynamic indices show co-linearity in some scenarios (R2
values are not significantly distinctive), resulting in difficulty in decision making for dose
selection. The predictive capacity of empirical pharmacodynamic indices has been
evaluated by Nielsen using semi-mechanism PK/PD model.19 The results suggest that
the efficacy of moxifloxacin can be described by both fCmax/MIC (R2=0.93) and
9
fAUC/MIC (R2=0.99) and the efficacy of erythromycin can be characterized by both
fAUC/MIC (R2=0.93) and fT>MIC (R2=0.90). The dose fractionation study conducted by
Andes demonstrated that the correlations of AUC/MIC, T>MIC with bacterial count were
82% and 57%, respectively, for linezolid against Streptococcus pneumoniae and 75%
and 74%, respectively, for linezolid against Staphylococcus aureus.18 Although the
author claimed that the narrow insufficient difference between effective and ineffective
dosing regimens may lead to indistinguishable pharmacodynamic parameters against S.
aureus, the result still left us questioning whether the empirical pharmacodynamic
parameters derived from animal infection model may be bacteria dependent and
whether the information provides complementary information of drug efficacy from
different perspectives. A new pharmacodynamic index, weighted AUC/MIC, was
proposed by Corvaisier et al. in order to take into account both the concentration-
dependent part and the concentration-independent part of the antibiotic efficacy.20 As
the selection of the appropriate PD index as a surrogate marker of efficacy is important,
the magnitude of these indices that maximizes efficacy is equally important if not more
important. It was previously suggested that the PD index determined in rodent studies
can be extrapolated to clinical efficacy.14 Many of the current dosing regimens in the
clinic were based on PD indices that were determined from mice. Table 1 lists the target
PD indices for several antimicrobial agents. As in the example from Figure 1, the fT>MIC
of at least 60% of the dosing interval for ceftazidime is associated with the lowest log10
cfu at 24 hour post-dose. Vancomycin dosing regimen was optimized to achieve the
target AUC/MIC ratio of 400 h in treating ventilator-associated S. aureus pneumonia.21,

22
The vancomycin nomogram of dosing regimen based on body weight and renal
10
function was designed to achieve a target trough concentration of 15-20 mg/L to avoid
vancomycin-related nephrotoxicity and at the same time targeting an AUC/MIC ratio of
400 h.23, 24 Interestingly, AUC/MIC is a PD surrogate index actually possessing a time
dimension (h). Thus, the ratio of 400 is computed over a 24 h period at steady state.
Toutain et al. suggested a more universal metric by dividing the AUC/MIC by the time
interval of interest to permit the assessment of a truly dimensionless ratio and offer
direct clinical interpretation, based on which the target concentration of vancomycin at
steady state would be 16.6 fold MIC.25 There are some indications that these PD indices
are not affected by bacterial strain. In the study of gatifloxacin against Salmonella
enterica serotype Typhi, where two isolates consisting of both susceptible and resistant
strains, the PD index was the same against both strains, regardless of the MIC value.26
Studies of colistin PD index, on the other hand, seems to indicate that the magnitude of
the index value is bacteria- and strain-dependent. Dudhani et al. reported fAUC/MIC
value between 23 and 46 for colistin against Pseudomonas aeruginosa 27, 28whereas
Guyonnet et al. reported a much lower index value against Escherichia coli for its
bactericidal effect characterized by a reduction of at least 3 log unit in cfu.29 Bergen et
al. reported an index value between 6.81 and 35.7 for several strains of P. aeruginosa.30
2.2 Pharmacodynamic Indices in Antimicrobial Combinations
2.2.1 β-lactam – β-lactamase combination
As multidrug-resistant infections have contributed to a rise in mortality in the
hospital setting, many antimicrobial agents are combined with another agent that
counterattacks the bacteria’s drug resistance mechanism. β-lactamases, which are
11
responsible for the bacteria’s resistance to β-lactams, threatened the utility of the class
of β-lactams. Consequently, β-lactamase inhibitors were developed and introduced to
clinical practice. These inhibitors when combined with a partnering β-lactam would
confer susceptibility of the bacteria to β-lactams and restore the antibacterial activity of
a partnering β-lactam. The clinically available combinations include amoxicillin-
clavulanate, ticarcillin-clavulanate, ampicillin-sulbactam, piperacillin-tazobactam,
ceftolozane-tazobactam and ceftazidime-avibactam, listed in the format, β-lactam – β-

lactamase inhibitor. With the exception of ceftazidime-avibactam combination which
recently received marketing approval from the US Food and Drug Administration, drug
resistance to β-lactam – β-lactamase inhibitor combinations has been documented in
the literature.31-34 The drug resistance was primarily due to β-lactamases that were not
covered by these inhibitors. For example, TEM-30 and SHV-10 β-lactamases are
resistant to clavulanic acid, sulbactam and tazobactam.35 Table 2 shows the molecular
classification of β-lactamases, the corresponding β-lactam substrates and the β-
lactamase inhibitors that inhibit the specific class of enzymes. The molecular
classification is based on the conserved and distinct amino acid motifs. The β-
lactamases in classes A, C and D hydrolyzes their substrate by forming an acyl enzyme
through their serine active site whereas those in class B are metalloenzymes that use
the zinc active site for hydrolysis of β-lactams. There are no commercially available β-
lactamase inhibitors for the class B metalloenzymes, whereas the available inhibitors
have only limited coverage of the class D serine-based enzymes. The competitive
landscape indicates a commercial opportunity for broader spectrum inhibitors or
antibiotics that can evade β-lactam resistance through a different mechanism of action.
12
For the past several decades, β-lactamase inhibitors have been used clinically
but very limited information were found in the literature on optimizing dosing regimens of
these inhibitors using PD indices. The PD index for β-lactam–β-lactamase inhibitor
combination is based on the established T>MIC for the β-lactam and T>threshold concentration
(or T>TC) for the β-lactamase inhibitor. The threshold concentration is the lowest
concentration of the β-lactamase inhibitor at which a significant difference between MIC
of the partnering β-lactam as monotherapy and the MIC of the combination can be
observed in the resistant strain bacteria. The threshold concentration for tazobactam
varied in several articles. An early study reported a threshold concentration of 4 mg/L
tazobactam in an in vitro model of infection that was carried out in piperacillin-
susceptible E. coli J53 and P. aeruginosa ATCC 27853 and piperacillin-resistant
Staphylococcus aureus 7176 and isogenic TEM-3 construct in E. coli J53.36 Their
approach is based on the proposal that the activities of various β-lactam – β-lactamase
combinations be determined by assessment of the concentration of inhibitor needed to
bring the susceptibilities of individual organisms to below accepted susceptibility
breakpoints for the β-lactam alone or even to the susceptibilities for non-β-lactamase
wildtype strains.37 The resulting T>4 mg/L tazobactam was 50 and 38% for 3g/0.375g q6h and
4g/0.5g q8h piperacillin/tazobactam, respectively; both dosing regimens of the
combination were highly active against all strains resulting in at least 4 log kill.36 In two
later studies, the process of determination of threshold proposed for the ceftolozane-
tazobactam combination utilized a different algorithm wherein the threshold candidate
concentrations of 0.01 to 1 and 0.25 to 4 mg/L were evaluated in time-kill kinetic
studies.38, 39 In one study, their analysis returned a much lower concentration values of
13
0.05 and 0.25 mg/L tazobactam concentration for the low to moderate and high β-
lactamase expression E. coli strain consisting of blaCTX-M-15 constructs, respectively.39 In
another study, the same authors determined that the threshold concentration for
tazobactam ranged from 0.5 to 4 mg/L in four E. coli and three Klebsiela pneumoniae
clinical isolates.38
The relationships between three tazobactam exposure measures, AUC, Cmax and
T>TC, and the change in log10 cfu after 24 h therapy in a PK-PD in vitro infection model
were examined.39 The dose fractionation schedules were q6h, q8h, q12h and q24h. The
relationship between T>TC and microbiological outcome resulted in an R2 value of 0.938.
The authors concluded that the magnitudes of T >TC for tazobactam associated with net
stasis (no change in log10 cfu), and a 1- and 2-log10 cfu reduction based on the model-
predicted data at 24 h were 35, 50 and 70%, respectively, regardless of the level of
transcription efficiency of the β-lactamase enzyme.39
The optimal PD index for avibactam determined from dose fractionation
experiments was also T>TC.40 The T>TC for avibactam was approximately 60% to 80%,
corresponding to 14.8 h and at most 19.2 h in which the partnering ceftaroline at 600
mg q8h was used and the threshold avibactam concentration of ≥4 mg/L was
assumed.40 In the ceftazidime-avibactam combination, a threshold avibactam
concentration of 0.5 mg/L was required to suppress regrowth of Enterobacteriaceae
expressing AmpC β-lactamase.41
In the dose-finding trials, the threshold avibactam concentration was 1 mg/L.42
The information presented by the developer of ceftazidime-avibactam combination at
14
the EMA dose finding symposium indicated that the same duration (as percentage of
the dosing interval) for avibactam as that for ceftazidime to be maintained above the
MIC was required to maintain continued suppression of β-lactamase activity, which in
this case was 50% fT>ceftazidime-avibactam MIC and 50% fT>avibactam TC.
2.2.2 β-lactam – aminoglycoside/fluoroquinolone combination.
Combination therapy, comprised of a β-lactam and an aminoglycoside or a

fluoroquinolone, is highly recommended to treat patients with serious infections caused
by P. aeruginosa according to a guideline from the Infectious Diseases Society of
America (IDSA).43 Combination chemotherapy has the potential to provide the highest
probability of attaining optimal organism kill by suppressing resistance development and
the lowest probability of side effect by decreasing the levels of both components. The
study published by Louie evaluated combination chemotherapy of meropenem and
tobramycin for P. aeruginosa in a murine pneumonia model.44 The results show the
near-maximal effect was reached at 60 mg/kg/day and 50 mg/kg/day of meropenem
and tobramycin, which produced a T>MIC of 35.25% and an AUC/MIC ratio of 80.1 h in
epithelial lining fluid (ELF). The similar effect requires an AUC/MIC ratio in the epithelial
lining fluid of 240.3 h when tobramycin was administered as a single agent, implying the
advantage of combination therapy over monotherapy. However, Joly-Guillou compared
the in vivo activity of levofloxacin alone and in combination with imipenem in a mouse
model of Acinetobacter baumannii pneumoniae and observed no efficacy enhancement
with combination therapy.45 The benefit of combination therapy of a β-lactam and an
aminoglycoside was also questioned by several papers based on meta-analyses in
some special populations, demonstrating that all-cause mortality rates are comparable,
15
whereas combination treatment carries a significant risk of nephrotoxicity compared to
monotherapy of β-lactam.46, 47 Clinically, combination therapy has been widely practiced
in treatment of infections caused by multidrug-resistant organism (MDRO) with the
expectation of introducing synergism to reduce the selection of mutant bacterial
resistance to each antimicrobial component. The PD indices could serve as a good
efficacy surrogate to assess the drug-drug interactive effect for antimicrobial

combination and help dose selection in the future clinical study design.
2.3 Issues with MIC-based approach
The MIC is a measure of the potency of an antimicrobial drug. However, MIC is
usually determined in vitro by aerobic incubation in a protein-free liquid medium at pH
7.2. The conditions are highly distinct from those at the site of infection, where the
environment is anaerobic, acidic and surrounded with protein.48 Thus, the MIC values
derived from artificial fluids do not necessarily indicate accurate drug efficacy in vivo in
biological fluids. A MIC comparison of oxytetracycline was conducted by Brentnall in
Cation Adjusted Mueller Hinton Broth (CA-MHB) and calf serum.49 The results showed
that the MIC value was 19-fold higher in serum than in broth, indicating a significant loss
of potency of oxytetracycline in calf serum. Protein binding may have contributed to the
higher MIC obtained in serum as protein binding of 18.6% to 72% has been reported for
cattles,50, 51 which would only account for partial of the ratio. The difference of calcium
and magnesium concentrations in two matrices may also affect the MIC value. A further
assessment is required to provide grounded explanations. On the other hand, Buyck
found that the susceptibility of P. aeruginosa to macrolides and ketolides in eukaryotic
cell culture media and biological fluid would be dramatically enhanced compared to CA-
16
MHB due to decreased expression of oprM and increased outer-membrane
permeability. The work opens new perspectives that macrolides may exert in vivo
antibacterial activity against P. aeruginosa that would not be predicted on the basis of
conventional susceptibility testing system.52 Pruul also examined the interaction of
azithromycin and other macrolides with normal human serum in relation to MIC.53 The
result elucidated that the MIC of azithromycin was decreased by twenty-six-fold for
serum resistant E. coli and fifteen-fold for S. aureus in presence of 40% serum with low
protein binding characteristics (8.5% at a concentration of 0.01 nM in 95% serum).
Erythromycin had a similar susceptibility pattern, while roxithromycin was less active
against S. aureus in the presence of serum.53 In general, free unbound drug
concentrations at the site of action are responsible for the pharmacodynamic effect of
anti-infective agents. Although many other factors may have certain impact on efficacy
of antimicrobial drugs, MIC-based approach is still a well-established method offering a
profound understanding of why a particular dosing regimen achieves clinical success.
In addition, one of the widely-accepted antimicrobial susceptibility testing
methods is macrobroth or tube dilution method. This procedure involved preparing two-
fold dilutions of antibiotics in a liquid growth medium dispensed in test tubes.54 The MIC
determined by 2-fold dilution is considered as a relatively crude index since the “true”
MIC is a point between the lowest test concentration that inhibits the growth of the
bacteria and the next lower test concentration. To improve accuracy of MIC, Aliabadi
and his colleagues used five overlapping sets of doubling dilution and their result
illustrated that the MICs of danofloxacin were 0.03 μg/mL in broth, sheep serum and
exudate and 0.035 μg/mL in transudate.55 Disk diffusion and gradient diffusion are also
17
reliable manual methods that provide reproducible result and convenience of
preparation. Currently, four automated instrument instruments have been approved by
the FDA for use in the United State to produce susceptibility test results in a shorter
period than manual reading; these instruments have significantly reduced the manual
labor involved in running susceptibility testings.7
2.4 In vitro Time-Course-Based Approach

The in vitro time-course of bacterial response to both static and dynamic drug
concentration characterized by time-kill assays has been used in pharmaceutical drug
development of antimicrobial agents. The time-course approach provides greater detail
of how the colony population of bacteria reacts to an antimicrobial challenge over time.
Depending on the objective of the study, the drug concentration in the in vitro time-kill
experiment can be constant in the static setting or dynamically changing to mimic the in
vivo half-life of the drug in humans. The constant or static exposure is achieved by not
replacing the medium, while the dynamic assay is accomplished by changing the
medium with fresh ones that do not contain the antibiotic. A loss of bacteria due to the
experimental setting, which is observed in some models, may have a substantial
influence on the results. By using semi-permeable membranes, loss of bacteria can be
avoided during replacement of medium that mimics the loss of drug or decreasing drug
concentrations.56
The information from static time-kill experiments are often used to develop a
mathematical model that links the free drug concentrations to the bacterial response
whereas the dynamic time-kill data can be used to validate the model or to evaluate the
18
outcome in the clinic using humanized dosing regimens.57 The dynamic kill-curve also
provides an alternative for evaluating PK-PD relationships, as it simulates the time
course of the free drug concentrations in the plasma based on the characteristic half-life
of the drug.2 The effects of different dosing regimens, drug half-lives and even starting
inoculum sizes can be simulated to study their effects on the bacterial population
dynamics over a specific time period, such as 24, 48 or 72 h.58-60

The common type of models that are often developed for the purpose of
establishing PK-PD relationship are the semi-mechanistic models. As opposed to
mechanistic models, the semi-mechanistic models are empirical models that capture the
broad general characteristics of bacterial growth and drug effects, as well as
development of bacterial resistance. These models are often developed from in vitro
information that comes from both static and dynamic time-kill experimental data.
The most common type of semi-mechanistic models in the literature to describe
the PK-PD relationship of antimicrobial agents and bacterial population dynamics is the
logistic growth model, which was introduced by Pierre-François Verhulst in 1838 to
study human population:61
1
( )
where N is the population number in unit of cfu/mL, kgrowth is the growth rate constant
with units often in h-1, and Nmax is the maximum population size that is supported by the
environment, also known as the carrying capacity and in unit of cfu/mL. The important
characteristic of this differential equation is that there is a limit as time approaches
19
( )
infinity for its analytical solution, which is ( ) , where N0 is the
[ ( ) ]
initial population size. This limit is Nmax (i.e. ( ) ). In the in vitro bacterial
system, the bacterial cfu is restricted from growing indefinitely and reaches a plateau,
where there is no net growth. For this reason, the logistic growth model is suitable for
describing this behavior.
The effect of antimicrobial intervention on the capacity limited growth model in

equation 1 is incorporated to the model with a function that describes the drug effect:
2
( ) ( )
where the ( ) is a function of drug concentration and is often
represented by an Emax or sigmoidal Emax model,
3
( )
wherein Emax is also termed as kkill,max with unit of h-1 and EC50 has the same unit as the
drug concentration.
We note that the MIC, EC50 and the stationary concentration are not parameters
but rather variables as their values are dependent on the inoculum size. 62 The
stationary concentration or the concentration at which the growth rate equals the kill rate
where no net change in the number of bacteria is occurring can be derived by assuming
a linear growth at the initial growth phase where and equation 2 becomes
( ) .63 The analytical solution for this differential equation is
20
( ) [( ) ]. The stationary concentration occurs when
( ) ( )
( ) or . By taking the natural log of , this value is 0. Thus,
. Solving for C, one obtain the following equation:
4
( )
[ ]
( )
( )
( )
When there is no net change in the number of bacteria, the term approaches 0
and the static concentration (SC) is:
5
[ ]
It is not to be confused between SC and MIC. Because at SC, we assumed that
( ) , the initial inoculum size is approximately 5  105 cfu/mL, which is not visible
to the human eye. The visible bacterial density is approximately 10 7 to 108 cfu/mL. Thus
a correction factor to the SC equation is required to obtain the MIC:63
6
[ ]
( )
The value of 0.29 came from the assumption that ( ) reached 108 cfu/mL at 18 h with
an initial inoculum of 5  105 cfu/mL. This correction factor is dependent on the specific
system that the drug is being tested.
21
Further modifications to the logistic growth factor included incorporating an
adaptation factor to the EC50 parameter and “delay functions” to both the growth and
drug action part of the model. Both of these modifications allow the model to adapt to
the bacterial regrowth that is often observed in time-kill kinetic studies. The adaptation
factor to the EC50 parameter has the following form:64
7
( )
( )
wherein α is defined as:
( ) 8
where is the exponent of the adaptation factor and is the maximum value for the
adaptation, since the range of values for the function is between 0 and 1. A
baseline EC50 value at time 0 would either increase or decrease over time to a maximal
value of , depending if is positive or negative, respectively.
The delay function has the form, , which behaves like a cumulative
density function starting from 0 at time 0 to a maximum value of 1 as time approaches
infinity. The equation 2 incorporating two delay functions is shown in equation 9:
9
[ ( )( )
( )( )]
22
This model was applied to model bacterial dynamics of S. pneumoniae, Haemophilus
influenza and Moraxella catarrhalis in response to azithromycin.65
The logistic growth model was recently adapted for combination therapy utilizing
Loewe additivity to evaluate whether a combination of an aminoglycoside and a β-
lactam would result in synergistic activities,66 since both agents have antimicrobial
activities with different mechanism of actions. Greco proposed a generalized sigmoidal

Emax equation utilizing Loewe additivity model:67
10
⁄ ⁄
( ) ( )
( ⁄ ⁄ )
( )
The first two terms describe the additive effect of the two agents while the third term is
an interaction term, wherein the parameter indicates synergism-antagonism
interaction. The interaction is considered additive if the 95% confidence interval of the
estimate overlaps the zero value. If or , the interaction is either synergistic or
antagonistic, respectively. An additional interaction evaluating the initial killing rate (k max)
was included:
11
The final Emax model with the adaptation factor to evaluate the combination of these two
agents was:
23
12
( )
( )
where refers to the adaptation factor mentioned in equation 8. This empirical
approach described well the combined effects of an aminoglycoside and a β-lactam
against P. aeruginosa.66
In the examples above, these models were fitted to the data. It is important to
consider whether the parameters estimated are identifiable and do not have co-linearity
with other parameters. In cases of increasing model complexity where parameters
outnumber the available data points to estimate the parameters precisely, non-
identifiability of parameters can arise. Strategies to detect non-identifiability in
parameters include slight perturbation to a parameter and evaluation of which
parameters are affected. Co-linearity can be detected by correlation or by plotting the
perturbed parameter values against other parameters estimated by the model. 68
Algorithms to detect non-linear manifolds have been developed69 but are not very
sensitive. When co-linearity arises, one can consider fixing specific parameters to
literature values.
To overcome parameter identifiability problems with increasing model complexity,
for example in mechanistic models, simulation approach rather than fitting model to data
is often undertaken. The simulation approach requires more efforts in collecting
literature information on the parameter values. The complex modeling of networks
24
involved in resistance development is one such example of highly complex models. 70, 71
In later section, models for resistance development in bacteria are discussed.
2.5 Measurement of free unbound drug concentrations for accurate
characterization of antimicrobial efficacy
The free unbound drug concentration in the interstitial fluid at the target site has
been shown to be better correlated with the drug’s antimicrobial efficacy. In contrast,
total plasma drug concentration is often a poor descriptor of the drug’s activities at the
site of action, particularly for antibiotics, as most infections do not occur in the plasma
but rather in the tissue sites. Given that tissues are not homogeneous compartments,
the use of protein binding as proportion of total drug concentration does not extend to
the free drug concentrations in the tissue. To measure free drug concentration in the
interstitial spaces, in vivo microdialysis is used to characterize drug distribution in the
tissues.72 This technique involves implanting a dialysis probe into the tissue or organ of
interest and the probe is constantly flushed with a compatible fluid for example saline
solution at a controlled flow rate. The protein-free drug in the interstitial fluid enters the
probe’s semipermeable membrane by passive diffusion. The equilibrium between
concentration gradient inside and outside the probe is established relatively rapidly. The
free drug concentration in the tissue is then computed based on the relative recovery
determined depending on the particular method such as retrodialysis and no net flux.
The important assumption for the method is that the recovery is constant over time.
Problems could arise implementing this procedure as the recovery can be influenced by
factors such as the membrane area of the probe, molecular weight cutoff for substance
entering the semipermeable membrane, flow rate of the fluid used to flush the probe,
25
perfusate and temperature.73 Thus, a pilot study evaluating the feasibility and
compatibility of the drug with the microdialysis procedure is often performed in vitro prior
to implementing microdialysis technique in human test subjects.
Many applications of microdialysis have been in antimicrobial therapies. The
prophylactic use of 1.5 g short-term infusion of cefuroxime in morbidly obese patients
during abdominal surgery was investigated by evaluating whether subcutaneous and

muscle drug concentration provides sufficient coverage against methicillin-suspectible
S. aureus and E. coli.74 Their study showed that this dosing regimen achieved free drug
concentrations in the respective tissues for over 40% of the dosing interval; cefuroxime
is a β-lactam whose pharmacodynamics index is characterized by fT>MIC. The
pharmacokinetics of ertapenem was monitored in the interstitium of healthy uninfected
tissue and infected tissue from patients suffering from diabetic foot infections to assess
the sufficiency of antibiotic concentrations at the target site. The results suggested that
the free interstitial ertapenem concentrations were higher than in inflamed tissue of
diabetic feet than in healthy adipose tissue, indicating good tissue penetration in
diabetic foot infections. However, 1 g of ertapenem daily given intravenously was
unable to reach fT>MIC higher than 40% in more than half of the patients in the
interstitium of infected tissue. Higher daily dose might be considered in patients with
diabetic foot infection to optimize bactericidal effect.75 The levofloxacin penetration into
the lung tissue after cardiac surgery was measured using microdialysis technique in six
patients receiving a dose of 500 mg via intravenous administration in addition to the
standard antibiotic prophylaxis regimen. The median fAUC/MIC in lung tissue was 2.4
(ranging from 1.3 to 4.2) for P. aeruginosa, demonstrating that the dose was borderline
26
sufficient for the treatment of nosocomial pneumonia caused by K. pneumoniae and
insufficient for the treatment of pneumonia caused by P. aeruginosa. An fAUCtissue/MIC
ratio of 30 to 40 is associated with high rates of bacterial killing and thus maximizes the
efficacies of fluoroquinolones.76
3. Modeling Approach
The model-based development approach is particularly useful for development of

antimicrobial agents, because the classification of antimicrobial classes based on the
PD indices are well established and the translational value of in vitro time-kill kinetics to
the clinical practice has been proven to work. This section will discuss modeling
strategies that can be incorporated into the development of antimicrobial agents.
3.1 PK-PD models based on static MIC and time-kill curves
In the MIC-based approach, the PK exposure parameters come from the serum
antimicrobial concentration and the PD parameter is the MIC. The goal is to find a dose
where the index is attained or exceeded. For example, if the antimicrobial agent is
concentration-dependent, one would first establish the ratio of fCmax/MIC that achieves
the optimized kill from either animal studies or in vitro dynamic time-kill studies. Once
the PD index is established, the human PK information would then come from phase I
ascending dose studies to determine which dose would result in the required free drug
Cmax (i.e. protein-binding-corrected Cmax) to achieve or exceed the target fCmax/MIC
ratio. The human PK is modeled by a population PK model so that simulations of
different dosing regimens can be done for the purpose designing future trials. The
population PK model consists of both deterministic and stochastic components.5 The
27
deterministic model takes the form of the classical compartmental model. The stochastic
component describes the variability between individuals as well as determines the
sources of inter-subject variability through covariate models. The second level of
variability is within-subject variability which can be described by the residual error model
and inter-occasion variability.5
The covariate model quantifies the source of inter-subject variability contributing

to a PK parameter. For example, the covariate model may include body weight as a
covariate of the volume of distribution and drug clearance, sometimes utilizing allometric
scaling. Many antimicrobial agents such as vancomycin, amikacin are dosed by body
weight.21, 77 For many antibiotics, the renal function has an important impact on the
clearance of the drug. Creatinine clearance, in this case, would be an important
covariate of the population PK model. These covariate models are important and should
be considered during model development.
3.2 Monte Carlo PK-PD simulations
Once the population PK model is established for humans and the PD indices
determined from animal or in vitro studies, several dosing regimens can be simulated
with virtual individuals to determine which dose and dosing interval are most optimized
to achieve the target PD index. In the example of drug-drug interaction study of
efavirenz and para-aminosalicylic acid, efavirenz was shown to induce the clearance of
para-aminosalicylic acid, which is used as companion drug to prevent further
development of drug resistance in tuberculosis infection.78, 79 This drug-drug interaction
was evaluated because many tuberculosis patients in the African continent also have
28
HIV co-infection and often are on highly active antiretroviral regimen containing
efavirenz in addition to their multiple-drug therapy for tuberculosis. In that study,
several dosing regimens of para-aminosalicylic acid were simulated to determine which
doses and dosing intervals would not achieve the target PD index of maintaining the
free trough para-aminosalicylic acid concentration above the MIC of 1 mg/L with and
without concomitant efavirenz. The simulations were stratified by whether concomitant

efavirenz medication was present or not. The population PK model-based exposure
parameters generated from the simulations were 24-h steady-state AUC, peak, trough
and the free trough concentrations. The results indicate that at least 4g twice-daily
dosing regimen ensures free drug concentrations exceeding MIC over the entire dosing
interval, even in HIV-infected patients who received efavirenz, whereas once-daily
dosing even as high as 12 g would risk the free trough concentration falling below the
MIC in some patients.78
Once both the population PK model and the PK-PD model for the in vitro time-kill
kinetics are developed, the time course of bacterial cfu can be simulated to study which
dosing strategy would give a favorable microbiological outcome. In the study by Zhuang
et al (2015),66 the prediction of the antibacterial activity of an aminoglycoside in
development for three dosing regimens, namely, 3 mg/kg QD, 5 mg/kg QD and 2.5
mg/kg BID were simulated, as shown in Figure 4. The simulations were based on the
PK model using the population parameter mean values. The population parameter
mean values without including the intersubject variability were used for the simulations
because the in vitro semi-mechanistic PD model has its inherent error. If one were to
include intersubject variability in the PK model, the range of values in the antibacterial
29
activities would be amplified and the pattern of bacterial kill could become unclear due
to the exaggerated variability in the response. The same approach using PK-PD model
simulation can be utilized in drug development for dose optimization in the clinic. The
PK-PD model is often developed immediately before going into the phase II trial, since
the PK information from the phase I trial is used for the population PK model
development. The modeling and simulation approach can give better confidence to the
dose selection for the phase II and III trials.
3.3 Probability of target attainment and cumulative fraction of
response
The probability of target attainment is the percentage of the simulated individual’s
profiles that are over the threshold or target PD index. Very often, approximately 10,000
individual drug concentration-time profiles are simulated using a population PK model
developed from PK studies. The large number of simulations is required to characterize
accurately the probability values. The distribution of PD index becomes the basis for
determining the likelihood of achieving a specific target attainment. In the previous
example for para-aminosalicylic acid,78 we estimated the probability of target attainment
to evaluate the dosing regimens that would achieve free trough concentrations above
MIC. The PTA is computed in a statistical program by ranking the simulated trough
concentrations and estimating the percentiles that these values are greater than a range
of MIC values.
The alternative prediction of clinical outcome using Monte Carlo simulation is the
cumulative fraction of response (CFR), which is defined as the expected population PTA
30
given a population of microorganisms for a specific dosing regimen.1, 13 The equation to
compute CFR is as follows:
13
( ) ∑
wherein CFR is the sum of the PTA at a specific MIC multiplied by the frequency (F) of
bacterial isolates with that MIC over the range of MIC values. In this case, a population
distribution of MIC values rather than a single MIC value is used and the expected
probability of success of a dosing regimen is estimated for the population distribution of
MICs instead of estimating for one MIC value. This approach is said to be more useful
for community medicine than for drug development, as the situation may be such that
the pathogen susceptibility may not have been determined at the time of patient
evaluation. A key point is the selection of the adequate range of MIC values, as the
pathogen susceptibility may vary between countries, areas and hospitals, as well as
over time.1 Thus, CFR is often specific for an institution or hospital.80
4. Translational Approach to Bridge In vitro Information to the Clinic
Examples are presented in this section to show how the in vitro information is
utilized to shed light on dosing strategies in humans and how modeling was applied to
optimize dosing regimens in special population.
4.1 Modeling approach to determine pharmacodynamic indices
In the example of ceftazidime, the time-course of bacterial response to the drug
from the in vitro time-kill curves was used to infer why the PD index fT>MIC of at least
31
50% is required for bactericidal effect in vivo.81 The logistic-growth model was fitted to
the time-kill data. Using the in vitro PK-PD model, the hypothetical in vivo bacterial
response over time after 1 mg every two hours to 256 mg every 8 hour was simulated to
evaluate the dosing regimen that would be able to provide sufficient antibacterial
activities. The time-course of drug concentration profile was simulated from the PK
model for both humans and mice. The dosing regimens that achieved a predicted static
effect such that the cfu at 24 h is less than the cfu at 0 h were then evaluated. The
corresponding fT>MIC for that dosing regimen was subsequently determined. The
authors estimated that fT>MIC of at least 50% is required in order to achieve a 2 log10 kill
after 24 hours. This is one example of how modeling and simulation can be used to
derive a target PD index.
Another group used a semi-mechanistic PK-PD model to predict the PD indices
of several antibiotics.19 Even though the simulations using the in vitro information can be
used to predict target PD indices, the author cautioned that the suitable PD index for a
specific antimicrobial is sensitive to study conditions including dosing frequency and
uncertainty in the MIC determination.
4.2 PK-PD simulation to evaluate dosing regimens in patients with
end stage renal disease
Patients with end-stage renal disease have deteriorating chronic kidney disease
wherein their renal function are <10% of the normal capacity.82 Repeated hemodialysis
is required to prevent the accumulation of fluid, electrolytes and toxins. Infection is the
32
second leading cause of mortality in end-stage renal disease patients.83 Thus
antimicrobial agents are highly used in this patient population.
Dosing of antimicrobial agents in end-stage renal disease patients is still quite
controversial as these patients tend to have altered drug clearance and the dialysis
procedure tends to remove majority of the drug in the system.83 We note that drugs with
a rather small steady state volume of distribution are readily removed by dialysis. The
current guideline for dosing gentamicin is to administer half of the full dose immediately
after hemodialysis; this recommendation is being challenged by several studies that
argue for administration of full dose an hour before hemodialysis.84-87 The predialysis
dosing argument is based on PD index of fCmax/MIC that requires Cmax to be 8 to 10-fold
higher than MIC,86 without considering the area under the curve which is also
associated with the efficacy of gentamicin.
Zhuang and colleagues utilized a semi-mechanistic model developed from in
vitro time-kill curves and the PK model of gentamicin in end-stage renal disease
patients undergoing hemodialysis to compare the antimicrobial activities of
administering 120 mg gentamicin immediately after hemodialysis compared with
activities associated with 240 mg given 1 hour before hemodialysis.88 The results of the
simulation exercise show that the half dose of gentamicin at the end of hemodialysis
would result in similar microbiological outcome as that when a full dose of gentamicin is
administered 1 hour before hemodialysis. The end-stage renal disease patients present
a case wherein PK-PD indices may not give sufficient information about how
hemodialysis is affecting the time-course of action of the antibiotic that a modeling and
simulation exercise can provide a bigger picture of the time course.
33
4.3 Humanized dosing in animal models to evaluate efficacy
Translational strategy to verify the dosing regimen in the clinic was applied to the
recently approved β-lactamase inhibitor with a partnering β-lactam, ceftazidime-
avibactam. The study evaluated the efficacy of human-simulated plasma exposure of 2
g ceftazidime and 0.5 g avibactam every 8 h administered as a 2-h infusion in
neutropenic and immunocompetent mice using both thigh and lung infection model of
several P. aeruginosa clinical isolates.89, 90 The PK of the drug was based on that
reported in humans.91 Given that the half-lives of the drugs are different in the mice from
that in the humans, human-like drug clearance was achieved by administering 5-10
mg/kg uranyl nitrate 3 days prior to treatment to induce a stable and reversible renal
impairment sufficient enough to delay drug elimination. Drug infusion was adjusted to
mimic the exposure in humans including peak drug concentration and half-life. The
bacterial densities in the animal studies were evaluated after 24 hours and the change
in log10 cfu/mL was determined against the starting density. The efficacies were
compared to ceftazidime alone.
In the murine lung infection model, the time at which the serum free drug
concentrations remains above MIC (fT>MIC) in the epithelial lining fluid (ELF) was also
evaluated in the neutropenic mice.90 Again, the efficacy was evaluated based on the
change in bacterial density after 24 hours. The study found that for isolates with
ceftazidime MIC of 32 μg/mL, the corresponding ELF fT>MIC was ≥ 12% and bacterial
reductions were observed. No reduction in bacterial density was observed in isolates
with ceftazidime MIC of 64 μg/mL. This example shows how simulation of human drug
PK in the murine model can be used to evaluate and extrapolate efficacy.
34
4.4 Modeling strategies to counter development of bacterial
resistance
Though the approach using PD surrogate indices, such as AUC24/MIC, has been
used to guide dosing of antimicrobial agents, some investigators have suggested that
these indices may not be appropriate for prediction of bacterial resistance.92-94 The
surrogate indices approach assumes that the MIC is stationary, which is not the case,
as bacteria will acquire resistance and adaptation when faced with antibiotic therapeutic
pressure. Lee et al. showed that few highly resistant mutants improve the survival of the
population’s susceptible constituents by emitting signal molecules to its surrounding to
turn on protective mechanisms, including upregulation of drug efflux pumps, during an
antimicrobial stressed environment.95 When bacteria are exposed to insufficient drug
concentrations to eradicate the resistant mutants, the population will acquire resistance
that result in a shift towards higher MIC.96 For example, the AmpC β-lactamase is
induced when the bacteria are exposed to β-lactams.97 Thus, simplification of dosing
schemes based on the PD surrogate indices may foster emergence of resistant
bacterial populations.
Baquero suggested a dangerous zone of drug concentration that potentially
confers survival advantage to these resistant mutants;92, 93 this zone is called the mutant
selection window (MSW).98-100 The MSW is the range of drug concentrations between
the MIC of the susceptible bacteria and the mutant prevention concentration, which is
the concentration that inhibits the growth of resistant mutants when evaluated at high
inoculum (>109 cfu/mL) using agar dilution method. Firsov and colleagues have shown
that the time spent within the MSW (TMSW) is a better predictor of emergence of
35
resistance in S. aureus following exposure to fluroquinolones.101, 102 For TMSW >20% of
the dosing interval, susceptibility of S. aureus against gatifloxacin, levofloxacin,
moxifloxacin and ciprofloxacin declined regardless of the AUC24/MIC ratio whereas
there was no change in bacterial susceptibility at TMSW<20% of dosing interval.101
Khachman et al. utilized fTMSW ≤ 20% and fTMSW ≤ 30% to estimate the probability of
attainment for various ciprofloxacin dosing regimens against several Gram-negative
pathogens in the intensive care unit (ICU).94 Their mutant prevention concentrations
were assumed to be 4- to 6-fold of MIC. With a population pharmacokinetic model of
ciprofloxacin in ICU patients, their simulations have shown that the PTA for fTMSW ≤ 20%
were less than 40% for MIC between 0.25 and 1 mg/L against P. aeruginosa and
Acinetobacter baumannii and the authors recommend against using ciprofloxacin for
these two bacteria types.94
Models of time-kill kinetics depicting both susceptible (S) and resistant (R)
bacteria are accomplished by setting two compartments for the two susceptibility states.
The overall total number of bacteria would then be the sum of these two state
populations. The assumption is that resistant bacteria come from de novo spontaneous
mutations.103-105 The mutation frequency is low at a rate of 10-8 to 10-6 per cycle and the
R/S ratio is set to 10-6 as their initial value in the model.106 Nielsen presented a two-
compartment model to describe the in vitro effect of moxifloxacin, vancomycin,
benzylpenicillin, cefuroxime and erythromycin against Streptococcus pyogenes.107 In
their assumption, the antibiotics have no effect on the persistent population. In a follow-
up study, Mohamed et al. introduced two additional compartments to regulate
resistance development in E. coli against gentamicin.108
36
In characterizing antibiotic effect on rise of resistant bacteria in piglets, Nguyen et
al. utilized the logistic growth model to describe both the S and R bacteria.109 They
argued that the rise in ciprofloxacin-resistant bacteria more likely comes from the
environment (i.e. ingestion of resistant bacteria). The difference in fitness between the S
and R was taken into account by assuming that the rate of growth of S is greater than
that of the R. The altruistic efforts of resistant bacteria to confer resistance to its
susceptible constituents comes at a cost to its own fitness.95 Nguyen and colleagues
found that the best fit to their data was when the model assumed that ciprofloxacin had
no activity on the resistant bacteria; alternative assumptions were such that the ratio of
EC50 values of ciprofloxacin against resistant and against susceptible populations were
4, 16 and 100. Their study recommends reducing intestinal exposure of piglets to
antibiotic residues in order to minimize the excretion and dissemination of resistant
bacteria to the environment in the animal feces.109
Other investigators have developed technique to quantify resistant bacterial
subpopulation from an in vitro time-kill experiment by plating the bacteria in agar plates
containing drug concentration three times or greater than the MIC, which ensures that
susceptible bacteria would not survive in the drug-containing agar plate.110 Tam et al.
applied the PK-PD model to describe the dynamics of the response of garenoxacin-
sensitve and resistant subpopulations of P. aeruginosa and S. aureus to fluctuating
quinolone drug concentrations.110, 111 The results of their study indicated that exposure
below a specific breakpoint allowed resistant subpopulation to grow rapidly. Jumbe et
al. utilized simulation approach in estimating the proliferation of resistant population in
37
insufficient antimicrobial therapy and evaluated dosing regimens required to prevent in
vivo amplification of resistant subpopulation.112
Other mechanistic models utilized three-state susceptibility, consisting of
susceptible, intermediate and resistant states, plus a fourth compartment to account for
the difference between the initial total bacterial burden and the initial conditions of the
other three states; this model was used to investigate colistin effect on P. aeruginosa.113
Their model also considered the ability of colistin to displace Mg2+ and Ca2+
competitively from the binding sites in the outer membrane; this cation displacement is
the mechanism of colistin killing. The fractional occupancy of the aggregate cations was
also defined in their model to take into account the relative dissociation rates of the
cations and colistin. Common with increasing complexity of models is that very often
parameters are fixed and simulation rather than fitting is often the approach taken, as
we discussed in previous section.
5. Conclusion
The PK-PD of antimicrobial agents and the tools available to evaluate drug
activities are well-established and tested through various candidates that eventually
became commercially available drug products. As there are tremendous unmet medical
needs for drug discovery and development programs targeting suppression of
resistance selection and eradication of multidrug resistant infection, PK-PD modeling
and simulation is well positioned to tackle the challenges of getting the right dosing
strategy for various patient populations. As we have shown here, many of the current
38
dosing regimens for the newer antimicrobial agents benefitted tremendously from
understanding of the PK-PD relationship between the drug and the bacterial infection.
6. Expert Opinion
In this article, we established the fundamental principles in antimicrobial PK and
PD, including modeling and simulation strategies. Table 3 summarizes these
methodologies, objectives of the approach, advantages and disadvantages and their

application in dose optimization. Many of the development of antimicrobial agents are
still based on establishing the PD surrogate index while modeling and simulation
approach is still underutilized. Empirical, mechanistic and semi-mechanistic PK-PD
models presented in the literature have shown good predictive value and are aptly
capable of iterative optimization of trial designs. We show examples wherein modeling
and simulation approach can give more information than the MIC-based PD indices,
which is based on the assumption that the MIC is stationary over time. It has been
shown that MIC can shift resulting from bacteria acquiring resistance, especially when
bacteria are exposed to a low concentration of drug that is insufficient to eradicate
them.96 Relying on a “snapshot” view of MIC for defining the PK-PD relationship for the
entire treatment duration can be misleading and also foster emergence of resistance.
Complementing the MIC-based approach, drug developers should also consider
utilizing in vitro time-kill curve information through modeling and simulation to define and
refine the PK-PD relationship.
Another trend that we have seen is that many of the refinement and optimization
of dosing regimens in special population are done by clinicians and researchers, outside
39
of drug development and after the drug has already been approved for marketing. Some
of the antimicrobial agents where dosing regimens in special population were
determined long after the drug was approved include polymixin B and collistin.114-116
Polymixin and colistin were antibiotics available in the 1950s but became out of use
when other antibiotics with better safety profiles were approved. These two drugs are
making a comeback recently due to diminishing therapeutic options in face of multidrug-
resistant Gram-negative bacteria.115 Recent report on population PK of polymyxin B in

critically ill patients indicates that the drug is best scaled by total body weight and that
renal function has no impact on polymyxin PK.114 Another example of antibiotic
combination where modeling and simulation in special population have been applied as
post-approval analysis is the combination of piperacillin and tazobactam in patients with
renal disease117-119 and in obesity.120-122 The population PK models in these populations
started to appear in the literature as late as 2010, even though the combination therapy
was originally approved in 1993. Even with the recently approved ceftolozane-
tazobactam combination, the literature on PK-PD model that utilizes the in vitro time-kill
kinetic information is still not available.
Thus, we see many opportunities moving forward to utilize state-of-the-art tools
to further our understanding of dosage optimization during drug development. Both
theories and applications examined here provide an overall understanding of how these
tools can be used to streamline drug development process of antimicrobial agents and
help make crucial decisions including clinical trial design. The search for regimens and
drug combinations for effective antibacterial activities can benefit tremendously from
modeling and simulation approach. More progress is needed to maximize the benefits
40
to the patients by implementing evidence-based treatment programs that evaluate and
incorporate all available information from in vitro studies, animal models of infections
and clinical trials.
Declaration of Interest
SKB Sy is currently an employee of Nektar Therapeutics. L Zhang discloses that the
work contributed to this manuscript took place while at the University of Florida and prior
to joining the US Food and Drug Administration. The opinions presented here are those
of the authors and no official support or endorsement by the FDA is intended or should
be inferred. The authors have no other relevant affiliations or financial involvement with
any organization or entity with a financial interest in or financial conflict with the subject
matter or materials discussed in the manuscript apart from those disclosed.
41
Abbreviations
ADME Absorption, distribution, metabolism and elimination
AUC Area under the concentration-time curve
CFR Cumulative fraction of response
cfu Colony forming unit
Cmax Maximum drug concentration within a dosing interval

Cmin Minimum drug concentration within a dosing interval
f Free drug concentration
ICU Intensive care unit
MIC Minimum inhibitory concentration
MSW Mutant selection window
PK-PD Pharmacokinetic-Pharmacodynamic
PTA Probability of target attainment
R Resistant
S Susceptible
SC Static concentration
T>MIC Time above MIC as a percentage of the dosing interval
T>TC Time above threshold concentration
TMSW Time spent within the mutant selection window
42
Bibliography
Papers of special note have been highlighted as:

* of interest
** of considerable interest
1. Asin-Prieto E, Rodriguez-Gascon A, Isla A. Applications of the
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Chemother 2015;45:504-11.
2. Sy SK, Derendorf H. Pharmacometrics in bacterial infections. In: Schmidt S,
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* This article provides a concise overview of pharmacokinetic modeling approach
in general.
3. Nielsen EI, Friberg LE. Pharmacokinetic-pharmacodynamic modeling of
antibacterial drugs. Pharmacol Rev 2013;65:1053-90.
** This article is a very thorough review of antimicrobial PK-PD research up until
2013.
4. Heerdink ER, Urquhart J, Leufkens HG. Changes in prescribed drug doses after
market introduction. Pharmacoepidemiol Drug Saf 2002;11:447-53.
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Table 1: Pharmacodynamic indices and their target values by antimicrobial class and
agents. Table modified from Asin-Prieto et al. (2015)1.
Antimicrobial Agent PD index Target value Ref

86
Aminoglycosides Cmax/MIC 10
β-lactams
123
Penicillins fT>MIC 50-60%
123
Cephalosporins fT>MIC 60-70%
123
Carbapenems fT>MIC 40-50%
124
Monobactams fT>MIC 60-70%
Clindamycin
27-30
Colistin fAUC/MIC 6 – 46 (variable depending on
bacteria type and strain)
Glycopeptides/lipopeptides
125
Daptomycin AUC/MIC 666
126, 127
Teicoplanin Cmin/MIC ≥10 for serious Gram-positive
≥20
128
Televancin AUC/MIC 219
129
Vancomycin AUC/MIC 400
Macrolides
130
Azithromycin fAUC/MIC 25
130
Clarithromycin fAUC/MIC 25
131
Teilithromycin fAUC/MIC 30
Oxazolidinones
132
Linezolid AUC/MIC 100
133, 134
Quinolones
135, 21
Gatifloxacin AUC/MIC 140
136, 22
Ciprofloxacin AUC/MIC 125
135, 21
Levofloxacin AUC/MIC 100
137, 23
Moxifloxacin AUC/MIC 95
Tetracyclines
138
Tigecycline fT>MIC 25
62
Table 2: Molecular classification (Ambler) of β-lactamases. Table modified from Bush et al. (2010)35.
Molecular β-lactam substrates Inhibitors Representative Comments

class (active enzymes
site)
A (serine) Penicillins, extended Clavulanic TEM, SHV, Hydrolysis of benzylpenicillin and
spectrum acid, CTX, KPC cephalosporins, TEM-30, TEM-50 and

cephalosporins, tazobactam, SHV-10 are resistant to sulbactam,
monobactams, sulbactam, tazobactam and clavulanic acid
carbenicillin avibactam
B (zinc) Carbapenems None NDM, IMP, VIM, Monobactams are impervious to IMP-1,
IND VIM-1 and IND-1
C (serine) Cephalosporins Avibactam AmpC, CMY Increased hydrolysis of cephalosporins
than benzylpenicillin, oxyimino-β-
lactams
D (serine) Carbapenems, Variable‡ OXA Hydrolyzes cloxacillin or oxacillin,
extended-spectrum oxyimino-β-lactams and carbapenems
cephalosporins,
cloxacillin
‡
The commercially available β-lactamase inhibitors have limited coverage of class D enzymes.
63
Table 3: Summary of methodologies in characterizing antimicrobial agents PK-PD properties
Method/Approach Purpose Advantages/Disadvantages Applications to Dose Optimization
MIC and PD To determine PD Advantages: the methodology is relatively straight forward and the PD surrogate index is an important factor to
surrogate index surrogate index of processes to characterize PD surrogate index are well established. consider for dosing intervals, infusion
an agent Disadvantages: the empirical PD surrogate indices for an durations, as well as the dose of the
antimicrobial agent often show co-linearity, which can make dose antimicrobial agent. For newer antimicrobial
selection quite difficult. MIC in broth may not be representative of true agents, the characterization of their PD
MIC in biological fluids. surrogate index is mandatory.
Static time-kill To characterize the Advantages: The static time-kill curves are used to develop the PK- PK-PD models are developed from the
kinetics time-course of PD model, utilizing the information on changes in bacterial density in information from static time-kill kinetics for
bacterial response response to an antibiotic challenge maintained at constant the purpose of simulating bacterial
to antimicrobial concentration. response to non-static antibiotic
challenge at fixed Disadvantages: The static time-kill curves are very labor intensive. In concentration that mimics the kinetic
drug concentration combination studies, the number of these curves to characterize well behavior of the drug in vivo.
the combined drug effect can increase exponentially.
Dynamic time-kill To evaluate the Advantages: The dynamic time-kill kinetic studies mimic human Several dosing regimens can be evaluated
kinetics time-course of pharmacokinetic behavior of the drug. This set-up allows various with the dynamic time-kill kinetics.
bacterial density dosing regimens to be simulated in vitro. The characteristic PD
change when the surrogate index for a drug can be determined by evaluating several
antibiotic dosing regimens in the dynamic time-kill experiments.
concentration is Disadvantages: The hollow fiber equipment that automates the
changing according process of dynamic time-kill experiment is very expensive. Mere
to its half-life in information from dynamic time-kill experiments is insufficient for
humans model development. Thus, the data from dynamic time-kill kinetics
are often modeled together with data from the static time-kill kinetics.
Additionally, dynamic time-kill kinetics requires information on drug
half-life in humans. Consequently, the human pharmacokinetics of
the drug has to be available by the time the dynamic time-kill kinetic
studies are planned.
Microdialysis To determine time- Advantages: Microdialysis allows for direct measurement of the free There are a number of literatures that
course of free drug concentration at the site of infection. PD surrogate index based looked at the free drug concentration at the
unbound drug on microdialysis-collected information is more relevant especially if tissue sites. Evaluating the PD surrogate
concentration at the the PD surrogate index is determined from time-kill kinetic study. index based on the free drug concentration
tissue site Disadvantages: Validating the microdialysis procedure for the specific at the site of infection is a more direct
antimicrobial agent is a tedious process. Feasibility study is required method to evaluate whether sufficient
prior to running the microdialysis trial in humans. antimicrobial activities are occurring at the
site of infection.
64
Animal models of To determine the in Advantages: Humanized dosing in neutropenic or immunocompetent This procedure can be used to determine
humanized dose vivo efficacy using mouse thigh- or lung-infected model is pertinent to evaluate in vivo the duration of therapy as well as to
human-like drug efficacy of antimicrobial agents. One can evaluate human-like validate the dose and dosing interval
disposition pharmacokinetics in the animal model of infection. This procedure determined to achieve optimized PD
can provide information on the duration and length of therapy surrogate index target.
required to clear the infection.
Disadvantages: The procedure is predictive for drugs that are renally
cleared. For drugs with extensive hepatic metabolism, the human-like

pharmacokinetic may be difficult to mimic.
Modeling To develop PK-PD Advantages: Once the population pharmacokinetic model is This is a translational research application
model that developed in the relevant human population, predictions of the time- utilizing the information of human
describes the course of different dosing regimens and the extent of variability pharmacokinetic and linking in vitro
relationship between individuals can be done. The development of the PK-PD information of bacteria response to
between drug model can provide insights into how drug disposition in humans is pertinent drug concentrations.
concentration and affecting the dynamics of bacteria growth and kill.
its effect on Disadvantages: Human pharmacokinetic data is required for building
bacterial density the population pharmacokinetic model. The pharmacokinetics in
diseased patients may not be the same as healthy volunteers.
Model simulations To predict the Advantages: Simulations allow for investigation of the effect of Simulations are performed to provide better
bacteria dynamics various dosage regimens on the hypothetical growth and kill of confidence of the dosage regimen and
in various proposed bacteria in vivo, before designing or running a clinical trial. insights of the trial design, especially for a
dosing regimens Simulations can provide better confidence on the decided dosage phase III trial. In the clinic and hospital
regimen and to some extent the required duration of therapy before setting, simulations are used to predict the
running a trial. Simulations are also utilized to determine the best next dose for therapeutic drug
optimized “next” dose for a patient in a therapeutic drug monitoring monitoring of patients to achieve a specific
setting. target drug concentration.
Disadvantages: Prior to running a simulation, one needs to have a
model developed.
Probability of target To determine the Advantages: PTA provides statistical quantification of whether a Once the human pharmacokinetics is
attainment (PTA) percentage of the dosage regimen can achieve the PD surrogate index for the target determined including interindividual
patient population population. One can also evaluate the PTA for increasing MIC values variability, the utility of several dosage
that will achieve the and understand the limit of the dosage regimen against drug resistant regimens in attaining the required PD
required PD bacteria population. surrogate index can be evaluated and
surrogate index for Disadvantages: There are some indications that PTA values are not compared. PTA of a dosage regimen is
a specific dosage representative of the clinical outcomes. plotted against increasing MIC values.
regimen
65
Figure 1: Pharmacodynamic indices (24-hr AUC/MIC, Peak/MIC and Time above MIC) for ceftazidime in relationship to
the 24-hour log10 cfu of K. pneumoniae in the lung-infected neutropenic mice model. AUC, area under the curve; MIC,
minimum inhibitory concentration; cfu, colony forming unit.
Adapted with permission from Andes and Craig (2002)16.

66
Figure 2: Patterns of bactericidal activity of tobramycin, ciprofloxacin and ticarcillin in 6-hr constant concentration time-kill
kinetic studies. cfu, colony forming unit; MIC, minimum inhibitory concentration.
Adapted with permission from Craig and Ebert (1990)17.

67
Figure 3: Determination of pharmacodynamic index of linezolid by fitting a sigmoidal Emax model and determining the
highest R2 value for the relationship between microbiological outcome and pharmacokinetic parameter. MIC, minimum
inhibitory concentration; cfu, colony forming unit; AUC, area under the curve; %T>MIC, percentage of the dosing interval
wherein drug concentration is above MIC.
Adapted with permission from Andes et al. (2002)18.
68
Figure 4: Prediction of antibacterial activity of vertilmicin, an aminoglycoside in development, under three dosing
regimens. The top graphs represent drug concentration of vertilmicin and the bottom graphs are hypothetical P.
aeruginosa response to the three dosing regimens. QD, once daily dosing; BID, twice daily dosing.
Adapted with permission from Zhuang et al (2015)66.
69

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Expert Opinion on Drug Metabolism & Toxicology

ISSN: 1742-5255 (Print) 1744-7607 (Online) Journal homepage: http://www.tandfonline.com/loi/iemt20

Pharmacokinetics and Pharmacodynamics in

Sherwin K. B. Sy, Luning Zhuang & Hartmut Derendorf

To link to this article: http://dx.doi.org/10.1517/17425255.2016.1123250

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Journal: Expert Opinion on Drug Metabolism & Toxicology

Sherwin K. B. Sy1,2, Luning Zhuang1,3 and Hartmut Derendorf1‡

Hartmut Derendorf, PhD

development of antibiotics. Suboptimal regimens and inappropriate choice of drug give

commercially available antimicrobial agents. Strategies to optimize therapy of

antimicrobial candidates to speed up the development process are urgently needed.

Areas covered: We examined pharmacokinetics and pharmacodynamics of

based on minimum inhibitory concentration to evaluate antimicrobial dosing strategy is

bacterial response to a particular dosing regimen. Animal studies of dosing regimens

that mimic human pharmacokinetics are another option to evaluate antimicrobial

efficacy. Empirical, semi-mechanistic and mechanistic models of bacterial dynamics and

development of drug resistance in response to drug therapy are discussed.

more rigorous integration of PK-PD modeling approaches even at preclinical stage to

strategies in relevant populations where the drug is mostly used.

Keywords: PK-PD modeling, antibiotics, in vitro models, translational research

 We identified and summarized PK-PD approaches that are routinely practiced in

drug development of antimicrobial agents

 The MIC-based approach is the most extensively used approach to identify PD

index of antibiotic that is predictive of microbiological efficacy and outcome

can be more rigorously applied in the evaluation of drug candidates

information related to the time-course of antibacterial activities as well as

development of drug resistance in bacteria

 PK-PD has been successfully applied in optimizing antimicrobial dosing regimens

The utility of pharmacokinetic-pharmacodynamic (PK-PD) approach in

antimicrobial drug development is very well established and now acknowledged by

existing commercially available antimicrobial agents to combat global rise in drug-

due to change in susceptibility towards higher drug resistance in the target

microorganism.4 Therefore, it is important that drug developers determine the optimal

concepts are broadly applicable to drug development in other therapeutic areas.

Once an antimicrobial agent is administered to a patient, there are processes

of distribution, bioavailability and protein binding. These parameters allow one to

course of the pharmacological effect, which in turn is dependent on the time-course of

the PK behavior and the dosing regimen.

In antimicrobial therapy, the time-course of the pharmacological effect, which is

concentration (MIC) can provide valuable information on the susceptibility of the

response against the infection; development of multi-drug resistant bacteria in response

drug’s PK and PD properties.

2.1 MIC-Based Approach

activity in rodents and the concentration-dependent killing of streptomycin and

administered as continuous infusion while concentration-dependent agents are better

given as intravenous bolus to achieve high maximum concentrations.10, 11 In the 90s,

Craig expanded on the concept of PD indices through a number of rodent studies

2.1.1 Pharmacodynamic Indices

The quantitative relationship between PK parameter and microbiological

time-kill kinetic studies, is referred to as a PD surrogate index. The characterization of

antimicrobial killing effect are summarily characterized as either time-dependent or

concentration-dependent killing effects. Some investigators included a third

classification as concentration-independent killing with prolonged post-antibiotic

is exposed to the drug until the survivors start to multiply rapidly.14

“concentration-independent” or “time-dependent” kill. That is because the efficacy of β-

postantibiotic effect against gram-negative bacteria.14, 15 A classic illustration of β-lactam

exhibiting time-dependent killing effect is shown in Figure 1. In this example of

ceftazidime against Klebsiella pneumoniae ATCC 43816 in the lungs of neutropenic

dosing interval achieves the greatest bacterial kill.