Understanding Synergy

Understanding synergy
Nori Geary
Am J Physiol Endocrinol Metab 304:E237-E253, 2013. First published 4 December 2012;

doi: 10.1152/ajpendo.00308.2012
You might find this additional info useful...
This article cites 161 articles, 37 of which you can access for free at:
http://ajpendo.physiology.org/content/304/3/E237.full#ref-list-1
This article has been cited by 3 other HighWire-hosted articles:
http://ajpendo.physiology.org/content/304/3/E237#cited-by
Updated information and services including high resolution figures, can be found at:
http://ajpendo.physiology.org/content/304/3/E237.full
This information is current as of August 2, 2016.
American Journal of Physiology - Endocrinology and Metabolism publishes results of original studies about
endocrine and metabolic systems on any level of organization. It is published 24 times a year (twice monthly) by the
American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright 2013 the American
Physiological Society. ESSN: 1522-1555. Visit our website at http://www.the-aps.org/.
Downloaded from http://ajpendo.physiology.org/ by guest on August 2, 2016
Additional material and information about American Journal of Physiology - Endocrinology and
Metabolism can be found at:
http://www.the-aps.org/publications/ajpendo
Am J Physiol Endocrinol Metab 304: E237E253, 2013.

First published December 4, 2012; doi:10.1152/ajpendo.00308.2012.
Review
Understanding synergy
Nori Geary
Zielackerstrasse 10, 8603 Schwerzenbach, Switzerland
Submitted 15 June 2012; accepted in final form 3 December 2012
cooperative effect synergy; energy homeostasis; food intake; Loewe additivity;

response surface methodology
One and one is what Im telling you . . . Debbie Harry (71)

INTRODUCTION
What is Synergy?
INTERACTION AMONG DIFFERENT ENDOCRINE CONTROLS and among
endocrine and nervous controls is a fundamental characteristic of
physiological regulation. Interactions are evident at all levels of
organization, from genetic to organismic (34, 67, 78, 85, 104,
160). For example, the secretion of most hormones is under both
endocrine and neural control, and many endocrine target cells as
well as neurons in a variety of brain sites express receptors to
multiple hormones (34, 67, 85, 104). At the organismic level, the
coordinated actions of nerves and hormones acting in several
tissues are vital to many regulatory functions, including blood
glucose homeostasis, fluid and mineral balance, and energy homeostasis, which provides many of the examples in this review (7,
54, 68, 83, 85, 110, 151). That many endocrine and metabolic
diseases are treated with combination therapies also reflects the
importance of interactions in physiological regulation (10, 29, 85).
Administration of combinations of drugs often produces unexpectedly large responses. This is known as synergy. It is often
described as 1 1 2, or supra-additive synergy. This is an apt
description because, as shown below, addition causes the most
Address for correspondence: N. Geary, Zielackerstrasse 10, 8603 Schwerzenbach, Switzerland (e-mail: ndg47@hotmail.com).
http://www.ajpendo.org
complications and misunderstandings in synergy analyses. (Note

that I couch the discussion in terms of drugs for simpicity; what
is meant is any quantifiable manipulation, including doses of
hormones, neurotransmitters, neuromodulators, pharmaceutical
agents, etc., as well as amounts of activity, hours of food deprivation, etc.).
What is the Problem?
Despite the apparent simplicity of the concept of supra-additive
synergy, translating it into a specific quantitative methodology
with formal validity is a thorny problem for which many solutions
have been offered (for a sense of the variety of approaches, see
Refs. 12, 64, and 121). Furthermore, few investigators appreciate
the prerequisites and limitations of supra-additive synergy methodologies. These problems are exacerbated by the different evolution of the concept in different fields, by the relative paucity of
theoretical accounts of synergy that are not forbiddingly quantitative, by the failure of most theorists to clearly distinguish their
approaches from those of others, by some widely propagated
errors in the existing accounts, and by the momentum of the
misdirected literature.
Synergy analyses in energy homeostasis research provide an
unfortunate example of inappropriate or incomplete synergy
analyses. Review of the energy homeostasis literature identified 44 studies, including four of my own, that used the
response additivity approach to supra-additive synergy (2, 8,
0193-1849/13 Copyright 2013 the American Physiological Society
E237
Geary N. Understanding synergy. Am J Physiol Endocrinol Metab 304: E237

E253, 2013. First published December 4, 2012; doi:10.1152/ajpendo.00308.2012.
Analysis of the interactive effects of combinations of hormones or other manipulations with qualitatively similar individual effects is an important topic in basic
and clinical endocrinology as well as other branches of basic and clinical research
related to integrative physiology. Functional, as opposed to mechanistic, analyses
of interactions rely on the concept of synergy, which can be defined qualitatively
as a cooperative action or quantitatively as a supra-additive effect according to
some metric for the addition of different dose-effect curves. Unfortunately, doseeffect curve addition is far from straightforward; rather, it requires the development
of an axiomatic mathematical theory. I review the mathematical soundness, face
validity, and utility of the most frequently used approaches to supra-additive
synergy. These criteria highlight serious problems in the two most common synergy
approaches, response additivity and Loewe additivity, which is the basis of the
isobole and related response surface approaches. I conclude that there is no
adequate, generally applicable, supra-additive synergy metric appropriate for endocrinology or any other field of basic and clinical integrative physiology. I
recommend that these metrics be abandoned in favor of the simpler definition of
synergy as a cooperative, i.e., nonantagonistic, effect. This simple definition avoids
mathematical difficulties, is easily applicable, meets regulatory requirements for
combination therapy development, and suffices to advance phenomenological basic
research to mechanistic studies of interactions and clinical combination therapy
research.
Review
E238
UNDERSTANDING SYNERGY
Why is Understanding Synergy Important?

Improving research practice by understanding synergy is
important for at least three reasons. First, if synergy were better
understood, researchers would make fewer innocent errors in
searching for supra-additive synergy. Second, there would be
fewer misguided attempts to understand the physiological
bases of what are in fact artifactual synergies. Third, there
would be fewer misinformed efforts to translate apparently
promising but erroneous basic research synergies into clinical
research. These improvements in research practice would lead
to substantial savings in human, animal, and financial resources.
What is the Solution?
My review of supra-additive synergy in RESPONSE ADDITIVITY
and OTHER ACTIVITIES leads
me to conclude that there is no metric to detect supra-additive
synergy, i.e., 1 1 2, with adequate formal validity, face
validity, and general applicability. Therefore, I recommend
adoption of a simpler definition of synergy as a cooperative
effect. As I describe in COOPERATIVE EFFECT SYNERGY, such
synergy is defined simply as a response to a drug combination
that is greater than the responses to the drugs individually.
Addition is not involved. Figure 1 provides some examples of
cooperative effect synergy and its utility in furthering basic and
clinical research.
SYNERGY, LOEWE ADDITIVITY SYNERGY,
SUPRA-ADDITIVE SYNERGY IS A FORMAL QUANTITATIVE

THEORY
Mathematical Nature of Synergy

As is often emphasized (19, 49, 65, 142), quantitative synergy
analyses are formal mathematical exercises, not wet physiology.
There are two main reasons. First, there is not sufficient mechanistic knowledge to enable quantitative predictions of the effects
of drug combinations. If, for example, two drugs each bound
reversibly to a single receptor with known kinetics and if the
drugs individual effects depended only on the number of receptors bound, then their individual and interactive effects could be
computed using the laws of mass action (12, 30, 64, 139, 143,
155). Similarly, interactive effects could be computed if they were
shown to result directly from the frequency of action potentials in
some accessible population of neurons. However, such quantitative mechanistic knowledge is not yet available in endocrinology
or in other branches of integrative physiology. The second reason
that synergy is intrinsically mathematical is that physiological
measurements cannot be used to test synergy metrics. The theorems derived in Euclidian geometry can be compared with the
characteristics of existing objects to determine whether the theory
corresponds to our direct experience of physical reality. In contrast, supra-additive synergy metrics lead to mathematical predictions that have no directly observable physiological counterparts.
This has the implication that, unlike more mechanistic models, it
is difficult or impossible to apply the criteria of construct and
predictive validity to synergy. How then to judge synergy metrics? I suggest the criteria of 1) face validity, 2) mathematical
soundness, and 3) utility with respect to furthering basic and
clinical research.
Although my approach to synergy is quantitative, and many
important points can be made only mathematically, the mathematical content of the article is minimal. I present mathematical derivations in the appendices in this article, and I illustrate
many points graphically.
Mathematical Notation
The mathematical notation is as follows: a and b denote
particular amounts or doses of drugs A and B, which might
be hormones or any other quantifiable manipulations; EA and
EB are the dose effect functions of drugs A and B, respectively,
although the subscripts are omitted in unambiguous situations,
situations; E(a) or EA(a) and E(b) or EB(b) are the effects of
dose a of A and b of B when applied individually, and E(a
b) is the effect of a and b applied simultaneously. EADD(a b)
is the theoretical additive effect of the a b combination. For
clarity, I occasionally denote the effect of a or b alone as E(a
0) or E(0 b). I assume that the dose-effect curves of both A
and B increase (or decrease) monotonically to a maximum.
Other situations bring special problems (4, 5, 26, 27, 157).
RESPONSE ADDITIVITY SYNERGY
According to the response or effect additivity synergy metric, the additive or zero-interactive effect of a drug combination is the sum of the individual effects,
EADDa b EAa EBb ,
(1)
and synergy occurs when the observed E(a b) is greater than

this sum (12, 76, 82, 170). This metric is ad hoc based neither
AJP-Endocrinol Metab doi:10.1152/ajpendo.00308.2012 www.ajpendo.org
15, 17, 23, 36, 40, 43, 44, 46 48, 53, 59, 61, 70, 75, 80, 86, 87,
103, 109, 111, 112, 115, 116, 127129, 132, 135, 138, 144,
145, 147, 149, 158, 161163, 166 168, 171). None of these 44
studies attempted to establish the validity of this approach, and,
as explained in RESPONSE ADDITIVITY SYNERGY, it is almost never
valid. A simple thought experiment shows why. If a drug has
a linear log dose-effect curve, as many do through much of
their dynamic ranges, then adding a dose of the drug to itself
will not double the effect, as predicted by response additivity,
but increase it only by a factor of 0.3 (i.e., by a factor of log 2).
Doubling the log dose is required to double the response. This
suggests that many response additivity studies that report
additivity may in fact have shown synergy.
Eleven energy homeostasis studies that used a linear Loewe
additivity approach were identified (9, 13, 50, 84, 91, 131, 134,
136, 137, 161, 169). Again, none of these studies attempted to
establish the validity of this approach, and, as explained in
Loewe Additivity Synergy, it is rarely valid. Other fields have
more successfully shunned response additivity but still suffer
from misunderstandings of Loewe addivity. For example, although nowhere is the concept of synergy more important and
more discussed than in anesthesiology (45, 72, 77, 108, 142,
143), one can find recent examples of uncritical application of
linear Loewe additivity there as well (e.g., Refs. 38, 73, and
79).
The gravity of incorrect applications of supra-additive synergy metrics can hardly be overstated. For example, as shown
in Interdeterminate Loewe Additivity Solutions, uncritical use
of linear Loewe additivity can completely reverse the interpretation of combination data, turning what should be antagonism
into synergy.
Review
E239
VOLUME CONSUMED (ml)
1 ml gastric saline load

20
15
10
*
5
10 pmol i.a. CCK
CCK
GAS
CCK
0
0
15
30
45
60
1 ml gastric saline load + 10 pmol i.a. CCK
CONTROL
GASTRIC LOAD
CCK ANALOG
10 s
GASTRIC LOAD +
CCK ANALOG
20
D
15
Intake (g)
0
VEH/VEH
CCK/VEH
VEH/LEP
DIO Prone
-2
-4
-6
Vehicle
Leptin
Amylin
Amylin+Leptin
-8
-10
0
Time (d)
10
# #
12
14
fak/fak
Ad-lacZ
CCK/LEP
10
E
Change in body weight (% vehicle-corrected)
Veh
CCK
Change in body weight from enrollment (%)
MEAN 30 MINUTE DIET INTAKE (ml)
fak/fak
Ad-LEPR-B
Pramlintide
Monotherapy or
Pretreatment Combination Treatment
Metrelepin
Pramlintide
Pramlintide+Metreleptin
-5
#
-10
###
##
###
##
-15
-4
12
16
20
Time (wk)
TIME (min)
Fig. 1. Examples of the utility of synergy studies

in energy homeostasis. Three examples demonstrate translations of initial basic research findings
suggesting synergy among inhibitory controls of
eating (A, C, and E) to mechanistic (B and D) and
clinical (F) studies. Note that in each case, a
simple definition of synergy as cooperative action
would suffice as well as supra-additive definitions
to motivate followup. A and B: the synergistic
effect of gastric loads (GAS) and cholecystokinin
agonism (CCK analog) on food intake (141)
prompted a neurophysiological study that demonstrated that this interaction occurs at the level of
abdominal vagal afferents (data are extracellular
recordings of action potentials) (140). i.a., Intraarterial; VEH, control vehicle injections. *Significantly different from control. C and D: the synergistic effect of peripheral CCK and central leptin
(LEP) injections on food intake (41) prompted a
molecular genetic investigation of the site of the
leptin receptors mediating this effect (113). fak/fak,
Koletsky rats, which lack functional leptin receptors; Ad-LEPR-B, adenovirus expressing these receptors; Ad-lacZ, control adenovirus expressing a
reporter gene. Ad-LEPR-B and Ad-lacZ were delivered to the arcuate nucleus of the hypothalamus
6 days prior to the effect of intraperitoneal injection of CCK on food intake being tested. In
C: *significantly different from VEH/VEH and
significantly different from CCK/VEH. In
D: *significantly different from VEH injection in
wild-type rats (data not shown) and significantly
different from fak/fak Ad-lacZ. E and F: the synergistic effect of chronic amylin and leptin treatment on body weight in rats susceptible to dietary
obesity (DIO Prone) (133) prompted a clinical
study that demonstrated a similar phenomenon in
obese humans (pramlintide and metreleptin are
amylin and leptin agonists, respectively) (133). In
E: #significantly different from amylin, P 0.05.
in F: significantly different from metreleptin and
pramlintide; P 0.01; ##P 0.01; ###P
0.001. All parts of the figure reprinted with permission.
Review
E240
additivity in the threshold region violates the principle of sham

combinations. This is shown schematically in Fig. 2. The
mistaken belief that two effects must differ if one is significant
and the other is not, which is widespread in neuroscience (117),
may contribute to the attraction of subthreshold response
additvity.
More generally, the principle of sham combinations emphasizes the critical point that, in the context of synergy analyses,
addition refers not simply to the addition of two numbers but
to the addition of dose response curves. Thus, supra-additive
synergy requires the development of a theory of addition of
dose-effect curves.
It is also worth noting that factorial analysis of variance of the
individual and combination effects of particular doses of drugs A
and B [i.e., EA(a), EB(b) and E(a b), respectively] is inappropriate for assessing synergy (28). The problem is that, in factorial
analysis of variance, deviations from additivity are indicated by
significant interaction effects, i.e., EA(a) EB(b) E(a b).
This is the equivalent of testing Eq. 1 or simple response additivity; i.e., it is not a valid approach to synergy if one accepts the
principle of sham combinations.
Finally, it is important to note that not all theorists agree on
the necessity of fulfilling the principle of sham combinations.
This is the case, for example, in a recent response additvity
approach developed for the detection of synergy in antimicrobial effects (20, 92, 159) as well as in the Bliss additivity
approach (please see Bliss Aditivity).
LOEWE ADDITIVITY SYNERGY
Theory and Validity

5
EFFECT
4
3
2
1
0
DOSE
Observed: E(2) = 1
Predicted: E(2) + E(2) = 2
Observed: E(4) = 3.8
Fig. 2. Response additivity violates the principle of sham combinations by

producing nonsensical predictions for drugs with curvilinear dose-effect
curves. In the example shown, the effect of dose 2 is 1, so according to the
principle of sham combinations, the predicted effect of dose 2 plus dose 2 is
1 1 2. In fact, due to the concave-up shape of the dose-effect curve in that
range, the observed effect of dose 4 is almost 4. Thus, response additivity
indicates that low doses of this drug synergize potently with themselves. The
same problem occurs when using response additivity to analyze the effects of
2 subthreshold drug doses. If the threshold for a statistically significant effect
under the conditions tested is 3, then the effect of dose 2 will be nonsignificant, and adding 2 individually nonsignificant doses of 2 will result in a
significant effect of 3; thus, according to response additivity, drugs will
synergize with themselves around the dose range for detecting statistically
significant effects.
Originally, Loewe additivity referred to a graphic way to

analyze drug interactions that was introduced by Fraser (51,
52) and developed in detail by Loewe and Muischnek (9396).
Loewes most fundamental contribution was to discern the
principles underlying the method and recognize that they could
lead to a variety of nonlinear forms of drug additivities. The
principles are the sham combination principle and the drug
dose equivalence principle.
Grabovsky and Tallarida (63), Tallarida (153156), and Tallarida and Raffa (157) articulated these principles into an axiomatic mathematical model. Loewe additivtiy is sometimes called the
isobolographic method, dose additivity, or, in the toxicology literature, concentration additivity (76, 119, 126). It is also the basis of
comparison in most response surface synergy metrics. Despite the
methods popularity, its prerequisites and shortcomings are not
widely understood. Therefore, I describe them here in detail.
The principles of sham combinations and drug dose equivalence combine to produce additive predictions as follows:
dose a of drug A is equivalent to dose ba of drug B if a and ba
have equal effects (principle of dose equivalence), and ba can
be added to any other dose b of drug B to give the additive
effect of the (a b) combination (principle of sham combinations). Dose equivalence can be computed on the basis of
transforming either from the dose-effect curve of A to that of B
(A B) or from B to A (B A). That is,
EADDa b EBba b EAa ab ,
(2)
where EB is measured on the dose-effect curve of drug B and

EA is measured on the dose-effect curve of drug A. This is
shown schematically in Fig. 3.
on physiology nor on formal axioms. Nevertheless, the notions simplicity has intuitive appeal and is used frequently. I
listed 44 examples above from the energy homeostasis literature; examples are also easily found in anesthesiology (e.g.,
Refs. 38, 73, and 79) and other fields.
The problem with the method is that it usually violates the
principle of sham combinations, which was introduced by
Loewe and Muischnek (93, 94, 96) but independently conceived
by others (e.g., see Ref. 69). The principle is simply that a drug
cannot synergize with itself (or, equivalently, it must add to itself
according to the additivity metric). This seems self-evident. Response additivity violates this principle, because for any drug with
a curvilinear dose-effect curve sham combinations indicate synergy in concave-up parts of the dose-effect curve (i.e., 2nd
derivative of the dose effect equation 0) and indicate infraadditivity in concave-down parts (2nd derivative 0). Figure 2
depicts this graphically. Only if both drugs have linear dose-effect
curves with zero intercepts is response additivity synergy valid
with respect to this principle. Of course, this is very rarely the
case. Rather, most dose-effect curves in pharmacology and physiology are curvilinear (35, 37, 105, 130). Thus, response additivity
synergy is generally invalid if one accepts the principle of sham
combinations.
Synergy is sometimes claimed if combinations of subthreshold doses of two drugs yield significant effects. This form of
response additivity is also invalid. The dose-effect curve of any
drug with a threshold is empirically concave-up in the region of
the threshold because all subthreshold effects are indistinguishable from zero and thus lie on a horizontal line. Thus, response
Review
E241
EFFECT
0
1
10
100
1000
DOSE of A or B; Log scale

Dose 10A = Dose 100B
EADD(10A+ 100 B) is either
Linear Isoboles are a Rarity
= EA(10 + 10) = EA(20) = 1.3

= EB(100 + 100) = EB(200) = 1.3
Fig. 3. Graphic computation of equivalent doses and Loewe additive predictions. Dose 10 of drug A has effect 1, which is also given by dose 100 of drug
B (dotted lines). Thus, doses 10A and 100B are equivalent. The Loewe additive
prediction for combination of doses 10A and 100B [i.e., EADD(10A 100B)]
can be solved by either B A or A B trasnforms (dashed lines). In the
former case, EADD(10A 100B) EA(10 10) EA(20) 1.3; in the latter
case, EADD(10A 100B) EB(100 100) EB(200) 1.3. Please see text
for further details.
The method is usually used to derive pairs of dose a of drug

A and dose b of drug B that should add to a constant effect, i.e.,
if both dose Ax of drug A and dose Bx of drug B have effect X
(a b), dose combinations for which
EADDa b EAAx EBBx X.
(3)
The plot of doses that fulfill Eq. 3 on a graph whose x-axis

is the dose of drug A and whose y-axis is the dose of drug B is
the isobole for effect level X. Synergy (or supra-additivity)
occurs if combinations of doses a and b that fall on the isobole
produce an effect greater than X or, alternatively, if combinations of doses a and b that lie below the isobol produce effect
X. If (a b) combinations that lie on the isobol produce effect
X, they are additive; if they produce an effect less than X, they
are infra-additive.
If the dose-effect curves of drugs A and B are characterized
mathematically, then formulas for equivalent dose of one drug
in terms of the other and additive predictions can be sometimes
be derived algebraically, as discussed in Linear Isoboles are a
Rarity and Curvilinear Isoboles. However, if the form of the
dose-effect curve is complex, a solution may not be possible, as
discussed in Indeterminate Loewe Additivity Solutions and
Boundary Conditions.
Loewe addition has good face validity. It suggests that, at the
level of intracellular postreceptor effects or postsynaptic neuronal processing, the representations of diverse stimuli that
synergize are transformed, at least in part, into a single representation. This is exactly how we understand the integrative
actions of intracellular and interneuronal signaling. It is a
logical generalization of the classical Sherringtonian principle
of neural signal processing (25, 146) that if two stimuli elicit
Only in a limited number of situations are isoboles straight

lines. The simplest case is if both drugs have linear dose-effect
curves with zero y-intercepts and without maxima in the region
studied. The isobol for effect level X is the follwing line
1 a Ax b Bx,
(4)
shown in Fig. 4 (again, doses Ax and Bx alone each lead to

effect level X). The derivation of Eq. 4 from the principles of
drug dose equivalence and sham combinations in this simple
case is given in APPENDIX 1. As mentioned above, synergy (or
supra-additivity) occurs if combinations of doses a and b that
fall on the isobole produce an effect greater than X or, alternatively, if combinations of doses a and b that lie below the
isobol produce effect X.
Isoboles are also straight lines for dose-effect curves that
approximate rectangular hyperbolas with equal maxima. Rectangular hyperbolas have the form
b = Bx
1 = a/Ax + b/Bx
b = Bx/2
0
0
a = Ax/2
a = Ax
DOSE off A ((a))

Fig. 4. The equation and graph (solid line) of the linear isobole. The isobole
describes drug combinations that lead to the same effect. Dose Ax of drug A
and dose Bx of drug B as well as all (a b) combinations that fall on the
isobole lead to identical effects or display Loewe additivity. The isobole is
linear as depicted here only if the relative potency of the 2 drugs is constant.
Constant relative potency has the consequence that the effects of (a b)
combinations (Ax Bx) for which 1 have the same effect as Ax
alone or Bx alone. The dotted line shows this for 0.5.
the same reflex, they must converge into a single final common
pathway. From an information-processing perspective, the input signals generated by the two stimuli lose their individual
identities at the point of convergence; i.e., the output could be
driven by either stimulus, which corresponds to the principle of
drug dose equivalence. In addition, the integrated output signal
can be increased identically by appropriate increases in either
input signal, which corresponds to the principle of sham combinations. A response in the Sherringtonian context is an
abstraction referring to an elemental neural reflex, such as
the iconic scratch reflex. However, it can be extended to any
elemental neuroendocrine response, for example, the secretion of insulin by the pancreatic -cells or the momentary
rate of licking liquid food by a rat. By extension, more
integrated responses that are driven in part by the elemental
responses, such as blood glucose level or daily food intake,
fit the same analysis.
DOSE of B (b)
Review
E242
EAa EAMaxa a A50 ,
(5)
where EA(a) is the effect of dose a of drug A, EAMax is its

maximal effect, and A50 is its rate or potency constant, which
is equal to dose producing the half-maximal effect. If drugs A
and B have rectangular hyperbolic dose-effect curves with
equal maxima (i.e., EAMax EBMax), then the isobole is again
linear and described by Eq. 4, as shown in Appendix 2 (156,
157). Figure 5 shows typical rectangular hyperbolic dose-effect
curves. Although many physiological dose-effect curves are
E = 100D/(D + 1)
E= 100D/(D
( + 10))
E = 100D/(D + 100)
E = 150D/(D + 100)
150
EFFECT (E)
125
W, X
100
75
b B50 A50 a A50 ,
50
25
0
200
400
600
800
1000
DOSE (D)
150
EFFECT (E)
125
100
W
X
75
50
25
0
-2
-1
LOG DOSE (D)

Fig. 5. Examples of rectangular hyperbolic dose-effect curves, showing that
constant relative potency is associated with parallel log dose-effect curves.
A: formulas for 4 rectangular hyperbolic dose-effect curves, drugs WZ. The
formulas have the form E EMAX D /(D D50), where EMAX is the maximum
effect, D is the dose, and D50 is a rate or potency constant that equals the dose
yielding half-maximal effect. B: the 4 dose-effect curves in untransformed
coordinates. C: the same dose-effect curves with dose on a log scale. Note that
for drugs W, X, and Y, EMAX 100, the rate constants vary by factors of 10,
and the nearly linear midranges of their log dose-effect curves are parallel and
separated by 1 log unit. This means that their relative potencies are constant;
the effect of any dose of drug W is also the effect of dose 10W of drug X and
dose 100W of drug Z. Note also that drug Z has the same rate constant as drug
Y, but a larger EMAX, so that the midranges of their log dose-effect curves are
not parallel and their relative potencies vary; for small effects, drug Y is nearly
as potent as drug Z, but for larger effects, drug Y is much less potent than
drug Z.
(6)
which rearranges by simple algebra to Eq. 4. Thus, this formulation is not generally valid. Rather, it is valid only for
dose-effect curves for which the principles of dose equivalence
and sham combination lead to Eq. 4, linear Loewe additivity.
As described in more detail below, isoboles for many,
probably most, drug combinations do not follow Eq. 4 but are
curvilinear (19, 63, 99, 100, 155, 156, 172). For example,
rectangular hyperbolic dose-effect curves with different maxima produce curvilinear isoboles (please see Curvilinear Isoboles). Equation 4, and therefore linear isoboles, results only
when the relative potency of drugs A and B is constant; i.e.,
dose-effect curves for which a/b is a constant for all doses a of
drug A and b of drug B for which the (a b) combination leads
to the same effect. If the relative potency of drugs A and B is
variable, then the isobole is not linear.
Relative potency is most conveniently tested using log
dose-effect curves. Drugs with parallel log dose-effect curves
have constant relative potency. Therefore, if the slopes of two
log dose-effect curves are not significantly different, it is
appropriate to analyze synergy with linear isoboles (or the
resulting response surfaces; please Loewe Additvity Response
Surfaces). This criterion is rarely, if ever, applied. Furthermore, parallel log dose-effect curves seem to be the exception
rather than the rule; for example, I have found no such
instances in interaction studies in energy homeostasis research.
Even rather small deviations from constant relative potency
can lead to curvilinear isobolograms (please see Curvilinear
Isoboles) and worse, indeterminate solutions (please see Indeterminate Loewe Additvity Solutions). Without consideration of
the individual dose-effect curves, deviations from the linear
predictions indicate only that the two drugs are not identical
and are not both agonists of a single receptor (139, 155). They
do not indicate that they are supra-additive or synergistic.
The misunderstanding of the applicability of linear isoboles
stems in large part from influential reviews by Berenbaum (11,
W:
X:
Y:
Z:
well described by rectangular hyperbolas (35, 37, 105, 130),

the constraint that the two drugs under consideration have the
same maximal effects substantially reduces the generality of
the linear isobole.
It is possible, of course, to equate different maximum effects
by expressing data as percent maximal effect, but this is not
advisable in situations where transformation changes the relationship of the variables change to the underlying physiologically relevant variables. For example, if one drugs maximum
weight loss effect is 10 kg and anothers is 20 kg, then 1%
maximum response for the former represents only half as much
weight loss in kilograms as 1% maximum response for the
latter. Synergy analyses in percent transforms in such situations seems senseless.
To my knowledge, the only other dose-effect curves that
result in linear isoboles are the probit and logit functions (153),
which are commonly used to describe proportions of successes
in a given number of cases.
Additivity is often defined as the ability of doses of two
drugs to substitute for each other in proportion to their potencies to produce the half-maximal effect (recall that potencies
are the doses producing the half-maximal effects A50 and B50);
that is, for predicted additivity, the dose b of drug B to add with
a particular dose a of drug A is
Review
E243
In contrast to the situations described above, nonparallel log

dose-effect curves generate curvilinear isoboles. This appears
to occur much more frequently than do linear isobols (e.g.,
Refs. 63, 99, 100, 155, 156, and 172 and the examples below).
It is the case, for example, for rectangular hyperbolic doseeffect curves with different maxima, such as dose-effect curves
W and Z in Fig. 5. For such curves, Grabovsky and Tallarida
(63) derived the following equivalent dose formula
(8)
Eq. 8 leads to curvilinear isoboles in which convex or

concave arcs connect points (Ax, 0) and (Bx, 0).
Indeterminate Loewe Additivity Solutions
Perhaps the greatest disadvantage of Loewe additivity is that
often no clear prediction can be generated. Such indeterminancy occurs when the additive prediction based on using the
B-equivalent dose of A differs from that based on the A-equivalent dose of B so that drug-dose combinations between the
two solutions are supra-additive according to one transformation and infra-additive according to the other.
Indeterminant Loewe additivity solutions occur frequently. Tallarida (155, 156) gives an example of indeterminate Loewe
additivity based on the Hill equation (or Hill-Langmuir equation).
The Hill equation is a three-parameter equation that results in
sigmoid functions that provide good fits to a wide range of pharmacological and physiological data. It is considered the basis of
quantitative pharmacology (33, 58, 62). The form of the equation is
EAa AAMaxap ap Ap50
X: E= 100D0.5/(D
( 0.5 + 500.5)
Y: E = 100D/(D + 50)
Z: E = 100D3/(D3 + 503)
(9)
where a is the dose of drug A, EAMax is the maximum effect, A50

is dose yielding the half-maximal effect, and p is a constant, often
100
Y
75
50
25
0
0
(7)
which results in curvilinear isoboles for all effect levels X,

1 a Ax A50 AxEAMax EBMax1 B50 b 1 .
200
400
600
800
1000
DOSE (D)
100
X
Y
EFFECT (E)
a ba A50 EAMax EBMax1 B50 b 1 ,
EFFECT (E)
Curvilinear Isoboles
called the slope parameter (note that if p 1, Eq. 9 becomes the

rectangular-hyperbola equation, Eq. 5). Examples are shown in
Fig. 6.
If the dose-effect curves of two drugs are given by Hill
equations with different values of p, solving for the isobole
leads to two different curvilinear solutions, depending on
whether one uses the A B or B A transformation (i.e.,
whether one uses the B-equivalent dose of A or the A-equivalent dose of B). This is shown in Fig. 7. Figure 8 shows
graphically how this can result from combinations of a drug
with a linear dose-effect curve and a drug with a rectangular
hyperbolic dose-effect curve.
75
50
25
0
-1
LOG DOSE (D)

Fig. 6. Examples of Hill equation dose-effect curves. The formulas have the
same form as the rectangular hyperbolic equation in Fig. 5, except that each
term is raised to an exponent, p. This results in the possibility of independently
varying the slope, maximal effect (EMAX), and half-maximal dose (A50), thus
greatly increasing the number of dose-effect curves that can be well modeled.
A: formulas for three Hill equation dose-effect curves, drugs XZ. The
formulas have the form E EMAX Dp/(Dp D50p), where EMAX is the
maximum effect, D is the dose, D50 is the dose yielding the half-maximal
effect, and p is a constant causing the slope to vary. B: the dose-effect curves
in untransformed coordinates. C: the same dose-effect curves with dose on a
log scale.
12) that included derivations supposedly establishing that Eq. 4

is true regardless of the form of the drugs dose-effect curves.
This derivation is incorrect (please see APPENDIX 3). The proof
is in fact limited to dose-effect curves whose relative potency
is constant. Either this was not recognized or its implications
were not understood, and the linear-isobole (and linear response surface method described in the next section) quickly
became the gold standard for synergy studies (21, 55).
Although he did not compute additive effects mathematically, Loewe (94) understood and stated clearly that linear
isoboles are valid only if the dose-effect curves of the drugs
considered have constant relative potency and that this is
rarely the case (. . . the overwhelming probability is that
the isobole deviates from the endpoint diagonal with either
SW- or NE-convexity and that . . . heterodynamic isoboles usually have two isoboles for the same endpoint, one
deviating to NE and the other to SW from the endpoint
diagonal; the endpoint diagonal is the linear isobole; NE
and SW are compass directions relative to the y-axis, which
is north; heterodynamic means variable relative potency).
Many major reviews of synergy (11, 12, 19, 21, 56, 57, 60,
122, 170) cite Loewe (94) without mentioning these points.
How this could be is a puzzlement.
Review
E244
ect BX)
DOSE B (dose 22 yields effe
additivity energy homeostasis studies also appeared to differ by

20% (9, 50, 91, 131, 134, 136, 161, 169), suggesting that correct
analyses would also change the interpretation of these studies.
25
20
Boundary Conditions
15
10
b
b
aa
0
0
10
20
30
40
50
60
DOSE A (dose 60 yields effect AX)
EFFECT
1 A = 3.2 B
2
1 B = 0.2 A
0
0
B3
EFFECT
The interpretation different outcomes of A B or B A

transformations is problematic. Tallarida (155) argues that,
because each solution represents additivity, the region contained between them is a region of additivity. But each solution
represents additivity for only one of the two equally valid
transformations and represents infra- or supra-additivity for the
other. Thus, it seems more reasonable to interpret both solutions and the area between them as indeterminate outcomes.
This counterintuitive result can be described as a situation in
which EA(a) EB(ba) and EB(b) EA(ab) but EA(a ab)
EB(ba b). Synergy is then restricted to the area below both
solutions and antagonism to the area above both.
Loewe addition frequently leads to such indeterminate regions. Furthermore, the region of indeterminancy is often
large. In a series of studies of combinations of various antiepileptic drugs in rat models, Luszczki and colleagues (98
101) and Wojda et al. (172) found that A B and B A
transformations of the data resulted in indeterminate areas that
filled 2575% of the total dose space [i.e., the area bounded
by the 4 points (0, 0), (Ax, 0), (0, Bx), and (Ax, Bx)]. Lorenzo
and Snchez-Marn (97) concluded that the gravity and frequency of this problem suffice to invalidate Loewe additivity
as a general solution to synergy.
Combinations of two drugs with linear log dose-effect curves
that have different slopes also lead to indeterminate outcomes.
The energy homeostasis literature exemplifies this. The 11 Loewe
additivity energy homeostasis studies that I know of all used a
linear approach (9, 13, 50, 84, 91, 131, 134, 136, 137, 161, 169).
An example is shown in Fig. 9. Note that the slopes of the log
dose-effect curves of cholecystokinin (CCK) alone and amylin
alone differed by 25%. Correct calculation of Loewe additivity
indicated that this difference is sufficient to change the interpretation of some of the CCK-amylin combination effects from
synergy, as these authors concluded, to antagonism. The individual log dose-effect curves in eight of the 10 remaining Loewe
DOSE (A or B)
E(1A + 1B) is equivalent to either:
EA(1 + 0.2) = EA(1.2) = 2.5
EB(3.2 + 1) = EB(4.2) = 2.8
Fig. 8. Graphic application of the dose equivalence principle yielding indeterminate Loewe additive solutions. A: dose 1 of drug A is equivalent to dose 3.2
of drug B (dashed lines), and dose 1 of drug B is equivalent to dose 0.2 of drug
A (dotted lines), as explained in Fig. 3 and in the text. B: different additive
predictions are generated for combination of dose 1 of drug A and dose 1 of
drug B when the drug A-equivalent dose of drug B is used (dashed lines, 1.2
units of drug A yields effect 2.5) than when the drug B-equivalent dose of drug
A is used (dotted lines, 3.3 units of drug B yields effect 2.8).
Fig. 7. Loewe additivity often leads to indeterminate isobolar solutions. The

dotted lines are equally valid alternate solutions for the isobole for the effect
of 30% EMAX (yielded by doses Ax and Bx) for drugs A and B with dose-effect
curves given by the Hill equation, Eq. 6, with the same EMAX but different
exponents (i.e., with relative potencies that change as effect level changes).
The upper dotted line results from transforming doses of drug A to their
equivalent doses of drug B, and the lower dotted line results from transforming
doses of drug B to their equivalent doses of drug A. The area between the lines
is supra-additive according to the A B transformation solution and infraadditive according to the B A transformation. Please see text for further
details. From Ref. 155, used with permission.
Boundary conditions pose additional problems for Loewe additivity. If dose equivalence is computed graphically, there is no
exact transform for subthreshold doses. A reasonable solution is to
use linear interpolation between zero and the smallest dose with a
measureable effect. If dose equivalence is computed mathematically, below-threshold doses are not a problem.
More difficult is when doses of drug A lead to effects exceeding
the maximum effect of drug B (i.e., drug B is a partial agonist).
Such doses of drug A cannot be transformed to equivalent doses
of drug B either graphically or mathematically, leaving the upper
border of Loewe additivity undefined. The problem of different
maxima is common. One example is provided by leptin. As
shown in Fig. 1, C and E, exogenous leptin alone often has only
modest effects on food intake and body weight in rats but
apparently large interactive effects when combined with CCK or
Review
De
ecrease in 30
0 min Food In
ntake (%)
Amylin
+ CCK
100
80
CCK
60
Amylin
40
n 25%
20
Zero
0.1
10
100
Dose (nmol/kg)
COMBINATION EFFECTS
DOSE
(Amylin, CCK;
nmol/kg)
LOEWE
ADDITIVITY
(%)
0.77, 0.09
0.26, 0.88
0.77, 0.88
0.26, 2.62
17 19
30 42
42 56
57 62
OBSERVED
EFFECT
(%)
33 13
58 10
54 10
39 11
Fig. 9. An example of indeterminate Loewe additivity solutions and erroneous

interpretations arising from improper application of Loewe additivity. A: the
acute eating-inhibitory effects of intraperitoneal injections of amylin () and
cholecystokinin (CCK; ). The authors smoothed the raw data with 4-parameter logistic fits, which they used to interpolate isoeffective doses of amylin
and CCK. Whether the observed combination effects (o) were synergistic was
assessed with linear isoboles and response surfaces based on the isoeffective
dose estimations; this analysis suggested that almost all amylin-CCK dose
combinations resulted in synergy. However, note that the amylin and CCK log
dose-effect curves are nonparallel; the slopes differ (n) by 25%. Thus,
analysis of the combination data with linear isoboles and response surfaces was
inappropriate. Graph from Ref. 13, reproduced with permission. B: graphic
estimation of Loewe additivity predictions for dose combinations near the
curves linear portions indicates that additive solutions were indeterminate for
considerable effect range. For example, for combination of 0.26 nmol/kg
amylin with 0.88 nmol/kg CCK, Loewe additivity was indeterminate from
30 to 42% decrease, and for combination of 0.77 nmol/kg amylin with 0.88
nmol/kg CCK, Loewe additivity was indeterminate from 42 to 56%. As a
result, although some dose combinations indeed appeared synergistic (e.g.,
combinations of 0.77 nmol/kg amylin with 0.09 nmol/kg CCK and 0.26
nmol/kg amylin with 0.88 nmol/kg CCK), others were infra-additive rather
than synergistic (e.g., combination of 0.26 nmol/kg amylin with 2.62 nmol/kg)
or indeterminate (e.g., combination of 0.77 nmol/kg amylin with 0.88 nmol/kg
CCK). Note also that the maximum effect of CCK exceeded that of amylin,
which also poses a problem for the computation of Loewe additivtiy. The
effects described in this article prompted a study suggesting that 7-day
infusions of amylin-CCK combinations elicited a synergistic increase in
weight loss in dietary obese rats (162). A cooperative effect synergy analysis
would also indicate that most dose combinations were synergistic and would
presumably also have sufficed to motivate this followup.
amylin. As discussed in Linear Isoboles are a Rarity, normalizing

to equate maxima, for example, by percent maximum transformations, is an inadequate solution. Another possibility is to develop an alternative supra-additivity metric. Howard and Webster
(76), for example, recently proposed a generalized concentration

addition method that was based on the inverse functions of the
drugs dose-effect curves {function f1(x) is the inverse of function f(x) if f1[f(x)] x}. Although this clever mathematical ploy
avoids the partial agonist problem, it abandons the axiomatic
foundation of Loewe additivity.
A conceptually similar situation is when in one drug is completely inactive. Much of the synergy literature in clinical
anesthesiology is designed to meet just this problem (e.g., Refs.
45, 74, 77, 102, 108, 142, and 143). The basic approach of
contemporary anesthesiology is to capitalize on the potent
synergistic effects of combinations of a hypnotic drug with an
analgesic drug that alone has little or no hypnotic potency; i.e.,
it is a partial agonist (e.g., Refs. 74 and 102). Loewe additivity
cannot handle these situations because there are no equivalent
doses. In response, anesthesiologists have developed a variety
of alternative additivity metrics (45, 74, 77, 102, 108, 142,
143), frequently not emphasizing that these are no longer true
Loewe additivity analyses. Tallarida (153) suggested a solution
based on an ad hoc formula to generate pseudoequivalent
doses. Another is to abandon supra-additive synergy, as described more in Response Surface Modeling and Cooperative
Effect Synergy.
Loewe Additivity Response Surfaces
The response surface approach extends the analysis of synergism from a single-effect level to a range of effect levels. In
simple cases, the surface is a three-dimensional contour describing the Loewe additive effects of continuous ranges of
doses derived mathematically from the principles of sham
addition and dose equivalence in a manner analogous to the
derivation of isoboles (153). Doses of drugs A and B are
plotted on the x- and z-axes, and the predicted E(a b) is
plotted on the y-axis. The intersection of the response surface
with a horizontal plane at height z X is isobole for effect
level X. If the dose-effect curves of drugs A and B are linear
with zero z-intercepts, rectangular hyperbolas with equal maxima, or parallel logits or probits, these intersections will be
linear. An example is shown in Fig. 10.
Synergy can be assessed in two ways. If the observed E(a b)
is added to the surface plot, then synergy is indicated by points
above the surface and infra-additivity by points below the
surface. Alternatively, the observed data can be fit to a surface
using second- or higher-order polynomials (57, 64, 108, 153).
The observed contour is then compared with the additive
predictions. Dose combinations under areas of the observed
contour that lie above the additive prediction are synergistic.
Response surfaces are more complex, and indeed are not
surfaces in the usual sense at all, if the drugs dose-effect
curves lead to indeterminate solutions, as discussed in Indeterminate Loewe Additvity Solutions. In such cases, single-dose
combinations will approach as a limit the additive solutions of
many effect levels, not just one. This can be envisioned with
reference to Fig. 7, which shows the additive solutions for
effect X, which is obtained with dose 60 of drug A alone and
dose 22 of drug B alone. If one now imagines the additive
solutions for effect 0.8X given by dose 50 of drug A alone and
dose 18 of drug B alone, it should be clear that the lower
additivity solution for X and the upper additivity solution for
0.8X will intersect; i.e., the dose combinations represented by
-20
E245
Review
E246
OTHER ADDITIVITIES
A number of other supra-additive synergy models have been

advanced (30, 32, 42, 74, 76, 92, 148, 150, 174). I briefly
review four, the first three of which are closely related to
Loewe additivity.
P
1.00
The Greco and Minto Models

0.67
0.33
120
80
0.00
120
40
80
40
Drug 2 (mg/kg)
Drug 1 (mg/kg)
1 a Ax Ex1p b Bx Ex1q
(10)
ab Ax Bx Ex12p12q
120
where Ex is the effect of Ax and of Bx normalized to the

0-dose effect ECON [i.e., Ex E/(ECON E)], p and q are
the slope parameters for drugs A and B, respectively, and
is the interaction index. Note that the two left terms of Eq. 10
are similar to Eq. 4. Synergy is indicated by 0, and
additivity is indicated by 0, i.e., if the right term of Eq. 10
drops out. Several modified forms of the original model have been
proposed (16, 24, 74).
The quite different approach by Minto et al. (108) is based
on the dose-effect curves of combinations of fixed-dose ratios
of drugs A and B, i.e., combinations for which a/b is constant.
The dose ratios, called , were expressed with respect to the
drugs half-maximal effects, i.e., a/A50 and b/B50. That is,
80
40
a A50 a A50 b B50 ,

0
0
40
80
120
Drug 1 (mg/kg)
Fig. 10. Loewe additivity response surface analysis. A: the estimated response
surface describing the loss of the righting reflex for 30 min (%mice
responding) produced by sham combinations of sodium hexobarbitol with
itself (56). A logistical model was used to render the data continuous and
estimate the response surface. Because the relative potency of hexobarbitol as
drug 1 and hexobarbitol as drug 2 was a constant 1, the linear response surface
model is valid. The data fit the model well (2 0.95). Note that intersections
of the surface with horizontal planes are linear isoboles for the effect level of
the height of the plane. B: the estimated linear isobole for 50% efficacy and its
95% confidence interval generated from the same data. Note that 78 mg/kg
hexobarbitol as drug 1 and 84 mg/kg hexobarbitol as dose 2 led to 50%
responses. Reprinted with permission.
these points are additive for both effect X and effect 0.8X.
Isoboles for many more effect levels will also intersect the X
isobole. This cannot be graphed in three dimensions because
single (x, y) dose combinations require numerous z-values.
This situation is analogous to what are known as multiplevalued mathematical functions, for example y x0.5. Such
functions are one source of four- and higher-dimensional
Riemann surfaces. Thus, Loewe additivity response surfaces
are highly complex mathematical structures.
(11)
where ranges from 0, a mixture of all drug B, to 1, a mixture

of all drug A. Each value was treated as a new drug whose
dose-effect curve is a new Hill equation. Minto et al. (108) then
derived fourth-order polynomial equations to estimate the three
Hill equation parameters (i.e., the maximal effect, half-maximal
dose, and slope parameter) as functions of . These polynomials
involved five coefficients, 0 4, but 0 and 1 could be
expressed in terms of and eliminated. Finally, the type of
interaction is reflected by values of the remaining three -coefficients. If all three 0, the drugs are additive; if 2 0 and
3 4 0, the drugs synergize; if two or three 0, the drugs
have complex interactions, for example, interactions including
both synergy and antagonism.
Both Greco and colleagues (64, 66) and Minto et al. (108)
accepted Berenbaums assertion (11, 12) of the generality of
Eq. 4 and, therefore, that the criterion for additivity is the linear
isobole. Unfortunately, as discussed above (Linear Isoboles
are a Rarity; also see APPENDIX 3), Berenbaumss assertion is
incorrect, and true Loewe additivity isoboles generated for Hill
equation dose-effect curves using the principles of dose equivalence and sham addition are to a great extent indeterminate
(Indeterminate Loewe Additvity Solutions). Thus, rather than
being mathematically valid applications of the axioms of
Loewe additivity, both the Greco and colleagues (64, 66) and
Minto et al. (108) algorithms are ad hoc supra-additive synergy
Drug 2 (mg/kg)
Greco and colleagues (64, 66) and Minto et al. (108) proposed interaction models for drugs whose dose-effect curves fit
the Hill equation, Eq. 9, which was discussed in Indeterminate
Loewe Additvity Solutions. Greco and colleagues (64, 66)
starting point was to accept the assertion by Berenbaum (11,
12) that the linear isobole, Eq. 4, is generally valid. They then
used it and a form of Eq. 9 for inhibitory effects to generate an
interaction index that expressed the difference between the
observed combination effects and the linear isobole
Review
E247
Bliss additivity is often used in the analysis of synergy in

antimicrobiology, toxicology, radiation medicine, and other fields
(20, 39, 60, 64, 123, 124, 152, 174, 177). According to Yeh et al.
(176), an advantage of Bliss additivity is that it is exactly analogous to the definition of epistasis used in molecular biology. The
additivity algorithm used by the GraphPad statistical package is
also based on Bliss additivity (see http://www.graphpad.com/faq/
viewfaq.cfm?faq991; GraphPad Software, La Jolla, CA).
The Chou Model
COOPERATIVE EFFECT SYNERGY
Chou (30) and Chou and Talalay (31, 32) described a synergy
model based on the derivation of a generalized equation representing the unified general theory for the Michaelis-Menten, Hill,
Henderson-Hasselbalch, and Scatchard equations that they derived:
Definition and Advantages
f a 1 1 D m D m ,
(12)
where fa is the fractional response, D is dose, Dm is the median

effective dose, and m is a shape parameter (m 1 for
hyperbolic dose-response curves and m 1 for sigmoidal
dose-response curves). This equation was used to generate
equations for the sum of the effects of two or more drugs,
which in turn was used in conjunction with Eq. 4, the linear
isobologram, to generate an expression for deviations from
Loewe additivity. Lee and Kong (88) recently developed a
method to estimate the confidence intervals of the Chou interaction index.
There are several problems with Chou and Talalays approach for integrative physiology. First, the dose-response
curves are transformed to fractional or percent maximum
response, which, as discussed in Linear Isoboles are a Rarity,
is not appropriate when the maximal effects differ. Second,
although Chou and Talalay do not emphasize it, the method is
limited to drugs with constant relative potencies. The calculations do not apply to mutually nonexclusive drugs, i.e., those
whose relative potency is not constant. Therefore, Chou (30)
simply posits that nonexclusivity indicates synergy (in
Chous words, the author integrated the nonexclusive condition as an intrinsic contribution to the synergistic effect).
From the perspective of formal theory, this is an indefensible
ad hoc maneuver. Third, Greco et al. (64) pointed out a number
of mathematical weaknesses in Chous derivations.
Bliss Additivity
Bliss (14) additivity is an axiomatic supra-additive synergy
model based on the principle that drug effects are outcomes of
probabilistic processes so that zero interaction is equivalent to
probabilistic independence. Thus, combination effects are
quantified as joint probabilities according to the familiar rule
PEa or Eb PEa PEb PEa and Eb ,
(13)
in which P represents the probability of observing the effect

indicated (Ea, etc.). The most common criticism of Bliss
addtivity is that, except in the case of exponential dose-effect
curves (11, 12, 152), it violates the principle of sham combinations; i.e., combinations of a drug with itself lead to synergy
or antagonism. Another criticism from the perspective of
integrative physiology is that interaction seems to be the
antithesis of independence.
The simplest quantitative concept of synergy is that it

reflects an increase in effect over the agents alone. The quantitative definition is that dose a of drug A and dose b of drug
B synergize if E(a b) E(a) and E(a b) E(b) (recall that
drugs A and B are drugs with qualitatively similar overt
effects). The analogous definition of antagonism is that doses a
and b are antagonists if E(a b) E(a) or E(a b) E(b)
(for example, inverse agonists fit this definition). Drug combinations with intermediate effects may be termed functionally
neutral. Cooperative effect synergy is also called superior yield,
highest single agent, or therapeutic synergy (3, 18, 90, 120, 152a).
Cooperative effect synergy has much to recommend it. The
lack of any addition metric is a major advantage, as it obviates
the need for an axiomatic mathematical theory for the addition
of dose-effect curves and the complications that come along
with it, as described above. This also enables synergy to be
assessed for particular doses without characterization of the
drugs dose-effect curves. Thus, simple experiments suffice to
identify interesting results that encourage further mechanistic
or translational research, as exemplified in Fig. 1.
Another major advantage of cooperative effect synergy is
that it meets the criteria recently adopted by the US Food and
Drug Administration (FDA) for evaluation of combination
therapies; i.e., two or more drugs may be combined in a single
dosage . . . to enhance the safety or effectiveness of the principal active component [Code of Federal Regulations of the
USA, Title 21, Volume 5, Section 300.50(a)(1), April 1, 2011].
This is an important shift away from the previous regulatory
guideline that combination therapies demonstrate some form of
supra-additive synergy (for discussions related to the evolution
of the FDAs stance, see Refs. 118, 125, 164, and 173). This
change should influence the designs of both basic discovery
and clinical research.
Response Surface Modeling and Cooperative Effect Synergy
Response surface analysis is a powerful general method to
analyze multivariate data (22, 114) that can be profitably applied
to cooperative action synergy. In drug discovery and other fields,
cooperative effect synergy based on response surface analysis is
considered an excellent strategy for high-throughput drug discovery (1, 3, 18, 90, 120, 178).
Response surface analysis is based on modeling the topology
of response magnitudes. This is usually done empirically, i.e.,
with no assumptions about the shape of the surface. Secondorder polynomial equations, which contain linear, quadratic,
and interaction terms, usually suffice to provide good fits to the
data. If several dose combinations are tested, even small
amounts of data (n 5 or so per dose combination) usually
metrics. Few of the many discussions of these models mention

this problem (1, 16, 24, 74, 81, 89, 102, 106, 107, 139, 165).
However, it should be noted that this criticism applies only to
the interpretation in terms of synergy, not to the goodness of fit
of the Hill equation response surfaces to the experimental data.
Thus, both models could be used in general response surface
analyses of combination effects, as described in COOPERATIVE
EFFECT SYNERGY (see, for example, Ref. 74).
Review
E248
CONCLUSION
Functional analyses of the effects of combinations of drugs

or other manipulations that invoke a notion of additivity are
mathematical models that must be developed and validated
mathematically. Unfortunately, this is rarely recognized, much
less done. As a result, much of the theoretical and empirical
synergy literature propagates fundamental misunderstandings
of Loewe additivity. True Loewe additivity is a formally valid
axiomatic mathematical theory, with face validity as a model
of integrative physiological processes. Unfortunately, however, it does not solve the synergy problem. Rather, in most
situations involving integrative physiology, including applications in energy homeostasis, endocrinology, and anesthesiology, the shapes of the dose-response curves complicate the
application of Loewe additivity and prevent clear additivity
solutions. Other supra-additive synergy models are no more
satisfactory (Table 1).
The elusive nature of good quantitative synergy solutions
has long been a concern. In 1953, Loewe (94) wrote, Although, according to this study, the terms synergism and
antagonism . . . have no definable place in the treatment of
combination problems and should be eliminated from the field
because of the menace of confusion, the greater probability is
that they will live on as so many other undefined and undefinable terms. In 1996, the view of Greco et al. (65) was that
good synergy assessment and interpretation will never be
simple and likened the search for such a proper and easy
Table 1. Comparison of several synergy metrics

Metric
Supra-Additive?
Face
Validity?
Formal
Validity?
Recommended?
Response additivity
Loewe additivity
Greco model
Minto model
Chou model
Bliss additivity
Cooperative effect
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
Yes
Yes
Yes
No
Yes
No
Yes
No
No
No
Yes
N/A
No
No
No
No
No
No
Yes
N/A, not applicable. Supra-additive indicates that synergy is assessed

against an additive or zero-interactive prediction. Face validity is based on
whether the metric distinguishes a drug from itself (the principle of sham
combinations) and other considerations described in the text. Formal validity
is indicated if the metric generates additive predictions based on an axiomatic
mathematical theory of dose-response curve addition; this is not applicable for
cooperative effect synergy because it does not involve addition. Loewe
additivity is not recommended because it is often complex, often leads to
indeterminate solutions for many or most drug combinations, and cannot be
applied to some boundary conditions. Please see text for further details.
solution to Dorothys escape from Oz in that we are waiting

for some wizard to tell us the secret. And in 2012, Shafer
(142), in discussing eight synergy models in anesthesia, concluded that, although useful, all models are wrong.
Supra-additive synergy approaches also fail to provide
unique clues as to mechanism. Yates (175) enumerated nine
characteristics of a good mathematical model of physiological
processes. Neither Loewe additivity nor the other supra-additivity approaches described here seem to fulfill any of them
and, therefore, seem unlikely to be guides to understanding the
mechanisms of integrative responses.
For these reasons, I conclude that the disadvantages and
difficulties of supra-additive synergy models far outweigh their
benefits and recommend that they be abandoned.
Nevertheless, synergy is an important physiological and
clinical issue worthy of pursuit. I believe that the simple
definition of synergy as cooperative, i.e., nonantagonistic,
action is adequate both for basic discovery research and clinical research. The designs and analyses are far simpler than
those of supra-additive synergy and avoid the many problems
described above. Cooperative effect synergy suffices to identify good candidates for further mechanistic or clinical research
(Fig. 1). The definition meets regulatory requirements for
establishing combination therapies. Finally, it has proven productive in other fields.
APPENDIX 1: LOEWE ADDITIVITY EQUATION FOR LINEAR
DOSE-EFFECT CURVES
The simplest application of the theory of Loewe additivity is to

generate an additive formula for linear dose-effect curves with zero
intercepts and with maxima ignored. In this case, the effects E(a) and
E(b) for doses a and b of two drugs (or other manipulations), drugs A
and B, are given by:
Ea nAa and Eb nBb,
(A1.1)
where nA and nB are the slopes of the two lines. The equations can be
used to generate equivalent doses of drugs A and B. If E(a) E(b),
then nAa nBb and
a nB nAb.
(A1.2)
This equation also indicates that the relative potency of drug A with
respect to drug B is nB/nA. Note that, unlike the situation in APPENDIX 3, 1)
permit rough estimation of the equation parameters. This

enables predictions of dosages for maximum effects or for
particular effect levels, for example, using algorithms designed
to determine paths of steepest ascent toward the maximum or
desired response (i.e., lines normal to the fitted surface contours directed toward the desired end point). Additional experiments targeted to restricted ranges of combinations or computer simulations can then be performed to better characterize
the response surface. Such methods have been extended to
quite complexly contoured surfaces. Multiple responses can be
studied simultaneously, for example, to optimize therapeutic
windows, i.e., identify dose combinations with the largest
difference between therapeutic and undesired responses (90).
An important advantage of response surface analysis of
cooperative effect synergy is that it need not make assumptions
about the dose-effect curves of the individual drugs. Thus, drug
combinations involving drugs with variable relative potency or
drugs with different maximal effects or one drug that alone has
no measurable effect do not pose modeling problems.
Response surfaces with theoretical bases can also be used.
For example, one study of opioid-sedative anesthetic interactions fit the data to a compound logistic dose-effect curve (102)
and another to a hierarchical function that modeled the ascending neural mechanisms of analgesia and hypnosis (74). The
former study also exemplifies the predictive use of response
surface analysis. That is, Manyam et al. (102) first computed an
area on the response surface that identified sedative-hypnotic
dose combinations that were optimal for anesthesia and then
used the drugs individual clearance times and a stepwise
iterative simulation procedure to identify the dose combinations that were predicted to lead to the most rapid recovery
from anesthesia.
Review
E249
Eqs. A1.1 and A1.2 are true at all effect levels, 2) the relative potency
of drugs A and B is derived, not asserted, and 3) nB/nA is constant.
Next, consider an effect level X that is given by doses Ax and Bx
alone. What combinations of dose a of drug A and dose b of drug B
also give effect X? For any such (a b) pair, the dose a and the drug
A-equivalent dose of drug B must add to Ax because Ax has the
desired effect X. Substituting the formula in Eq. A1.2 for equivalent
doses, this yields
a nB nAb Ax.
(A1.3)
Dividing by Ax gives
a/Ax nB nAb/Ax 1.
(A1.4)
Finally, again from Eq. A1.2 above, the dose of drug B that is
equivalent to Ax is (nB/nA)Bx. Substituting this into Eq. A1.4 gives
a/Ax nB nAb/nB nABx 1,
(A1.5)
a/Ax b/Bx 1,
(A1.6)
the linear-isobole equation (Eq. 4 in the main text). Thus, for the
particular situation of two linear dose-effect curves with zero intercepts, the additive prediction for dose combinations yielding a constant effect that is generated by the dose-equivalence principle is the
linear isobole equation.
APPENDIX 2: LOEWE ADDITIVITY EQUATION FOR
RECTANGULAR-HYPERBOLIC DOSE-EFFECT CURVES WITH
IDENTICAL MAXIMUM EFFECTS
This proof was presented by Tallarida (156) and Tallarida and

Raffa (157). If both drugs A and B have rectangular hyperbolic
dose-effect curves, the effects E(a) and E(b) of doses a and b of drugs
A and B are
Ea EAMax a a A50 and Eb EBMaxb b B50 ,
(A2.1)
where EAMax and EBMax are constants describing the maximal effects
of drugs A and B and A50 and B50 are rate constants, or potencies or
half-maximal effects, of drugs A and B. If the maximal effects are
equal, EAMax EBMax EMAX. If the effects of doses a and b are
equal, then
EMaxa a A50 EMaxb b B50 .
(A2.2)
Dividing by EMAX gives

a a A50 b b B50 .
Berenbaum (12) claims that the following derivation is a general

validation of the linear-isobole synergy metric because Its derivation
took no account, either explicitly or implicitly, of the shapes of
dose-response curves of the agents . . . In fact, as shown below, the
derivation does take account of the shapes of dose-response curves by
implicitly assuming that the two agents have a constant relative
potency. Therefore, its validity is limited to that situation.
The notation is that E(a) and E(b) are the individual effects of
various doses a and b of drugs A and B, respectively, and effect level
X is given by doses Ax of A and Bx of B. Berenbaum (12) begins by
considering a sham dose of drug A, called dose A= that has the same
effect as Bx, namely X. Because dose Ax is isoeffective with dose Bx,
then the sham dose A= must equal the dose BX times the relative
potency of Ax and Bx at effect level X
A Ax BxBx.
This is straightforward application of the principle of dose equivalence, although Berenbaum (12) did not recognize that as a general
principle. Next consider that (a b) dose combination also yield
effect X, i.e., doses that have an additive effect according to the
linear-isobole method. Berenbaum (12) wishes to construct an equivalent dose a= of drug A that can be substituted for dose b. He writes,
Now, the sham combination is, in fact, a of A plus (Ax/Bx) b of A
(italics added). That is, Berenbaum substitutes dose a= for drug A= and
dose b for Bx in Eq. A3.1 to generate the dose a= with an effect
equivalent to that of b units of B,
a Ax Bxb.
Ea of A Ax Bxb of A EAx .
(A2.4)
Because all the dose terms are in units of A, the doses on each side
of the equation must be equal; thus,
a Ax Bxb Ax.
(A2.5)
which is the dose of b that is the dose equivalent to a. Note that, as in

the linear example above, A50/B50 describes the two drugs relative
potencies and is constant. Finally, for an effect level X that is given by
doses Ax and Bx alone, what combinations of dose a of drug A and
dose b of drug B also give effect X? Again, for any such pair, dose a
and the drug A-equivalent dose of drug B must add to Ax because Ax
has the desired effect X. Expressing dose b as its drug A-equivalent
dose, given in the formula in Eq. A2.5, yields
a bA50 B50 Ax.
(A3.2)
This substitution is incorrect. There is no reason that (Ax/Bx) b of

A should have the same effect as b. Recall that Ax/Bx is the relative
potency of A and B at effect level X. No information is available
about the relative magnitudes of A and B that lead to other effect
levels. For example, 0.5 Ax need not have the same effect as 0.5 Bx.
Thus, Eq. A3.2 is invalid, and no equivalent dose of drug B in terms
of drug A can be generated.
Berenbaum (12) completes the derivation by equating the effect of
the sham combination with that of Ax
Subtracting ab from each side and dividing by B50 gives

a bA50 B50 ,
(A3.1)
(A2.3)
Multiplying by (a A50) (b B50) gives

ab aB50 ab bA50 ,
APPENDIX 3. BERENBAUMS GENERAL VALIDATION

OF THE ISOBOLE METHOD BASED ON THE
SHAM-COMBINATION PRINCIPLE
(A2.6)
Except for the names of the constants expressing relative potency,

this is identical to Eq. A1.3 and again can be transformed to the
linear-isobole equation (i.e., Eqs. 4 and A1.4). Thus, for two hyperbolic dose-effect curves with equal maxima, the Loewe additive
(A3.3)
(A3.4)
Finally, dividing by Ax would yield the linear-isobole equation

(Eq. 4).
a Ax b/Bx 1.
(A3.5)
ACKNOWLEDGMENTS
I thank Dr. Lori Asarian, Institute for Veterinary Physiology, University of
Zurich, Zurich, Switzerland, Dr. Anders Lehman, AstraZeneca, Mlndal,
Sweden, Dr. Timothy Moran, Department of Psychiatry, Johns Hopkins
University, Baltimore, MD, Dr. Jonathan Roth, Amylin, San Diego, CA, and
Dr. Gerard P. Smith, Department of Psychiatry, Weill Medical College of
Cornell University, New York, NY, for helpful discussions. I also thank the
reviewers for their helpful comments.
DISCLOSURES
I have no conflicts of interest, financial or otherwise, to declare.
in which the potency ratio nB/nA cancels, leaving
prediction for dose combinations yielding a constant effect is the

linear-isobole equation.
Review
E250
AUTHOR CONTRIBUTIONS
N.G. drafted the manuscript.
24.
25.
REFERENCES
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
1. Afeltra J, Vitale RG, Mouton JW, Verweij PE. Potent synergistic in

vitro interaction between nonantimicrobial membrane-active compounds
and itraconazole against clinical isolates of Aspergillus fumigatus resistant to itraconazole. Antimicrob Agents Chemother 48: 13351343, 2004.
2. Air EL, Benoit SC, Clegg DJ, Seeley RJ, Woods SC. Insulin and leptin
combine additively to reduce food intake and body weight in rats.
Endocrinology 143: 2449 2452, 2002.
3. Albritton RL, Coen DM, Gola DE. Types of drug interactionssynergy,
additivity and antagonism In: Principles of PharmacologyThe Pathophysiologic Basis of Drug Therapies, edited by Golan DA, Tashjian AH Jr,
Armstrong EJ, Galanter JM, Armstrong AW, Arnaout RA, and Rose HS.
Baltimore, MD: Lippincott, Williams & Wilkens, 2005, p. 605606.
4. Antin J, Gibbs J, Smith GP. Cholecystokinin interacts with pregastric
food stimulation to elicit satiety in the rat. Physiol Behav 20: 6770,
1978.
5. Asarian L, Geary N. Prior pregastric food stimulation and gastrinreleasing peptide127 (GRP) synergize to inhibit sham feeding. Peptides
20: 731736, 1999.
7. Asarian L, Gloy V, Geary N. Homeostasis. In: Encyclopedia of Human
Behavior (2nd ed.), edited by Ramachandran VS. San Diego, CA:
Elsevier, 2012, p. 324 333.
8. Barrachina MD, Martinez V, Wang L, Wei JY, Tache Y. Synergistic
interaction between leptin and cholecystokinin to reduce short-term food
intake in lean mice. Proc Natl Acad Sci USA 94: 1045510460, 1997.
9. Bello NT, Kemm MH, Ofeldt EM, Moran TH. Dose combinations of
exendin-4 and salmon calcitonin produce additive and synergistic reductions in food intake in nonhuman primates. Am J Physiol Regul Integr
Comp Physiol 299: R945R952, 2010.
10. Bennett WL, Maruthur NM, Singh S, Segal JB, Wilson LM, Chatterjee R, Marinopoulos SS, Puhan MA, Ranasinghe P, Block L,
Nicholson WK, Hutfless S, Bass EB, Bolen S. Comparative effectiveness and safety of medications for type 2 diabetes: an update including
new drugs and 2-drug combinations. Ann Intern Med 154: 602613,
2011.
11. Berenbaum MC. The expected effect of a combination of agents: the
general solution. J Theor Biol 114: 413431, 1985.
12. Berenbaum MC. What is synergy? Pharmacol Rev 41: 93141, 1989.
13. Bhavsar S, Watkins J, Young A. Synergy between amylin and cholecystokinin for inhibition of food intake in mice. Physiol Behav 64:
557561, 1998.
14. Bliss CI. The toxicity of poisons applied jointly. Annal Appl Biol 26:
585615, 1939.
15. Bojanowska E, Nowak A. Interactions between leptin and exendin-4, a
glucagon-like peptide-1 agonist, in the regulation of food intake in the
rat. J Physiol Pharmacol 58: 349 360, 2007.
16. Bol CJ, Vogelaar JP, Tang JP, Mandema JW. Quantification of
pharmacodynamic interactions between dexmedetomidine and midazolam in the rat. J Pharmacol Exp Ther 294: 347355, 2000.
17. Boozer CN, Leibel RL, Love RJ, Cha MC, Aronne LJ. Synergy of
sibutramine and low-dose leptin in treatment of diet-induced obesity in
rats. Metabolism 50: 889 893, 2001.
18. Borisy AA, Elliott PJ, Hurst NW, Lee MS, Lehar J, Price ER,
Serbedzija G, Zimmermann GR, Foley MA, Stockwell BR, Keith
CT. Systematic discovery of multicomponent therapeutics. Proc Natl
Acad Sci USA 100: 79777982, 2003.
19. Bosgra S, van Eijkeren JC, Slob W. Dose addition and the isobole
method as approaches for predicting the cumulative effect of noninteracting chemicals: a critical evaluation. Crit Rev Toxicol 39: 418
426, 2009.
20. Boucher AN, Tam VH. Mathematical formulation of additivity for
antimicrobial agents. Diagn Microbiol Infect Dis 55: 319 325, 2006.
21. Bovill JO. Analysis of drug interactions. Ball Clin Anesth 12: 153168,
1998.
22. Box GEP, Draper NR. Response Surfaces, Mixtures and Ridge Analysis.
Hoboken, NJ: John Wiley & Sons, 2007.
23. Brennan IM, Feltrin KL, Horowitz M, Smout AJ, Meyer JH,
Wishart J, Feinle-Bisset C. Evaluation of interactions between CCK
and GLP-1 in their effects on appetite, energy intake, and antropylo-
roduodenal motility in healthy men. Am J Physiol Regul Integr Comp

Physiol 288: R1477R1485, 2005.
Brun YF, Greco WR. Characterization of a three-drug nonlinear mixture response model. Front Biosci (Schol Ed) 2: 454 467, 2010.
Burke RE. Sir Charles Sherringtons the integrative action of the
nervous system: a centenary appreciation. Brain 130: 887894, 2007.
Calabrese EJ. Hormesis and medicine. Br J Clin Pharmacol 66: 594
617, 2008.
Calabrese EJ, Baldwin LA. Hormesis: the dose-response revolution.
Annu Rev Pharmacol Toxicol 43: 175197, 2003.
Caudle RM, Williams GM. The misuse of analysis of variance to detect
synergy in combination drug studies. Pain 55: 313317, 1993.
Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA,
Izzo JL Jr, Jones DW, Materson BJ, Oparil S, Wright JT Jr,
Roccella EJ; National Heart, Lung, and Blood Institute Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure; National High Blood Pressure Education Program Coordinating Committee. The Seventh Report of the
Joint National Committee on Prevention, Detection, Evaluation, and
Treatment of High Blood Pressure: the JNC 7 report. JAMA 289:
2560 2572, 2003.
Chou TC. Theoretical basis, experimental design, and computerized
simulation of synergism and antagonism in drug combination studies.
Pharmacol Rev 58: 621681, 2006.
Chou TC, Talalay P. Generalized equations for the analysis of inhibitions of Michaelis-Menten and higher-order kinetic systems with two or
more mutually exclusive and nonexclusive inhibitors. Eur J Biochem
115: 207216, 1981.
Chou TC, Talalay P. Quantitative analysis of dose-effect relationships:
the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme
Regul 22: 2755, 1984.
Colquhoun D. The quantitative analysis of drug-receptor interactions: a
short history. Trends Pharmacol Sci 27: 149 157, 2006.
Cone RD, Low MJ, Elmquist JK, Cameron JL. Neuroendocrinology.
In: Williams Textbook of Endocriniology (10th ed.), edited by Larsen PR,
Kronenberg HM, Melmed S, and Polonsky K. Philadelphia, PA: Saunders, 2003, p. 81176.
Cooper JR, Bloom FE, Roth RH. The Biochemical Basis of Neuropharmacology. New York: Oxford University, 2003.
Day JW, Ottaway N, Patterson JT, Gelfanov V, Smiley D, Gidda J,
Findeisen H, Bruemmer D, Drucker DJ, Chaudhary N, Holland J,
Hembree J, Abplanalp W, Grant E, Ruehl J, Wilson H, Kirchner H,
Lockie SH, Hofmann S, Woods SC, Nogueiras R, Pfluger PT, PerezTilve D, DiMarchi R, Tschp MH. A new glucagon and GLP-1
co-agonist eliminates obesity in rodents. Nat Chem Biol 5: 749 757,
2009.
DeLean A, Munson PJ, Rodbard D. Simultaneous analysis of families
of sigmoidal curves: application to bioassay, radioligand assay, and
physiological dose-response curves. Am J Physiol Endocrinol Metab
Gastrointest Physiol 235: E97E102, 1978.
Eger EI 2nd, Tang M, Liao M, Laster MJ, Solt K, Flood P, Jenkins
A, Raines D, Hendrickx JF, Shafer SL, Yasumasa T, Sonner JM.
Inhaled anesthetics do not combine to produce synergistic effects regarding minimum alveolar anesthetic concentration in rats. Anesth Analg 107:
479 485, 2008.
Einav S, Sobol HD, Gehrig E, Glenn JS. The hepatitis C virus (HCV)
NS4B RNA binding inhibitor clemizole is highly synergistic with HCV
protease inhibitors. J Infect Dis 202: 6574, 2010.
Ellacott KL, Lawrence CB, Rothwell NJ, Luckman SM. PRL-releasing peptide interacts with leptin to reduce food intake and body weight.
Endocrinology 143: 368 374, 2002.
Emond M, Schwartz GJ, Ladenheim EE, Moran TH. Central leptin
modulates behavioral and neural responsivity to CCK. Am J Physiol
Regul Integr Comp Physiol 276: R1545R1549, 1999.
Fang HB, Ross DD, Sausville E, Tan M. Experimental design and
interaction analysis of combination studies of drugs with log-linear dose
responses. Stat Med 27: 30713083, 2008.
Ferrer-Lorente R, Cabot C, Fernndez-Lpez JA, Alemany M.
Combined effects of oleoyl-estrone and a beta3-adrenergic agonist
(CL316,243) on lipid stores of diet-induced overweight male Wistar rats.
Life Sci 77: 20512058, 2005.
Ferrer-Lorente R, Cabot C, Fernndez-Lpez JA, Alemany M.
Effects of combined oleoyl-estrone and rimonabant on overweight rats. J
Pharm Sci 104: 176 182, 2007.
Review
69. Grossman MI. Potentiation: a reply. Gastroenterology 56: 815816,

1969.
70. Gutzwiller JP, Degen L, Matzinger D, Prestin S, Beglinger C. Interaction between GLP-1 and CCK-33 in inhibiting food intake and appetite
in men. Am J Physiol Regul Integr Comp Physiol 287: R562R567, 2004.
71. Harry D, Destri J. Picture this. In: Blondie, Parallel Lines. Hollywood,
CA: Chrysalis Records, 1978.
72. Hemmings HC Jr, Antognini JF. Do general anesthetics add up?
Anesthesiology 104: 1120 1122, 2006.
73. Hendrickx JF, Eger EI 2nd, Sonner JM, Shafer SL. Is synergy the
rule? A review of anesthetic interactions producing hypnosis and immobility. Anesth Analg 107: 494 506, 2008.
74. Heyse B, Proost JH, Schumacher PM, Bouillon TW, Vereecke HE,
Eleveld DJ, Luginbuhl M, Struys MM. Sevoflurane remifentanil interaction: comparison of different response surface models. Anesthesiology
116: 311323, 2012.
75. Hinton V, Rosofsky M, Granger J, Geary N. Combined injection
potentiates the satiety effects of pancreatic glucagon, cholecystokinin,
and bombesin. Brain Res Bull 17: 615619, 1986.
76. Howard GJ, Webster TF. Generalized concentration addition: a method
for examining mixtures containing partial agonists. J Theor Biol 259:
469 477, 2009.
77. Johnson KB, Syroid ND, Gupta DK, Manyam SC, Egan TD, Huntington J, White JL, Tyler D, Westenskow DR. An evaluation of
remifentanil propofol response surfaces for loss of responsiveness, loss
of response to surrogates of painful stimuli and laryngoscopy in patients
undergoing elective surgery. Anesth Analg 106: 471 479, table of contents, 2008.
78. Joyner MJ, Pedersen BK. Ten questions about systems biology. J
Physiol 589: 10171030, 2011.
79. Kang S, Kim CH, Lee H, Kim DY, Han JI, Chung RK, Lee GY.
Antinociceptive synergy between the cannabinoid receptor agonist WIN
55,212-2 and bupivacaine in the rat formalin test. Anesth Analg 104:
719 725, 2007.
80. Kastin AJ, Pan W, Akerstrom V, Hackler L, Wang C, Kotz CM.
Novel peptide-peptide cooperation may transform feeding behavior.
Peptides 23: 2189 2196, 2002.
81. Kern SE, Xie G, White JL, Egan TD. A response surface analysis of
propofol-remifentanil pharmacodynamic interaction in volunteers. Anesthesiology 100: 13731381, 2004.
82. Klaasen CD. Principles of toxicology and treatment of drug poisoning.
In: Goodman & Gilmans The Pharmacological Basis of Therapeutics
(10th ed.), edited by Hardman JG and Limbird LE. New York: McGraw
Hill, 2001, p. 67 80.
83. Kohan DE, Rossi NF, Inscho EW, Pollock DM. Regulation of blood
pressure and salt homeostasis by endothelin. Physiol Rev 91: 177, 2011.
84. Kulkosky PJ, Glazner GW. Dose-additive inhibition of intake of
ethanol by cholecystokinin and bombesin. Alcohol Clin Exp Res 12:
277281, 1988.
85. Larsen PR, Kronenberg HM, Melmed S, Polonsky KS. Williams
Textbook of Endocrinology. Philadelphia, PA: Saunders, 2003.
86. Le Sauter J, Geary N. Pancreatic glucagon and cholecystokinin synergistically inhibit sham feeding in rats. Am J Physiol Regul Integr Comp
Physiol 253: R719 R725, 1987.
87. Le Sauter J, Geary N. Redundant vagal mediation of the synergistic
satiety effect of pancreatic glucagon and cholecystokinin in sham feeding
rats. J Auton Nerv Syst 30: 1322, 1990.
88. Lee JJ, Kong M. Confidence Intervals of Interaction Index for Assessing
Multiple Drug Interaction. Stat Biopharm Res 1: 4 17, 2009.
89. Lee SI. Drug interaction: focusing on response surface models. Korean
J Anesthesiol 58: 421434, 2010.
90. Lehr J, Krueger AS, Avery W, Heilbut AM, Johansen LM, Price
ER, Rickles RJ, Short GF 3rd, Staunton JE, Jin X, Lee MS, Zimmermann GR, Borisy AA. Synergistic drug combinations tend to
improve therapeutically relevant selectivity. Nat Biotechnol 27: 659
666, 2009.
91. Liang NC, Bello NT, Moran TH. Additive feeding inhibitory and
aversive effects of naltrexone and exendin-4 combinations. Int J Obes
(Lond). In press.
92. Lim TP, Ledesma KR, Chang KT, Hou JG, Kwa AL, Nikolaou M,
Quinn JP, Prince RA, Tam VH. Quantitative assessment of combination antimicrobial therapy against multidrug-resistant Acinetobacter baumannii. Antimicrob Agents Chemother 52: 2898 2904, 2008.
45. Fidler M, Kern SE. Flexible interaction model for complex interactions
of multiple anesthetics. Anesthesiology 105: 286 296, 2006.
46. Field BC, Wren AM, Peters V, Baynes KC, Martin NM, Patterson
M, Alsaraf S, Amber V, Wynne K, Ghatei MA, Bloom SR. PYY336
and oxyntomodulin can be additive in their effect on food intake in
overweight and obese humans. Diabetes 59: 16351639, 2010.
47. Figlewicz DP, Sipols AJ, Seeley RJ, Chavez M, Woods SC, Porte D
Jr. Intraventricular insulin enhances the meal-suppressive efficacy of
intraventricular cholecystokinin octapeptide in the baboon. Behav Neurosci 109: 567569, 1995.
48. Figlewicz DP, Stein LJ, West D, Porte D Jr, Woods SC. Intracisternal
insulin alters sensitivity to CCK-induced meal suppression in baboons.
Am J Physiol Regul Integr Comp Physiol 250: R856 R860, 1986.
49. Fitzgerald JB, Schoeberl B, Nielsen UB, Sorger PK. Systems biology
and combination therapy in the quest for clinical efficacy. Nat Chem Biol
2: 458 466, 2006.
50. Foltin RW, Woolverton WL, Schuster CR. Effects of psychomotor
stimulants, alone and in pairs, on milk drinking in the rat after intraperitoneal and intragastric administration. J Pharmacol Exp Ther 226:
411418, 1983.
51. Fraser TR. An experimental research on the antagonism between the
actions of physostigma and atropia. Proc Royal Soc (Edinburgh) 7:
506 511, 1870 1871.
52. Fraser TR. Lecture on the Antagonism between the Actions of Active
Substances. Br Med J 2: 485487, 1872.
53. Geary N, Kissileff HR, Pi-Sunyer FX, Hinton V. Individual, but not
simultaneous, glucagon and cholecystokinin infusions inhibit feeding in
men. Am J Physiol Regul Integr Comp Physiol 262: R975R980, 1992.
54. Geary N, Moran TH. Basic science of appetite. In: Comprehensive
Textbook of Psychiatry (9th ed.), edited by Sadock BJ, Sadock VA, and
Ruiz P. Philadelphia, PA: Wolters Kluwer/Lippincott, Wiliams & Wilkens, 2009, p. 375387.
55. Gebhart GF. Comments on the isobole method for analysis of drug
interactions. Pain 51: 381; author reply 383388, 1992.
56. Gennings C, Carter WH Jr, Campbell ED, Staniswalis JG, Martin
TJ, Martin BR, White KL Jr. Isobolographic characterization of drug
interactions incorporating biological variability. J Pharmacol Exp Ther
252: 208 217, 1990.
57. Gessner PK. Isobolographic analysis of interactions: an update on
applications and utility. Toxicology 105: 161179, 1995.
58. Gesztelyi R, Zsuga J, Kemeny-Beke A, Varga B, Juhasz B, Tosaki A.
The Hill equation and the origin of quantitative pharmacology. Arch Hist
Exact Sci 66: 427438, 2012.
59. Gibbs J, Kulkosky PJ, Smith GP. Effects of peripheral and central
bombesin on feeding behavior of rats. Peptides 2, Suppl 2: 179 183,
1981.
60. Goldoni M, Johansson C. A mathematical approach to study combined
effects of toxicants in vitro: evaluation of the Bliss independence criterion and the Loewe additivity model. Toxicol In Vitro 21: 759 769,
2007.
61. Gourcerol G, Wang L, Wang YH, Million M, Tache Y. Urocortins and
cholecystokinin-8 act synergistically to increase satiation in lean but not
obese mice: involvement of corticotropin-releasing factor receptor-2
pathway. Endocrinology 148: 61156123, 2007.
62. Goutelle S, Maurin M, Rougier F, Barbaut X, Bourguignon L, Ducher
M, Maire P. The Hill equation: a review of its capabilities in pharmacological modelling. Fundam Clin Pharmacol 22: 633648, 2008.
63. Grabovsky Y, Tallarida RJ. Isobolographic analysis for combinations
of a full and partial agonist: curved isoboles. J Pharmacol Exp Ther 310:
981986, 2004.
64. Greco WR, Bravo G, Parsons JC. The search for synergy: a critical
review from a response surface perspective. Pharmacol Rev 47: 331385,
1995.
65. Greco WR, Faessel H, Levasseur L. The search for cytotoxic synergy
between anticancer agents: a case of Dorothy and the ruby slippers? J
Natl Cancer Inst 88: 699 700, 1996.
66. Greco WR, Park HS, Rustum YM. Application of a new approach for
the quantitation of drug synergism to the combination of cis-diamminedichloroplatinum and 1-beta-D-arabinofuranosylcytosine. Cancer Res
50: 5318 5327, 1990.
67. Griffin JE, Ojeda SR. Textbook of Endocrine Physiology. New York:
Oxford University, 2000.
68. Grill HJ. Leptin and the systems neuroscience of meal size control.
Front Neuroendocrinol 31: 6178, 2010.
E251
Review
E252
114. Myers RM, Montgomery DC. Response Surface Methodology: Process
and Product Optimization Using Designed Experiments. Hoboken, NJ:
John Wiley & Sons, 2002.
115. Neary NM, McGowan BM, Monteiro MP, Jesudason DR, Ghatei
MA, Bloom SR. No evidence of an additive inhibitory feeding effect
following PP and PYY 336 administration. Int J Obes (Lond) 32:
1438 1440, 2008.
116. Neary NM, Small CJ, Druce MR, Park AJ, Ellis SM, Semjonous NM,
Dakin CL, Filipsson K, Wang F, Kent AS, Frost GS, Ghatei MA,
Bloom SR. Peptide YY336 and glucagon-like peptide-1736 inhibit
food intake additively. Endocrinology 146: 5120 5127, 2005.
117. Nieuwenhuis S, Forstmann BU, Wagenmakers EJ. Erroneous analyses of interactions in neuroscience: a problem of significance. Nat
Neurosci 14: 11051107, 2011.
118. Parks M, Rosebraugh C. Weighing risks and benefits of liraglutide
the FDAs review of a new antidiabetic therapy. N Engl J Med 362:
774 777, 2010.
119. Payne J, Rajapakse N, Wilkins M, Kortenkamp A. Prediction and
assessment of the effects of mixtures of four xenoestrogens. Environ
Health Perspect 108: 983987, 2000.
120. Petersen JL, Mahaffey KW, Hasselblad V, Antman EM, Cohen M,
Goodman SG, Langer A, Blazing MA, Le-Moigne-Amrani A, de
Lemos JA, Nessel CC, Harrington RA, Ferguson JJ, Braunwald E,
Califf RM. Efficacy and bleeding complications among patients randomized to enoxaparin or unfractionated heparin for antithrombin therapy in
non-ST-Segment elevation acute coronary syndromes: a systematic overview. JAMA 292: 89 96, 2004.
121. Plaa GL, Venzina M. Factors to consider in the design and evaluation
of chemical interaction studies in laboratory animals. In: Toxic Interactions, edited by Goldstein RS, Hewitt WR, and Hook JB. New York:
Academic, 1990, p. 330.
122. Plummer JL, Short TG. Statistical modeling of the effects of drug
combinations. J Pharmacol Methods 23: 297309, 1990.
123. Poch G, Dittrich P, Reiffenstein RJ, Lenk W, Schuster A. Evaluation
of experimental combined toxicity by use of dose-frequency curves:
comparison with theoretical additivity as well as independence. Can J
Physiol Pharmacol 68: 1338 1345, 1990.
124. Poch G, Reiffenstein RJ, Unkelbach HD. Application of the isobologram technique for the analysis of combined effects with respect to
additivity as well as independence. Can J Physiol Pharmacol 68: 682
688, 1990.
125. Podolsky SH, Greene JA. Combination drugs hype, harm, and hope.
N Engl J Med 365: 488 491, 2011.
126. Rajapakse N, Silva E, Kortenkamp A. Combining xenoestrogens at
levels below individual no-observed-effect concentrations dramatically
enhances steroid hormone action. Environ Health Perspect 110: 917
921, 2002.
127. Reidelberger RD, Haver AC, Apenteng BA, Anders KL, Steenson
SM. Effects of exendin-4 alone and with peptide YY(336) on food
intake and body weight in diet-induced obese rats. Obesity (Silver
Spring) 19: 121127, 2011.
128. Riedy CA, Chavez M, Figlewicz DP, Woods SC. Central insulin
enhances sensitivity to cholecystokinin. Physiol Behav 58: 755760,
1995.
129. Romero MM, Esteve M, Alemany M. Combined effects of oral oleoylestrone and limited food intake on body composition of young overweight male rats. Int J Obes (Lond) 30: 1149 1156, 2006.
130. Ross EM, Kenakin TP. Pharmacodynamics. In: Goodman and Gilmans
The Pharmacological Basis of Therapeutics (10th ed.), edited by Hardman JG, Limbird LE, and Gilman AG. New York: McGraw-Hill, 2001,
p. 31 43.
131. Roth JD, Coffey T, Jodka CM, Maier H, Athanacio JR, Mack CM,
Weyer C, Parkes DG. Combination therapy with amylin and peptide
YY[336] in obese rodents: anorexigenic synergy and weight loss additivity. Endocrinology 148: 6054 6061, 2007.
132. Roth JD, DSouza L, Griffin PS, Athanacio J, Trevaskis JL, Nazarbaghi R, Jodka C, Hoyt J, Forood B, Parkes DG. Interactions of
amylinergic and melanocortinergic systems in the control of food intake
and body weight in rodents. Diabetes Obes Metab 14: 608 615, 2012.
133. Roth JD, Roland BL, Cole RL, Trevaskis JL, Weyer C, Koda JE,
Anderson CM, Parkes DG, Baron AD. Leptin responsiveness restored
by amylin agonism in diet-induced obesity: evidence from nonclinical
and clinical studies. Proc Natl Acad Sci USA 105: 72577262, 2008.
93. Loewe S. Die Mischarznei. Versuch einer allgemeinen Pharmakologie

der Arzneiombinationen. Klinische Wochenschrift 6: 10771084, 1927.
94. Loewe S. The problem of synergism and antagonism of combined drugs.
Arzneimittelforschung 3: 285290, 1953.
95. Loewe S. Randbemerkungen zur quantitativen Pharmakologie der Kombinationen. Arzneimittelforschung 9: 449 456, 1959.
96. Loewe S, Muischnek H. ber Kombinationswirkungen. Archiv fr
experimentelle Pathologie 114: 313326, 1926.
97. Lorenzo JI, Snchez-Marn P. Comments on Isobolographic analysis
for combinations of a full and partial agonist: curved isoboles. J
Pharmacol Exp Ther 316: 476 478; author reply 479, 2006.
98. Luszczki J, Krzyzanowski M, Swiader MJ. Additive interaction of
tiagabine with phenobarbital against pentylenetetrazole-induced clonic
seizures: an isobolographic analysis for non-parallel dose-response relationship curves. Annales Univerisitatis Mariae Curie-Sklodowska 64:
2231, 2009.
99. Luszczki JJ. Interactions of tiagabine with ethosuximide in the mouse
pentylenetetrazole-induced seizure model: an isobolographic analysis for
non-parallel dose-response relationship curves. Naunyn Schmiedebergs
Arch Pharmacol 378: 483492, 2008.
100. Luszczki JJ. Isobolographic analysis of interaction between drugs with
nonparallel dose-response relationship curves: a practical application.
Naunyn Schmiedebergs Arch Pharmacol 375: 105114, 2007.
101. Luszczki JJ, Wu JZ, Raszewski G, Czuczwar SJ. Isobolographic
characterization of interactions of retigabine with carbamazepine, lamotrigine, and valproate in the mouse maximal electroshock-induced
seizure model. Naunyn Schmiedebergs Arch Pharmacol 379: 163179,
2009.
102. Manyam SC, Gupta DK, Johnson KB, White JL, Pace NL, Westenskow DR, Egan TD. Opioid-volatile anesthetic synergy: a response
surface model with remifentanil and sevoflurane as prototypes. Anesthesiology 105: 267278, 2006.
103. Matson CA, Reid DF, Ritter RC. Daily CCK injection enhances
reduction of body weight by chronic intracerebroventricular leptin infusion. Am J Physiol Regul Integr Comp Physiol 282: R1368 R1373,
2002.
104. McEwen BS. Stress, sex, and neural adaptation to a changing environment: mechanisms of neuronal remodeling. Ann NY Acad Sci 1204 Suppl:
E38 E59, 2010.
105. Meddings JB, Scott RB, Fick GH. Analysis and comparison of sigmoidal curves: application to dose-response data. Am J Physiol Gastrointest
Liver Physiol 257: G982G989, 1989.
106. Meletiadis J, Mouton JW, Meis JF, Verweij PE. In vitro drug interaction modeling of combinations of azoles with terbinafine against
clinical Scedosporium prolificans isolates. Antimicrob Agents Chemother
47: 106 117, 2003.
107. Mertens MJ, Olofsen E, Engbers FH, Burm AG, Bovill JG, Vuyk J.
Propofol reduces perioperative remifentanil requirements in a synergistic
manner: response surface modeling of perioperative remifentanil-propofol interactions. Anesthesiology 99: 347359, 2003.
108. Minto CF, Schnider TW, Short TG, Gregg KM, Gentilini A, Shafer
SL. Response surface model for anesthetic drug interactions. Anesthesiology 92: 16031616, 2000.
109. Moon HS, Chamberland JP, Diakopoulos KN, Fiorenza CG, Ziemke
F, Schneider B, Mantzoros CS. Leptin and amylin act in an additive
manner to activate overlapping signaling pathways in peripheral tissues:
in vitro and ex vivo studies in humans. Diabetes Care 34: 132138, 2011.
110. Moran TH. Gut peptides in the control of food intake. Int J Obes (Lond)
33, Suppl 1: S7S10, 2009.
111. Moran TH, Carrigan TS, Schwartz GJ, Ladenheim EE. Bombesin
and cholecystokinin differentially affect ingestive microstructural variables whether given alone or in combination. Behav Neurosci 110:
1110 1116, 1996.
112. Moriya R, Mashiko S, Ishihara A, Takahashi T, Murai T, Ito J,
Mitobe Y, Oda Z, Iwaasa H, Takehiro F, Kanatani A. Comparison of
independent and combined chronic anti-obese effects of NPY Y2 receptor agonist, PYY(336), and NPY Y5 receptor antagonist in diet-induced
obese mice. Peptides 30: 1318 1322, 2009.
113. Morton GJ, Blevins JE, Williams DL, Niswender KD, Gelling RW,
Rhodes CJ, Baskin DG, Schwartz MW. Leptin action in the forebrain
regulates the hindbrain response to satiety signals. J Clin Invest 115:
703710, 2005.
Review
157. Tallarida RJ, Raffa RB. The application of drug dose equivalence in the
quantitative analysis of receptor occupation and drug combinations.
Pharmacol Ther 127: 165174, 2010.
158. Talsania T, Anini Y, Siu S, Drucker DJ, Brubaker PL. Peripheral
exendin-4 and peptide YY(336) synergistically reduce food intake
through different mechanisms in mice. Endocrinology 146: 3748 3756,
2005.
159. Tam VH, Schilling AN, Lewis RE, Melnick DA, Boucher AN. Novel
approach to characterization of combined pharmacodynamic effects of
antimicrobial agents. Antimicrob Agents Chemother 48: 43154321,
2004.
160. Thackray VG, Mellon PL, Coss D. Hormones in synergy: regulation of
the pituitary gonadotropin genes. Mol Cell Endocrinol 314: 192203,
2010.
161. Trevaskis JL, Coffey T, Cole R, Lei C, Wittmer C, Walsh B, Weyer
C, Koda J, Baron AD, Parkes DG, Roth JD. Amylin-mediated restoration of leptin responsiveness in diet-induced obesity: magnitude and
mechanisms. Endocrinology 149: 5679 5687, 2008.
162. Trevaskis JL, Turek VF, Griffin PS, Wittmer C, Parkes DG, Roth
JD. Multi-hormonal weight loss combinations in diet-induced obese rats:
therapeutic potential of cholecystokinin? Physiol Behav 100: 187195,
2010.
163. Turek VF, Trevaskis JL, Levin BE, Dunn-Meynell AA, Irani B, Gu
G, Wittmer C, Griffin PS, Vu C, Parkes DG, Roth JD. Mechanisms
of amylin/leptin synergy in rodent models. Endocrinology 151: 143152,
2010.
164. Unger EF. Weighing benefits and risksthe FDAs review of prasugrel.
N Engl J Med 361: 942945, 2009.
165. Voigt W, Kegel T, Weiss M, Mueller T, Simon H, Schmoll HJ.
Potential activity of paclitaxel, vinorelbine and gemcitabine in anaplastic
thyroid carcinoma. J Cancer Res Clin Oncol 131: 585590, 2005.
166. Wang L, Martinez V, Barrachina MD, Tache Y. Fos expression in the
brain induced by peripheral injection of CCK or leptin plus CCK in
fasted lean mice. Brain Res 791: 157166, 1998.
167. Weintraub M, Hasday JD, Mushlin AI, Lockwood DH. A doubleblind clinical trial in weight control. Use of fenfluramine and phentermine alone and in combination. Arch Intern Med 144: 11431148, 1984.
168. Wellman PJ, Jones SL, Miller DK. Effects of preexposure to dexfenfluramine, phentermine, dexfenfluramine-phentermine, or fluoxetine on
sibutramine-induced hypophagia in the adult rat. Pharmacol Biochem
Behav 75: 103114, 2003.
169. Wellman PJ, Tow S, McMahon L. Isobolographic assessment of the
effects of combinations of phenylpropanolamine and fenfluramine on
food intake in rats. Pharmacol Biochem Behav 50: 287291, 1995.
170. Wessinger WD. Approaches to the study of drug interactions in behavioral pharmacology. Neurosci Biobehav Rev 10: 103113, 1986.
171. White NE, Dhillo WS, Liu YL, Small CJ, Kennett GA, Gardiner JV,
Ghatei MA, Bloom SR. Co-administration of SR141716 with peptide
YY336 or oxyntomodulin has additive effects on food intake in mice.
Diabetes Obes Metab 10: 167170, 2008.
172. Wojda E, Wlaz A, Patsalos PN, Luszczki JJ. Isobolographic characterization of interactions of levetiracetam with the various antiepileptic
drugs in the mouse 6 Hz psychomotor seizure model. Epilepsy Res 86:
163174, 2009.
173. Woodcock J, Griffin JP, Behrman RE. Development of novel combination therapies. N Engl J Med 364: 985987, 2011.
174. Yan H, Zhang B, Li S, Zhao Q. A formal model for analyzing drug
combination effects and its application in TNF-alpha-induced NFkappaB
pathway. BMC Syst Biol 4: 50, 2010.
175. Yates FE. Good manners in good modeling: mathematical models and
computer simulations of physiological systems. Am J Physiol Regul
Integr Comp Physiol 234: R159 R160, 1978.
176. Yeh P, Tschumi AI, Kishony R. Functional classification of drugs by
properties of their pairwise interactions. Nat Genet 38: 489 494, 2006.
177. Zaider M. The interaction of two agents revisited: response to M.C.
Berenbaum. Radiat Res 126: 266 268, 1991.
178. Zimmermann GR, Lehr J, Keith CT. Multi-target therapeutics: when
the whole is greater than the sum of the parts. Drug Discov Today 12:
34 42, 2007.
134. Roth JD, Rowland NE. Anorectic efficacy of the fenfluramine/phentermine combination in rats: additivity or synergy? Eur J Pharmacol 373:
127134, 1999.
135. Roth JD, Trevaskis JL, Wilson J, Lei C, Athanacio J, Mack C, Kesty
NC, Coffey T, Weyer C, Parkes DG. Antiobesity effects of the beta-cell
hormone amylin in combination with phentermine or sibutramine in
diet-induced obese rats. Int J Obes (Lond) 32: 12011210, 2008.
136. Rowland NE, Marshall M, Robertson K. Anorectic effect of dehydroepiandrosterone combined with dexfenfluramine or thionisoxetine. Eur J
Pharmacol 419: 6164, 2001.
137. Rowland NE, Marshall M, Roth JD. Comparison of either norepinephrine-uptake inhibitors or phentermine combined with serotonergic agents
on food intake in rats. Psychopharmacology (Berl) 149: 7783, 2000.
138. Rushing PA, Lutz TA, Seeley RJ, Woods SC. Amylin and insulin
interact to reduce food intake in rats. Horm Metab Res 32: 6265, 2000.
139. Schumacher PM, Dossche J, Mortier EP, Luginbuehl M, Bouillon
TW, Struys MM. Response surface modeling of the interaction between
propofol and sevoflurane. Anesthesiology 111: 790 804, 2009.
140. Schwartz GJ, McHugh PR, Moran TH. Gastric loads and cholecystokinin synergistically stimulate rat gastric vagal afferents. Am J Physiol
Regul Integr Comp Physiol 265: R872R876, 1993.
141. Schwartz GJ, Netterville LA, McHugh PR, Moran TH. Gastric loads
potentiate inhibition of food intake produced by a cholecystokinin analogue. Am J Physiol Regul Integr Comp Physiol 261: R1141R1146,
1991.
142. Shafer SL. All models are wrong. Anesthesiology 116: 240 241, 2012.
143. Shafer SL, Hendrickx JF, Flood P, Sonner J, Eger EI 2nd. Additivity
versus synergy: a theoretical analysis of implications for anesthetic
mechanisms. Anesth Analg 107: 507524, 2008.
144. Shapiro A, Matheny M, Zhang Y, Tumer N, Cheng KY, Rogrigues E,
Zolotukhin S, Scarpace PJ. Synergy between leptin therapy and a
seemingly negligible amount of voluntary wheel running prevents progression of dietary obesity in leptin-resistant rats. Diabetes 57: 614 622,
2008.
145. Shen L, Tso P, Woods SC, Sakai RR, Davidson WS, Liu M. Hypothalamic apolipoprotein A-IV is regulated by leptin. Endocrinology 148:
26812689, 2007.
146. Sherrington CS. The Integrative Action of the Nervous System. New
Haven, CT: Yale University, 1920.
147. Sipols AJ, Stuber GD, Klein SN, Higgins MS, Figlewicz DP. Insulin
and raclopride combine to decrease short-term intake of sucrose solutions. Peptides 21: 13611367, 2000.
148. Steel GG, Peckham MJ. Exploitable mechanisms in combined radiotherapy-chemotherapy: the concept of additivity. Int J Radiat Oncol Biol
Phys 5: 8591, 1979.
149. Stein LJ, Woods SC. Cholecystokinin and bombesin act independently
to decrease food intake in the rat. Peptides 2: 431436, 1981.
150. Straetemans R, Bijnens L. Application and review of the separate ray
model to investigate interaction effects. Front Biosci (Elite Ed) 2:
266 278, 2010.
151. Stricker EM, Verbalis JG. Water intake and bodily fluids. In: Fundamental
Neuroscience (2nd ed.), edited by Squire LR, Bloom FE, McConnell SK,
Roberts JR, Spitzer NC, and Zigmond MJ. San Diego, CA: Academic, 2003,
p. 10111029.
152. Suhnel J. Parallel dose-response curves in combination experiments.
Bull Math Biol 60: 197213, 1998.
152a.SYNERGY Executive Committee. Superior Yield of the New strategy of Enoxaparin, Revascularization and GlYcoprotein IIb/IIIa
inhibitors. The SYNERGY trial: study design and rationale. Am Heart J
143: 952960, 2002.
153. Tallarida RJ. Drug Synergism and Dose-Effect Data Analysis. Boca
Raton, FL: Chapman & Hall/CRC, 2000.
154. Tallarida RJ. Drug synergism: its detection and applications. J Pharmacol Exp Ther 298: 865872, 2001.
155. Tallarida RJ. Interactions between drugs and occupied receptors. Pharmacol Ther 113: 197209, 2007.
156. Tallarida RJ. An overview of drug combination analysis with isobolograms. J Pharmacol Exp Ther 319: 17, 2006.
E253

Understanding Synergy

Încărcat de

Informații document

Drepturi de autor

Formate disponibile

Partajați acest document

Partajați sau inserați document

Opțiuni de partajare

Vi se pare util acest document?

Este necorespunzător acest conținut?

Drepturi de autor:

Formate disponibile

Understanding Synergy

Încărcat de

Drepturi de autor:

Formate disponibile

Understanding synergy

Am J Physiol Endocrinol Metab 304:E237-E253, 2013. First published 4 December 2012;

This information is current as of August 2, 2016.

Downloaded from http://ajpendo.physiology.org/ by guest on August 2, 2016

Am J Physiol Endocrinol Metab 304: E237E253, 2013.

cooperative effect synergy; energy homeostasis; food intake; Loewe additivity;

One and one is what Im telling you . . . Debbie Harry (71)

complications and misunderstandings in synergy analyses. (Note

0193-1849/13 Copyright 2013 the American Physiological Society

Downloaded from http://ajpendo.physiology.org/ by guest on August 2, 2016

Geary N. Understanding synergy. Am J Physiol Endocrinol Metab 304: E237

Why is Understanding Synergy Important?

SUPRA-ADDITIVE SYNERGY IS A FORMAL QUANTITATIVE

Mathematical Nature of Synergy

and synergy occurs when the observed E(a b) is greater than

AJP-Endocrinol Metab doi:10.1152/ajpendo.00308.2012 www.ajpendo.org

Downloaded from http://ajpendo.physiology.org/ by guest on August 2, 2016

VOLUME CONSUMED (ml)

1 ml gastric saline load

10 pmol i.a. CCK

1 ml gastric saline load + 10 pmol i.a. CCK

Change in body weight from enrollment (%)

MEAN 30 MINUTE DIET INTAKE (ml)

AJP-Endocrinol Metab doi:10.1152/ajpendo.00308.2012 www.ajpendo.org

Downloaded from http://ajpendo.physiology.org/ by guest on August 2, 2016

Fig. 1. Examples of the utility of synergy studies

additivity in the threshold region violates the principle of sham

Theory and Validity

Fig. 2. Response additivity violates the principle of sham combinations by

Originally, Loewe additivity referred to a graphic way to

where EB is measured on the dose-effect curve of drug B and

AJP-Endocrinol Metab doi:10.1152/ajpendo.00308.2012 www.ajpendo.org

Downloaded from http://ajpendo.physiology.org/ by guest on August 2, 2016

DOSE of A or B; Log scale

Linear Isoboles are a Rarity

= EA(10 + 10) = EA(20) = 1.3

The method is usually used to derive pairs of dose a of drug

The plot of doses that fulfill Eq. 3 on a graph whose x-axis

Only in a limited number of situations are isoboles straight

shown in Fig. 4 (again, doses Ax and Bx alone each lead to

DOSE off A ((a))

AJP-Endocrinol Metab doi:10.1152/ajpendo.00308.2012 www.ajpendo.org

Downloaded from http://ajpendo.physiology.org/ by guest on August 2, 2016

EAa EAMaxa a A50 ,

where EA(a) is the effect of dose a of drug A, EAMax is its

b B50 A50 a A50 ,

LOG DOSE (D)

AJP-Endocrinol Metab doi:10.1152/ajpendo.00308.2012 www.ajpendo.org

Downloaded from http://ajpendo.physiology.org/ by guest on August 2, 2016

well described by rectangular hyperbolas (35, 37, 105, 130),

In contrast to the situations described above, nonparallel log

Eq. 8 leads to curvilinear isoboles in which convex or

where a is the dose of drug A, EAMax is the maximum effect, A50

which results in curvilinear isoboles for all effect levels X,

a ba A50 EAMax EBMax1 B50 b 1 ,

called the slope parameter (note that if p 1, Eq. 9 becomes the

LOG DOSE (D)

AJP-Endocrinol Metab doi:10.1152/ajpendo.00308.2012 www.ajpendo.org

Downloaded from http://ajpendo.physiology.org/ by guest on August 2, 2016

12) that included derivations supposedly establishing that Eq. 4

additivity energy homeostasis studies also appeared to differ by

DOSE A (dose 60 yields effect AX)

The interpretation different outcomes of A B or B A

AJP-Endocrinol Metab doi:10.1152/ajpendo.00308.2012 www.ajpendo.org