On the Influence of Protein Binding on Pharmacological

Activity of Drugs

LEONID M. BEREZHKOVSKIY
Genetech Inc., 1 DNA Way, South San Francisco, California 94080

Received 6 July 2009; accepted 26 August 2009

Published online 13 October 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21958

ABSTRACT: The effect of variable protein binding (taken as independent parameter) on

pharmacological activity of drugs is considered in terms of the exposure or the steady
state concentration of unbound drug at targeted tissue. Based on the application of the
parallel tube or dispersion models it is shown that for the most common case of orally
administered drugs eliminated mainly by hepatic metabolism the increase of protein
binding may be beneficial for drug action. In contrary, consideration of this case using
the well-stirred model suggests that changes in protein binding do not influence drug
efficiency. The relatively simplistic well-stirred model appears not accurate enough to
reveal the influence of variation in protein binding on drug exposure. The conclusion in
favor of the predictions based on parallel tube or dispersion models is supported by
experimental data. In case of the oral dosing of drugs that are subjected to nonhepatic
elimination as well as for parenteral drug administration with arbitrary routes of
elimination the decrease in protein binding would lead to the increase of unbound drug
exposure and thus may enhance drug efficiency. An advanced approach to evaluation of
drug activity based on the assumption of the necessity to exceed certain minimal drug
concentration at action site is implied. Such a consideration leads to the conclusion that
there should be an optimal value of protein binding which provides maximum drug
activity. The case when drug action is determined by binding to targeted receptors is
discussed in terms of equilibrium binding and kinetics. ß 2009 Wiley-Liss, Inc. and the
American Pharmacists Association J Pharm Sci 99:2153–2165, 2010
Keywords: protein binding; drug exposure; drug efficiency; bioavailability; well-
stirred model; parallel tube model; dispersion model; tissue–plasma partitioning;
clearance; drug–receptor binding

INTRODUCTION tion as independent parameter), the mechanism of

The influence of protein binding on drug pharma-
cokinetics and pharmacological effects is widely
recognized, and appears to be a common con-
sideration in drug discovery and development.1 In
general, to assess the impact of protein binding on
drug activity (assuming the unbound drug frac-
Correspondence to: Leonid M. Berezhkovskiy (Telephone:
650-225-1798; Fax: 650-467-4836;
E-mail: berezhkovskiy.leo@gene.com)
Journal of Pharmaceutical Sciences, Vol. 99, 2153–2165 (2010)
ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
drug action should be known. A relevant PK/
PD model needs to be developed to study the
contribution of different factors (including dosing
regiment) on achieving the optimal drug per-
formance. A simplified consideration of reaching
greater exposure, AUCu,t, of the unbound drug in
the targeted tissue for a given fixed drug dose may
be applied at the early screening stage of drug
development. The advanced models may also
consider the drug affinity to the target receptor
and possibly kinetics of drug–receptor binding.
The assumption that drug transfer to the
targeted tissue (or organ) is provided by a passive

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010 2153

2154 BEREZHKOVSKIY
diffusion allows reducing the evaluation of
the compound efficiency to consideration of the
exposure, AUCu, of the unbound drug in plasma.
Indeed, for the passive diffusion from plasma to
tissue, the quantity of drug in tissue, At(t), is
described as
dAt ðtÞ
¼ PS½Cu ðtÞ  Cu;t ðtÞ (1)
dt
where Cu(t) and Cu,t(t) are the concentrations of
unbound drug in plasma and tissue extracellular
water, respectively, PS is the product of per-
meability coefficient, P, and the plasma–tissue
contact surface area, S. Integrating Eq. (1) over
time from t ¼ 0 to infinity with the account that
At(t ¼ 0) ¼ At(t ! 1) ¼ 0 yields
AUCu;t ¼ AUCu (2)
The common measurements of the total drug
plasma concentration, C(t), and the unbound
drug fraction in plasma, fu, yield the exposure
of unbound drug in plasma
AUCu ¼ fu AUC (3)
where AUC is the exposure of the total drug
in plasma. Since AUCu,t ¼ AUCu (Eq. 2), Eq. (3)
eventually provides the exposure of the unbound
drug concentration in tissue. Protein binding
equilibration almost always occurs virtually
instantaneously, so that Cu(t) ¼ fuC(t) is a usual
assumption.2 Though if binding kinetics is linear
and protein bound drug is not eliminated, Eq. (3)
holds even if there is no instantaneous equilibra-
tion, that is, Cu(t) 6¼ fuC(t) during the time course
of drug in plasma.3 In case the target tissue
eliminates a drug, or there is efflux transport, an
extra term kVtCu,t is added into the right-hand
side of Eq. (1), where k is the rate constant
of the process and Vt is the tissue volume. The
term kVt can be considered as intrinsic tissue
clearance, Clint,t ¼ kVt, corresponding either to
drug elimination or efflux. Thus Eq. (1) becomes
dAt ðtÞ
¼ PS½Cu ðtÞ  Cu;t ðtÞ  Clint;t Cu;t ðtÞ (4)
dt
Integration of Eq. (4) (similarly as done with Eq. 1)
results in
AUCu
AUCu;t ¼ ¼ AUCu ð1  Et Þ (5)
1 þ Clint;t =ðPSÞ
where Et ¼ Clint,t/(Clint,t þ PS) is the tissue extrac-
tion ratio. Therefore the exposure of unbound
drug in tissue is proportional to the exposure
AUCu of the unbound drug in plasma. Thus
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
obtaining the higher value of AUCu due to the
variation of the unbound drug fraction fu, may be
assumed as a criterion for the evaluation of the
influence of protein binding on drug efficiency.
Some limitations of this approach are considered
in the Discussion section. It is assumed further
that the contribution of possible drug elimination
by the targeted tissue to the total body clearance is
negligible.
Drug protein binding depends on the concen-
tration of plasma proteins, which can be altered
due to various diseases, or, not that often, due to
possible protein binding displacement by another
drug (competitive protein binding),4 or due to
relatively high drug concentration.5 It was pub-
licized (based on the application of well-stirred
model) that for the most common case of the orally
administered drugs subjected mainly to hepatic
elimination, that AUCu is independent of protein
binding.6 Thus for such a consideration of this
case, changes in plasma protein binding may have
little clinical relevance.
The goal of this article is to show that a more
detailed consideration actually suggests that the
increase of protein binding can be beneficial for
drug action (in terms of increasing AUCu) in the
particular case of the orally administered drugs,
which are eliminated by hepatic metabolism
only. It is also shown that for the case of oral
drug administration and nonhepatic elimination,
and for the case of parenteral administration
and both hepatic or nonhepatic elimination, the
decrease in protein binding may enhance the
drug pharmacological activity. It is pointed out
that the same conclusions about the influence of
protein binding on drug effectiveness are valid
when a continuous drug administration is con-
sidered and the value of the steady state unbound
drug concentration in plasma, Cu,ss, is taken a
marker of drug activity. In addition, the use of
apparently more accurate criteria of drug effi-
ciency, rather than AUCu, is considered, which
leads to the conclusion that there should be an
optimal value of protein binding resulting in
highest drug activity.
DETERMINATION OF DRUG EXPOSURE
IN PLASMA
The total body clearance (calculated with respect
to drug plasma concentration) is defined as
Cl ¼ FD/AUC, where FD is the quantity of drug
that entered the circulation, D is the drug dose
DOI 10.1002/jps

and F is bioavailability. For the intravenous
administration of dose Div (assuming the absence
of the first-pass lung metabolism), D ¼ Div and
F ¼ 1, and D ¼ Dpo for the oral administration of
dose Dpo. Consequently the total exposure can be
calculated as AUC ¼ FD/Cl, and according to
Eq. (3) the exposure of unbound drug in plasma is
FD
AUCu ¼ fu (6)
Cl
In general, the total clearance is the sum of
clearances provided by each elimination route
(organ or tissue), and the model of elimination
organ is applied to obtain its clearance (commonly
considering linear models and using the steady
state approach7). The clearance value obtained
from the steady state condition of eliminating
organs is actually the same as the mean value
D/AUC obtained from the plasma concentration
time course after intravenous input of dose D. This
appears to be an intrinsic property of linear
pharmacokinetic system. Indeed, at steady state
of the linear system corresponding to iv drug
infusion at constant rate R, the steady state
plasma concentration, Css, is provided by the
general equation Css(D/AUC) ¼P R. The rate of
elimination at steady state, Ess ¼ Ess,i is equal to
the rate of infusion, that is, Ess ¼ R, where Ess,i are
the steady state elimination rates of individual
organs. Thus Ess/Css ¼ R/Css ¼ D/AUC, which
denotes the equality of the mean D/AUC and
the
P sum of the steady state organ clearances
Ess,i/Css ¼ Ess/Css.
The oral bioavailability (assuming negligible
lung first pass effect) is calculated as
F ¼ Fa Fg Fh (7)
where Fa is the fraction of administered drug that
is absorbed into the gut wall, Fg is the fraction of
the absorbed drug that gets through the gut wall
unchanged, and the hepatic bioavailability Fh is
the fraction of drug which subsequently passes
unchanged through the liver into the systemic
circulation.
Oral Drug Dosing, Hepatic Elimination
Let us first consider the common case of oral drug
administration, and hepatic clearance as being
the only route of drug elimination. For oral dosing,
according to Eq. (6)
FDpo
AUCu ¼ fu (8)
Cl
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PHARMACOLOGICAL ACTIVITY OF DRUGS 2155
Hepatic clearance, calculated with respect to drug
plasma concentration, is provided as
Clh ¼ rQEh ¼ rQð1  Fh Þ (9)
where r is the equilibrium blood–plasma concen-
tration ratio, Q is the rate of liver blood flow, and
Eh ¼ 1  Fh is the drug hepatic extraction ratio.
For the case of only hepatic elimination, taking
Cl ¼ Clh in Eq. (8) and applying Eq. (7) for F yields
Fa Fg Fh Dpo
AUCu ¼ fu (10)
rQEh
Let us apply the two commonly used models for
the calculation of Clh by Eq. (9). According to the
well-stirred model
Clint fu
Eh ¼ (11)
rQ þ Clint fu
and according to the parallel tube model
 
Clint fu
Eh ¼ 1  exp  (12)
rQ
where Clint is the intrinsic hepatic clearance.8
Substitution of Eq. (11) for Eh and Fh ¼ 1  Eh in
Eq. (10) yields for a well-stirred model
Fa Fg Dpo
AUCu ¼ (13)
Clint
Thus Eq. (13), which does not contain the term fu,
leads to the conclusion that AUCu is independent
of protein binding. Therefore the changes in
protein binding have no clinical relevance in this
case, that is, no dose adjustment is needed to
maintain the same exposure AUCu (and even-
tually sustain the drug action) to account in
regard to the variation of protein binding.6 As
considered in the Discussion section, the inde-
pendence of AUCu on fu is a mathematical artifact
of the equation for clearance given by the well-
stirred model.
On the other hand, if hepatic clearance is
obtained from Eq. (12) for parallel tube model, we
get
Fa Fg Dpo exp½Clint fu =ðrQÞ
AUCu ¼ fu (14)
rQf1  exp½Clint fu =ðrQÞg
For such consideration, Eq. (14) shows a strong
dependence of the exposure AUCu on the value
of unbound drug fraction fu. This is shown in
Figure 1A for AUCu calculated using well-stirred
and parallel tube models. AUCu in Figure 1A is
normalized by the factor N ¼ FaFgDpo/Clint to
bring data to the same scale, so that according
to Eq. (10) AUCu/N ¼ (Clint/rQ)( fuFh/Eh) ! 1 when
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2156 BEREZHKOVSKIY
Figure 1. Case of oral drug administration and
elimination by hepatic metabolism. Comparison of
AUCu dependence on the unbound drug fraction fu for
calculations based on well-stirred (Eq. 13) and parallel
tube (Eq. 14) models at different values of intrinsic
clearance. (Panel A) AUCu is normalized by the
factor N ¼ FaFgDpo/Clint. - - -, Calculation by well-stirred
model (does not depend on Clint/rQ); —, calculation by
parallel tube model: (1) Clint/(rQ) ¼ 0.2, (2) Clint/
(rQ) ¼ 0.5, (3) Clint/(rQ) ¼ 1, (4) Clint/(rQ) ¼ 2, (5) Clint/
(rQ) ¼ 5, (6) Clint/(rQ) ¼ 10. (Panel B) AUCu is normal-
ized by the factor K ¼ FaFgDpo/(rQ). - - -, Calculation by
well-stirred model, —, calculation by parallel tube
model: (1) Clint/(rQ) ¼ 0.2, (2) Clint/(rQ) ¼ 0.5, (3) Clint/
(rQ) ¼ 1, (4) Clint/(rQ) ¼ 5.
4a
Fh ¼
ð1 þ aÞ exp½ða  1Þ=2DN   ð1  aÞ2 exp½ða þ 1Þ=2DN 
2
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
fu ! 0 for both models. Only in the limiting case of
low extraction ratio drug Clintfu << rQ Eq. (14) for
parallel tube model yields the same value of AUCu
as that calculated using well-stirred model
equation (13). For moderate and especially high
extraction ratio drug the change of AUCu pre-
dicted by the parallel tube model is quite
significant. For instance for the drug with
Clint/(rQ) ¼ 10 the decrease of AUCu is 156-fold
when the unbound fraction changes from 0.01
(99% protein binding) to 0.7 (30% protein binding).
Of course, there is a strong dependence of AUCu
on drug intrinsic clearance, which is shown in
Figure 1B, where AUCu/( FaFgDpo/rQ) ¼ fuFh/Eh
(Eq. 10) is plotted. The higher stability (smaller
value of Clint/(rQ)) results in substantial increase
of AUCu.
What is really important, is that AUCu
increases with the increase of protein binding
(decrease of fu). Thus the consideration of parallel
tube model results in the conclusion that for orally
administered drugs which are mainly eliminated
by liver, the increase of protein binding is
beneficial for drug action (in terms of getting
higher exposure AUCu). It means that in drug
development high protein binding should not be a
concern in the case of oral drug administration
and elimination by hepatic metabolism only.
Though (depending on the mechanism of drug
action) a very high protein binding could become
problematic issue, which is considered in the
Discussion section. In terms of clinical relevance,
the decrease of protein binding due to the
reduction of protein content of plasma (related
to disease) or due to drug–drug interaction would
require to increase the drug dose compared to that
given to subjects with normal levels of fu.
There is obviously a huge difference in the
prediction of AUCu by the well-stirred and parallel
tube models. The well-stirred model yields
the highest value of AUCu (independent of fu),
which coincides with the prediction of parallel
tube model in two limiting cases fu ! 0 and
Clint/(rQ) ! 0 (as seen in Fig. 1). The concentra-
tion of drug is rather changing along the organ (as
given by parallel tube model) than being constant
due to instantaneous uniformed distribution
(well-stirring). Both models are the extreme cases
of a general dispersion model,7 which gives
(15)
DOI 10.1002/jps

where RN ¼ fuClint/(rQ) is the efficiency number,
DN is the axial dispersion number, and a ¼
(1 þ 4RNDN)1/2. A well-stirred model corresponds
to DN ! 1 and the parallel tube model corre-
sponds to DN ! 0. Calculation of AUCu by Eq. (10)
using the dispersion model (Eq. 15) with
DN ¼ 0.17 9 is shown in Figure 2. As expected,
the dispersion model provides a significant
dependence of AUCu on the unbound drug
fraction, though less strong than that calculated
based on parallel tube model.
The direct experimental data of Diaz-Garcia
et al.10 on the kinetics of diazepam hepatic
elimination by the isolated perfused rat liver
allowed us to figure out the relevance of the
predictions made based on the well-stirred
and parallel tube models. The values of hepatic
bioavailability measured at different levels of
unbound drug fraction fu in perfusate were
provided in Table 1 of the article.10 We used
this data to calculate the factor fuFh/Eh,
which according to Eq. (10) provides AUCu as
fuFh/Eh ¼ AUCu/[FaFgDpo/(rQ)] (as plotted in
Fig. 1B). The experimental results10 shown in
Figure 3 are fitted using the dispersion model
with Clint/(rQ) ¼ 7.4, DN ¼ 0.18 and the parallel
tube model with Clint/(rQ) ¼ 6.7. These experi-
mental data are obviously in reasonably good
Figure 2. Case of oral drug administration and
elimination by hepatic metabolism. Comparison of
AUCu dependence on the unbound drug fraction fu for
calculations based on parallel tube (Eq. 14) and disper-
sion (Eqs. 10 and 15) models at different values of
intrinsic clearance. AUCu is normalized by the factor
N ¼ FaFgDpo/Clint. - - -, Calculation by dispersion model,
—, calculation by parallel tube model: (1) Clint/
(rQ) ¼ 0.2, (2) Clint/(rQ) ¼ 1, (3) Clint/(rQ) ¼ 3, (4) Clint/
(rQ) ¼ 10.
DOI 10.1002/jps
PHARMACOLOGICAL ACTIVITY OF DRUGS 2157
Figure 3. Comparison of the experimental data for
the dependence of fuFh/Eh ¼ AUCu/K, K ¼ FaFgDpo/(rQ)
(Eq. 10) on the unbound drug fraction fu with fuFh/Eh
calculated by dispersion and parallel tube models. &,
Experimental data of Diaz-Garcia et al.10 —, Calcula-
tion by dispersion model with Clint/(rQ) ¼ 7.4, DN ¼ 0.18,
- - -, calculation by parallel tube model with Clint/
(rQ) ¼ 6.7.
agreement with predictions of dispersion model
and definitely do not confirm that fuFh/Eh is
constant independent of fu as suggested by the
well-stirred model. The predictions of parallel
tube model are not as accurate as that provided
by the dispersion model, but exhibit the right
tendency in the dependence of fuFh/Eh on fu.
The use of a well-stirred model (which does not
seem quite physiological) both for eliminating
and noneliminating organs in the simulations of
drug pharmacokinetics is dictated by a relative
simplicity of this approach. Each tissue (organ) is
characterized by a single drug concentration in
this case, while for a parallel tube model there
should be a concentration profile along the organ
(assuming one-dimensional consideration, that is,
the existence of instantaneous radial equilibra-
tion8). The current profile is determined by the
concentration of drug in blood that entered
the organ at earlier instants of time. Just a
simple two-compartment model of a well-stirred
plasma and the eliminating organ considered in
a parallel tube mode yields quite complicated
solution for the plasma concentration–time
course.11
Oral Drug Dosing, Nonhepatic Elimination
Let us consider the orally dosed drugs that are not
eliminated by liver. In this case Fh ¼ 1 in Eq. (7)
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2158 BEREZHKOVSKIY

for oral bioavailability, so that F ¼ FaFg. We drugs application of the parallel tube model
consider that drug elimination is provided by a (Eq. 23) results in the same equations for the
single organ (most likely kidneys) and the organ exposure (Eqs. 21 and 22) as that obtained using
clearance, Clo, is described either by well-stirred the well-stirred model. The graphs, which show
or parallel tube model, which incorporates plasma the dependence of AUCu on protein binding
protein binding calculated using well-stirred and parallel tube
Clo ¼ rQo Eo (16) models, are shown in Figure 4. Both models
exhibit the same tendency of increasing AUCu
where Qo is the blood flow to the organ and Eo is with decrease of protein binding (slightly more
the organ extraction ratio. For a well-stirred pronounced according to well-stirred model).
model
Clint;o fu
Eo ¼ (17) Parenteral Drug Administration
rQo þ Clint;o fu
To obtain the exposure of a drug administered
and for parallel tube model
  intravenously as bolus dose or as infusion,
Clint;o fu assuming that the first-pass lung bioavailability
Eo ¼ 1  exp  (18)
rQo Flung ¼ 1, according to Eq. (6) we get
where Clint,o is the organ intrinsic clearance. Div
Applying the general equation (8) for AUCu with AUCu ¼ fu (24)
Cl
F ¼ FaFg and Cl ¼ Clo given by Eq. (16), results in
For a drug given by other parenteral route
Fa Fg Dpo
AUCu ¼ fu (19) Div should just be replaced by FparDpar in
rQo Eo the equations of this section, where Dpar is the
If a well-stirred model for elimination organ is parenteral drug dose and Fpar is parenteral
applied, then according to Eqs. (17) and (19), we bioavailability. Considering the elimination by
get either nonhepatic or hepatic route (Eq. 17 or
Eq. 18 depending on model choice) yields the
Fa Fg Dpo ðrQo þ Clint;o fu Þ
AUCu ¼ (20) same equation for AUCu as for the case of
rQo Clint;o
oral administration and nonhepatic elimination
Eq. (20) indicates that the increase of protein (Eqs. 20 and 23), but with FaFgDpo replaced by Div.
binding (smaller fu) leads to the decrease of the Thus for the well-stirred model
exposure AUCu. This is especially pronounce for
Div ðrQo þ Clint;o fu Þ
the high extraction ratio drugs (Clint,ofu rQo), so AUCu ¼ (25)
that AUCu is directly proportional to fu rQo Clint;o

Fa Fg Dpo and for the parallel tube model

AUCu ’ fu (21)
rQo
Div fu
For low extraction ratio drugs (Clint,ofu  rQo) AUCu ¼ (26)
rQo f1  exp½Clint;o fu =ðrQo Þg
Eq. (20) provides
Fa Fg Dpo To calculate AUCu for the case of a drug which
AUCu ’ (22) is eliminated by hepatic metabolism, the para-
Clint;o
meters Qo and Clint,o should be, respectively,
so that AUCu does not depend on protein binding replaced by Q and Clint in Eqs. (25) and (26). Same
in this case. equations for the asymptotic limits of high and low
For consideration of elimination organ by the extraction ratios (Eqs. 21 and 22 with FaFgDpo
parallel tube model Eqs. (18) and (19) yield replaced by Div) are valid for the intravenous
Fa Fg Dpo fu administration.
AUCu ¼ (23) Eventually the conclusion is that the larger
rQo f1  exp½Clint;o fu =ðrQo Þg
value of AUCu is obtained when protein binding
Eq. (23) provides that the increase of protein is lower (larger fu). Figure 4 (for consideration
binding leads to smaller values of the exposure of oral drug administration with nonhepatic
AUCu (same as for well-stirred model). For the elimination) also illustrates the dependence of
limiting cases of high and low extraction ratio AUCu on protein binding for the case of parenteral

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Figure 4. Case of oral drug administration and non-
hepatic elimination. Comparison of AUCu dependence
on the unbound drug fraction fu for calculations
based on well-stirred (Eq. 20) and parallel tube
(Eq. 23) models at different values of intrinsic clearance.
AUCu is normalized by the factor K ¼ FaFgDpo/(rQ).
(1) Clint,o/(rQo) ¼ 0.2, (2) Clint,o/(rQo) ¼ 0.5, (3) Clint,o/
(rQo) ¼ 2, (4) Clint,o/(rQo) ¼ 10; – – –, calculation by
well-stirred model, —, calculation by parallel tube
model.
drug administration (taking Div instead of
FaFgDpo in normalization factor).
DISCUSSION
According to the consideration of the previous
section both scenarios that are the increase and
decrease of the exposure AUCu with the increase
of fu, are possible depending on the routes of drug
administration and elimination. For oral dosing
AUCu is determined by the quantity of drug that
was absorbed from intestine and got through
the first-pass into systemic circulation, and by the
following elimination. Considering liver as the
only eliminating organ, the increase of fu leads to
the decrease of the first-pass unchanged drug
quantity, but to the increase of AUCu conse-
quently generated by this quantity (as finally
reflected in Eq. 10 by the factor fuFh/Eh).
Eventually, as supported by experimental data,
the combination of the parameters fu, Fh, and Eh
(as fuFh/Eh) results in the increase of AUCu after
oral administration with the increase of protein
binding (smaller fu). The simplest well-stirred
model is not accurate enough to exhibit the change
of AUCu with the change of fu, while apparently
DOI 10.1002/jps
PHARMACOLOGICAL ACTIVITY OF DRUGS 2159
more realistic models (dispersion and parallel
tube) show a strong influence of fu on AUCu value.
Interestingly, the well-stirred and parallel tube
models are in a reasonably good agreement in
calculation of hepatic clearance (within 1–1.3-fold
greater values provided by parallel tube model),
though lead to contradictory conclusions on the
prediction of AUCu for the case of orally adminis-
tered drugs which are eliminated mainly by
hepatic metabolism. Such a singularity of well-
stirred model in prediction of AUCu after oral drug
administration and hepatic metabolic elimination
only is actually embedded in the form of Eq. (11)
for the extraction ratio Eh. According to the
general equation (10), for this case AUCu would be
independent of fu if the factor fuFh/Eh were
constant. Taking
fu Fh fu ð1  Eh Þ
¼ ¼B
Eh Eh
where B is an arbitrary constant yields
fu =B
Eh ¼
1 þ fu =B
When B is taken as rQ/Clint, the equation above
exactly coincides with the equation for Eh
provided by the well-stirred model (Eq. 11), which
can be written as
fu =ðrQ=Clint Þ
Eh ¼
1 þ fu =ðrQ=Clint Þ
Thus the form of Eq. (11) given by the well-
stirred model for the hepatic extraction ratio Eh
underlies the independence of AUCu on fu for
a drug given orally and eliminated by liver
metabolism only.
If orally administered drug is eliminated mainly
by nonhepatic routes, the increase of the exposure
AUCu is expected with the reduction of protein
binding. This is also valid for parenteral drug
administration regardless the route of elimina-
tion. Both the well-stirred and parallel tube modes
are in agreement in prediction of the dependence
of AUCu on fu in this cases, though a little stronger
influence of the increase of fu on the increase of
AUCu is expected according to well-stirred model
(Fig. 4).
The same conclusions about the influence of
protein binding on drug effectiveness are valid
when a continuous drug administration is con-
sidered and the value of the steady state unbound
drug concentration in plasma, Cu,ss, is taken a
marker of drug activity. Indeed the steady
state unbound drug concentration in tissue
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2160 BEREZHKOVSKIY

Cu,t,ss ¼ Cu,ss (as follows from Eq. 1), or according According to the provided analysis, for the most
to Eq. (4) Cu,t,ss ¼ Cu,ss(1  Et) if the targeted common case of orally administered drugs that
tissue eliminates a drug. In any case the are eliminated mainly due to hepatic metabolism,
steady state unbound drug concentration in the increase of protein binding leads to larger
tissue is proportional to Cu,ss in plasma. Thus to AUCu and thus can be considered beneficial for
maximize Cu,ss could be a goal in developing better drug activity. The consideration of AUCu as the
drug, so that the same concentration level Cu,ss determinant of drug action may not be quite
would be reached with less drug dose. The steady adequate. Most likely there is a certain minimal
state drug plasma concentration, Css, correspond- concentration of unbound drug in the targeted
ing to a continuous constant rate drug adminis- tissue Cu,t,min (and the corresponding value for
tration (considering linear phamacokinetics) is plasma Cu,min) below which the drug activity can
calculated as be assumed negligible. Thus it would be relevant
to consider that the area under the curve,
FI AUCu,t,eff, in the time interval (t1, t2) when Cu,t(t)
Css ¼ (27)
Cl is above Cu,t,min (as shown in Fig. 5A), determines
where I is the rate of continuous drug adminis-
tration (infusion for iv dosing). Same as for a
single dose F ¼ 1 for iv administration (assuming
Flung ¼ 1) and F ¼ FaFgFh for oral dosing. Since
Cu,ss ¼ fuCss, from Eq. (27) we get
FI
Cu;ss ¼ fu (28)
Cl
The right-hand side of Eq. (28) differs from that of
Eq. (6) by just having the infusion rate I instead of
dose D. Therefore the analysis of the factor Ffu/Cl
for the dependence of Cu,ss on fu should be
performed, which is exactly the same as for the
analysis of the dependence of AUCu on fu. Thus
the appropriate equations above will be valid
when AUCu is replaced by Cu,ss and the dose D is
replaced by the rate of continuous administration
I (Div ! Iiv, Dpo ! Ipo).
In theory the parameter fu may be considered as
independent variable. This approach is relevant
for the estimation of the influence of protein
binding changes (due to decrease of plasma
protein content, or interaction with other drug)
on the efficiency of the given drug, that is, for
assessing the clinical relevance of the variation in
protein binding.6 For drug development, though fu
may be considered as independent parameter, this
is not technically feasible. If some modification of
the potentially active compound is done to alter its
protein binding, most likely it will be reflected in Figure 5. Same unbound drug exposure in tissue
the changes of other parameters that determine (or plasma) may or may not be sufficient for drug
physiological activity. (Panel A) Exposure of the
AUCu (or Cu,ss), which are Fa, Fg, Clint,o and r.
unbound drug in tissue only at concentrations Cu,t(t),
The biological activity of the compound can
which are above certain minimal value Cu,t,min, may
possibly change as well. Eventually the advantage determine the drug action. (Panel B) Unbound drug
provided by improved protein binding can be concentration in tissue Cu,t(t) does not exceed the mini-
cancelled out by the changes in other parameters mal value Cu,t,min, so that the drug may not be active
(most likely Fg and Clint,o). This makes the process regardless the same exposure in tissue AUCu,t (or in
of optimizing protein binding very complicated. plasma AUCu) as in Panel A.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010 DOI 10.1002/jps

the drug action
Zt2
AUCu;t;eff ¼ Cu;t ðtÞ dt
t1
Depending on mechanism of drug activity it could
be more adequate to define AUCu,t,eff as
Zt2
AUCu;t;eff ¼ ðCu;t ðtÞ  Cu;t;min Þ dt
t1
The case when during the drug time course
Cu,t(t) < Cu,t,min indicates the lack of drug pharma-
cological activity. AUCu,t,eff is nonexistent in this
case (i.e., t1 and t2 are not found), and can be
accepted as AUCu,t,eff ¼ 0. Using this criterion
(Eq. 29 or Eq. 30) for evaluation of drug efficiency
makes the analysis quite complicated. It would
require the knowledge of the drug time course
Cu,t(t) in the targeted tissue, so that a detailed
pharmacokinetic model needs to be developed to
obtain the concentration time curves, rather than
using the integrated values of areas under the
curves. An adequate determination of the minimal
concentration Cu,t,min (based on the mechanism of
drug action) seems also quite complicated. This
parameter may strongly influence the outcome of
AUCu,t,eff calculations, especially when the max-
imum of Cu,t(t) and Cu,t,min are of the same order of
magnitude. Apparently AUCu,t,eff would charac-
terize the dependence of drug activity on the given
dose more accurately than AUCu,t (which is just
dose proportion for a linear pharmacokinetics).
AUCu,t,eff would be especially dose sensitive at
relatively low levels of administered dose when
the highest values of Cu,t(t) 0 Cu,t,min.
In case of oral dosing of a highly protein bound
drug ( fu ! 0) which is stable in the blood stream,
the elimination and tissue distribution are quite
slow, so that (assuming relatively fast absorption)
the concentration of drug in plasma may be close
to maximum possible value of Cmax ¼ FDpo/Vp,
where Vp is the plasma volume. If this drug is
eliminated by hepatic metabolism only, the
value of AUCu will be reaching the maximum
value (Fig. 1). The unbound drug concentration in
plasma Cu(t) will be limited by the maximum
possible value fuCmax. According to Eq. (1) the
unbound drug concentration in tissue Cu,t(t) 
Cu(t), so that Cu,t(t)  fuCmax ¼ fuFDpo/Vp. Then for
the values of fu that at any instant yield
Cu,t(t)  Cu,t,min, that is, fuFDpo/Vp  Cu,t,min (or
DOI 10.1002/jps
PHARMACOLOGICAL ACTIVITY OF DRUGS 2161
fu  VpCu,t,min/(FDpo)), the drug unbound concen-
tration in tissue Cu,t(t) will be below Cu,t,min value,
as shown in Figure 5B. This will be the case when
(29) a strong protein binding makes a drug inefficient
(AUCu,t,eff ¼ 0 in Eq. 29 or in Eq. 30), though AUCu
will be very large. In other words protein binding
limits the drug distribution to the site of activity,
though sustain the low concentration there for a
long time. Eventually a large value of AUCu will
(30) not be sufficient for drug action. In terms of
continuous administration of drug in this case,
the high steady state concentration Cu,t,ss will be
achieved (assuming that linear pharmacokinetics
approach is not violated, so that Eqs. 27 and 28 are
valid). Though it will take a very long time to
reach the steady state concentration in plasma
and possibly even much longer in tissue.12
Practically this is not very convenient, and such
property of a drug is not desirable. In general, the
calculation of drug concentration–time course in
tissue is needed for an advanced consideration of
drug efficacy (i.e., using AUCu,t,eff), which is
discussed further.
The assumption of the necessity of reaching
certain minimal concentration of drug at action
site leads to the conclusion that the rate of drug
absorption after oral dosing may be crucial for
drug action. Indeed, if absorption is relatively
slow, the concentration of drug in plasma C(t) may
not be high enough to provide the tissue concen-
tration Cu,t(t) above Cu,t,min (especially if drug
elimination is relatively fast). The area under the
curve AUCu could be relatively large due to a long
drug time course (dictated by slow absorption),
but not efficient for drug action since the condition
Cu,t(t) > Cu,t,min will not be met. The total absorbed
quantity of drug is taken into account by Eq. (10)
through the integrated value of AUCu (which is
reflected by oral bioavailability F ¼ FaFgFh).
Eq. (10) though does not provide the information
on the kinetics of absorption in terms of the
plasma concentration levels C(t) during the drug
time course. In other words the same value of
AUCu may be achieved through different concen-
tration profiles C(t), which depend on drug
absorption kinetics (though having not only the
same value of protein binding fu, but even the
same values of all parameters in the right-
hand side of Eq. 10). Eventually the condition
Cu,t(t) > Cu,t,min may or may not be met. Thus the
advanced consideration of possible criteria of
drug pharmacological activity (Eq. 29 or Eq. 30)
indicates that the rate of drug absorption followed
by the oral dose should be taken into account and
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
2162 BEREZHKOVSKIY
may become a limiting step in developing better
drug.
In terms of protein binding, for an orally
administered drug that is eliminated by liver
metabolism only, a strong protein binding
could be beneficial for obtaining higher levels of
AUCu (Fig. 1), but results in low concentration
levels in tissue (Cu,t(t) < Cu,t,min when fu ! 0). This
is the case when a strong protein binding
limits the delivery of a drug to the site of activity.
On the other hand a week protein binding
would lead to smaller AUCu but higher levels of
drug in tissue (possibly above Cu,t,min, so that
a drug would be pharmacologically active).
Therefore there should be an optimal value or
interval of fu (considering it as independent
parameter) that would result in the best drug
performance.
For drugs administered intravenously a weak
protein binding is beneficial for obtaining larger
values of AUCu (Eqs. 25 and 26). In this case a
drug is eliminated relatively fast (compared to the
case of strong protein binding). Thus it may turn
out that reaching the targeted tissue may be
limited by a slow drug transfer (combined with
fast elimination), so that in the worst case Cu,t(t)
may not reach Cu,t,min (though AUCu, as well as
AUCu,t, would be relatively large, as shown in
Fig. 5B). On the other hand a strong protein
binding leads to smaller values of AUCu, but
prolongs the duration of drug time course,
which eventually may yield higher concentration
levels in targeted tissue. Therefore the unbound
drug fraction fu could have an optimal value (or
interval) that provides the highest drug efficiency.
The average time a drug molecule spends
as unbound in the tissue extracellular water,
MRTt,u, is calculated as13
Z1 Z1
1 Vt;w
MRTt;u ¼ Au;t ðtÞ dt ¼ Cu;t ðtÞ dt
D D
0 0
Vt;w AUCu;t
¼
D the border line Cu,t,min in Figure 5 will be
where Au,t(t) is the quantity of unbound to drug in
tissue extracellular water at instant t, and Vt,w is
the volume of tissue extracellular water. As
considered previously AUCu,t ¼ AUCu (Eq. 2) or
AUCu,t is proportional to AUCu (Eq. 5). This leads
to the conclusion that AUCu is proportional to the
average time MRTt,u that a drug molecule spends
in the tissue extracellular water as unbound.
Thus the requirement to maximize AUCu actually
corresponds to the achieving the longest average
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
time that a drug molecule would spend in
the tissue extracellular water in the unbound
state. Similarly, reaching the largest value of
AUCu,t,eff (Eq. 29) for best pharmacological effect
corresponds to the achieving the longest average
time that a drug molecule would spend as
unbound in the tissue extracellular water when
concentrations Cu,t(t) are above Cu,t,min.
The action of drug may occur due to the
interaction of the free drug in tissue with the
targeted receptors. This interaction is often
described in terms of the equilibrium of a simple
binding reaction
D þ T ¼ DT (31)
where D denotes the unbound drug in tissue
(which has the concentration Cu,t(t)), T denotes
the targeted receptor, and DT is the drug–
target complex. The equilibrium dissociation
constant Kd determines the concentration of
bound receptor at equilibrium. According to the
mass action law
Cu;t ðTo  Tb Þ
Kd ¼ (32)
Tb
where To is the total receptor concentration, Tb is
the concentration of drug–receptor complexes
DT (bound receptors), so that To  Tb is the
concentration of free receptors. Thus for the
target occupancy Tb/To (which is time dependent)
Eq. (32) yields
Tb ðtÞ 1
¼ (33)
To 1 þ Kd =Cu;t ðtÞ
For example, it can be assumed, based on
the drug action mechanism, that the drug is
physiologically active when at least half of
the targeted receptors are occupied (50% occu-
pancy), that is, when Tb/To  0.5. According
to the equation above this takes place when
Cu,t(t)  Kd. Thus for such a consideration
Cu,t,min ¼ Kd.
In general, to find the time interval (t1, t2)
in Figure 5A and perform further integration to
find AUCu,t,eff (Eq. 29 or Eq. 30) requires the
knowledge of the drug time course Cu,t(t) in tissue.
This can be obtained from Eq. (4) taking
At(t) ¼ Cu,t(t)Vt/fu,t, where Vt is the tissue volume
and fu,t is the unbound drug fraction in tissue,
which can be calculated based on physico-
chemical properties of a drug.14 Then Eq. (4)
DOI 10.1002/jps
 PHARMACOLOGICAL ACTIVITY OF DRUGS 2163

becomes where the concentration profile Cu,t(t) may be

dCu;t ðtÞ ðPS þ Clint;t Þfu;t
þ Cu;t ðtÞ
dt Vt
PSfu;t
¼ Cu ðtÞ
Vt
This type of equations has a general solution for
Cu,t(t) for the known (measured) plasma time-
concentration profile Cu(t).15 For instance, if the
unbound drug concentration in plasma is given
as Cu(t) ¼ a exp(at) þ b exp(bt), the solution of
Eq. (34) (with initial condition Cu,t(t ¼ 0) ¼ 0, that
is, a drug is not administered directly into the
tissue) is
 at
ae b ebt
Cu;t ðtÞ ¼ k1 þ advantage of this simplification is that we can just
k2  a k2  b
 
a b
 þ ek2 t 
k2  a k 2  b
where k1 ¼ PSfu,t/Vt and k2 ¼ (PS þ Clint,t)fu,t/Vt.
Such in vivo based pharmacokinetic model was
successfully applied to interpret the drug time
course of compounds in brain and find the rate
constants of blood–brain transfer.12 Thus having
the drug plasma concentration–time curve
Cu(t) ¼ fuC(t), the time course of drug in tissue
can be obtained by Eq. (34) and used to calculate
AUCu,t,eff in the interval Cu,t(t)  Cu,t,min (Eq. 29 or
Eq. 30). In case when the targeted tissue does not
eliminate a drug, Clint,t ¼ 0 should be used in
Eq. (34). The problem is that the parameter PS/Vt
is not readily available except for the tissue that
can be considered in terms of well-stirred perfu-
sion limited physiologically based pharmacoki-
netic model.
In case of drug action being determined by drug
binding to the targeted receptors in tissue, it
appears that the integrated occupancy level, L,
defined as
Z1
Tb ðtÞ
L¼ dt
To
0 ing. A more detailed consideration (Eq. 36 or
could be a more adequate criterion of drug action
than AUCu. The larger value of L would be an
indication of better drug efficacy. According to
Eq. (33) the integrated occupancy level can be
expressed as
Z1
dt
L¼
1 þ Kd =Cu;t ðtÞ
0 intricate case.
provided by Eq. (34). Depending on the mechan-
ism of drug action, it could be more appropriate to
perform integration in Eqs. (35) and (36) in the
time interval (t1, t2) when Cu,t(t)  Cu,t,min. The
(34)
larger value of Cu,t(t)/Kd in Eqs. (33) and (36)
results in a larger value of receptor occupancy
Tb(t)/To. This allows to simplify the criterion of
drug effectiveness and instead of Eq. (36) use the
following expression
Z1
Cu;t ðtÞ dt AUCu;t
¼
Kd Kd
0
With the account of Eq. (5), we obtain from the
equation above AUCu,t/Kd ¼ AUCu(1  Et)/Kd. The
use the integrated value of exposure in plasma
AUCu for drug evaluation. Eq. (36) would be more
relevant for the estimation of drug activity, but it
requires the knowledge of drug time course in
tissue, which is not routinely available. In most
cases the targeted tissue does not eliminate a drug
(i.e., Et ¼ 0), so that the calculation of
AUCu AUCu;t
¼ (37)
Kd Kd
as a key factor of drug action, which accounts for
both the exposure and the affinity for receptor,
appears to be pertinent for the case when drug
action is provided by the binding to the targeted
receptors. To apply Eq. (37) for the evaluation
of drug action rather than just considering AUCu
is obviously more relevant. In other words, if
two compounds were compared and had the
same exposure AUCu, the one with higher affinity
to receptor (smaller Kd) would appear to have
better potential since it had larger value of
AUCu/Kd.
Obviously using AUCu ¼ fuAUC as a criterion
for the evaluation of drug action in terms of the
exposure at activity site is very convenient as it
requires just a commonly measured drug plasma
(35)
concentration–time course and its protein bind-
Eq. 37, or possibly using some other equations
based on pharmacodynamic model of drug action)
could be more appropriate for the late stage
of drug development. Drug binding to the
receptor may not necessarily be very fast, so that
binding kinetics (rather than instantaneous
equilibrium, Eq. 33) could be considered for the
(36) calculation of target occupancy in this more

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
2164 BEREZHKOVSKIY
The occupancy of the receptors at equilibrium is
determined by the affinity Kd. It is suggested that
a longer average time interval, tb, that a drug
molecule spends bound to the receptor may be
beneficial for drug action.16 Compounds may have
the same values of affinity to the receptor, but
different values of tb. The affinity is the ratio of
the reaction rate constants, that is, Kd ¼ koff/kon,
where and kon and koff are respectively the on- and
off-rate (dissociation rate) constants. The average
bound time is determined by the off-rate constant
and is calculated as
1 4. Christensen H, Baker M, Tucker GT, Rostami-
tb ¼
koff
At equilibrium the average time, tu, between each
binding to the receptor (‘‘unbound time’’) can be
found using the interpretation of unbound and
bound drug fractions in terms of kinetics, so that
tb/tu ¼ Tb/Cu,t. Using Eqs. (33) and (38) we get
ð1 þ Cu;t =Kd Þ
tu ¼
kon To
In terms of achieving longer bound time tb for the
same value of affinity Kd ¼ koff/kon, the better
compound would have smaller value of koff (as
provided by Eq. 38), as well as smaller value of
the on-rate constant kon. Thus to find the best
compound it terms of optimal equilibrium and
kinetic features would include both the reaching
of higher exposure–affinity ratio AUCu/Kd
(or integrated occupancy, Eq. (36), for a more
elaborate consideration) along with possibly
smaller value of dissociation rate constant of
drug–receptor complex.
It appears that there is also a problem in finding
the quantitative criteria that could adequately
reflect the influence of bound time tb on drug
action. As mentioned earlier, in theory it is
possible to consider the unbound drug fraction
(protein binding) as independent variable that can
be optimized to achieve better drug action.
Though most likely that the change of fu would
result in the change of bound time tb. Therefore
there should be suggested a criterion that
adequately accounts for the influence on drug
performance of both the integrated receptor
occupancy and the average time a drug molecule
resides on the receptor. This appears to be a
formidable problem to approach in terms of both
physiological and mathematical considerations.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
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