Animal models and conserved processes

Greek and Rice Theoretical Biology and Medical Modelling 2012, 9:40
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.- THEORETICAL BIOLOGY AND
MEDICAL MODELLING
^'


[RESEARCH Op


Animal models and conserved processes

Ray Greek" and Mark J Rice2


I AC* D R k


seIreopon fence r ay reey @
gmail com
Americans For Medical Advancement
(wwwAFMA-curediseaseorg), 2251
Refugio Rd, Goleta, CA 93117, USA
Full list of author information is
available at the end of the article


0 2012 Greek and Rice, licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Biolled Central Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.


Abstract

Background: The concept of conserved processes presents unique opportunities for
using nonhuman animal models in biomedical research. However, the concept must
be examined in the context that humans and nonhuman animals are evolved,
complex, adaptive systems. Given that nonhuman animals are examples of living
systems that are differently complex from humans, what does the existence of a
conserved gene or process imply for inter-species extrapolation?
Methods: We surveyed the literature including philosophy of science, biological
complexity, conserved processes, evolutionary biology, comparative medicine,
anti-neoplastic agents, inhalational anesthetics, and drug development journals in
order to determine the value of nonhuman animal models when studying conserved
processes.
Results: Evolution through natural selection has employed components and
processes both to produce the same outcomes among species but also to generate
different functions and traits. Many genes and processes are conserved, but new
combinations of these processes or different regulation of the genes involved in
these processes have resulted in unique organisms. Further, there is a hierarchy of
organization in complex living systems. At some levels, the components are simple
systems that can be analyzed by mathematics or the physical sciences, while at
other levels the system cannot be fully analyzed by reducing it to a physical system.
The study of complex living systems must alternate between focusing on the parts
and examining the intact whole organism while taking into account the connections
between the two. Systems biology aims for this holism. We examined the actions of
inhalational anesthetic agents and anti-neoplastic agents in order to address what
the characteristics of complex living systems imply for inter-species extrapolation of
traits and responses related to conserved processes.
Conclusion: We conclude that even the presence of conserved processes is
insufficient for inter-species extrapolation when the trait or response being studied is
located at higher levels of organization, is in a different module, or is influenced by
other modules. However, when the examination of the conserved process occurs at
the same level of organization or in the same module, and hence is subject to study
solely by reductionism, then extrapolation is possible.
Keywords: Anesthesia, Animal models, Cancer, Complexity, Conserved processes,
Systems biology






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Background
Marc Kirschner and John Gerhart introduced the concept of facilitated variation and
conserved core processes in their book, The Plausibility of Life [1], in order to explain
how novelty arises in evolution. Motivated by advances in evolutionary and developmental
biology (evo devo), these investigators proposed that conserved processes are ubiquitous in
eukaryotes but pointed out that by using conserved processes differently, for example by
differently regulating the genes that code for the processes, expressing the genes differently,
varying the sequences or combination of genes or transcription factors, novelty can arise.
Mutations in the genes that regulate the conserved processes can accomplish this novelty.
Moreover, by adjusting the regulatory genes, the organism can evolve with fewer mutations
than would be the case if a trait had to arise de novo or from mutations in structural genes.
This has implications for using nonhuman animals (hereafter referred to simply as animals)
as models for humans in biomedical research. One should expect to discover information
regarding conserved processes in humans by studying animal models. We sought to deter-
mine whether limits exist on this method and if so what those limits are.


Methods
We surveyed the relevant literature including philosophy of science, biological complexity,
conserved processes, evolutionary biology, comparative medicine, anti-neoplastic
agents, inhalational anesthetics, and drug development journals in order to determine
the appropriate role for animal models when studying conserved processes. Philosophy
of science is relevant to our discussion as it includes the premises and assumptions on
which research is then based. A study or method can be methodologically sound but if
the premises are incorrect, then the study loses much if not all of its value. The drug
development literature was searched because the final application of much research is
targeted intervention via drugs hence that literature can inform regarding the success of
a practice or modality. The literature concerning biological complexity and conserved
processes was surveyed as it directly relates to the issue being explored. All of this must
be placed into the context of evolutionary biology in order to better explain the findings.
We chose inhalational anesthetics and anti-neoplastic agents as examples because of the
well-known conserved nature of these agents.


Results
Animal models
The use of models has a long history in science, which led philosopher of science Richard
Braithwaite to warn that: "The price of employment of models is eternal vigilance" [2]. In
this section, we will explore what animal models are, how they can be used in scientific
investigation, including biomedical research, and discuss classification schemes. In this art-
icle, we will address the use of predictive animal models in light of the concepts of complex
systems, personalized medicine and pharmacogenomics, and evolutionary biology. We will
then explore what this implies when using animal models to study conserved processes.
Models are important for scientific pursuits and can take the form of abstract models,
computational models, heuristic models, mathematical models, physical models such as
scale models, iconic models, and idealized models. Models can also be divided on the
basis of whether they are used to replicate a portion of the item being modeled or are used






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to test hypotheses or interpret aspects of a theory. Examples of historically important
models include Watson and Crick's physical model of DNA, Pauling's model of chemical
bonds, Bohr's solar system model of the atom, and the billiard ball model of gases. More
recent models include the computer model of the brain, mathematical models of disease
spread, and Lorenz's model of the atmosphere.
Robert Hinde observed that models:

Should be different from the thing being modeled, because if it is not, the modeler
might assume that all properties demonstrated by the model exist in the thing being
modeled;
Are usually less complicated than the thing being modeled;
Are more readily available than the thing being modeled, and;
"pose questions, suggest relations, or can be manipulated in ways not possible with
the original" [3].

In light of the importance of models, some philosophers of science assert that the
study of models per se has been neglected by the philosophy of science community.
Frigg and Hartmann [4] state: "What fills in the blank in 'M represents T if and only
if __; where M is a model and T a target system?" Moreover, how one classifies
models and what criteria must be fulfilled in order for M to be considered a specific
type of model has arguably not been adequately addressed by the philosophy of science
community. Yet another problem with the philosophy of models is the relationship between
theory and model [4]. We maintain that this lack of scholarly attention to models has
played a role in what we see as the confusion surrounding the use of animals as models.
Animal models are physical models and can be further classified based on various
features and uses. For example, they can be distinguished by the phylogenetic distance
of the model species from humans. Animal models can also be classified based on fidel-
ity-how well the model resembles humans-as well as based on validity-how well
what you think you are measuring corresponds to what you really are measuring.
Animal models can also be considered based on reliability-the precision and accuracy
of the measurement [5]. Hau explains that animal models can be categorized as spon-
taneous, induced, transgenic, negative and orphan. Hau states: "The majority of labora-
tory animal models are developed and used to study the cause, nature, and cure of
human disorders" [[6] p3]. This is important as Hau further states that animal models
can be used to predict human responses: "A third important group of animal models is
employed as predictive models. These models are used with the aim of discovering and
quantifying the impact of a treatment, whether this is to cure a disease or to assess
toxicity of a chemical compound. The appropriateness of any laboratory animal model
will eventually be judged by its capacity to explain and predict the observed effects in
the target species" [6]. Others agree that predicting human response is a common use
for animal models [7-12]. For example, Heywood stated: "Animal studies fall into two
main categories: predictive evaluations of new compounds and their incorporation into
schemes designed to help lessen or clarify a recognized hazard" [13].
Animals are utilized for numerous scientific purposes (see ]Table 1) and one of the
authors (Greek) has addressed these various uses in previous publications [14-20]. One
cannot have a meaningful discussion regarding the utility of animal models unless one







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Table 1 Categories of animal use in science and research [16]
1. As predictive models for human disease
2. As predictive models to evaluate human exposure safety in the context of pharmacology
and toxicology (e.g., in drug testing)
3. As sources of 'spare parts' (e.g., aortic valve replacements for humans)
4. As bioreactors (e.g., as factories for the production of insulin, or monoclonal antibodies, or
the fruits of genetic engineering)
5. As sources of tissue in order to study basic physiological principles
6. For dissection and study in education and medical training
7. As heuristic devices to prompt new biological/biomedical hypotheses
8. For the benefit of other nonhuman animals
9. For the pursuit of scientific knowledge in and of itself


specifies the category under discussion. For example, areas in which animal models
have been successfully employed include the evaluation of a phenomenon that can be
described by the physicochemical properties of the organism, the study of basic physiologic
functions, and the study of other traits that can be described by the use of conversion
factors based on the body surface area of the organism. In general, animal models can be
successfully employed in categories 3-9 in Table 1. However, animal models have failed to
be predictive modalities for human response to drugs and disease [13-16,18,21-41], depicted
by categories 1 and 2 in Table 1. (The authors have addressed this failure in numerous
publications and, because an exploration for this failure is not the purpose of the article, we
refer the reader to those publications [14-20,23] even though we realize that some view this
position as controversial [7,11,42-44].) This is not to say that a species can never be found
in retrospect that mimics an outcome in humans. Such a species usually can be identified,
however retrospective correlation is obviously not the same as prediction [45-47]. Moreover,
any process or modality claiming to be predictive can be evaluated by use of the binomial
classification table and equations in Table 2 (as illustrated in Table 3 [48]). Such calculations
are commonly used in science [49-53].

Table 2 Binary classification test
Gold standard
GS+ GS-
Test T+ TP FP
T- FN TN
Sensitivity TP/(TP + FN)
Specificity TN/(FP + TN)
Positive Predictive Value -TP/(TP+ FP)
Negative Predictive Value -TN/(FN+TN)
T- -Test negative
T + Test positive
FP -False positive
TP True positive
FN False negative
TN -True negative
GS- Gold standard negative
GS+- Gold standard positive
The binary classification test allows calculations for determining how well a test or practice compares with reality or the
gold standard.







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Table 3 Example of binary classification values
Gold standard (human)
GS+ GS-
Test T+ 22 26
T- 22 30
Sensitivity 22/(22 + 22) 0.5
Specificity 30/(26 + 30) 0.54
Positive Predictive Value -22/(22+26) -0.46
Negative Predictive Value 30/(22 + 30) 0.58
Binary classification values for cardiovascular toxicity test in monkeys from 25 compounds also tested in humans [48].
Note the values are approximately what would be expected from a coin toss.


When judging the predictive value of a modality, one is not using the term predict
in the same sense as when describing how hypotheses generate predictions to be
tested. The predictive value of a commonly used modality usually is known, or can be
ascertained, for example the positive and negative predictive value of x-ray computed
tomography (commonly referred to as a CT scan) for diagnosing pneumothorax (a rupture
of, or interference in, the pleural membrane which allows air to enter the pleural space and
thus interferes with breathing) approaches 1.0 (is accurate for diagnosing the condition in
100% of cases).
Animal models as used in biomedical research, can also be categorized as causal
analogical models (CAMs) or as heuristic or hypothetical analogical models (HAMs)
[54-59]. The use of animal models to predict human response to drugs and disease, in
accordance with categories 1 and 2 in Table 1, would be an example of using animals
as CAMs. Analogical models in general include the hydraulic model of economies and
the computer model of the brain and can be further divided based on various criteria [4].
Causalism or causal determinism dates to Aristotle who stated: "what is called Wisdom is
concerned with the primary causes and principles." Causalism can be summarized
succinctly, as "everything has a cause." This notion of causation was the basis for animal
models as can be appreciated by the writings of Claude Bernard [60], considered the father
of animal modeling since the 19th century. Bernard's thoughts on animal models are an
extension of Aristotle via the determinism of Descartes and Newton [61]. Causal deter-
minism and the principle of uniformity led to the concept, still accepted by many animal
modelers today, that the same cause would result in the same effect in qualitatively similar
systems. This line of thinking was in keeping with the creationist thinking of 19th century
French physiologists, including Bernard, who rejected Darwin's Theory of Evolution
[60,62,63]. The notion of causal determinism and the principle of uniformity combined
with the rejection of evolution led to the belief in the interchangeability of parts. There-
fore, if one ascertained the function of the pancreas in a dog, he could directly extrapolate
that knowledge to the function of the pancreas in humans, once scaling for size had been
factored in [14,63,64]. Unfortunately, this linear thinking persists as the baboon heart
transplant to Baby Fae illustrates. The operation was performed by the creationist surgeon
Leonard Bailey of Loma Linda University in 1984 [[65] p162-3].
We acknowledge that the concept of causation is problematic [66]. Russell suggested
it be abandoned in 1913 [67] and it is clearly more useful for linear systems than
complex systems. While an exhaustive explanation and discussion of the controversies
surrounding causation would occupy more space than is available for this article







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(see Bunge [61] for such an analysis) we should note that a more current explanation for
causation is that of a "first order approximation." Causation is usually discussed in the
context of a chain of causes. Bunge summarizes current thinking: "neodeterminism ..
asserts in this connection that causation is only one among several interrelated categories
concurring in real processes" [61]. This principle is appreciated even more fully in
complex systems. Current thinking notwithstanding, the use of animal models assumes
the Cartesian concept of causation in that a causal model assumes a deterministic causal
relationship between variables. We will explore this thinking and show that even in the
traditional context there are problems with using animal models to discover "causal"
relationships. These problems are increased exponentially when placed in the context of
complex systems.
Based on the writings of LaFollette and Shanks [[58]p63], we suggest the following in
order for a model to be considered a CAM. X (the model) and Y (the subject being
modeled) share properties {a.. .e}. In X, these properties are associated with, and
thought relevant to, state Sl. S1 has not been observed directly in Y, but Y likely also
has would exhibit S1 under the same conditions as X. This concept is illustrated in
Table 4. LaFollette and Shanks [58] state that, "there should be no causally-relevant
disanalogies between the model and the thing being modeled." Unfortunately, causally
relevant disanalogies do exist among species and even within a species, which leads to
different states or outcomes, as illustrated in Table 4. We again paraphrase LaFollette
and Shanks [[58] p112] and suggest that two more conditions must be met for a model
to qualify as a CAM: the shared properties {a,...,e} must have a causal relationship with
state S1 and be the only causally relevant properties associated with Sl. As Table 4
illustrates, the commonalities between the humans and chimpanzees are insufficient to
qualify chimpanzees as CAMs for human response to HIV infection. (For more on


Table 4 Causal analogical models
X, the model Y, the system Shared properties Perturbation Outcome Outcome in system
being modeled between X and Y to the model in model being modeled
Animal system Human system
(for example,
Pan troglodytes)
a. Genes. >90% of Exposure State St. AIDS. State S1 is
nucleotide sequences to HIV. Mild illness not shared despite
identical, of limited the presence of
duration, shared, relevant
properties.
b. Immune system.
Many commonalities.
Constructed on
generally the same
plan.
c. White blood cells
present and function
similarly.
d. Receptors on white
blood cells also present
and function similarly.
e. Shared intracellular
components of white
blood cells.
Shared properties a ... e for humans and chimpanzee do not result in state S7 also being shared.






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animal models of HIV/AIDS see [14,68].) As we will show, animals and humans are
evolved complex systems and as such exhibit the properties of robustness and redundancy;
hence numerous "causes" can result in the same effect and the same perturbation can
result in different outcomes. Because of this and other properties of complex systems,
we should expect different species to exhibit different causal relationships.
Correspondingly, Giere, Bickle, and Mauldin [69] note that some question the use of
causal models in the study of humans because humans are complex systems whereas
casual models assume a deterministic system: an outcome in a simple system is fixed by
the variables. The problems of determining causation are further explored by Bunge [61]
in his neodeterminism explanation alluded to above and his analysis is highly relevant to
this discussion. While we will attempt to contrast the traditional deterministic view of
causality in light of complexity science, this article will not do justice the current thinking
on causation and we refer the reader to Bunge [61] for a fuller explanation.
Giere, Bickle, and Mauldin suggest a probabilistic relationship instead of a 100%
causal relationship for the model: "C is a positive causal factor (probabilistic) for E in
an individual, I, characterized by residual state, S, if in I the probability of E given C is
greater than the probability of E given Not-C." Likewise, LaFollette and Shanks raise
the question as to whether animal models can be weak CAMs: "Begin with two systems
Si and S2. Si has causal mechanisms {a,b,c,d,e}, S2 has mechanisms {a,b,c,x,y}. When
we stimulate sub-system {a,b,c} of Si with stimuli sf response rf regularly occurs. We
can therefore infer that were we to stimulate sub-systems {a,b,c}of S2 with sf rf would
probably occur" [[58] p141]. LaFollette and Shanks then explain that this outcome will
be highly probable if and only if {a,b,c} are causally independent of {d,e} and {x,y}.
Again we anticipate problems in using animal models as weak CAMs, even in the
traditional deterministic-causation view, because, as we shall discuss, various properties
of complex systems will likely give rise to difficulties in isolating subsystems, which
would be required for an animal model to be a weak CAM. These problems have been
referred to as causal/functional asymmetry and mandates caution in extrapolating data
between species. Kirschner and Gerhart give an example of this:

The case of the octopus and the human camera eye has been looked into, and the
lessons are clear. Underneath the gross anatomical similarities are many differences.
The eye derives from different tissues by different developmental means. Although
both structures use the same pigment (rhodopsin) for photoreception, and both send
electrical signals to the brain, we now know that the intervening circuitry is
completely different [[1] p240-01].

Independent evolution has also produced spindle neurons in species as diverse as
humans and cetaceans. Spindle neurons connect parts of the brain involved in higher
cognition and were thought to only occur in primates but have recently been discovered
in cetaceans, such as humpback whales and fin whales, as well as elephants [70-72].
Convergent evolution, the acquisition of the same trait in different lineages, is also
important when considering the role of animal models.

Evolved complex systems
Reductionism is a method of study that seeks to break a system down into its compo-
nent parts, study each part individually, and then reach a conclusion about the system






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as a whole or at least the role of the individual part. Descartes introduced the concept
and it has proven effective for ascertaining many facts about the material universe.
Conversely, the clockwork universe of Descartes has not held up to scrutiny on all
levels. Quantum mechanics, relativity, chaos, and complexity have revealed the stochastic
nature of the supposedly clockwork, deterministic universe. Regrettably, while physicists
recognized the limitations of reductionism, biologists were uncritically embracing it. Francis
Crick extended reductionism to all aspects of biology when he stated: "The ultimate aim of
the modern movement in biology is to explain all biology in terms of physics and chemistry"
[73]. Biological reductionism arguably reached its zenith in the Human Genome Project
(HGP) [74,75] and, ironically, the consequences of the HGP-that humans have a relatively
small number of genes-have, in large part, been responsible for a re-examination of the
role of reductionism in biology. This has been especially true for human pathophysiology
where animals are used as models for humans.
Systems can be categorized as simple or complex. The world of Newton and Descartes
was largely confined to simple systems hence reductionism functioned well for discovery.
At some levels, the components of a complex system can be simple systems and thus are
subject to study by reductionism while at other levels these simple systems combine to
make complex systems thus necessitating study of the intact whole. Mazzocchi
points out that when reductionism takes a component out of its natural environment
it has consequences for extrapolating the results back to the organism as a whole:
"But this extrapolation is at best debatable and at worst misleading or even hazardous. The
failure of many promising drug candidates in clinical research shows that it is not always
possible to transfer results from mice or even primates to humans" [76].
While evolution is defined as a change in allele frequency over time, complexity
science can be defined as "the study of the behaviour of large collections of simple,
interacting units, endowed with the potential to evolve with time" [77,78]. Living
organisms are complex systems that have highly variable evolutionary histories and as
such are best modeled using nonlinear differential equations. The difficulty with this
approach is that the values for many of the factors are unknown; hence solving the
equation is impossible [49,77].
Animals and humans are examples of living complex adaptive systems and as such
exhibit the following properties [79-97]:

1. Complex systems are composed of many components. Some of these components
may be simple systems, but many are complex systems. These components exist on many
scales and interact extensively with each other. A complex system is a "system of systems."
2. The components can be grouped as modules. For example, the following could be
considered as modules: the cell; the various processes in a cell; gene networks; gene-
gene interactions; gene-protein interactions; protein-protein interactions; organs; and
all the factors that influence the natural history of a disease. However, failure in one
module does not necessarily spread demise to the system as a whole as redundancy
and robustness (see #s 5 and 6 below) also exist and the various modules also
communicate with each other.
3. The different components of a complex system are linked to and affect one
another in a synergistic manner. There is positive and negative feedback in a complex
system [93].






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4. A complex system demonstrates hierarchal levels of organization [98,99]. These
levels range from the subatomic to the molecular to the whole individual to collections
of individuals [100]. Emergence (see # 13 below) occurs at each level; therefore, even a
complete understanding of the lower level is insufficient for explaining the upper level.
The various levels interact such that there is both upward causation and downward
causation. In order to understand a particular level, one must alternate between
looking at the components and looking at the whole while taking into account the
connections between each [76,101]. Moreover, the various levels may respond
differently to the same perturbation.

The various levels of organization are important when considering which responses
to specific perturbations can be extrapolated among species. Living complex systems
have numerous properties that can be studied without consideration of the fact that the
whole, intact organism is a complex system. Some systems or components follow only
the laws of physics, or even simple geometry, while others are best described by their
physicochemical properties or just by chemistry. Some properties of complex systems
can be described simply by math formulas. Growth, for example, can be described as
geometrical in some cases and exponential in others. The surface area of a body
increases by the square of the linear dimensions while the volume increases by the
cube. This is a consequence of geometry and is important in physiology, in part,
because heat loss is proportional to surface area while heat production is proportional
to volume. Haldane stated: "Comparative anatomy is largely the story of the struggle to
increase surface in proportion to volumes" [102]. For example, chewing increases the
surface area of food, the rate the small bowel absorbs nutrients and other chemicals
depends in part on the surface area of the small bowel, and air sacs in the lungs rely on
surface area for gas exchange, as do capillaries.
Allometry is the study of the relationship of body size to shape. Examples of allometric
laws include Kleiber's law: qo -~M where qo is metabolic rate and is proportional to M,
body mass, raised to the % power. The rate t, of breathing and heart contractions are
proportional to M, body mass, raised to % power: t -M". Further, many physiological
functions affect or depend on surface area.
Levels of organization can also be described based on whether they are primarily
chemical reactions and hence subject to analysis by chemistry. Reactions or perturba-
tions that involve the denaturation of proteins should affect all systems, be they simple
or complex, similarly because at this level of organization other factors do not come
into play. Exactly what effects sulfuric acid would have in a person over an extended
period of time are irrelevant as it denatures protein more or less immediately. Perhaps
species differences would manifest if small amounts of H2SO4 were infused over long
periods of time, but the immediate effects are the same across species because of the
chemical properties of the acid.
Animals can be successfully used for numerous purposes in science (see 3-9 in
Table 1). One of the purposes for which animals can be successfully used is to evaluate
phenomena that can be described by the physicochemical properties of the organism.
The same applies to basic physiologic functions. There are physiological parameters
that can be applied across species lines by the use of conversion factors based on the
weight or surface area of the organism. There are also properties of organisms that can






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be anticipated by the physical or chemical properties of the substance acting on the
organism. All of these are instances of successfully treating a complex system as if it
were a simple system. Problems arise however, as the level of organization under study
increases. Allometric scaling based on body surface area (BSA), for example, does not
include differences that manifest at higher levels of organization for example in the
elimination or metabolism of drugs. Different levels of organization can be acted on by
single factors or many factors but perturbations of simple systems, or systems that can
be described as simple on the level or organization being affected, should produce
similar results.

5. Complex systems are robust, meaning they have the capacity to resist change.
This can be illustrated by the fact that knocking out a gene in one strain of mouse
may produce negligible effects while being lethal to another strain. Gene pleiotropy is
an additional example [103].
6. Complex systems exhibit redundancy. For example, living systems exhibit
redundancy of some genes and proteins [103].
7. Complex systems are dynamic. They communicate with, and are acted on by,
their environment.
8. Complex systems exhibit self-organization, which allows adaptation to the
environment [85,104-106]. The intact cell is a prime example of this property.
9. Complex systems are dependent on initial conditions. The well-known example of
the butterfly flapping its wings and causing a weather catastrophe on the other side of
the earth-the butterfly effect-is an example of dependence on initial conditions. An
example in living complex systems would be that very small differences in genetic
makeup between two systems could result in dramatic differences in response to the
same perturbation. For example, monozygotic twins raised in the same environment
may have different predispositions to diseases such as multiple sclerosis and
schizophrenia [107-110]. Additionally, the above-mentioned observation that knocking
out a gene results in different outcomes in two stains of mice illustrates the concept
that small differences in initial conditions-genetic makeup-can mean the difference
between life and death [93,103,111,112].
10. The initial conditions of a complex living system are determined, in part, by
evolution. Various species have different evolutionary histories and thus are differently
organized complex systems. Initial conditions can be different, despite the exact same
genes, secondary to modifier genes, differences in regulation or expression of genes,
epigenetics, and mutations among others factors. For example, small epigenetic
changes probably account for the dissimilarities between monozygotic twins in terms
of disease susceptibility [107,108,113-116].
11. Perturbations to complex systems result in effects that are nonlinear [99]. Large
disturbances may result in no change to the system while minor perturbations may
cause havoc [76,105]. Efforts to describe complex systems in terms of linear cause
and effect relationships are prone to failure [117]. Extrapolating among complex
systems is even more problematic because of nonlinearity, along with the other
factors described.
12. The whole of a complex system is greater than the sum of the parts; hence, some
processes and or perturbations are not amenable to study by reductionism.






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13. Complex systems have emergent properties that cannot be predicted even in light
of full knowledge of the component parts.

Animal models have historically been utilized for the prediction of human responses
to drugs and disease and this use has also been the justification for animal use in
research in general [118,119]. But because various levels of organization and different
modules can be acted on by the same perturbation, in order to evaluate whether an
animal model can be used as a predictive modality, one needs to understand the levels
affected by the perturbation, what rules are being followed at those levels, and whether
the system is simple or complex at the respective levels. Empirical evidence, explained
and placed in context by theory developed from complexity science and evolutionary
biology, suggests animal models cannot predict human responses to drugs and disease
[14-16,18,57,58,120], despite the presence of shared physicochemical properties and
conserved processes.

Conserved processes
Theodosius Dobzhansky famously stated: "Nothing in biology makes sense except in
the light of evolution." We want to examine the consequences that various characteristics
of evolved complex systems, such as modules and different levels of organization, have
on processes conserved by evolution in terms of determining the response of whole
organisms to perturbations. Conserved processes and genes are the subject of much
interest today [1,121-133]. Kirschner and Gerhardt state: "all organisms are a mixture of
conserved and nonconserved processes (said otherwise, or changing and unchanging
processes)" [[1] p34-35]. Conserved processes are not reactions to the laws of physics or
the determination of properties of an organism as they relate to chemistry or geometry.
Nevertheless, conservation reaches across phyla and even kingdoms. Kirschner and
Gerhardt have pointed out that processes conserved include those involved in cell function
and organization, development, and metabolism and that these processes are similar in
animals, yeast, and bacteria. They note that novelty has been the result of using the
conserved processes in different ways rather than inventing completely new processes
[[1] p34-35]. This has critical implications for what can be learned from interspecies study.
Housekeeping genes in general perform the same function; make the same proteins,
in mice, frogs or humans. The role of FOX transcription factors is conserved among
species [134] as is the role of Sarco(endo)plasmic reticulum (SER) Ca2' ATPases
(SERCA) pumps [135]. Modules have also been conserved. The fin module of the
modern fish for example, arose roughly 400 million years ago and has been conserved
ever since [[1] p65].
Conserved processes include core genes like those in the homeobox that are involved in
the same developmental processes. Because these processes and genes are conserved
among species, we could reasonably expect the same outcome from the same perturbation,
regardless of the species containing these processes. But is this the case? In 1978 Lewis
[136] published his seminal work on the anterior-posterior layout of Drosophila. This was
followed in 1984 by the discovery of the homeobox by McGinnis et al. [137]. The field of
evo devo developed in large part from this work. In the last decade, enormous strides have
been made as a result of research in evo devo and the various genome projects. The results
of such research have revealed an enormous genetic similarity among mammals. At the






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level of the genes centrally involved in development, e.g., the homeobox genes, bilaterians
are virtually identical. The homeobox class of genes [138] are conserved across species
lines, functioning in early cellular organization and anterior-posterior body plan
layout [139]. There are important differences however. For example, there are nine
Hox genes in flies but thirty-nine in mammals. Pertinently, we understand how
modifications (gene duplications, deletions, changes in the regulatory processes and
so forth) to these conserved processes have resulted in the evolution of different
body types and indeed different species [138,140-142].
MicroRNA (miRNA) has been found in essentially all species from Caenorhabditis
elegans to humans and plays a large role in gene regulation [143-145]. Apparently, over
50% of miRNAs are conserved across species lines in vertebrates [145]. An important
consideration for drug development, however, is the fact that even though miRNA is
conserved, up to 50% of miRNAs differs among vertebrates. This is important when
considering the use of animals as predictive human models. Furthermore, miRNA
expression levels change when tissues deteriorate from a healthy state to a diseased
state [146-152]. Thus the exact role of miRNA may differ intra-individually depending
on age and disease. Hence, we see both inter-species and intra-individual differences
with respect to this conserved process.
It is well known that humans and nonhuman primates respond differently to infec-
tions. For example, untreated humans usually progress to AIDS when infected with
HIV, are susceptible to malaria (except those with sickle cell anemia), have different
reactions to hepatitis B and C than nonhuman primates and, appear more susceptible
to many cancers and Alzheimer's disease [153-155]. Barreiro et al. [154] studied gene
expression levels in monocytes from humans, chimpanzees, and rhesus macaques and
found that all three species demonstrated "the universal Toll-like receptor response"
when stimulated with lipopolysaccharide (LPS). However they also discovered that only
58% of genes identified in the Toll-like receptor response "showed a conserved regula-
tory response to stimulation with LPS," and only 31% of those genes demonstrated the
same conserved regulatory response when exposed to viruses or bacteria. Barreiro et al.
also discovered that 335 genes in humans are unique among the species in responding
to LPS, with 273 genes responding only in chimpanzees, and 393 only in rhesus maca-
ques [154]. Even in conserved processes, there are going to be significant differences
that influence the outcomes from disease perturbations. Significant differences in the
details of conserved processes (also illustrated by Figure 1 [156]) mean that there are
differences in the initial conditions of the complex system and this has major implica-
tions for inter-species extrapolation.
The implications of the various properties of complex systems also become apparent
when scientists study processes such as preimplantation embryonic development
(PED). PED is thought to be highly conserved among species which led Xie et al. [157]
to study gene expression profiles in embryos from humans, mice, and cows. They
found that: "40.2% orthologous gene triplets exhibited different expression patterns
among these species." Differences in expression profiles have implications for drug and
disease response.
The Cdcl4 gene was discovered in the yeast Saccharomyces cerevisiae and is classified
as a dual-specificity phosphatase. It has since been found in many organisms including
humans. Human Cdcl4B fulfills the role, in yeast, of the yeast gene Cdcl4. Because the






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A S
as


iglec
671 910


3

01 1 1 1
4





-










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4


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2^ J
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Human


Fluorescence Intensity


0 1o1 l 10e 10i 10e I
Fluorescence Intensity


% Positive
Figure 1 Variation in sialic acid (Sia)-recognizing Ig-superfamily lectins among primates. "Expression
of CD33rSiglecs on human and great ape lymphocytes. (A) Percentage of positive lymphocytes for each
Siglec Ab (staining above negative controls) for 16 chimpanzees, 5 bonobos, and 3 ,. are shown, as
well as data for 8 humans (the latter were tested on one or more occasions). Examples of flow cytometry
histograms of human (B) and chimpanzee (C) lymphocytes using Abs recognizing Siglec-3, Siglec-5, Siglec-7,
and Siglec-9 (y axis: normalized cell numbers expressed as percent of maximum cell number detected). In later
samples examined, low levels of Siglec-11 staining (<5% positive) were occasionally detected on lymphocytes
in both great apes and humans (data not shown)" [156].


yeast gene plays a role in regulating late mitosis, it was assumed the gene would have
the same role in mammals. In actuality, neither Cdcl4A nor Cdcl4B are necessary for
cell-cycle progression in humans [158]. Thus, we have a conserved gene but not a
conserved function.
Pyrin proteins have been found to be ubiquitous in mammals. Pyrin-only protein 2
(POP2) was found in humans and thought to be important in inflammatory diseases.
Atianand et al. [159] studied mice but did not find POP2. They then discovered that
POP2 was not in rodents or many other mammals but was present in chimpanzees
(Pan troglodytes) and rhesus macaques (Macaca mulatta). Moreover, the chimpanzee


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i






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POP2 was identical to humans POP2 at both the DNA and protein levels but the
macaque POP2 was not.
Conserved processes act, are affected by, or interact at multiple levels of organization.
As Cairns-Smith points out, proteins, catalysts, nucleic acids, membranes, and lipids
are interlocked and all are dependent on the others for their production. Cairns-Smith
summarizes by stating: "Subsystems are highly interlocked ... The inter-locking is tight
and critical. At the centre everything depends on everything" [[81] p39]. The same per-
turbation may result in different effects or outcomes for different levels of organization
in the same intact system. This further complicates our ability to predict outcomes
between two intact living complex systems. Thus it appears that a perturbation of
complex system S containing conserved process Pi resulting in outcome 01 will not
necessarily result in 01 in the very similar complex system S2 that also has Pi.
We will now examine in more detail the response of organisms to inhalational anesthetics
and anti-neoplastic agents in order to illustrate what can and cannot be extrapolated
between species knowing that species are acted on and affected by the fundamental
principles of geometry, chemistry, and physics as well as shared conserved processes.

Conserved processes in anesthesia
General anesthesia by means of inhalational anesthetics (IAs) provides us with an excellent
opportunity to examine where the effects of conserved processes can and cannot be extra-
polated between species. We expect to see various effects at different levels of organization
and in different modules. We also anticipate effects on emergent properties. Because
IAs act on the system as a whole, we expect to see effects that cannot be predicted from
reductionism. This has implications for what can be expected in terms of predicting human
response by studying a different species or perhaps even a different individual. Therefore,
both the primary effect of the anesthetic agent as well as the side effects may vary.
Broadly speaking, general anesthesia in humans and animals is defined by amnesia,
controlled insensitivity and consciousness, and immobility. It has been observed that most,
if not all, extant vertebrate species exhibit an anesthetic-like response to a wide variety of
chemicals that seemingly have little in common. This has been termed the universal
response. Multiple mechanisms for the universal response have been postulated and this is
an area of intense current research [160-164]. There seems to be general agreement that
ligand gated ion channel (LGIC) protein receptors are involved as well as possible effects on
the cellular membrane. Regardless of the exact details, the conservation of mechanisms can
be seen in that inhalational anesthetics (IAs) have observable effects on motor or motility
responses in vertebrates and invertebrates [165-168], tactile plants [169] and ciliated protests
[170]. (We note that this is probably an example of an exaptation, specifically a spandrel,
rather than an adaptation [171-173].) Interestingly, effects have even been observed in S.
cerevisiae (Baker's yeast) [174], suggesting that crucial aspects of the universal response go
beyond metazoans to include Eukaryotes. Moreover, IAs have been shown to have effects
on membrane composition in prokaryote species [163,175] e.g., A. laidlawii [176,177],
Bacillus halodurans [175] and E. coli [178] and the single-celled eukaryote tetrahymena
[179,180] (a ciliated protozoan). The universal response appears to date far back in evolu-
tionary time and strongly suggests that the mechanism has been conserved among species.
However, there are differences in outcomes with respect to IAs. Humphrey et al.
[181] studied genes in Caenorhabditis elegans and Drosophila melanogaster in order to






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assess the function of genes thought involved in the response to IAs. They found that a
gene in C elegans, unc-79, and a gene in Drosophila, narrow abdomen (na), were
related to each other and play a conserved role in response to anesthetics. However,
mutations in each gene produced unique changes in sensitivity to IAs. The sensitivity
to halothane, an IA, was increased but the sensitivity to enflurane, a different IA, was
unchanged or perhaps even lowered. This is perplexing because one would have
expected the two inhalational agents to be affected in a similar fashion by the mutation.
The gene unc-79 appears to be a post-transcriptional regulator of na, thus the genes
operate in the same pathway. Interestingly, both genes are also associated with similar
phenotypes regarding locomotion: "fainting" in C elegans and "hesitant walking" in
Drosophila.
Stimulation of the conserved processes controlling the universal response results in
clinically significant variability among humans, even though the minimum alveolar
concentration (MAC) for IAs for most species is approximately the same. MAC is the
most often used metric to assess IA potency. However, the concept of MAC implies
variability. MACso, simply called MAC in anesthesiology, is the minimum alveolar
concentration necessary to suppress movement in response to painful stimuli in 50% of
subjects [182]. MAC is significantly variable among humans depending on a number of
factors including age and sex. Why is this the case?
Sonner et al. reported, "one hundred forty-six statistically significant differences
among the 15 strains [of mice] were found for the three inhaled anesthetics (isoflurane,
desflurane, and halothane)" [164]. They concluded that multiple genes must be
involved in anesthetic potency. Wang et al. developed two strains of mice that mani-
fested different sensitivities to isoflurane [183]. MAC is an example of a phenomenon
controlled by quantitative trait loci [184], which may explain in part why, while one
can obtain a rough approximation of MAC by studying other species, there will still be
clinically significant differences.
IAs also function at different levels of organization and on modules in addition to the
one involved in the universal response. The side effects of the same chemical that
produce an effect on the conserved receptors or other processes vary greatly from
species to species and in some cases, even from person to person. A good example is
the case of isoflurane and coronary steal. In the 1980s, there was heated controversy
regarding the administration of the inhalation anesthetic isoflurane to patients with
heart disease. The controversy centered on research using canines that indicated that
the drug caused myocardial ischemia during certain situations in patients with coronary
disease. The phenomenon appeared to result from isoflurane causing dilation of the
normal coronary arteries, and thus blood being shunted away from the occluded coron-
ary arteries; the arteries and tissues that most needed it. This was called coronary steal.
Further, this situation was worsened by a decrease in blood pressure; a condition that
often occurs during general anesthesia with IAs. This supposed danger, based almost
entirely on studies in canines, was seized on by many in the anesthesiology community
as dogma [185,186].
This was an interesting reaction from clinicians for two reasons. First, experiments
with other species had failed to demonstrate coronary steal [187,188] and second,
anesthesiologists had not noticed ischemic changes associated with isoflurane despite
much use of the agent. The situation was also troublesome because isoflurane was a






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needed addition to an anesthesiologist's armamentarium when initially approved for
clinical practice. Six years after its introduction, it was the most frequently used IA, in
part because of the favorable properties of the drug [185]. Further studies continued to
demonstrate varying effects intra- and inter-species [189,190]. Ultimately, studies began
to appear that suggested isoflurane was in fact cardio-protective. The mechanism for
this protection was called preconditioningg and involves the opening of adenosine
triphosphate-dependent potassium channels" [191]. Isoflurane went from being contrain-
dicated in patients with coronary artery disease to being the drug of choice in such
patients. Studies from animals, specifically dogs, figured heavily in forming both, mutually
exclusive, conclusions.
Just as with the homeobox, miRNAs, and the response to inflammation, there are
differences among species in how the conserved process known as the universal
response to anesthesia manifests. Clinically, these differences are significant and limit
the amount of information that can be extrapolated between species even when the
underlying process is conserved. Inhalation anesthetics are also a good example of why,
when the level of organization or module being examined changes, extrapolation breaks
down. The same chemical that induces general anesthesia in a dog will probably result
in the same effect in humans but the dose may vary in a clinically significant fashion
and the side effects will most likely vary, as the conserved process does not dictate the
side effects. Differences in outcomes from perturbations like the ones we have seen
above have been explained by evolution-based species-specific differences, for example
background genes, mutations, expression levels, and modifier genes [192-209].

Anti-neoplastic drugs acting on mitosis
As discussed, a relationship exists between BSA and many physiological parameters
[210]. For example, Reagan-Shaw, Nihal, and Ahmad state: "BSA correlates well across
several mammalian species with several parameters of biology, including oxygen
utilization, caloric expenditure, basal metabolism, blood volume, circulating plasma
proteins, and renal function" [211]. Dosing algorithms for first-in-man (FIM) trials are
based on the assumption that there is a one-to-one dose scale between humans and
animals when BSA is taken into account [212]. The first study suggesting a relationship
between dose and body surface area was performed by Pinkel in 1958 [210] involving
anti-neoplastic agents, drugs where the effects and side effects are largely the same-
cell death. Subsequently, Freireich et al., [213] studied 18 anti-neoplastic drugs in six
animal species and concluded that the maximum tolerated dose (MTD) for humans
was 1/12 of the dose in mice that resulted in the death of 10% of the mice (LD10). They
also noted that the MTD was 1/7 of the LD10 in rats. These were also the ratios for
converting from a mg/kg dose to a dose based on BSA. Fifty anti-neoplastic drugs were
then studied using this formula and all were reportedly introduced into human trials
without incident [214,215]. The standard for FIM doses then became the 1/10th the
LD10 for mice. Actually Freireich recommended a starting dose of 1/3rd the LD10 not
1/10th but that changed over time. The 1/3rd recommendation was found to be too
large for FIM and was changed to 1/10th [216]. More studies appeared to confirm the
1/10th value [217].
The above makes a prima facie case that animal models can predict a starting dose
for humans in clinical trials for anti-neoplastics. Further substantiating this is the fact






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that anti-neoplastics are not always metabolized by the liver [218], thus possibly elimin-
ating a complex system from consideration. Cell division by mitosis is arguably the
most conserved process one can find in biology and the traditional drugs for treating
cancer act by interfering with mitosis. (Newer drugs act on targeted pathways as
opposed to the cell cycle.) Anti-neoplastics kill the cells that are dividing most rapidly
-the cancer cells. However, hair cells, cells in the bone marrow, and cells in the gut
also divide at a similar rate such that anti-neoplastics can affect them. Thus, in part,
the effects and side effects of anti-neoplastics are the same-cell death. The problem
with traditional anti-neoplastics is that they do not discriminate adequately.
Anti-neoplastic drugs are unique in medicine in that: 1) they are nonspecific; 2) long
term toxicities are anticipated and accepted because the patient frequently does not
have any other viable options; 3) the effects and side effects of the drugs are the same
-cell death; and 4) they act on a universally conserved process-mitosis. This is why
body surface area appears to be so effective for calculating FIM dose. Whereas, when
one is examining effects and side effects of drugs based on interactions at the level of
organization where complexity is relevant, for example metabolism [219-229], there are
simply too many other factors to allow for the expectation of one-to-one correlations.
Species-specific differences create perturbations in the complex system thus the differ-
ences among species outweigh the similarities [13-16,18,21-41].
However, in the final analysis even the FIM dose of the anti-neoplastic agents cannot
be reliably ascertained based on BSA. Most anti-neoplastics are effective only at doses
near the maximum tolerated dose and the drugs are given in an escalating fashion
during clinical trials. "Patients treated at the lower end of the dose escalation strategy
are unlikely to receive even a potentially therapeutic dose since most cytotoxic drugs
are only active at or near the MTD" [217]. Differences among species in dose response
for anti-neoplastics are due in part to differences in pharmacokinetics [217,230-232],
which cannot be accounted for based on BSA. Brennan et al. state that: "While proper
determination of drug doses can be complicated within the same species, it can be an
incredible challenge and burden between species" [233]. Brennan et al. continue by
pointing out that metabolism and clearance differ among species and that "...the liver,
kidneys and hematopoietic system between species may have significant differences in
their sensitivity to chemotherapeutic agents. None of these factors are taken into
account with the use of the species-specific dose calculations" [233]. They recommend
area under the curve (AUC) for calculating FIM dose but then concede: "However,
there are numerous examples in which the species-specific conversion dose varies
significantly from the AUC guided dose and/or far exceeds the animal's maximum
tolerated dose." They then list examples from pediatrics where the recommended and
actual doses differ significantly [233].
Horstmann et al. [234] reviewed 460 Phase I National Cancer Institute trials involving
11,935 adults that occurred between 1991 and 2002. Approximately 25% of the trials
were FIM trials. Horstmann et al. found that serious nonfatal effects occurred in 15%
of the patients undergoing single chemotherapy, with 58 deaths that were probably
treatment-related [234,235]. Concern has also been expressed that animal models have
derailed anti-neoplastics that would have been successful in humans [30,235-239].
FIM dose based on animal models is ineffective for predicting dose for other drug
classes as well-TGN1412 being a recent notable example [26,240,241]. An unnamed






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clinician, speaking of toxicity trials for new drugs in general in humans, was quoted in
Science, stating, "If you were to look in [a big company's] files for testing small-
molecule drugs you'd find hundreds of deaths" [242]. Chapman reinforced this stating:
". but other incidents of harm [besides TGN1412], even death, to participants in
Phase I trials, some then known and other unpublicized, had taken place" [235]. It is
also important to note that the 1/3rd or 1/10th safety factor is fabricated. Perlstein et al.
state: "Due to uncertainty in translating animal model findings to humans, particularly
for unprecedented mechanisms, a wide dose range (1000-fold) is expected to cover the
entire exposure-response curve" [243]. Extrapolating from species to species should
not require fudge factors if the process is truly science-based. In Phase I trials, where
FIM or first in human (FIH) occurs, scientists want to characterize the drug's PK prop-
erties and safety margins [244]. Wexler and Bertelsen summarize the situation when
they state: "Although allometric scaling techniques continue to provide poor predictive
estimates for human pharmacokinetic parameters, FIH starting doses are selected with
substantial safety factors applied to human equivalent dose, often in excess of regulatory
guidelines. Approaches that could enhance the predictive nature of a compound's
disposition and adaptive nature of FIH studies could provide a tremendous benefit for
drug development" [245]. FIM for all classes of drug could be easily accomplished using
microdosing [246-248] with the first dose of one nanogram [249,250] and increasing
subsequent doses to the desired endpoint.
Finally, one must recall that 95% [31,251,252] of anti-neoplastic agents fail in clinical trials.
Oncology drugs fail more frequently in clinical trials than most other categories [253,254]
and a higher percentage of anti-neoplastic drugs fail in Phase III trials than drugs from any
other category [255]. Reasons for the attrition include the fact that most of the effects and
side effects, even of the anti-neoplastic agents, when placed into the context of a complex
system, are not predicted from animal studies. Interfering in mitosis is a universal
phenomenon but the degree and success of that interference varies. The FIM dose estima-
tion is apparently successful because the level of organization in question is very basic and
conserved and because the dose is lowered even further by fudge factors. Picking a starting
dose based on the most toxic substances in nature [249,250] would be more scientific. The
apparent success also breaks down because the types of cancers in humans differ from those
in animals, the genetic background of humans varies from that in animals, and because the
reality of a complex system-the interactions of all the other systems (for example how the
drugs are eventually metabolized and eliminated and how those metabolites interact with
other systems and so on)-eventually appear. These are the problems that cannot be solved
by animal models and are why the attrition rate is 95%. Weinberg stated: "it's been well
known for more than a decade, maybe two decades, that many of these preclinical human
cancer models have very little predictive power in terms of how actual human beings-actual
human tumors inside patients-will respond ... preclinical models of human cancer, in large
part, stink ... hundreds of millions of dollars are being wasted every year by drug companies
using these [animal] models" [236]. Others have also pointed out the inadequacy of animal
models of cancer, including genetically modified animal models [41,214,252,256-261].

Conserved processes in light of systems biology
As the level of organization in a complex system increases, we expect to see an increase
in the number of emergent properties as well as more overall interactions. A gene or






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process that has been conserved will interact with the intact whole organism yielding
new processes and states. Perturbations of these conserved genes or processes will thus
likely result in new states not seen in other organisms that share the conserved
processes; perhaps not even in organisms of the same lineage (clade) or species.
The lack of appreciation for the differences between levels of organization and other
properties of complex systems is apparent in the following from Kardong [[262] p2],
writing in his textbook of comparative vertebrate anatomy: "For example, by testing a
few vertebrate muscles, we may demonstrate that they produce a force of 15 N newtonss)
per square centimeter of muscle fiber cross section. Rather than testing all vertebrate
muscles, a time-consuming process, we usually assume that other muscles of similar cross
section produce a similar force (other things being equal). The discovery of force production
in some muscles is extrapolated to others. In medicine, the comparative effects of drugs on
rabbits or mice are extrapolated to tentative use in humans." At the level of organization
where one studies the force generated by muscle fibers, no doubt inter-species extrapolation
is useful, but that is an entirely different level from where drug actions occur.
Indeed the successes from using animal models have been examples of perturbations
occurring at subsystems that can be described as simple systems and or outcomes or
characteristics that apply on the gross level of examination. For example, the Germ
Theory of Disease applies to humans and animals. The immune system reacts to
foreign entities in a manner that is grossly similar across species lines. The details of
immunity are clinically very different, for example HIV infection leads to AIDS in
humans but not chimpanzees [263-265]. Nevertheless, grossly, inflammation, white
blood cells, and antibodies are identifying characteristics of the immune system in the
phylum Chordata. Likewise, while the heart functions to circulate the blood in
mammals, the diseases various mammalian hearts are subject to differ considerably
[266-272]. The failures of animal models have occurred when attempting to extrapolate
data from higher levels of organization, levels where complexity is an important com-
ponent in the system or subsystem under consideration. For example, a drug that has
passed animal tests and is in Phase I human clinical trials has only an 8% chance of
making it to market [273]. Over 1,000 drugs have been shown to improve outcomes in
cerebral ischemia in animal models but none, save aspirin and thrombolysis, which
were not animal-based discoveries, have been successful in humans [35,274-277]. The
animal model for polio, monkeys, revealed a pathophysiology that was very different
from that of humans [278-281]. Extracranial-intracranial bypass for inoperable carotid
artery disease was successful in animals but results in net harm for humans [282-285].
Most diseases are multifactorial hence it should come as no surprise that conserved
processes play a small, although at times important role, in major diseases like heart
disease, cancer and stroke. The field of systems biology was formed in part in an
attempt to place the parts of molecular biology and genetics in the larger context of the
human system; the system that actually responds to drugs and disease. An editorial in
Nature asks: "What is the difference between a live cat and a dead one? One scientific
answer is 'systems biology'. A dead cat is a collection of its component parts. A live cat
is the emergent behaviour of the system incorporating those parts" [286]. According to
the Department of Systems Biology at Harvard Medical School: "Systems biology is the
study of systems of biological components, which may be molecules, cells, organisms
or entire species. Living systems are dynamic and complex, and their behavior may be






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hard to predict from the properties of individual parts" [287]. Systems biology [288]
takes a top-down approach as opposed to reductionism, which evaluates organisms
from the bottom-up. Systems biology is concerned more with networks than individual
components, although both are studied. It also recognizes the importance of emergent
phenomena. (See Figure 2 [79]). Such top-down approaches are used by the fields com-
monly referred to as "Omics," for example: interactomics, metabolomics, proteomics,
transcriptomics, and even fractalomics [289].
Nobel laureate Sydney Brenner, in 1998, emphasized that the interactions of components
was important in understanding an organism [290]. Only by studying proteins and
processes in the context of their systems can we expect to understand what happens to the
intact organisms as a result of these processes and genes. Further, evolution uses old
pathways and processes in different ways to create novelty [1,133]. Everything is context
dependent. Noble stresses that in order to predict how drugs will act, one must understand
"how a protein behaves in context" at higher levels of organization [291].
Heng [292], writing in JAMA states that, because of reductionism, biological scientists
have sought individual components in a disease process so they could intervene. A linear
cause and effect relationship was assumed to exist. Heng cites diabetes intervention in an
attempt to control blood glucose and cancer therapies as examples. He points out that
while this has worked well in many cases, very tight control of blood glucose was recently
found to increase the risk of death [293]. Along the same lines, chemotherapies for cancer
have decreased the size of the tumors but at the expense of an increase in frequency of
secondary tumors and a very adversely affected lifestyle. Furthermore, most chemotherapy
does not prolong life or result in a longer, high quality life [294-296]. Instead of focusing on
small modules or components of a system, complexity theory mandates that biomedical
science look at the system as a whole.
Closely related to systems biology are the concepts of personalized medicine and
pharmacogenomics [226,297-305]. It has long been appreciated that humans respond
differently to drugs and have different susceptibilities to disease. Based on studies of




Reductionism Systems Science


000
o % <10 =>

Components 0 Interrelationships,
Time / Dynamics
Space
Context

Medical Treatments Medical Treatments
Disease-driven Individualized
Disease-driven Multidimensional use of drugs
Aimed for normalcy (normal range) Time-sensitive
Additive Space-sensitive
Synergistic


Figure 2 Reductionism versus systems biology.






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twins, there appears to be a genetic component to susceptibility to leprosy, poliomyelitis
and hepatitis B, as well as response to opioids [306-309]. Other infectious diseases that
appear to have a genetic component to susceptibility include HIV, Hepatitis C, malaria,
dengue, meningococcal disease, variant Creutzfeldt-Jakob disease and perhaps tuberculosis
among others [310]. Differences in drug and disease response are manifest among ethnic
groups [311-319] and sexes [320-326]. Even monozygotic twins manifest differences in
response to such perturbations [107,108,113-116]. Rashmi R Shah, previous Senior Clinical
Assessor, Medicines and Healthcare products Regulatory Agency, London stated in 2005:
"During the clinical use of a drug at present, a prescribing physician has no means of
predicting the response of an individual patient to a given drug. Invariably, some patients
fail to respond beneficially as expected whereas others experience adverse drug reactions
(ADRs)" [327].
Similarly, Allen Roses, then-worldwide vice-president of genetics at GlaxoSmithKline
(GSK), said fewer than half of the patients prescribed some of the most expensive drugs
derived any benefit from them: "The vast majority of drugs more than 90% only
work in 30 or 50% of the people." Most drugs had an efficacy rate of 50% or lower
[328]. Because of differences in genes, like SNPs, all children may not currently be
protected by the same vaccine [329,330]. It is estimated that "between 5 and 20 per
cent of people vaccinated against hepatitis B, and between 2 and 10 per cent of those
vaccinated against measles, will not be protected if they ever encounter these viruses"
[330]. In the future such children may be able to receive a personalized shot. Currently,
numerous drugs have been linked to genetic mutations and alleles. See Table 5 [303]
and Table 6 [331]. The number of personalized medicine products has increased from
13 in 2006 to 72 as of 2012 [332].
When animals were being used as models in the 19th century, many of the scientists
who were using them had not accepted evolution and believed that animal parts were
interchangeable with their human counterparts [60,62,63]. Given that we now under-
stand that intra-human variation results in such markedly different responses to drugs
and disease, attempting to predict human response from animal models, even for
perturbations acting on conserved processes, seems unwarranted. Yet, despite the
implications of personalized medicine [22], some scientists continue to commit the
fallacy described by Burggren and Bemis: "Yet the use of 'cockroach as insect; 'frog as
amphibian; or 'the turtle as reptile' persists, in spite of clear evidence of the dangers of
this approach. Not surprisingly, this type of comparative physiology has neither
contributed much to evolutionary theories nor drawn upon them to formulate and test
hypotheses in evolutionary physiology" [[333] p206]. Comparative research will yield a
nice comparison of the trait or process among species or phyla. However, one simply
cannot assume that the outcome from a specific perturbation in, say the cockroach, will
be seen in insects in general and this concept becomes even more important when
relying on animal models for medical interventions in humans.

Conclusion
A perturbation of living complex system S containing conserved process P1 resulting
in outcome 01 will not result in 01 in the very similar living complex system S2 that
also has Pi often enough to qualify S as a predictive modality for S2 when the trait or
response being studied is located at higher levels of organization, is in a different







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Table 5 Examples of drugs with genetic information in thier labels
Drug Sponsor Indication Gene or Effect of
genotype genotype
Abacavir GlaxoSmithKline HIV-1 HLA-B*5701 Hypersensit
(Ziagen)


Clinical directive on
label
ivity Black-box warning.
"Prior to initiating


therapy with abacavir,
screening for the HLA-
B*5701 allele is
recommended." "Your
doctor can determine
with a blood test if
you have this gene
variation."


Azathioprine
(Imuran)


Prometheus Renal allograft
transplantation,
rheumatoid


Carbamazepine Novartis
(Tegretol)


Epilepsy,
trigeminal
neuralgia


TPT 2TPT*3Aand Severe
TPMT*3C myeloxicity









HLA-B*1502 Stevens-
Johnson
syndrome c
toxic epider
necrolysis


"TPTgenotyping or
phenotyping can help
identify patients who
are at an increased
risk for developing
Imuran toxicity."
"Phenotyping and
genotyping methods
are commercially
available."
Black-box warning:
"Patients with
,r ancestry in genetically
mal at-risk populations
should be screened
for the presence of
HLA-B*;502 prior to
initiating treatment
with Tegretol. Patients
testing positive for
the allele should not
be treated with
Tegretol." "For
genetically at-risk
patients, high-
resolution HLA-B*1502
typing is
recommended."


KRAS mutations Efficacy "Retrospective subset
analyses of metastatic
or advanced
colorectal cancer trials
have not shown a
treatment benefit for
Erbitux in patients
whose tumors had
KRAS mutations in
codon 12 or 13. Use
of Erbitux is not
recommended for the
treatment of
colorectal cancer with
mutations."


Clopidogrel Bristol-Myer
(Plavix) Squibb


Anticoaculation CYP2C19*2*3


:acy "Tests are a available tc
identify a patient's
CYP2C19 genotype;
these tests can be
used as an aid in
determining
therapeutic strategy.
Consider alternative
treatment or
treatment strategies
in patients identified


Page 22 of 33


Cetuximab
(Erbitux)


Imclone


Metastatic
colorectal
cancer







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Table 5 Examples of drugs with genetic information in thier labels (Continued)
as CYP2C19 poor
metabolizer."
Irinotecan Pfizer Metastatic UGTIA *28 Diarrhea "A reduction in the
(Camptosar) colorectal neutropenia starting dose by at
cancer least one level of
Camptosar should be
consider for patients
knows to be
homozygous for the
UGT1 Al*28 allele. "A
laboratory test is
available to determine
the UGT1A1 status of
patients."
Pantumumab Amgen Metastatic KRAS mutations Efficacy "Retrospective subset
(Vectibix) colorectal analyses of metastatic
cancer colorectal cancer trials
have not shown a
treatment benefit for
Vectibix in patients
whose tumors had
KRAS mutations in
codon 12 or 13. Use
of Vectibix is not
recommended for the
treatment of
colorectal cancer with
these mutations."
Transtuzumab Genetech HER2-positive HER2 expression Efficacy "Detection of HER2
(Herceptin) breastcancer protein
overexpression is
necessary for
selection of patients
appropriate for
Herceptin therapy
because these are the
only patients studied
and for whom benefit
has shown." "Several
FDA-approved
commercial assays are
available to aid in the
selection of breast
cancer and metastatic
cancer patients for
Herptin therapy."
Wafarin Bristol-Myer Venous CYP2C9*2*3 and Bleeding Includes the following
(Coumadin) Squibb thrombosis VKORC1 variants complications table: Range of
Expected Therapeutic
Warfarin Doses Based
on CYP2CP and
VKORC1 Genotypes.
*AII drug labels were accessed through Drugs @FDA at www.accessdata.fda.gov/scripts/cder/drugsatfda. HIV-1 denotes
human immunodeficiency virus type 1, TPMTthiopurine methyltransferase, UGTIA UDP glucuronosyltransferanse 1
family polypeptide Al, and VKORCI vitamins K epoxide reductase complex subunit 1


module, or is influenced by other modules. However, when the examination of the
conserved process occurs at the same or lower level of organization or in the same
module, and hence is subject to study solely by reductionism, then extrapolation is
possible. We believe this is a valuable principle.
Our current understanding of evo devo, evolution in general, complexity science, and
genetics allows us to generalize regarding trans-species extrapolation, even when
conserved processes are involved. Shanks and Greek:







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Table 6 The most significant genetic predictors of drug response
Organ or system involved Associated gene/allele Drug/drug response phenotype
RInod


Red blood cells
Neutrophils


Plates
Coagulation
Brain and peripheral nerve
CNS depression
Anaesthesia
Peripheral nerves
Drug hypersesitivity







Drug-induced liver injury


G6PD
TMPT*2
UGTIA 1 28
CYP2C 1972
CY2C9*2, *3, VKORC1
us system
CYP2D6*N
Butyrylcholinesterase
NAT-2
HLA-B*5701
HLA-B*1502

HLA-A*3101

HLA-B*5801
HLA-B*5701
HLA-DR81* 1501-DQ81 70602
HLA-DR81* 1501-DQ81 70602
HLA-BR81 07-DOAI 02
HLA-DQA1 0201


Primaquine and others
Azathioprine/6MP-induced neutropenia
Irintotecan-induced neutropenia
Stent thrombusis
Warfarin dose-requirement


Codeine-related sedation and respiratory depression
Prolonged apnoea
Isoniazid-induced peripheral neuropathy
Abacavir hypersensitivity
Carbamazepine-induced Steve Johnson syndrome
(in some Asian groups )
Carbamazepine-induced hypersensitivity in Causians
and Japanese
Allopurinol-induced serious cutaneous reactions
Flucloxacillin
Co-amoxiclav
Lumiracoxib
Ximelagatran
Lapatinib


Infection
HIV-1 infection
Hepatitis C infection
Malignancy
Breast cancer
Chronic myeloid leukaemia
Colon cancer
GI stromal tumours
Lung cancer


Malignant melanoma


Maraviroc efficacy
Interferon-alpha efficacy


CYP2DA
BCR-ABL
KRAS
c-kit
EGFR
EML4-ALK
BRAF V600E


Response to tamoxifen
Imatinib and other tyrosine kinase inhibitors
Cetuximab efficacy
Imatinib efficacy
Gefinib efficacy
Crizotinib efficacy
Vemurafenib efficacy


Living complex systems belonging to different species, largely as a result of the
operation of evolutionary mechanisms over long periods of time, manifest different
responses to the same stimuli due to: (1) differences with respect to genes
present; (2) differences with respect to mutations in the same gene (where one
species has an ortholog of a gene found in another); (3) differences with
respect to proteins and protein activity; (4) differences with respect to gene
regulation; (5) differences in gene expression; (6) differences in protein-protein
interactions; (7) differences in genetic networks; (8) differences with respect to
organismal organization (humans and rats may be intact systems, but may be
differently intact); (9) differences in environmental exposures; and last but not
least; (10) differences with respect to evolutionary histories. These are some of
the important reasons why members of one species often respond differently to


Page 24 of 33







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drugs and toxins, and experience different diseases. Immense empirical evidence
supports this position ([14] p358).


The failures of animal models as a predictive modality for human response to disease
and drugs, even when such perturbations are acting on conserved processes, can be
explained in the context of evolved complex systems. One does not need to study every
such perturbation in every species in order to conclude that the animal model will not
be a predictive modality for humans when perturbations occur at higher levels of
organization or involve different modules or affect the system as a whole. This is not to
deny that animal models, as characterized by 3-9 in Table 1, have contributed and will
continue to contribute to scientific advancements.


Competing interests
The authors declare that they have no competing interests

Authors' contributions
The authors contributed equally to this paper

Authors' information
Ray Greek, MD has been on faculty in the Department of Anesthesiology at the University of Wisconsin-Madison and
at Thomas Jefferson University in Philadelphia He is currently president of the not-for-profit Americans For Medical
Advancement (www AFMA-curediseaseorg)
Mark Rice, MD is currently on faculty at the University of Florida (UF) He is chief of the liver transplant division at UF
Department of Anesthesiology, has seven US patents, and reviews for several major journals

Acknowledgements
None

Author details
Americans For Medical Advancement (wwwAFMA-curediseaseorg), 2251 Refugio Rd, Goleta, CA 93117, USA 2Department of
Anesthesiology, University of Florida College of Medicine, PO Box 100254, Gainesville, FL 32610-0254, USA

Received: 30 July 2012 Accepted: 31 August 2012 Published: 10 September 2012

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doi:10.1186/1742-4682-9-40
Cite this article as: Greek and Rice Animal models and conserved processes. Theoretical Biology and Medical
Modelling 2012 940

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RESEARCHOpenAccessAnimalmodelsandconservedprocessesRayGreek1*andMarkJRice2*Correspondence: DrRayGreek@ gmail.com1AmericansForMedicalAdvancement (www.AFMA-curedisease.org),2251 RefugioRd,Goleta,CA93117,USA Fulllistofauthorinformationis availableattheendofthearticleAbstractBackground: Theconceptofconservedprocessespresentsuniqueopportunitiesfor usingnonhumananimalmodelsinbiomedicalresearch.However,theconceptmust beexaminedinthecontextthathumansandnonhumananimalsareevolved, complex,adaptivesystems.Giventhatnonhumananimalsareexamplesofliving systemsthatare differentlycomplex fromhumans,whatdoestheexistenceofa conservedgeneorprocessimplyforinter-speciesextrapolation? Methods: Wesurveyedtheliteratureincludingphilosophyofscience,biological complexity,conservedprocesses,evolutionarybiology,comparativemedicine, anti-neoplasticagents,inhalationalanesthetics,anddrugdevelopmentjournalsin ordertodeterminethevalueofnonhumananimalmodelswhenstudyingconserved processes. Results: Evolutionthroughnaturalselectionhasemployedcomponentsand processesbothtoproducethesameoutcomesamongspeciesbutalsotogenerate differentfunctionsandtraits.Manygenesandprocessesareconserved,butnew combinationsoftheseprocessesordifferentregulationofthegenesinvolvedin theseprocesseshaveresultedinuniqueorganisms.Further,thereisahierarchyof organizationincomplexlivingsystems.Atsomelevels,thecomponentsaresimple systemsthatcanbeanalyzedbymathematicsorthephysicalsciences,whileat otherlevelsthesystemcannotbefullyanalyzedbyreducingittoaphysicalsystem. Thestudyofcomplexlivingsystemsmustalternatebetweenfocusingontheparts andexaminingtheintactwholeorganismwhiletakingintoaccounttheconnections betweenthetwo.Systemsbiologyaimsforthisholism.Weexaminedtheactionsof inhalationalanestheticagentsandanti-neoplasticagentsinordertoaddresswhat thecharacteristicsofcomplexlivingsystemsimplyforinter-speciesextrapolationof traitsandresponsesrelatedtoconservedprocesses. Conclusion: Weconcludethateventhepresenceofconservedprocessesis insufficientforinter-speciesextrapolationwhenthetraitorresponsebeingstudiedis locatedathigherlevelsoforganization,isinadifferentmodule,orisinfluencedby othermodules.However,whentheexaminationoftheconservedprocessoccursat thesameleveloforganizationorinthesamemodule,andhenceissubjecttostudy solelybyreductionism,thenextrapolationispossible. Keywords: Anesthesia,Animalmodels,Cancer,Complexity,Conservedprocesses, Systemsbiology 2012GreekandRice;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.GreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40 http://www.tbiomed.com/content/9/1/40

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BackgroundMarcKirschnerandJohnGerhartintroducedtheconceptoffacilitatedvariationand conservedcoreprocessesintheirbook, ThePlausibilityofLife [1],inordertoexplain hownoveltyarisesinevolution.Motivatedbya dvancesinevolutionaryanddevelopmental biology(evodevo),theseinvestigatorsproposedthatconservedprocessesareubiquitousin eukaryotesbutpointedoutthatbyusingconservedprocessesdifferently,forexampleby differentlyregulatingthegenesthatcodeforthe processes,expressingthegenesdifferently, varyingthesequencesorcombinationofgenesortranscriptionfactors,noveltycanarise. Mutationsinthegenesthatregulatetheconservedprocessescanaccomplishthisnovelty. Moreover,byadjustingtheregulatorygenes,theorganismcanevolvewithfewermutations thanwouldbethecaseifatraithadtoarise denovo orfrommutationsinstructuralgenes. Thishasimplicationsforusingnonhumananimals(hereafterreferredtosimplyasanimals) asmodelsforhumansinbiomedicalresearch.Oneshouldexpecttodiscoverinformation regardingconservedprocessesinhumansbystudyinganimalmodels.Wesoughttodeterminewhetherlimitsexistonthismethodandifsowhatthoselimitsare.MethodsWesurveyedtherelevantliteratureincluding philosophyofscience,biologicalcomplexity, conservedprocesses,evolutionarybiology,comparativemedicine,anti-neoplastic agents,inhalationalanesthetics,anddrugdevelopmentjournalsinordertodetermine theappropriateroleforanimalmodelswhenstudyingconservedprocesses.Philosophy ofscienceisrelevanttoourdiscussionasitincludesthepremisesandassumptionson whichresearchisthenbased.Astudyormethodcanbe methodologically soundbutif thepremisesareincorrect,thenthestudylosesmuchifnotallofitsvalue.Thedrug developmentliteraturewassearchedbecausethefinalapplicationofmuchresearchis targetedinterventionviadrugshencethatliteraturecaninformregardingthesuccessof apracticeormodality.Theliteratureconcerningbiologicalcomplexityandconserved processeswassurveyedasitdirectlyrelatestotheissuebeingexplored.Allofthismust beplacedintothecontextofevolutionarybiologyinordertobetterexplainthefindings. Wechoseinhalationalanestheticsandanti-neoplasticagentsasexamplesbecauseofthe well-knownconservednatureoftheseagents.ResultsAnimalmodelsTheuseofmodelshasalonghistoryinscience, whichledphilosopherofscienceRichard Braithwaitetowarnthat: “ Thepriceofemploymentofmo delsiseternalvigilance ” [2].In thissection,wewillexplorewhatanimalmodelsare,howtheycanbeusedinscientific investigation,includingbiomedicalresearch,a nddiscussclassificationschemes.Inthisarticle,wewilladdresstheuseofpredictiveanimalmodelsinlightoftheconceptsofcomplex systems,personalizedmedicineandpharmac ogenomics,andevolutionarybiology.Wewill thenexplorewhatthisimplieswhenusinga nimalmodelstostudyconservedprocesses. Modelsareimportantforscientificpursuitsandcantaketheformofabstractmodels, computationalmodels,heuristicmodels,mathematicalmodels,physicalmodelssuchas scalemodels,iconicmodels,andidealizedmodels.Modelscanalsobedividedonthe basisofwhethertheyareusedtoreplicateaportionoftheitembeingmodeledorareusedGreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page2of33 http://www.tbiomed.com/content/9/1/40

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totesthypothesesorinterpretaspectsofatheory.Examplesofhistoricallyimportant modelsincludeWatsonandCrick ’ sphysicalmodelofDNA,Pauling ’ smodelofchemical bonds,Bohr ’ ssolarsystemmodeloftheatom,andthebilliardballmodelofgases.More recentmodelsincludethecomputermodelofthebrain,mathematicalmodelsofdisease spread,andLorenz ’ smodeloftheatmosphere. RobertHindeobservedthatmodels:Shouldbedifferentfromthethingbeingmodeled,becauseifitisnot,themodeler mightassumethatallpropertiesdemonstratedbythemodelexistinthethingbeing modeled;Areusuallylesscomplicatedthanthethingbeingmodeled;Aremorereadilyavailablethanthethingbeingmodeled,and;“ posequestions,suggestrelations,orcanbemanipulatedinwaysnotpossiblewith theoriginal ” [ 3 ]. Inlightoftheimportanceofmodels,somephilosophersofscienceassertthatthe studyofmodels perse hasbeenneglectedbythephilosophyofsciencecommunity. FriggandHartmann[4]state: “ Whatfillsintheblankin ‘ M represents T ifandonly if____, ’ where M isamodeland T atargetsystem? ” Moreover,howoneclassifies modelsandwhatcriteriamustbefulfilledinorderfor M tobeconsideredaspecific typeofmodelhasarguablynotbeenadequatelyaddressedbythephilosophyofscience community.Yetanotherproblemwiththe philosophyofmodels istherelationshipbetween theoryandmodel[4].Wemaintainthatthislackofscholarlyattentiontomodelshas playedaroleinwhatweseeastheconfusionsurroundingtheuseofanimalsasmodels. Animalmodelsarephysicalmodelsandcanbefurtherclassifiedbasedonvarious featuresanduses.Forexample,theycanbedistinguishedbythephylogeneticdistance ofthemodelspeciesfromhumans.Animalmodelscanalsobeclassifiedbasedonfidelity — howwellthemodelresembleshumans — aswellasbasedonvalidity — howwell whatyouthinkyouaremeasuringcorrespondstowhatyoureallyaremeasuring. Animalmodelscanalsobeconsideredbasedonreliability — theprecisionandaccuracy ofthemeasurement[5].Hauexplainsthatanimalmodelscanbecategorizedasspontaneous,induced,transgenic,negativeandorphan.Haustates: “ Themajorityoflaboratoryanimalmodelsaredevelopedandusedtostudythecause,nature,andcureof humandisorders ” [[6]p3].ThisisimportantasHaufurtherstatesthatanimalmodels canbeusedtopredicthumanresponses: “ Athirdimportantgroupofanimalmodelsis employedas predictive models.Thesemodelsareusedwiththeaimofdiscoveringand quantifyingtheimpactofatreatment,whetherthisistocureadiseaseortoassess toxicityofachemicalcompound.Theappropriatenessofanylaboratoryanimalmodel willeventuallybejudgedbyitscapacitytoexplainandpredicttheobservedeffectsin thetargetspecies ” [6].Othersagreethatpredictinghumanresponseisacommonuse foranimalmodels[7-12].Forexample,Heywoodstated: “ Animalstudiesfallintotwo maincategories:predictiveevaluationsofnewcompoundsandtheirincorporationinto schemesdesignedtohelplessenorclarifyarecognisedhazard ” [13]. Animalsareutilizedfornumerousscientificpurposes(see]Table1)andoneofthe authors(Greek)hasaddressedthesevarioususesinpreviouspublications[14-20].One cannothaveameaningfuldiscussionregardingtheutilityofanimalmodelsunlessoneGreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page3of33 http://www.tbiomed.com/content/9/1/40

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specifiesthecategoryunderdiscussion.Forexample,areasinwhichanimalmodels havebeensuccessfullyemployedincludetheevaluationofaphenomenonthatcanbe describedbythephysicochemicalpropertieso ftheorganism,thestudyofbasicphysiologic functions,andthestudyofothertraitsth atcanbedescribedbytheuseofconversion factorsbasedonthebodysurfaceareaoftheorganism.Ingeneral,animalmodelscanbe successfullyemployedincategories3 – 9inTable1.However,animalmodelshavefailedto bepredictivemodalitiesforhumanresponseto drugsanddisease[13-16,18,21-41],depicted bycategories1and2inTable1.(Theauthors haveaddressedthisfailureinnumerous publicationsand,becauseanexplorationfort hisfailureisnotthepurposeofthearticle,we referthereadertothosepublications[14-20,23]eventhoughwerealizethatsomeviewthis positionascontroversial[7, 11,42-44].)Thisisnottosaythataspeciescanneverbefound inretrospectthatmimicsanoutcomeinhuman s.Suchaspeciesusuallycanbeidentified, howeverretrospectivecorrelationisobviouslynotthesameasprediction[45-47].Moreover, anyprocessormodalityclaimingtobepredict ivecanbeevaluatedbyuseofthebinomial classificationtableandequationsinTable2(as illustratedinTable3[48]).Suchcalculations arecommonlyusedinscience[49-53]. Table1Categoriesofanimaluseinscienceandresearch[16]1.Aspredictivemodelsforhumandisease 2.Aspredictivemodelstoevaluatehumanexposuresafetyinthecontextofpharmacology andtoxicology(e.g.,indrugtesting) 3.Assourcesof ‘ spareparts ’ (e.g.,aorticvalvereplacementsforhumans) 4.Asbioreactors(e.g.,asfactoriesfortheproductionofinsulin,ormonoclonalantibodies,or thefruitsofgeneticengineering) 5.Assourcesoftissueinordertostudybasicphysiologicalprinciples 6.Fordissectionandstudyineducationandmedicaltraining 7.Asheuristicdevicestopromptnewbiological/biomedicalhypotheses 8.Forthebenefitofothernonhumananimals 9.Forthepursuitofscientificknowledgeinandofitself Table2BinaryclassificationtestGoldstandard GS+GSTestT+TPFP T-FNTN Sensitivity=TP/(TP+FN) Specificity=TN/(FP+TN) PositivePredictiveValue=TP/(TP+FP) NegativePredictiveValue=TN/(FN+TN) T-=Testnegative T+=Testpositive FP=Falsepositive TP=Truepositive FN=Falsenegative TN=Truenegative GS-=Goldstandardnegative GS+=GoldstandardpositiveThebinaryclassificationtestallowscalculationsfordetermininghowwellatestorpracticecompareswithrealityorthe goldstandard.GreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page4of33 http://www.tbiomed.com/content/9/1/40

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Whenjudgingthepredictivevalueofamodality,oneisnotusingtheterm predict inthesamesenseaswhendescribinghowhypothesesgeneratepredictionstobe tested.Thepredictivevalueofacommonl yusedmodalityusuallyisknown,orcanbe ascertained,forexamplethepositiveandne gativepredictivevalueofx-raycomputed tomography(commonlyreferredtoasaCTscan )fordiagnosingpneumothorax(arupture of,orinterferencein,thepleuralmembrane whichallowsairtoenterthepleuralspaceand thusinterfereswithbreathing )approaches1.0(isaccuratefordiagnosingtheconditionin 100%ofcases). Animalmodelsasusedinbiomedicalresearch,canalsobecategorizedascausal analogicalmodels(CAMs)orasheuristicorhypotheticalanalogicalmodels(HAMs) [54-59].Theuseofanimalmodelstopredicthumanresponsetodrugsanddisease,in accordancewithcategories1and2inTable1,wouldbeanexampleofusinganimals asCAMs.Analogicalmodelsingeneralincludethehydraulicmodelofeconomiesand thecomputermodelofthebrainandcanbefurtherdividedbasedonvariouscriteria[4]. CausalismorcausaldeterminismdatestoAristotlewhostated: “ whatiscalledWisdomis concernedwiththeprimarycausesandprinciples. ” Causalismcanbesummarized succinctly,as “ everythinghasacause. ” Thisnotionofcausationwasthebasisforanimal modelsascanbeappreciatedbythewritingsofClaudeBernard[60],consideredthefather ofanimalmodelingsincethe19thcentury.Bernard ’ sthoughtsonanimalmodelsarean extensionofAristotleviathedeterminismofDescartesandNewton[61].Causaldeterminismandtheprincipleofuniformityledtotheconcept,stillacceptedbymanyanimal modelerstoday,thatthesamecausewouldresultinthesameeffectinqualitativelysimilar systems.Thislineofthinkingwasinkeepingwiththecreationistthinkingof19thcentury Frenchphysiologists,includingBernard,whorejectedDarwin ’ sTheoryofEvolution [60,62,63].Thenotionofcausaldeterminismandtheprincipleofuniformitycombined withtherejectionofevolutionledtothebeliefintheinterchangeabilityofparts.Therefore,ifoneascertainedthefunctionofthepancreasinadog,hecoulddirectlyextrapolate thatknowledgetothefunctionofthepancreasinhumans,oncescalingforsizehadbeen factoredin[14,63,64].Unfortunately,thislinearthinkingpersistsasthebaboonheart transplanttoBabyFaeillustrates.Theoperationwasperformedbythecreationistsurgeon LeonardBaileyofLomaLindaUniversityin1984[[65]p162-3]. Weacknowledgethattheconceptof causation isproblematic[66].Russellsuggested itbeabandonedin1913[67]anditisclearlymoreusefulforlinearsystemsthan complexsystems.Whileanexhaustiveexplanationanddiscussionofthecontroversies surroundingcausationwouldoccupymore spacethanisavailableforthisarticle Table3ExampleofbinaryclassificationvaluesGoldstandard(human) GS+GSTestT+2226 T-2230 Sensitivity=22/(22+22)=0.5 Specificity=30/(26+30)=0.54 PositivePredictiveValue=22/(22+26)=0.46 NegativePredictiveValue=30/(22+30)=0.58Binaryclassificationvaluesforcardiovasculartoxicitytestinmonkeysfrom25compoundsalsotestedinhumans[ 48 ]. Notethevaluesareapproximatelywhatwouldbeexpectedfromacointoss.GreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page5of33 http://www.tbiomed.com/content/9/1/40

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(seeBunge[61]forsuchananalysis)weshouldnotethatamorecurrentexplanationfor causationisthatofa “ firstorderapproximation. ” Causationisusuallydiscussedinthe contextofa chain ofcauses.Bungesummarizescurrentthinking: “ neodeterminism... assertsinthisconnectionthatcausationisonlyoneamongseveralinterrelatedcategories concurringinrealprocesses ” [61].Thisprincipleisappreciatedevenmorefullyin complexsystems.Currentthinkingnotwithstanding,theuseofanimalmodelsassumes theCartesianconceptofcausationinthatacausalmodelassumesadeterministiccausal relationshipbetweenvariables.Wewillexplorethisthinkingandshowthateveninthe traditionalcontextthereareproblemswithusinganimalmodelstodiscover “ causal ” relationships.Theseproblemsareincreasedexponentiallywhenplacedinthecontextof complexsystems. BasedonthewritingsofLaFolletteandShanks[[58]p63],wesuggestthefollowingin orderforamodeltobeconsideredaCAM. X (themodel)and Y (thesubjectbeing modeled)shareproperties{ a ... e }.InX,thesepropertiesareassociatedwith,and thoughtrelevantto,state S1 S1 hasnotbeenobserveddirectlyin Y ,but Y likelyalso haswouldexhibit S1 underthesameconditionsasX.Thisconceptisillustratedin Table4.LaFolletteandShanks[58]statethat, “ thereshouldbenocausally-relevant disanalogiesbetweenthemodelandthethingbeingmodeled. ” Unfortunately,causally relevantdisanalogiesdoexistamongspeciesandevenwithinaspecies,whichleadsto differentstatesoroutcomes,asillustratedinTable4.WeagainparaphraseLaFollette andShanks[[58]p112]andsuggestthattwomoreconditionsmustbemetforamodel toqualifyasaCAM:thesharedproperties{ a, ... ,e }musthaveacausalrelationshipwith state S1 andbetheonlycausallyrelevantpropertiesassociatedwith S1 .AsTable4 illustrates,thecommonalitiesbetweenthehumansandchimpanzeesareinsufficientto qualifychimpanzeesasCAMsforhumanresponsetoHIVinfection.(Formoreon Table4CausalanalogicalmodelsX,themodelY,thesystem beingmodeled Sharedproperties betweenXandY Perturbation tothemodel Outcome inmodel Outcomeinsystem beingmodeled Animalsystem (forexample, Pantroglodytes ) Humansystem a.Genes.>90%of nucleotidesequences identical. Exposure toHIV. State S1 Mildillness oflimited duration. AIDS.State S1 is not shareddespite thepresenceof shared,relevant properties. b.Immunesystem. Manycommonalities. Constructedon generallythesame plan. c.Whitebloodcells presentandfunction similarly. d.Receptorsonwhite bloodcellsalsopresent andfunctionsimilarly. e.Sharedintracellular componentsofwhite bloodcells.Sharedproperties a...e forhumansandchimpanzeedonotresultinstate S1 alsobeingshared.GreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page6of33 http://www.tbiomed.com/content/9/1/40

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animalmodelsofHIV/AIDSsee[14,68].)Aswewillshow,animalsandhumansare evolvedcomplexsystemsandassuchexhibitthepropertiesofrobustnessandredundancy; hencenumerous “ causes ” canresultinthesameeffectandthesameperturbationcan resultindifferentoutcomes.Becauseofthisandotherpropertiesofcomplexsystems, weshouldexpectdifferentspeciestoexhibitdifferentcausalrelationships. Correspondingly,Giere,Bickle,andMauldin[69]notethatsomequestiontheuseof causalmodelsinthestudyofhumansbecausehumansarecomplexsystemswhereas casualmodelsassumeadeterministicsystem:anoutcomeinasimplesystemisfixedby thevariables.TheproblemsofdeterminingcausationarefurtherexploredbyBunge[61] inhisneodeterminismexplanationalludedtoaboveandhisanalysisishighlyrelevantto thisdiscussion.Whilewewillattempttocontrastthetraditionaldeterministicviewof causalityinlightofcomplexityscience,thisarticlewillnotdojusticethecurrentthinking oncausationandwereferthereadertoBunge[61]forafullerexplanation. Giere,Bickle,andMauldinsuggestaprobabilisticrelationshipinsteadofa100% causalrelationshipforthemodel: “ Cisapositivecausalfactor(probabilistic)forEin anindividual,I,characterizedbyresidualstate,S,ifinItheprobabilityofEgivenCis greaterthantheprobabilityofEgivenNot-C. ” Likewise,LaFolletteandShanksraise thequestionastowhetheranimalmodelscanbe weakCAMs : “ Beginwithtwosystems S1andS2.S1hascausalmechanisms{a,b,c,d,e},S2hasmechanisms{a,b,c,x,y}.When westimulatesub-system{a,b,c}ofS1withstimulisfresponserfregularlyoccurs.We canthereforeinferthatwerewetostimulatesub-systems{a,b,c}ofS2withsfrfwould probablyoccur ” [[58]p141].LaFolletteandShanksthenexplainthatthisoutcomewill behighlyprobableifandonlyif{a,b,c}arecausallyindependentof{d,e}and{x,y}. AgainweanticipateproblemsinusinganimalmodelsasweakCAMs,eveninthe traditionaldeterministic-causationview,because,asweshalldiscuss,variousproperties ofcomplexsystemswilllikelygiverisetodifficultiesinisolatingsubsystems,which wouldberequiredforananimalmodeltobeaweakCAM.Theseproblemshavebeen referredtoas causal/functionalasymmetry andmandatescautioninextrapolatingdata betweenspecies.KirschnerandGerhartgiveanexampleofthis: Thecaseoftheoctopusandthehumancameraeyehasbeenlookedinto,andthe lessonsareclear.Underneaththegrossanatomicalsimilaritiesaremanydifferences. Theeyederivesfromdifferenttissuesbydifferentdevelopmentalmeans.Although bothstructuresusethesamepigment(rhodopsin)forphotoreception,andbothsend electricalsignalstothebrain,wenowknowthattheinterveningcircuitryis completelydifferent[[1]p240-01]. Independentevolutionhasalsoproducedspindleneuronsinspeciesasdiverseas humansandcetaceans.Spindleneuronsconnectpartsofthebraininvolvedinhigher cognitionandwerethoughttoonlyoccurinprimatesbuthaverecentlybeendiscovered incetaceans,suchashumpbackwhalesandfinwhales,aswellaselephants[70-72]. Convergentevolution,theacquisitionofth esametraitindifferentlineages,isalso importantwhenconsideringtheroleofanimalmodels.EvolvedcomplexsystemsReductionismisamethodofstudythatseekstobreakasystemdownintoitscomponentparts,studyeachpartindividually,andthenreachaconclusionaboutthesystemGreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page7of33 http://www.tbiomed.com/content/9/1/40

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asawholeoratleasttheroleoftheindividualpart.Descartesintroducedtheconcept andithasproveneffectiveforascertainingmanyfactsaboutthematerialuniverse. Conversely,theclockworkuniverseofDescarteshasnothelduptoscrutinyonall levels.Quantummechanics,relativity,chaos,andcomplexityhaverevealedthestochastic natureofthesupposedlyclockwork,determini sticuniverse.Regrettably,whilephysicists recognizedthelimitationsofreductionism,bio logistswereuncriticallyembracingit.Francis Crickextendedreductionismtoall aspectsofbiologywhenhestated: “ Theultimateaimof themodernmovementinbiologyistoexplaina llbiologyintermsofphysicsandchemistry ” [73].Biologicalreductionism arguablyreacheditszenithintheHumanGenomeProject (HGP)[74,75]and,ironically ,theconsequencesoftheHGP — thathumanshavearelatively smallnumberofgenes — have,inlargepart,beenresponsibleforare-examinationofthe roleofreductionisminbiology.Thishasbeenespeciallytrueforhumanpathophysiology whereanimalsareusedasmodelsforhumans. Systemscanbecategorizedassimpleorcomplex.TheworldofNewtonandDescartes waslargelyconfinedtosimplesystemshencereductionismfunctionedwellfordiscovery. Atsomelevels,thecomponentsofacomplexsystemcanbesimplesystemsandthusare subjecttostudybyreductionismwhileatotherlevelsthesesimplesystemscombineto makecomplexsystemsthusnecessitatin gstudyoftheintactwhole.Mazzocchi pointsoutthatwhenreductionismtakesacomponentoutofitsnaturalenvironment ithasconsequencesforextrapolatingth eresultsbacktotheorganismasawhole: “ Butthisextrapolationisatbestdebatablea ndatworstmisleadingorevenhazardous.The failureofmanypromisingdrugcandidatesinc linicalresearchshowsthatitisnotalways possibletotransferresultsfrommiceorevenprimatestohumans ” [76]. Whileevolutionisdefinedasachangeinallelefrequencyovertime,complexity sciencecanbedefinedas “ thestudyofthebehaviouroflargecollectionsofsimple, interactingunits,endowedwiththepotentialtoevolvewithtime ” [77,78].Living organismsarecomplexsystemsthathavehighlyvariableevolutionaryhistoriesandas sucharebestmodeledusingnonlineardifferentialequations.Thedifficultywiththis approachisthatthevaluesformanyofthefactorsareunknown;hencesolvingthe equationisimpossible[49,77]. Animalsandhumansareexamplesoflivingcomplexadaptivesystemsandassuch exhibitthefollowingproperties[79-97]: 1.Complexsystemsarecomposedofmanycomponents.Someofthesecomponents maybesimplesystems,butmanyarecomplexsystems.Thesecomponentsexistonmany scalesandinteractextensivelywitheachother.Acomplexsystemisa “ systemofsystems. ” 2.Thecomponentscanbegroupedasmodules.Forexample,thefollowingcouldbe consideredasmodules:thecell;thevariousprocessesinacell;genenetworks;genegeneinteractions;gene-proteininteractions;protein-proteininteractions;organs;and allthefactorsthatinfluencethenaturalhistoryofadisease.However,failureinone moduledoesnotnecessarilyspreaddemisetothesystemasawholeasredundancy androbustness(see#s5and6below)alsoexistandthevariousmodulesalso communicatewitheachother. 3.Thedifferentcomponentsofacomplexsystemarelinkedtoandaffectone anotherinasynergisticmanner.Thereispositiveandnegativefeedbackinacomplex system[ 93 ].GreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page8of33 http://www.tbiomed.com/content/9/1/40

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4.Acomplexsystemdemonstrateshierarchallevelsoforganization[ 98 99 ].These levelsrangefromthesubatomictothemoleculartothewholeindividualtocollections ofindividuals[ 100 ].Emergence(see#13below)occursateachlevel;therefore,evena completeunderstandingofthelowerlevelisinsufficientforexplainingtheupperlevel. Thevariouslevelsinteractsuchthatthereisbothupwardcausationanddownward causation.Inordertounderstandaparticularlevel,onemustalternatebetween lookingatthecomponentsandlookingatthewholewhiletakingintoaccountthe connectionsbetweeneach[ 76 101 ].Moreover,thevariouslevelsmayrespond differentlytothesameperturbation. Thevariouslevelsoforganizationareimportantwhenconsideringwhichresponses tospecificperturbationscanbeextrapolatedamongspecies.Livingcomplexsystems havenumerouspropertiesthatcanbestudiedwithoutconsiderationofthefactthatthe whole,intactorganismisacomplexsystem.Somesystemsorcomponentsfollowonly thelawsofphysics,orevensimplegeometry,whileothersarebestdescribedbytheir physicochemicalpropertiesorjustbychemistry.Somepropertiesofcomplexsystems canbedescribedsimplybymathformulas.Growth,forexample,canbedescribedas geometricalinsomecasesandexponentialinothers.Thesurfaceareaofabody increasesbythesquareofthelineardimensionswhilethevolumeincreasesbythe cube.Thisisaconsequenceofgeometryandisimportantinphysiology,inpart, becauseheatlossisproportionaltosurfaceareawhileheatproductionisproportional tovolume.Haldanestated: “ Comparativeanatomyislargelythestoryofthestruggleto increasesurfaceinproportiontovolumes ” [102].Forexample,chewingincreasesthe surfaceareaoffood,theratethesmallbowelabsorbsnutrientsandotherchemicals dependsinpartonthesurfaceareaofthesmallbowel,andairsacsinthelungsrelyon surfaceareaforgasexchange,asdocapillaries. Allometryisthestudyoftherelationshipofbodysizetoshape.Examplesofallometric lawsincludeKleiber ’ slaw: q0~ Mwhere q0ismetabolicrateandisproportionalto M, bodymass,raisedtothepower.Therate t, ofbreathingandheartcontractionsare proportionalto M ,bodymass,raisedtopower: t ~ M.Further,manyphysiological functionsaffectordependonsurfacearea. Levelsoforganizationcanalsobedescribedbasedonwhethertheyareprimarily chemicalreactionsandhencesubjecttoanalysisbychemistry.Reactionsorperturbationsthatinvolvethedenaturationofproteinsshouldaffectallsystems,betheysimple orcomplex,similarlybecauseatthisleveloforganizationotherfactorsdonotcome intoplay.Exactlywhateffectssulfuricacidwouldhaveinapersonoveranextended periodoftimeareirrelevantasitdenaturesproteinmoreorlessimmediately.Perhaps speciesdifferenceswouldmanifestifsmallamountsofH2SO4wereinfusedoverlong periodsoftime,buttheimmediateeffectsarethesameacrossspeciesbecauseofthe chemicalpropertiesoftheacid. Animalscanbesuccessfullyusedfornumerouspurposesinscience(see3 – 9in Table1).Oneofthepurposesforwhichanimalscanbesuccessfullyusedistoevaluate phenomenathatcanbedescribedbythephysicochemicalpropertiesoftheorganism. Thesameappliestobasicphysiologicfunctions.Therearephysiologicalparameters thatcanbeappliedacrossspecieslinesbytheuseofconversionfactorsbasedonthe weightorsurfaceareaoftheorganism.TherearealsopropertiesoforganismsthatcanGreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page9of33 http://www.tbiomed.com/content/9/1/40

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beanticipatedbythephysicalorchemicalpropertiesofthesubstanceactingonthe organism.Alloftheseareinstancesofsuccessfullytreatingacomplexsystemasifit wereasimplesystem.Problemsarisehowever,astheleveloforganizationunderstudy increases.Allometricscalingbasedonbodysurfacearea(BSA),forexample,doesnot includedifferencesthatmanifestathigherlevelsoforganizationforexampleinthe eliminationormetabolismofdrugs.Differentlevelsoforganizationcanbeactedonby singlefactorsormanyfactorsbutperturbationsofsimplesystems,orsystemsthatcan bedescribedassimpleonthelevelororganizationbeingaffected,shouldproduce similarresults. 5.Complexsystemsarerobust,meaningtheyhavethecapacitytoresistchange. Thiscanbeillustratedbythefactthatknockingoutageneinonestrainofmouse mayproducenegligibleeffectswhilebeinglethaltoanotherstrain.Genepleiotropyis anadditionalexample[ 103 ]. 6.Complexsystemsexhibitredundancy.Forexample,livingsystemsexhibit redundancyofsomegenesandproteins[ 103 ]. 7.Complexsystemsaredynamic.Theycommunicatewith,andareactedonby, theirenvironment. 8.Complexsystemsexhibitself-organization,whichallowsadaptationtothe environment[ 85 104 106 ].Theintactcellisaprimeexampleofthisproperty. 9.Complexsystemsaredependentoninitialconditions.Thewell-knownexampleof thebutterflyflappingitswingsandcausingaweathercatastropheontheothersideof theearth — thebutterflyeffect — isanexampleofdependenceoninitialconditions.An exampleinlivingcomplexsystemswouldbethatverysmalldifferencesingenetic makeupbetweentwosystemscouldresultindramaticdifferencesinresponsetothe sameperturbation.Forexample,monozygotictwinsraisedinthesameenvironment mayhavedifferentpredispositionstodiseasessuchasmultiplesclerosisand schizophrenia[ 107 110 ].Additionally,theabove-mentionedobservationthatknocking outageneresultsindifferentoutcomesintwostainsofmiceillustratestheconcept thatsmalldifferencesininitialconditions — geneticmakeup — canmeanthedifference betweenlifeanddeath[ 93 103 111 112 ]. 10.Theinitialconditionsofacomplexlivingsystemaredetermined,inpart,by evolution.Variousspecieshavedifferentevolutionaryhistoriesandthusaredifferently organizedcomplexsystems.Initialconditionscanbedifferent,despitetheexactsame genes,secondarytomodifiergenes,differencesinregulationorexpressionofgenes, epigenetics,andmutationsamongothersfactors.Forexample,smallepigenetic changesprobablyaccountforthedissimilaritiesbetweenmonozygotictwinsinterms ofdiseasesusceptibility[ 107 108 113 116 ]. 11.Perturbationstocomplexsystemsresultineffectsthatarenonlinear[ 99 ].Large disturbancesmayresultinnochangetot hesystemwhileminorperturbationsmay causehavoc[ 76 105 ].Effortstodescribecomplexsystemsintermsoflinearcause andeffectrelationshipsarepronetofailure[ 117 ].Extrapolating among complex systemsisevenmoreproblematicbecauseofnonlinearity,alongwiththeother factorsdescribed. 12.Thewholeofacomplexsystemisgreaterthanthesumoftheparts;hence,some processesandorperturbationsarenotamenabletostudybyreductionism.GreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page10of33 http://www.tbiomed.com/content/9/1/40

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13.Complexsystemshaveemergentpropertiesthatcannotbepredictedeveninlight offullknowledgeofthecomponentparts. Animalmodelshavehistoricallybeenutilizedforthepredictionofhumanresponses todrugsanddiseaseandthisusehasalsobeenthejustificationforanimalusein researchingeneral[118,119].Butbecausevariouslevelsoforganizationanddifferent modulescanbeactedonbythesameperturbation,inordertoevaluatewhetheran animalmodelcanbeusedasapredictivemodality,oneneedstounderstandthelevels affectedbytheperturbation,whatrulesarebeingfollowedatthoselevels,andwhether thesystemissimpleorcomplexattherespectivelevels.Empiricalevidence,explained andplacedincontextbytheorydevelopedfromcomplexityscienceandevolutionary biology,suggestsanimalmodelscannotpredicthumanresponsestodrugsanddisease [14-16,18,57,58,120],despitethepresenceofsharedphysicochemicalpropertiesand conservedprocesses.ConservedprocessesTheodosiusDobzhanskyfamouslystated: “ Nothinginbiologymakessenseexceptin thelightofevolution. ” Wewanttoexaminetheconsequencesthatvariouscharacteristics ofevolvedcomplexsystems,suchasmodulesanddifferentlevelsoforganization,have onprocessesconservedbyevolutionintermsofdeterminingtheresponseofwhole organismstoperturbations.Conservedprocessesandgenesarethesubjectofmuch interesttoday[1,121-133].KirschnerandGerhardtstate: “ allorganismsareamixtureof conservedandnonconservedprocesses(saidotherwise,orchangingandunchanging processes) ” [[1]p34-35].Conserved processes arenotreactionstothelawsofphysicsor thedeterminationofpropertiesofanorganismastheyrelatetochemistryorgeometry. Nevertheless,conservationreachesacrossphylaandevenkingdoms.Kirschnerand Gerhardthavepointedoutthatprocessesconservedincludethoseinvolvedincellfunction andorganization,development,andmetabolismandthattheseprocessesaresimilarin animals,yeast,andbacteria.Theynotethatnoveltyhasbeentheresultofusingthe conservedprocessesindifferentwaysratherthaninventingcompletelynewprocesses [[1]p34-35].Thishascriticalimplicationsfo rwhatcanbelearnedfrominterspeciesstudy. Housekeepinggenesingeneralperformthesamefunction;makethesameproteins, inmice,frogsorhumans.TheroleofFOXtranscriptionfactorsisconservedamong species[134]asistheroleofSarco(endo)plasmicreticulum(SER)Ca2+ATPases (SERCA)pumps[135].Moduleshavealsobeenconserved.Thefinmoduleofthe modernfishforexample,aroseroughly400millionyearsagoandhasbeenconserved eversince[[1]p65]. Conservedprocessesincludecoregeneslikethoseinthehomeoboxthatareinvolvedin thesamedevelopmentalprocesses.Becausetheseprocessesandgenesareconserved amongspecies,wecouldreasonablyexpectthesameoutcomefromthesameperturbation, regardlessofthespeciescontainingtheseprocesses.Butisthisthecase?In1978Lewis [136]publishedhisseminalworkontheanterior-posteriorlayoutof Drosophila .Thiswas followedin1984bythediscoveryofthehomeo boxbyMcGinnisetal.[137].Thefieldof evodevodevelopedinlargepartfromthiswork.Inthelastdecade,enormousstrideshave beenmadeasaresultofresearchinevodevoandthevariousgenomeprojects.Theresults ofsuchresearchhaverevealedanenormousgeneticsimilarityamongmammals.AttheGreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page11of33 http://www.tbiomed.com/content/9/1/40

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levelofthegenescentrallyinvolvedindevelo pment,e.g.,thehomeoboxgenes,bilaterians arevirtuallyidentical.Thehomeoboxclasso fgenes[138]areconservedacrossspecies lines,functioninginearlycellularorganizationandanterior-posteriorbodyplan layout[139].Thereareimportantdifferenc eshowever.Forexample,therearenine Hoxgenesinfliesbutthirty-nineinmammals.Pertinently,weunderstandhow modifications(geneduplicat ions,deletions,changesintheregulatoryprocessesand soforth)totheseconservedprocesseshav eresultedintheevolutionofdifferent bodytypesandindeeddifferentspecies[138,140-142]. MicroRNA(miRNA)hasbeenfoundinessentiallyallspeciesfrom Caenorhabditis elegans tohumansandplaysalargeroleingeneregulation[143-145].Apparently,over 50%ofmiRNAsareconservedacrossspecieslinesinvertebrates[145].Animportant considerationfordrugdevelopment,however,isthefactthateventhoughmiRNAis conserved,upto50%ofmiRNAsdiffersamongvertebrates.Thisisimportantwhen consideringtheuseofanimalsaspredictivehumanmodels.Furthermore,miRNA expressionlevelschangewhentissuesdeterioratefromahealthystatetoadiseased state[146-152].ThustheexactroleofmiRNAmaydifferintra-individuallydepending onageanddisease.Hence,weseebothinter-speciesandintra-individualdifferences withrespecttothisconservedprocess. Itiswellknownthathumansandnonhumanprimatesresponddifferentlytoinfections.Forexample,untreatedhumansusuallyprogresstoAIDSwheninfectedwith HIV,aresusceptibletomalaria(exceptthosewithsicklecellanemia),havedifferent reactionstohepatitisBandCthannonhumanprimatesand,appearmoresusceptible tomanycancersandAlzheimer ’ sdisease[153-155].Barreiroetal.[154]studiedgene expressionlevelsinmonocytesfromhumans,chimpanzees,andrhesusmacaquesand foundthatallthreespeciesdemonstrated “ theuniversalToll-likereceptorresponse ” whenstimulatedwithlipopolysaccharide(LPS).Howevertheyalsodiscoveredthatonly 58%ofgenesidentifiedintheToll-likereceptorresponse “ showedaconservedregulatoryresponsetostimulationwithLPS, ” andonly31%ofthosegenesdemonstratedthe sameconservedregulatoryresponsewhenexposedtovirusesorbacteria.Barreiroetal. alsodiscoveredthat335genesinhumansareuniqueamongthespeciesinresponding toLPS,with273genesrespondingonlyinchimpanzees,and393onlyinrhesusmacaques[154].Eveninconservedprocesses,therearegoingtobesignificantdifferences thatinfluencetheoutcomesfromdiseaseperturbations.Significantdifferencesinthe detailsofconservedprocesses(alsoillustratedbyFigure1[156])meanthatthereare differencesintheinitialconditionsofthecomplexsystemandthishasmajorimplicationsforinter-speciesextrapolation. Theimplicationsofthevariouspropertiesofcomplexsystemsalsobecomeapparent whenscientistsstudyprocessessuchaspreimplantationembryonicdevelopment (PED).PEDisthoughttobehighlyconservedamongspecieswhichledXieetal.[157] tostudygeneexpressionprofilesinembryosfromhumans,mice,andcows.They foundthat: “ 40.2%orthologousgenetripletsexhibiteddifferentexpressionpatterns amongthesespecies. ” Differencesinexpressionprofileshaveimplicationsfordrugand diseaseresponse. The Cdc14 genewasdiscoveredintheyeast Saccharomycescerevisiae andisclassified asadual-specificityphosphatase.Ithassincebeenfoundinmanyorganismsincluding humans.HumanCdc14Bfulfillstherole,inyeast,oftheyeastgene Cdc14 .BecausetheGreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page12of33 http://www.tbiomed.com/content/9/1/40

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yeastgeneplaysaroleinregulatinglatemitosis,itwasassumedthegenewouldhave thesameroleinmammals.Inactuality,neither Cdc14A nor Cdc14B arenecessaryfor cell-cycleprogressioninhumans[158].Thus,wehaveaconservedgenebutnota conservedfunction. Pyrinproteinshavebeenfoundtobeubiquitousinmammals.Pyrin-onlyprotein2 (POP2)wasfoundinhumansandthoughttobeimportantininflammatorydiseases. Atianandetal.[159]studiedmicebutdidnotfindPOP2.Theythendiscoveredthat POP2wasnotinrodentsormanyothermammalsbutwaspresentinchimpanzees ( Pantroglodytes )andrhesusmacaques( Macacamulatta). Moreover,thechimpanzee Figure1 Variationinsialicacid(Sia)-recognizingIg-superfamilylectinsamongprimates. “ Expression ofCD33rSiglecsonhumanandgreatapelymphocytes.( A )Percentageofpositivelymphocytesforeach SiglecAb(stainingabovenegativecontrols)for16chimpanzees,5bonobos,and3gorillasareshown,as wellasdatafor8humans(thelatterweretestedononeormoreoccasions).Examplesofflowcytometry histogramsofhuman( B )andchimpanzee( C )lymphocytesusingAbsrecognizingSiglec-3,Siglec-5,Siglec-7, andSiglec-9(yaxis:normalizedcellnumbersexpressedaspercentofmaximumcellnumberdetected).Inlater samplesexamined,lowlevelsofSiglec-11staining(<5%positive)wereoccasionallydetectedonlymphocytes inbothgreatapesandhumans(datanotshown) ” [156]. GreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page13of33 http://www.tbiomed.com/content/9/1/40

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POP2wasidenticaltohumansPOP2atboththeDNAandproteinlevelsbutthe macaquePOP2wasnot. Conservedprocessesact,areaffectedby,orinteractatmultiplelevelsoforganization. AsCairns-Smithpointsout,proteins,catalysts,nucleicacids,membranes,andlipids areinterlockedandallaredependentontheothersfortheirproduction.Cairns-Smith summarizesbystating: “ Subsystemsarehighlyinterlocked...Theinter-lockingistight andcritical.Atthecentreeverythingdependsoneverything ” [[81]p39].Thesameperturbationmayresultindifferenteffectsoroutcomesfordifferentlevelsoforganization inthesameintactsystem.Thisfurthercomplicatesourabilitytopredictoutcomes betweentwointactlivingcomplexsystems.Thusitappearsthataperturbationof complexsystemS1containingconservedprocessP1resultinginoutcomeO1willnot necessarilyresultinO1intheverysimilarcomplexsystemS2thatalsohasP1. Wewillnowexamineinmoredetailtherespons eoforganismstoinhalationalanesthetics andanti-neoplasticagentsinordertoillu stratewhatcanandcannotbeextrapolated betweenspeciesknowingthatspeciesareactedonandaffectedbythefundamental principlesofgeometry,chemistry,andphys icsaswellassharedconservedprocesses.ConservedprocessesinanesthesiaGeneralanesthesiabymeansofinhalationalane sthetics(IAs)providesuswithanexcellent opportunitytoexaminewheretheeffectsofc onservedprocessescanandcannotbeextrapolatedbetweenspecies.Weexpecttoseevariouseffectsatdifferentlevelsoforganization andindifferentmodules.Wealsoanticipateeffectsonemergentproperties.Because IAsactonthesystemasawhole,weexpecttoseeeffectsthatcannotbepredictedfrom reductionism.Thishasimplicationsforwhatcanbeexpectedintermsofpredictinghuman responsebystudyingadifferentspeciesorperhapsevenadifferentindividual.Therefore, boththeprimaryeffectoftheanestheticagentaswellasthesideeffectsmayvary. Broadlyspeaking,generalanesthesiainhumansandanimalsisdefinedbyamnesia, controlledinsensitivityandc onsciousness,andimmobility.Ithasbeenobservedthatmost, ifnotall,extantvertebratespeciesexhibita nanesthetic-likeresponsetoawidevarietyof chemicalsthatseeminglyhavelittleincommon.Thishasbeentermedthe universal response .Multiplemechanismsfortheuniversalresponsehavebeenpostulatedandthisis anareaofintensecurrentresearch[160-164 ].Thereseemstobegeneralagreementthat ligandgatedionchannel(LGIC)proteinreceptorsareinvolvedaswellaspossibleeffectson thecellularmembrane.Regardlessoftheexactdetails,theconservationofmechanismscan beseeninthatinhalationalanesthetics(IAs) haveobservableeffectsonmotorormotility responsesinvertebratesandinvertebrates[1 65-168],tactileplants[169]andciliatedprotists [170].(Wenotethatthisisprobablyanexampleofanexaptation,specificallyaspandrel, ratherthananadaptation[171-173].)Inter estingly,effectshaveevenbeenobservedin S. cerevisiae (Baker ’ syeast)[174],suggestingthatcrucialaspectsoftheuniversalresponsego beyondmetazoanstoincludeEukaryotes.M oreover,IAshavebeenshowntohaveeffects onmembranecompositioninprokaryotespecies[163,175]e.g., A.laidlawii [176,177], Bacillushalodurans [175]and E.coli [178]andthesingle-celle deukaryotetetrahymena [179,180](aciliatedprotozoan).Theuniversa lresponseappearstodatefarbackinevolutionarytimeandstronglysuggeststhatthem echanismhasbeenconservedamongspecies. However,therearedifferencesinoutcomeswithrespecttoIAs.Humphreyetal. [181]studiedgenesin Caenorhabditiselegans and Drosophilamelanogaster inordertoGreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page14of33 http://www.tbiomed.com/content/9/1/40

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assessthefunctionofgenesthoughtinvolvedintheresponsetoIAs.Theyfoundthata genein Celegans unc-79 ,andagenein Drosophila narrowabdomen(na) ,were relatedtoeachotherandplayaconservedroleinresponsetoanesthetics.However, mutationsineachgeneproduceduniquechangesinsensitivitytoIAs.Thesensitivity tohalothane,anIA,wasincreasedbutthesensitivitytoenflurane,adifferentIA,was unchangedorperhapsevenlowered.Thisisperplexingbecauseonewouldhave expectedthetwoinhalationalagentstobeaffectedinasimilarfashionbythemutation. Thegene unc-79 appearstobeapost-transcriptionalregulatorof na ,thusthegenes operateinthesamepathway.Interestingly,bothgenesarealsoassociatedwithsimilar phenotypesregardinglocomotion: “ fainting ” in C.elegans and “ hesitantwalking ” in Drosophila Stimulationoftheconservedprocessescontrollingtheuniversalresponseresultsin clinicallysignificantvariabilityamonghumans,eventhoughtheminimumalveolar concentration(MAC)forIAsformostspeciesisapproximatelythesame.MACisthe mostoftenusedmetrictoassessIApotency.However,theconceptofMACimplies variability.MAC50,simplycalledMACinanesthesiology,istheminimumalveolar concentrationnecessarytosuppressmovementinresponsetopainfulstimuliin50%of subjects[182].MACissignificantlyvariableamonghumansdependingonanumberof factorsincludingageandsex.Whyisthisthecase? Sonneretal.reported, “ onehundredforty-sixstatisticallysignificantdifferences amongthe15strains[ofmice]werefoundforthethreeinhaledanesthetics(isoflurane, desflurane,andhalothane) ” [164].Theyconcludedthatmultiplegenesmustbe involvedinanestheticpotency.Wangetal .developedtwostrainsofmicethatmanifesteddifferentsensitivitiestoisoflurane[183].MACisanexampleofaphenomenon controlledbyquantitativetraitloci[184] ,whichmayexplaininpartwhy,whileone canobtainaroughapproximationofMACbystudyingotherspecies,therewillstillbe clinicallysignificantdifferences. IAsalsofunctionatdifferentlevelsoforganizationandonmodulesinadditiontothe oneinvolvedintheuniversalresponse.Thesideeffectsofthesamechemicalthat produceaneffectontheconservedreceptorsorotherprocessesvarygreatlyfrom speciestospeciesandinsomecases,evenfrompersontoperson.Agoodexampleis thecaseofisofluraneandcoronarysteal.Inthe1980s,therewasheatedcontroversy regardingtheadministrationoftheinhalationanestheticisofluranetopatientswith heartdisease.Thecontroversycenteredonresearchusingcaninesthatindicatedthat thedrugcausedmyocardialischemiaduringcertainsituationsinpatientswithcoronary disease.Thephenomenonappearedtoresultfromisofluranecausingdilationofthe normalcoronaryarteries,andthusbloodbeingshuntedawayfromtheoccludedcoronaryarteries;thearteriesandtissuesthatmostneededit.Thiswascalled coronarysteal Further,thissituationwasworsenedbyadecreaseinbloodpressure;aconditionthat oftenoccursduringgeneralanesthesiawithIAs.Thissupposeddanger,basedalmost entirelyonstudiesincanines,wasseizedonbymanyintheanesthesiologycommunity asdogma[185,186]. Thiswasaninterestingreactionfromcliniciansfortworeasons.First,experiments withotherspecieshadfailedtodemonstratecoronarysteal[187,188]andsecond, anesthesiologistshadnotnoticedischemicchangesassociatedwithisofluranedespite muchuseoftheagent.ThesituationwasalsotroublesomebecauseisofluranewasaGreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page15of33 http://www.tbiomed.com/content/9/1/40

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neededadditiontoananesthesiologist ’ sarmamentariumwheninitiallyapprovedfor clinicalpractice.Sixyearsafteritsintroduction,itwasthemostfrequentlyusedIA,in partbecauseofthefavorablepropertiesofthedrug[185].Furtherstudiescontinuedto demonstratevaryingeffectsintra-andinter-species[189,190].Ultimately,studiesbegan toappearthatsuggestedisofluranewasinfactcardio-protective.Themechanismfor thisprotectionwascalled “ preconditioningandinvolvestheopeningofadenosine triphosphate-dependentpotassiumchannels ” [191].Isofluranewentfrombeingcontraindicatedinpatientswithcoronaryarterydiseasetobeingthedrugofchoiceinsuch patients.Studiesfromanimals,specificallydogs,figuredheavilyinformingboth,mutually exclusive,conclusions. Justaswiththehomeobox,miRNAs,andtheresponsetoinflammation,thereare differencesamongspeciesinhowtheconservedprocessknownastheuniversal responsetoanesthesiamanifests.Clinically,thesedifferencesaresignificantandlimit theamountofinformationthatcanbeextrapolatedbetweenspeciesevenwhenthe underlyingprocessisconserved.Inhalationanestheticsarealsoagoodexampleofwhy, whentheleveloforganizationormodulebeingexaminedchanges,extrapolationbreaks down.Thesamechemicalthatinducesgeneralanesthesiainadogwillprobablyresult inthesameeffectinhumansbutthedosemayvaryinaclinicallysignificantfashion andthesideeffectswillmostlikelyvary,astheconservedprocessdoesnotdictatethe sideeffects.Differencesinoutcomesfromperturbationsliketheoneswehaveseen abovehavebeenexplainedbyevolution-basedspecies-specificdifferences,forexample backgroundgenes,mutations,expressionlevels,andmodifiergenes[192-209].Anti-neoplasticdrugsactingonmitosisAsdiscussed,arelationshipexistsbetweenBSAandmanyphysiologicalparameters [210].Forexample,Reagan-Shaw,Nihal,andAhmadstate: “ BSAcorrelateswellacross severalmammalianspecieswithseveralparametersofbiology,includingoxygen utilization,caloricexpenditure,basalmetabolism,bloodvolume,circulatingplasma proteins,andrenalfunction ” [211].Dosingalgorithmsforfirst-in-man(FIM)trialsare basedontheassumptionthatthereisaone-to-onedosescalebetweenhumansand animalswhenBSAistakenintoaccount[212].Thefirststudysuggestingarelationship betweendoseandbodysurfaceareawasperformedbyPinkelin1958[210]involving anti-neoplasticagents,drugswheretheeffectsandsideeffectsarelargelythesame — celldeath.Subsequently,Freireichetal.,[213]studied18anti-neoplasticdrugsinsix animalspeciesandconcludedthatthemaximumtolerateddose(MTD)forhumans was1/12ofthedoseinmicethatresultedinthedeathof10%ofthemice(LD10).They alsonotedthattheMTDwas1/7oftheLD10inrats.Thesewerealsotheratiosfor convertingfromamg/kgdosetoadosebasedonBSA.Fiftyanti-neoplasticdrugswere thenstudiedusingthisformulaandallwerereportedlyintroducedintohumantrials withoutincident[214,215].ThestandardforFIMdosesthenbecamethe1/10ththe LD10formice.ActuallyFreireichrecommendedastartingdoseof1/3rdtheLD10not 1/10thbutthatchangedovertime.The1/3rdrecommendationwasfoundtobetoo largeforFIMandwaschangedto1/10th[216].Morestudiesappearedtoconfirmthe 1/10thvalue[217]. Theabovemakesa primafacie casethatanimalmodelscanpredictastartingdose forhumansinclinicaltrialsforanti-neoplastics.FurthersubstantiatingthisisthefactGreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page16of33 http://www.tbiomed.com/content/9/1/40

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thatanti-neoplasticsarenotalwaysmetabolizedbytheliver[218],thuspossiblyeliminatingacomplexsystemfromconsideration.Celldivisionbymitosisisarguablythe mostconservedprocessonecanfindinbiologyandthetraditionaldrugsfortreating canceractbyinterferingwithmitosis.(Newerdrugsactontargetedpathwaysas opposedtothecellcycle.)Anti-neoplasticskillthecellsthataredividingmostrapidly — thecancercells.However,haircells,cellsinthebonemarrow,andcellsinthegut alsodivideatasimilarratesuchthatanti-neoplasticscanaffectthem.Thus,inpart, theeffectsandsideeffectsofanti-neoplasticsarethesame — celldeath.Theproblem withtraditionalanti-neoplasticsisthattheydonotdiscriminateadequately. Anti-neoplasticdrugsareuniqueinmedicineinthat:1)theyarenonspecific;2)long termtoxicitiesareanticipatedandacceptedbecausethepatientfrequentlydoesnot haveanyotherviableoptions;3)theeffectsandsideeffectsofthedrugsarethesame — celldeath;and4)theyactonauniversallyconservedprocess — mitosis.Thisiswhy bodysurfaceareaappearstobesoeffectiveforcalculatingFIMdose.Whereas,when oneisexaminingeffectsandsideeffectsofdrugsbasedoninteractionsatthelevelof organizationwherecomplexityisrelevant,forexamplemetabolism[219-229],thereare simplytoomanyotherfactorstoallowfortheexpectationofone-to-onecorrelations. Species-specificdifferencescreateperturbationsinthecomplexsystemthusthedifferencesamongspeciesoutweighthesimilarities[13-16,18,21-41]. However,inthefinalanalysiseventheFIMdoseoftheanti-neoplasticagentscannot bereliablyascertainedbasedonBSA.Mostanti-neoplasticsareeffectiveonlyatdoses nearthemaximumtolerateddoseandthedrugsaregiveninanescalatingfashion duringclinicaltrials. “ Patientstreatedatthelowerendofthedoseescalationstrategy areunlikelytoreceiveevenapotentiallytherapeuticdosesincemostcytotoxicdrugs areonlyactiveatorneartheMTD ” [217].Differencesamongspeciesindoseresponse foranti-neoplasticsaredueinparttodifferencesinpharmacokinetics[217,230-232], whichcannotbeaccountedforbasedonBSA.Brennanetal.statethat: “ Whileproper determinationofdrugdosescanbecomplicatedwithinthesamespecies,itcanbean incrediblechallengeandburdenbetweenspecies ” [233].Brennanetal.continueby pointingoutthatmetabolismandclearancedifferamongspeciesandthat “ ... theliver, kidneysandhematopoieticsystembetweenspeciesmayhavesignificantdifferencesin theirsensitivitytochemotherapeuticagents.Noneofthesefactorsaretakeninto accountwiththeuseofthespecies-specificdosecalculations ” [233].Theyrecommend areaunderthecurve(AUC)forcalculatingFIMdosebutthenconcede: “ However, therearenumerousexamplesinwhichthespecies-specificconversiondosevaries significantlyfromtheAUCguideddoseand/orfarexceedstheanimal ’ smaximum tolerateddose. ” Theythenlistexamplesfrompediatricswheretherecommendedand actualdosesdiffersignificantly[233]. Horstmannetal.[234]reviewed460PhaseINationalCancerInstitutetrialsinvolving 11,935adultsthatoccurredbetween1991and2002.Approximately25%ofthetrials wereFIMtrials.Horstmannetal.foundthatseriousnonfataleffectsoccurredin15% ofthepatientsundergoingsinglechemotherapy,with58deathsthatwereprobably treatment-related[234,235].Concernhasalsobeenexpressedthatanimalmodelshave derailedanti-neoplasticsthatwouldhavebeensuccessfulinhumans[30,235-239]. FIMdosebasedonanimalmodelsisineffectiveforpredictingdoseforotherdrug classesaswell-TGN1412beingarecentnotableexample[26,240,241].AnunnamedGreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page17of33 http://www.tbiomed.com/content/9/1/40

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clinician,speakingoftoxicitytrialsfornewdrugsingeneralinhumans,wasquotedin Science, stating, “ Ifyouweretolookin[abigcompany ’ s]filesfortestingsmallmoleculedrugsyou ’ dfindhundredsofdeaths ” [242].Chapmanreinforcedthisstating: “ ...butotherincidentsofharm[besidesTGN1412],evendeath,toparticipantsin PhaseItrials,somethenknownandotherunpublicized,hadtakenplace ” [235].Itis alsoimportanttonotethatthe1/3rdor1/10thsafetyfactorisfabricated.Perlsteinetal. state: “ Duetouncertaintyintranslatinganimalmodelfindingstohumans,particularly forunprecedentedmechanisms,awidedoserange(1000-fold)isexpectedtocoverthe entireexposure – responsecurve ” [243].Extrapolatingfromspeciestospeciesshould notrequire fudgefactors iftheprocessistrulyscience-based.InPhaseItrials,where FIMorfirstinhuman(FIH)occurs,scientistswanttocharacterizethedrug ’ sPKpropertiesandsafetymargins[244].WexlerandBertelsensummarizethesituationwhen theystate: “ Althoughallometricscalingtechniquescontinuetoprovidepoorpredictive estimatesforhumanpharmacokineticparameters,FIHstartingdosesareselectedwith substantialsafetyfactorsappliedtohumanequivalentdose,ofteninexcessofregulatory guidelines....Approachesthatcouldenhancethepredictivenatureofacompound ’ s dispositionandadaptivenatureofFIHstudiescouldprovideatremendousbenefitfor drugdevelopment ” [245].FIMforallclassesofdrugcouldbeeasilyaccomplishedusing microdosing[246-248]withthefirstdoseofonenanogram[249,250]andincreasing subsequentdosestothedesiredendpoint. Finally,onemustrecallthat95%[31,251,252]ofa nti-neoplasticagentsfailinclinicaltrials. Oncologydrugsfailmorefrequentlyinclinical trialsthanmostothercategories[253,254] andahigherpercentageofanti-neoplasticdrugsfailinPhaseIIItrialsthandrugsfromany othercategory[255].Reasonsfortheattritionincludethefactthatmostoftheeffectsand sideeffects,evenoftheanti-neoplasticagents,whenplacedintothecontextofacomplex system,arenotpredictedfromanimalstud ies.Interferinginmitosisisauniversal phenomenonbutthedegreeandsuccessofthatinterferencevaries.TheFIMdoseestimationisapparentlysuccessfulbecausetheleve loforganizationinquestionisverybasicand conservedandbecausethedoseisloweredevenfurtherbyfudgefactors.Pickingastarting dosebasedonthemosttoxicsubstancesinnature[249,250]wouldbemorescientific.The apparentsuccessalsobreaksdownbecausethetypesofcancersinhumansdifferfromthose inanimals,thegeneticbackgroundofhumans variesfromthatinanimals,andbecausethe realityofacomplexsystem — theinteractionsofalltheothersystems(forexamplehowthe drugsareeventuallymetabolizedandeliminat edandhowthosemetabolitesinteractwith othersystemsandsoon) — eventuallyappear.Thesearetheproblemsthatcannotbesolved byanimalmodelsandarewhytheattritionrateis95%.Weinbergstated: “ it ’ sbeenwell knownformorethanadecade,maybetwodecades,thatmanyofthesepreclinicalhuman cancermodelshaveverylittlepredictivepowerintermsofhowactualhumanbeings — actual humantumorsinsidepatients — willrespond...preclinicalmodelsofhumancancer,inlarge part,stink...hundredsofmillionsofdollarsarebeingwastedeveryyearbydrugcompanies usingthese[animal]models ” [236].Othershavealsopointedouttheinadequacyofanimal modelsofcancer,includinggeneticallymodifiedanimalmodels[41,214,252,256-261].ConservedprocessesinlightofsystemsbiologyAstheleveloforganizationinacomplexsystemincreases,weexpecttoseeanincrease inthenumberofemergentpropertiesaswellasmoreoverallinteractions.AgeneorGreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page18of33 http://www.tbiomed.com/content/9/1/40

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processthathasbeenconservedwillinteractwiththeintactwholeorganismyielding newprocessesandstates.Perturbationsoftheseconservedgenesorprocesseswillthus likelyresultinnewstatesnotseeninotherorganismsthatsharetheconserved processes;perhapsnoteveninorganismsofthesamelineage(clade)orspecies. Thelackofappreciationforthedifferencesbetweenlevelsoforganizationandother propertiesofcomplexsystemsisapparentinthefollowingfromKardong[[262]p2], writinginhistextbookofcomparativevertebrateanatomy: “ Forexample,bytestinga fewvertebratemuscles,wemaydemonstratethattheyproduceaforceof15N(newtons) persquarecentimeterofmusclefibercrosssection.Ratherthantestingallvertebrate muscles,atime-consumingprocess,weusuallyassumethatothermusclesofsimilarcross sectionproduceasimilarforce(otherthingsbe ingequal).Thediscoveryofforceproduction insomemusclesisextrapolatedtoothers.Inmedicine,thecomparativeeffectsofdrugson rabbitsormiceareextrapolatedtotentativeuseinhumans. ” Attheleveloforganization whereonestudiestheforcegeneratedbymuscle fibers,nodoubtinter-speciesextrapolation isuseful,butthatisanentirelydifferentlevelfromwheredrugactionsoccur. Indeedthesuccessesfromusinganimalmodelshavebeenexamplesofperturbations occurringatsubsystemsthatcanbedescribedassimplesystemsandoroutcomesor characteristicsthatapplyonthegrosslevelofexamination.Forexample,theGerm TheoryofDiseaseappliestohumansandanimals.Theimmunesystemreactsto foreignentitiesinamannerthatisgrosslysimilaracrossspecieslines.Thedetailsof immunityareclinicallyverydifferent,forexampleHIVinfectionleadstoAIDSin humansbutnotchimpanzees[263-265].Nevertheless,grossly,inflammation,white bloodcells,andantibodiesareidentifyingcharacteristicsoftheimmunesysteminthe phylumChordata.Likewise,whiletheheartfunctionstocirculatethebloodin mammals,thediseasesvariousmammalianheartsaresubjecttodifferconsiderably [266-272].Thefailuresofanimalmodelshaveoccurredwhenattemptingtoextrapolate datafromhigherlevelsoforganization,levelswherecomplexityisanimportantcomponentinthesystemorsubsystemunderconsideration.Forexample,adrugthathas passedanimaltestsandisinPhaseIhumanclinicaltrialshasonlyan8%chanceof makingittomarket[273].Over1,000drugshavebeenshowntoimproveoutcomesin cerebralischemiainanimalmodelsbutnone,saveaspirinandthrombolysis,which werenotanimal-baseddiscoveries,havebeensuccessfulinhumans[35,274-277].The animalmodelforpolio,monkeys,revealedapathophysiologythatwasverydifferent fromthatofhumans[278-281].Extracranial-intracranialbypassforinoperablecarotid arterydiseasewassuccessfulinanimalsbutresultsinnetharmforhumans[282-285]. Mostdiseasesaremultifactorialhenceitshouldcomeasnosurprisethatconserved processesplayasmall,althoughattimesimportantrole,inmajordiseaseslikeheart disease,cancerandstroke.Thefieldofsystemsbiologywasformedinpartinan attempttoplacethepartsofmolecularbiologyandgeneticsinthelargercontextofthe humansystem;thesystemthatactuallyrespondstodrugsanddisease.Aneditorialin Nature asks: “ Whatisthedifferencebetweenalivecatandadeadone?Onescientific answeris'systemsbiology'.Adeadcatisacollectionofitscomponentparts.Alivecat istheemergentbehaviourofthesystemincorporatingthoseparts ” [286].Accordingto theDepartmentofSystemsBiologyatHarvardMedicalSchool: “ Systemsbiologyisthe studyofsystemsofbiologicalcomponents,whichmaybemolecules,cells,organisms orentirespecies.Livingsystemsaredynamicandcomplex,andtheirbehaviormaybeGreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page19of33 http://www.tbiomed.com/content/9/1/40

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hardtopredictfromthepropertiesofindividualparts ” [287].Systemsbiology[288] takesatop-downapproachasopposedtoreductionism,whichevaluatesorganisms fromthebottom-up.Systemsbiologyisconcernedmorewithnetworksthanindividual components,althoughbotharestudied.Italsorecognizestheimportanceofemergent phenomena.(SeeFigure2[79]).Suchtop-downapproachesareusedbythefieldscommonlyreferredtoas “ Omics, ” forexample:interactomics,metabolomics,proteomics, transcriptomics,andevenfractalomics[289]. NobellaureateSydneyBrenner,in1998,emphasizedthattheinteractionsofcomponents wasimportantinunderstandinganorgan ism[290].Onlybystudyingproteinsand processesinthecontextoftheirsystemscanweexpecttounderstandwhathappenstothe intactorganismsasaresultoftheseproces sesandgenes.Further,evolutionusesold pathwaysandprocessesindifferentwaystocr eatenovelty[1,133].Everythingiscontext dependent.Noblestressesthatinordertopr edicthowdrugswillact,onemustunderstand “ howaproteinbehavesincontext ” athigherlevelsoforganization[291]. Heng[292],writingin JAMA statesthat,becauseofreductionism,biologicalscientists havesoughtindividualcomponentsinadiseaseprocesssotheycouldintervene.Alinear causeandeffectrelationshipwasassumedtoexist.Hengcitesdiabetesinterventioninan attempttocontrolbloodglucoseandcancertherapiesasexamples.Hepointsoutthat whilethishasworkedwellinmanycases,verytightcontrolofbloodglucosewasrecently foundtoincreasetheriskofdeath[293].Alongthesamelines,chemotherapiesforcancer havedecreasedthesizeofthetumorsbutattheexpenseofanincreaseinfrequencyof secondarytumorsandaveryadverselyaffectedlifestyle.Furthermore,mostchemotherapy doesnotprolonglifeorresultinalonger,highqualitylife[294-296].Insteadoffocusingon smallmodulesorcomponentsofasystem,co mplexitytheorymandatesthatbiomedical sciencelookatthesystemasawhole. Closelyrelatedtosystemsbiologyaretheconceptsofpersonalizedmedicineand pharmacogenomics[226,297-305].Ithaslongbeenappreciatedthathumansrespond differentlytodrugsandhavedifferentsusceptibilitiestodisease.Basedonstudiesof Figure2 Reductionismversussystemsbiology. GreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page20of33 http://www.tbiomed.com/content/9/1/40

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twins,thereappearstobeageneticcomponenttosusceptibilitytoleprosy,poliomyelitis andhepatitisB,aswellasresponsetoopioids[306-309].Otherinfectiousdiseasesthat appeartohaveageneticcomponenttosusceptibilityincludeHIV,HepatitisC,malaria, dengue,meningococcaldisease,variantCreutzfeldt – Jakobdiseaseandperhapstuberculosis amongothers[310].Differencesindruganddiseaseresponsearemanifestamongethnic groups[311-319]andsexes[320-326].Evenmonozygotictwinsmanifestdifferencesin responsetosuchperturbations[107,108,113 -116].RashmiRShah,previousSeniorClinical Assessor,MedicinesandHealthcareproductsRegulatoryAgency,Londonstatedin2005: “ Duringtheclinicaluseofadrugatpresent,aprescribingphysicianhasnomeansof predictingtheresponseofanindividualpatienttoagivendrug.Invariably,somepatients failtorespondbeneficiallyasexpectedwhereasothersexperienceadversedrugreactions (ADRs) ” [327]. Similarly,AllenRoses,then-worldwidevice-presidentofgeneticsatGlaxoSmithKline (GSK),saidfewerthanhalfofthepatientsprescribedsomeofthemostexpensivedrugs derivedanybenefitfromthem: “ Thevastmajorityofdrugs-morethan90%-only workin30or50%ofthepeople. ” Mostdrugshadanefficacyrateof50%orlower [328].Becauseofdifferencesingenes,likeSNPs,allchildrenmaynotcurrentlybe protectedbythesamevaccine[329,330].Itisestimatedthat “ between5and20per centofpeoplevaccinatedagainsthepatitisB,andbetween2and10percentofthose vaccinatedagainstmeasles,willnotbeprotectediftheyeverencountertheseviruses ” [330].Inthefuturesuchchildrenmaybeabletoreceiveapersonalizedshot.Currently, numerousdrugshavebeenlinkedtogeneticmutationsandalleles.SeeTable5[303] andTable6[331].Thenumberofpersonalizedmedicineproductshasincreasedfrom 13in2006to72asof2012[332]. Whenanimalswerebeingusedasmodelsinthe19thcentury,manyofthescientists whowereusingthemhadnotacceptedevolutionandbelievedthatanimalpartswere interchangeablewiththeirhumancounterparts[60,62,63].Giventhatwenowunderstandthatintra-humanvariationresultsinsuchmarkedlydifferentresponsestodrugs anddisease,attemptingtopredicthumanresponsefromanimalmodels,evenfor perturbationsactingonconservedprocesses,seemsunwarranted.Yet,despitethe implicationsofpersonalizedmedicine[22],somescientistscontinuetocommitthe fallacydescribedbyBurggrenandBemis: “ Yettheuseof ‘ cockroachasinsect, ’‘ frogas amphibian, ’ or ‘ theturtleasreptile ’ persists,inspiteofclearevidenceofthedangersof thisapproach.Notsurprisingly,thistypeofcomparativephysiologyhasneither contributedmuchtoevolutionarytheoriesnordrawnuponthemtoformulateandtest hypothesesinevolutionaryphysiology ” [[333]p206].Comparativeresearchwillyielda nicecomparisonofthetraitorprocessamongspeciesorphyla.However,onesimply cannotassumethattheoutcomefromaspecificperturbationin,saythecockroach,will beseenininsectsingeneralandthisconceptbecomesevenmoreimportantwhen relyingonanimalmodelsformedicalinterventionsinhumans.ConclusionAperturbationoflivingcomplexsystemS1containingconservedprocessP1resulting inoutcomeO1willnotresultinO1intheverysimilarlivingcomplexsystemS2that alsohasP1oftenenoughtoqualifyS1asapredictivemodalityforS2whenthetraitor responsebeingstudiedislocatedathigherlevelsoforganization,isinadifferentGreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page21of33 http://www.tbiomed.com/content/9/1/40

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Table5ExamplesofdrugswithgeneticinformationinthierlabelsDrugSponsorIndicationGeneor genotype Effectof genotype Clinicaldirectiveon label Abacavir (Ziagen) GlaxoSmithKlineHIV-1 HLA-B*5701 HypersensitivityBlack-boxwarning. ” Priortoinitiating therapywithabacavir, screeningforthe HLAB*5701 alleleis recommended. ”“ Your doctorcandetermine withabloodtestif youhavethisgene variation. ” Azathioprine (Imuran) PrometheusRenalallograft transplantation, rheumatoid TPT*2TPT*3A and TPMT*3C Severe myeloxicity “ TPT genotypingor phenotypingcanhelp identifypatientswho areatanincreased riskfordeveloping Imurantoxicity. ” “ Phenotypingand genotypingmethods arecommercially available. ” Carbamazepine (Tegretol) NovartisEpilepsy, trigeminal neuralgia HLA-B*1502 StevensJohnson syndromeor toxicepidermal necrolysis Black-boxwarning: “ Patientswith ancestryingenetically at-riskpopulations shouldbescreened forthepresenceof HLA-B*1502 priorto initiatingtreatment withTegretol.Patients testingpositivefor thealleleshouldnot betreatedwith Tegretol. ”“ For geneticallyat-risk patients,highresolution HLA-B*1502 typingis recommended. ” Cetuximab (Erbitux) ImcloneMetastatic colorectal cancer KRAS mutationsEfficacy “ Retrospectivesubset analysesofmetastatic oradvanced colerectalcancertrials havenotshowna treatmentbenefitfor Erbituxinpatients whosetumorshad KRASmutationsin codon12or13.Use ofErbituxisnot recommendedforthe treatmentof colorectalcancerwith mutations. ” Clopidogrel (Plavix) Bristol-Myer Squibb AnticoagulationCYP2C19*2*3Efficacy “ Testsareavailableto identifyapatient ’ s CYP2C19genotype;thesetestscanbe usedasanaidin determining therapeuticstrategy. Consideralternative treatmentor treatmentstrategies inpatienrsidentified GreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page22of33 http://www.tbiomed.com/content/9/1/40

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module,orisinfluencedbyothermodules.However,whentheexaminationofthe conservedprocessoccursatthesameorlowerleveloforganizationorinthesame module,andhenceissubjecttostudysolelybyreductionism,thenextrapolationis possible.Webelievethisisavaluableprinciple. Ourcurrentunderstandingofevodevo,evolutioningeneral,complexityscience,and geneticsallowsustogeneralizeregardingtrans-speciesextrapolation,evenwhen conservedprocessesareinvolved.ShanksandGreek: Table5Examplesofdrugswithgeneticinformationinthierlabels (Continued)asCYP2C19poor metabolizer. ” Irinotecan (Camptosar) PfizerMetastatic colorectal cancer UGT1A1*28 Diarrhea neutropenia “ Areductioninthe startingdosebyat leastonelevelof Camptosarshouldbe considerforpatients knowstobe homozygousforthe UGT1A1*28allele. “ A laboratorytestis availabletodetermine theUGT1A1statusof patients. ” Pantumumab (Vectibix) AmgenMetastatic colorectal cancer KRAS mutationsEfficacy “ Retrospectivesubset analysesofmetastatic colorectalcancertrials havenotshowna treatmentbenefitfor Vectibixinpatients whosetumorshad KRASmutationsin codon12or13.Use ofVectibixisnot recommendedforthe treatmentof colorectalcancerwith thesemutations. ” Transtuzumab (Herceptin) GenetechHER2-positive breastcancer HER2expressionEfficacy “ DetectionofHER2 protein overexpressionis necessaryfor selectionofpatients appropriatefor Herceptintherapy becausethesearethe onlypatientsstudied andforwhombenefit hasshown. ”“ Several FDA-approved commercialassaysare availabletoaidinthe selectionofbreast cancerandmetastatic cancerpatientsfor Herptintherapy. ” Wafarin (Coumadin) Bristol-Myer Squibb Venous thrombosis CYP2C9*2*3and VKORC1variants Bleeding complications Includesthefollowing table:Rangeof ExpectedTherapeutic WarfarinDosesBased onCYP2CPand VKORC1Genotypes.*AlldruglabelswereaccessedthroughDrugs@FDAatwww.accessdata.fda.gov/scripts/cder/drugsatfda.HIV-1denotes humanimmunodeficiencyvirustype1, TPMT thiopurinemethyltransferase, UGT1A1 UDPglucuronosyltransferanse1 familypolypeptideA1,and VKORC1 vitaminsKepoxidereductasecomplexsubunit1GreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page23of33 http://www.tbiomed.com/content/9/1/40

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Livingcomplexsystemsbelongingtodifferentspecies,largelyasaresultofthe operationofevolutionarymechanismsoverlongperiodsoftime,manifestdifferent responsestothesamestimulidueto:(1)differenceswithrespecttogenes present;(2)differenceswithrespectt omutationsinthesamegene(whereone specieshasanorthologofagenefoundinanother);(3)differenceswith respecttoproteinsandproteinactivity;(4)differenceswithrespecttogene regulation;(5)differencesingeneexpres sion;(6)differencesinprotein-protein interactions;(7)differencesingeneticn etworks;(8)differenceswithrespectto organismalorganization(humansandratsmaybeintactsystems,butmaybe differentlyintact);(9)differencesinen vironmentalexposures;andlastbutnot least;(10)differenceswithrespecttoevo lutionaryhistories.Thesearesomeof theimportantreasonswhymembersofone speciesoftenresponddifferentlyto Table6ThemostsignificantgeneticpredictorsofdrugresponseOrganorsysteminvolvedAssociatedgene/alleleDrug/drugresponsephenotype Blood Redbloodcells G6PD Primaquineandothers Neutrophils TMPT*2 Azathioprine/6MP-inducedneutropenia UGT1A1*28 Irintotecan-inducedneutropenia Plates CYP2C19*2 Stentthrombusis Coagulation CY2C9*2,*3,VKORC1 Warfarindose-requirement Brainandperipheralnervoussystem CNSdepressionCYP2D6*NCodeine-relatedsedationandrespiratorydepression Anaesthesia Butyrylcholinesterase Prolongedapnoea Peripheralnerves NAT-2 Isoniazid-inducedperipheralneuropathy Drughypersesitivity HLA-B*5701 Abacavirhypersensitivity HLA-B*1502 Carbamazepine-inducedSteveJohnsonsyndrome (insomeAsiangroups) HLA-A*3101 Carbamazepine-inducedhypersensitivityinCausians andJapanese HLA-B*5801 Allopurinol-inducedseriouscutaneousreactions Drug-inducedliverinjury HLA-B*5701 Flucloxacillin HLA-DR81*1501-DQ81*0602 Co-amoxiclav HLA-DR81*1501-DQ81*0602 Lumiracoxib HLA-BR81*07-DOA1*02 Ximelagatran HLA-DQA1*0201 Lapatinib Infection HIV-1infection CCRS Maravirocefficacy HepatitisCinfection IL288 Interferon-alphaefficacy Malignancy Breastcancer CYP2D^ Responsetotamoxifen Chronicmyeloidleukaemia BCR-ABL Imatinibandothertyrosinekinaseinhibitors Coloncancer KRAS Cetuximabefficacy GIstromaltumours c-kit Imatinibefficacy Lungcancer EGFR Gefinibefficacy EML4-ALK Crizotinibefficacy Malignantmelanoma BRAFV600E Vemurafenibefficacy GreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page24of33 http://www.tbiomed.com/content/9/1/40

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drugsandtoxins,andexperiencedifferen tdiseases.Immenseempiricalevidence supportsthisposition([14]p358). Thefailuresofanimalmodelsasapredictivemodalityforhumanresponsetodisease anddrugs,evenwhensuchperturbationsareactingonconservedprocesses,canbe explainedinthecontextofevolvedcomplexsystems.Onedoesnotneedtostudyevery suchperturbationineveryspeciesinordertoconcludethattheanimalmodelwillnot beapredictivemodalityforhumanswhenperturbationsoccurathigherlevelsof organizationorinvolvedifferentmodulesoraffectthesystemasawhole.Thisisnotto denythatanimalmodels,ascharacterizedby3 – 9inTable1,havecontributedandwill continuetocontributetoscientificadvancements.Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors'contributions Theauthorscontributedequallytothispaper. Authors ’ information RayGreek,MDhasbeenonfacultyintheDepartmentofAnesthesiologyattheUniversityofWisconsin-Madisonand atThomasJeffersonUniversityinPhiladelphia.Heiscurrentlypresidentofthenot-for-profitAmericansForMedical Advancement(www.AFMA-curedisease.org). MarkRice,MDiscurrentlyonfacultyattheUniversityofFlorida(UF).HeischiefofthelivertransplantdivisionatUF DepartmentofAnesthesiology,hassevenUSpatents,andreviewsforseveralmajorjournals. Acknowledgements None. Authordetails1AmericansForMedicalAdvancement(www.AFMA-cured isease.org),2251RefugioRd,Goleta,CA93117,USA.2Departmentof Anesthesiology,UniversityofFloridaCollegeofMedicine,POBox100254,Gainesville,FL32610-0254,USA. Received:30July2012Accepted:31August2012Published:10September2012 References1.KirschnerMW,GerhartJC: ThePlausibilityofLife .NewHaven:YaleUniversityPress;2006. 2.BraithwaiteRB: Scientificexplanation:astudyofthefunctionoftheory,probabilityandlawinscience .Cambridge: CambridgeUniversityPress;1953. 3.HindeR: Animal-HumanComparisons .In TheOxfordCompaniontotheMind .EditedbyGregoryRL.Oxford: OxfordUniversityPress;1987:25 – 27. 4.FriggR,HartmannS: ScientificModels .In ThePhilosophyofScience:AnEncyclopediaVolume2N-Z .Editedby SarkarS,PfeiferJ.NewYork:Routledge;2012:740 – 749. 5.ShapiroK: AnimalModelResearch.TheApplesandOrangesQuandry. ATLA 2004, 32: 405 – 409. 6.HauJ:In AnimalModels ,HandbookofLaboratoryAnimalScienceSecondEditionAnimalModels,VolumeII.2nd edition.EditedbyHauJ,vanHoosierGKJr.BocaRotan:CRCPress;2003:1 – 9. 7.GadS: Preface .In AnimalModelsinToxicology .EditedbyGadS.BocaRotan:CRCPress;2007:1 – 18. 8. LongerTestsonLabAnimalsUrgedforPotentialCarcinogens .http://www.cspinet.org/new/200811172.html. 9.HuffJ,JacobsonMF,DavisDL: Thelimitsoftwo-yearbioassayexposureregimensforidentifyingchemical carcinogens. EnvironHealthPerspect 2008, 116: 1439 – 1442. 10.DevoyA,Bunton-StasyshynRKA,TybulewiczVLJ,SmithAJH,FisherEMC: Genomicallyhumanizedmice: technologiesandpromises. NatRevGenet 2012, 13: 14 – 20. 11.VassarR: Alzheimer'stherapy:aBACEinthehand? NatMed 2011, 17: 932 – 933. 12. THSCEOcriticizedforlinkstoanimaltesting .http://m.torontosun.com/2011/09/23/ths-ceo-criticized-for-links-toanimal-testing?noimage. 13.HeywoodR: ClinicalToxicity--Couldithavebeenpredicted?Post-marketingexperience .In AnimalToxicity Studies:TheirRelevanceforMan .EditedbyLumleyCE,WalkerS.Lancaster:Quay;1990:57 – 67. 14.ShanksN,GreekR: AnimalModelsinLightofEvolution .BocaRaton:BrownWalker;2009. 15.GreekR,GreekJ: Istheuseofsentientanimalsinbasicresearchjustifiable? PhilosEthicsHumanitMed 2010, 5: 14. 16.GreekR,ShanksN: FAQsAbouttheUseofAnimalsinScience:Ahandbookforthescientificallyperplexed .Lanham: UniversityPressofAmerica;2009. 17.ShanksN,GreekR: Experimentaluseofnonhumanprimatesisnotasimpleproblem. NatMed2008, 14: 807 – 808. 18.ShanksN,GreekR,GreekJ: Areanimalmodelspredictiveforhumans? PhilosEthicsHumanitMed 2009, 4: 2. 19.ShanksN,GreekR,NobisN,GreekJ: AnimalsandMedicine:DoAnimalExperimentsPredictHumanResponse? Skeptic 2007, 13: 44 – 51. 20.GreekR: Letter.Dogs,GenesandDrugs. AmSci 2008, 96: 4.GreekandRice TheoreticalBiologyandMedicalModelling 2012, 9 :40Page25of33 http://www.tbiomed.com/content/9/1/40

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Abstract
Background
The concept of conserved processes presents unique opportunities for using nonhuman animal models in biomedical research. However, the concept must be examined in the context that humans and nonhuman animals are evolved, complex, adaptive systems. Given that nonhuman animals are examples of living systems that are differently complex from humans, what does the existence of a conserved gene or process imply for inter-species extrapolation?
Methods
We surveyed the literature including philosophy of science, biological complexity, conserved processes, evolutionary biology, comparative medicine, anti-neoplastic agents, inhalational anesthetics, and drug development journals in order to determine the value of nonhuman animal models when studying conserved processes.
Results
Evolution through natural selection has employed components and processes both to produce the same outcomes among species but also to generate different functions and traits. Many genes and processes are conserved, but new combinations of these processes or different regulation of the genes involved in these processes have resulted in unique organisms. Further, there is a hierarchy of organization in complex living systems. At some levels, the components are simple systems that can be analyzed by mathematics or the physical sciences, while at other levels the system cannot be fully analyzed by reducing it to a physical system. The study of complex living systems must alternate between focusing on the parts and examining the intact whole organism while taking into account the connections between the two. Systems biology aims for this holism. We examined the actions of inhalational anesthetic agents and anti-neoplastic agents in order to address what the characteristics of complex living systems imply for inter-species extrapolation of traits and responses related to conserved processes.
Conclusion
We conclude that even the presence of conserved processes is insufficient for inter-species extrapolation when the trait or response being studied is located at higher levels of organization, is in a different module, or is influenced by other modules. However, when the examination of the conserved process occurs at the same level of organization or in the same module, and hence is subject to study solely by reductionism, then extrapolation is possible.
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ins Americans For Medical Advancement (www.AFMA-curedisease.org), 2251 Refugio Rd, Goleta, CA, 93117, USA
Department of Anesthesiology, University of Florida College of Medicine, PO Box 100254, Gainesville, FL, 32610-0254, USA
source Theoretical Biology and Medical Modelling
issn 1742-4682
pubdate 2012
volume 9
issue 1
fpage 40
url http://www.tbiomed.com/content/9/1/40
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cpyrt 2012collab Greek and Rice; licensee BioMed Central Ltd.note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
kwdg
kwd Anesthesia
Animal models
Cancer
Complexity
Conserved processes
Systems biology
abs
sec
st
Abstract
Background
The concept of conserved processes presents unique opportunities for using nonhuman animal models in biomedical research. However, the concept must be examined in the context that humans and nonhuman animals are evolved, complex, adaptive systems. Given that nonhuman animals are examples of living systems that are it differently complex from humans, what does the existence of a conserved gene or process imply for inter-species extrapolation?
Methods
We surveyed the literature including philosophy of science, biological complexity, conserved processes, evolutionary biology, comparative medicine, anti-neoplastic agents, inhalational anesthetics, and drug development journals in order to determine the value of nonhuman animal models when studying conserved processes.
Results
Evolution through natural selection has employed components and processes both to produce the same outcomes among species but also to generate different functions and traits. Many genes and processes are conserved, but new combinations of these processes or different regulation of the genes involved in these processes have resulted in unique organisms. Further, there is a hierarchy of organization in complex living systems. At some levels, the components are simple systems that can be analyzed by mathematics or the physical sciences, while at other levels the system cannot be fully analyzed by reducing it to a physical system. The study of complex living systems must alternate between focusing on the parts and examining the intact whole organism while taking into account the connections between the two. Systems biology aims for this holism. We examined the actions of inhalational anesthetic agents and anti-neoplastic agents in order to address what the characteristics of complex living systems imply for inter-species extrapolation of traits and responses related to conserved processes.
Conclusion
We conclude that even the presence of conserved processes is insufficient for inter-species extrapolation when the trait or response being studied is located at higher levels of organization, is in a different module, or is influenced by other modules. However, when the examination of the conserved process occurs at the same level of organization or in the same module, and hence is subject to study solely by reductionism, then extrapolation is possible.
bdy
Background
Marc Kirschner and John Gerhart introduced the concept of facilitated variation and conserved core processes in their book, The Plausibility of Life
abbrgrp
abbr bid B1 1
, in order to explain how novelty arises in evolution. Motivated by advances in evolutionary and developmental biology (evo devo), these investigators proposed that conserved processes are ubiquitous in eukaryotes but pointed out that by using conserved processes differently, for example by differently regulating the genes that code for the processes, expressing the genes differently, varying the sequences or combination of genes or transcription factors, novelty can arise. Mutations in the genes that regulate the conserved processes can accomplish this novelty. Moreover, by adjusting the regulatory genes, the organism can evolve with fewer mutations than would be the case if a trait had to arise de novo or from mutations in structural genes. This has implications for using nonhuman animals (hereafter referred to simply as animals) as models for humans in biomedical research. One should expect to discover information regarding conserved processes in humans by studying animal models. We sought to determine whether limits exist on this method and if so what those limits are.
Methods
We surveyed the relevant literature including philosophy of science, biological complexity, conserved processes, evolutionary biology, comparative medicine, anti-neoplastic agents, inhalational anesthetics, and drug development journals in order to determine the appropriate role for animal models when studying conserved processes. Philosophy of science is relevant to our discussion as it includes the premises and assumptions on which research is then based. A study or method can be methodologically sound but if the premises are incorrect, then the study loses much if not all of its value. The drug development literature was searched because the final application of much research is targeted intervention via drugs hence that literature can inform regarding the success of a practice or modality. The literature concerning biological complexity and conserved processes was surveyed as it directly relates to the issue being explored. All of this must be placed into the context of evolutionary biology in order to better explain the findings. We chose inhalational anesthetics and anti-neoplastic agents as examples because of the well-known conserved nature of these agents.
Results
Animal models
The use of models has a long history in science, which led philosopher of science Richard Braithwaite to warn that: “The price of employment of models is eternal vigilance”
B2 2
. In this section, we will explore what animal models are, how they can be used in scientific investigation, including biomedical research, and discuss classification schemes. In this article, we will address the use of predictive animal models in light of the concepts of complex systems, personalized medicine and pharmacogenomics, and evolutionary biology. We will then explore what this implies when using animal models to study conserved processes.Models are important for scientific pursuits and can take the form of abstract models, computational models, heuristic models, mathematical models, physical models such as scale models, iconic models, and idealized models. Models can also be divided on the basis of whether they are used to replicate a portion of the item being modeled or are used to test hypotheses or interpret aspects of a theory. Examples of historically important models include Watson and Crick’s physical model of DNA, Pauling’s model of chemical bonds, Bohr’s solar system model of the atom, and the billiard ball model of gases. More recent models include the computer model of the brain, mathematical models of disease spread, and Lorenz’s model of the atmosphere.Robert Hinde observed that models:indent 1 ● Should be different from the thing being modeled, because if it is not, the modeler might assume that all properties demonstrated by the model exist in the thing being modeled;● Are usually less complicated than the thing being modeled;● Are more readily available than the thing being modeled, and;● “pose questions, suggest relations, or can be manipulated in ways not possible with the original”
B3 3
.In light of the importance of models, some philosophers of science assert that the study of models per se has been neglected by the philosophy of science community. Frigg and Hartmann
B4 4
state: “What fills in the blank in ‘M represents T if and only if ____,’ where M is a model and T a target system?” Moreover, how one classifies models and what criteria must be fulfilled in order for M to be considered a specific type of model has arguably not been adequately addressed by the philosophy of science community. Yet another problem with the philosophy of models is the relationship between theory and model
4
. We maintain that this lack of scholarly attention to models has played a role in what we see as the confusion surrounding the use of animals as models.Animal models are physical models and can be further classified based on various features and uses. For example, they can be distinguished by the phylogenetic distance of the model species from humans. Animal models can also be classified based on fidelity—how well the model resembles humans—as well as based on validity—how well what you think you are measuring corresponds to what you really are measuring. Animal models can also be considered based on reliability—the precision and accuracy of the measurement
B5 5
. Hau explains that animal models can be categorized as spontaneous, induced, transgenic, negative and orphan. Hau states: “The majority of laboratory animal models are developed and used to study the cause, nature, and cure of human disorders” [
B6 6
p3]. This is important as Hau further states that animal models can be used to predict human responses: “A third important group of animal models is employed as predictive models. These models are used with the aim of discovering and quantifying the impact of a treatment, whether this is to cure a disease or to assess toxicity of a chemical compound. The appropriateness of any laboratory animal model will eventually be judged by its capacity to explain and predict the observed effects in the target species”
6
. Others agree that predicting human response is a common use for animal models
B7 7
B8 8
B9 9
B10 10
B11 11
B12 12
. For example, Heywood stated: “Animal studies fall into two main categories: predictive evaluations of new compounds and their incorporation into schemes designed to help lessen or clarify a recognised hazard”
B13 13
.Animals are utilized for numerous scientific purposes (see ]Table tblr tid T1 1) and one of the authors (Greek) has addressed these various uses in previous publications
B14 14
B15 15
B16 16
B17 17
B18 18
B19 19
B20 20
. One cannot have a meaningful discussion regarding the utility of animal models unless one specifies the category under discussion. For example, areas in which animal models have been successfully employed include the evaluation of a phenomenon that can be described by the physicochemical properties of the organism, the study of basic physiologic functions, and the study of other traits that can be described by the use of conversion factors based on the body surface area of the organism. In general, animal models can be successfully employed in categories 3–9 in Table 1. However, animal models have failed to be predictive modalities for human response to drugs and disease
13
14
15
16
18
B21 21
B22 22
B23 23
B24 24
B25 25
B26 26
B27 27
B28 28
B29 29
B30 30
B31 31
B32 32
B33 33
B34 34
B35 35
B36 36
B37 37
B38 38
B39 39
B40 40
B41 41
, depicted by categories 1 and 2 in Table 1. (The authors have addressed this failure in numerous publications and, because an exploration for this failure is not the purpose of the article, we refer the reader to those publications
14
15
16
17
18
19
20
23
even though we realize that some view this position as controversial
7
11
B42 42
B43 43
B44 44
.) This is not to say that a species can never be found in retrospect that mimics an outcome in humans. Such a species usually can be identified, however retrospective correlation is obviously not the same as prediction
B45 45
B46 46
B47 47
. Moreover, any process or modality claiming to be predictive can be evaluated by use of the binomial classification table and equations in Table T2 2 (as illustrated in Table T3 3
B48 48
). Such calculations are commonly used in science
B49 49
B50 50
B51 51
B52 52
B53 53
.
table
Table 1
caption
b Categories of animal use in science and research
16
tgroup align left cols 2
colspec colname c1 colnum colwidth 1*
c2
thead valign top
row rowsep
entry
tbody
1.
As predictive models for human disease
2.
As predictive models to evaluate human exposure safety in the context of pharmacology and toxicology (e.g., in drug testing)
3.
As sources of ‘spare parts’ (e.g., aortic valve replacements for humans)
4.
As bioreactors (e.g., as factories for the production of insulin, or monoclonal antibodies, or the fruits of genetic engineering)
5.
As sources of tissue in order to study basic physiological principles
6.
For dissection and study in education and medical training
7.
As heuristic devices to prompt new biological/biomedical hypotheses
8.
For the benefit of other nonhuman animals
9.
For the pursuit of scientific knowledge in and of itself
Table 2
Binary classification test
4
center
c3 3
c4
nameend namest
Gold standard
GS+
GS-
Test
T+
TP
FP
T-
FN
TN
tfoot
The binary classification test allows calculations for determining how well a test or practice compares with reality or the gold standard.
Sensitivity = TP/(TP + FN)
Specificity = TN/(FP + TN)
Positive Predictive Value = TP/(TP + FP)
Negative Predictive Value = TN/(FN + TN)
T- = Test negative
T + = Test positive
FP = False positive
TP = True positive
FN = False negative
TN = True negative
GS- = Gold standard negative
GS + = Gold standard positive
Table 3
Example of binary classification values
Gold standard (human)
GS+
GS-
Test
T+
22
26
T-
22
30
Binary classification values for cardiovascular toxicity test in monkeys from 25 compounds also tested in humans
48
. Note the values are approximately what would be expected from a coin toss.
Sensitivity = 22/(22 + 22) = 0.5
Specificity = 30/(26 + 30) = 0.54
Positive Predictive Value = 22/(22 + 26) = 0.46
Negative Predictive Value = 30/(22 + 30) = 0.58
When judging the predictive value of a modality, one is not using the term predict in the same sense as when describing how hypotheses generate predictions to be tested. The predictive value of a commonly used modality usually is known, or can be ascertained, for example the positive and negative predictive value of x-ray computed tomography (commonly referred to as a CT scan) for diagnosing pneumothorax (a rupture of, or interference in, the pleural membrane which allows air to enter the pleural space and thus interferes with breathing) approaches 1.0 (is accurate for diagnosing the condition in 100% of cases).Animal models as used in biomedical research, can also be categorized as causal analogical models (CAMs) or as heuristic or hypothetical analogical models (HAMs)
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. The use of animal models to predict human response to drugs and disease, in accordance with categories 1 and 2 in Table 1, would be an example of using animals as CAMs. Analogical models in general include the hydraulic model of economies and the computer model of the brain and can be further divided based on various criteria
4
. Causalism or causal determinism dates to Aristotle who stated: “what is called Wisdom is concerned with the primary causes and principles.” Causalism can be summarized succinctly, as “everything has a cause.” This notion of causation was the basis for animal models as can be appreciated by the writings of Claude Bernard
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, considered the father of animal modeling since the 19sup th century. Bernard’s thoughts on animal models are an extension of Aristotle via the determinism of Descartes and Newton
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. Causal determinism and the principle of uniformity led to the concept, still accepted by many animal modelers today, that the same cause would result in the same effect in qualitatively similar systems. This line of thinking was in keeping with the creationist thinking of 19th century French physiologists, including Bernard, who rejected Darwin’s Theory of Evolution
60
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. The notion of causal determinism and the principle of uniformity combined with the rejection of evolution led to the belief in the interchangeability of parts. Therefore, if one ascertained the function of the pancreas in a dog, he could directly extrapolate that knowledge to the function of the pancreas in humans, once scaling for size had been factored in
14
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. Unfortunately, this linear thinking persists as the baboon heart transplant to Baby Fae illustrates. The operation was performed by the creationist surgeon Leonard Bailey of Loma Linda University in 1984 [
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p162-3].We acknowledge that the concept of causation is problematic
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. Russell suggested it be abandoned in 1913
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and it is clearly more useful for linear systems than complex systems. While an exhaustive explanation and discussion of the controversies surrounding causation would occupy more space than is available for this article (see Bunge
61
for such an analysis) we should note that a more current explanation for causation is that of a “first order approximation.” Causation is usually discussed in the context of a chain of causes. Bunge summarizes current thinking: “neodeterminism asserts in this connection that causation is only one among several interrelated categories concurring in real processes”
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. This principle is appreciated even more fully in complex systems. Current thinking notwithstanding, the use of animal models assumes the Cartesian concept of causation in that a causal model assumes a deterministic causal relationship between variables. We will explore this thinking and show that even in the traditional context there are problems with using animal models to discover “causal” relationships. These problems are increased exponentially when placed in the context of complex systems.Based on the writings of LaFollette and Shanks [
58
p63], we suggest the following in order for a model to be considered a CAM. X (the model) and Y (the subject being modeled) share properties {a…e}. In X, these properties are associated with, and thought relevant to, state S1. S1 has not been observed directly in Y, but Y likely also has would exhibit S1 under the same conditions as X. This concept is illustrated in Table T4 4. LaFollette and Shanks
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state that, “there should be no causally-relevant disanalogies between the model and the thing being modeled.” Unfortunately, causally relevant disanalogies do exist among species and even within a species, which leads to different states or outcomes, as illustrated in Table 4. We again paraphrase LaFollette and Shanks [
58
p112] and suggest that two more conditions must be met for a model to qualify as a CAM: the shared properties {a,…,e} must have a causal relationship with state S1 and be the only causally relevant properties associated with S1. As Table 4 illustrates, the commonalities between the humans and chimpanzees are insufficient to qualify chimpanzees as CAMs for human response to HIV infection. (For more on animal models of HIV/AIDS see
14
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.) As we will show, animals and humans are evolved complex systems and as such exhibit the properties of robustness and redundancy; hence numerous “causes” can result in the same effect and the same perturbation can result in different outcomes. Because of this and other properties of complex systems, we should expect different species to exhibit different causal relationships.
Table 4
Causal analogical models
6
c5 5
c6
X, the model
Y, the system being modeled
Shared properties between X and Y
Perturbation to the model
Outcome in model
Outcome in system being modeled
Shared properties a e for humans and chimpanzee do not result in state S1 also being shared.
Animal system (for example, Pan troglodytes)
Human system
a. Genes. >90% of nucleotide sequences identical.
Exposure to HIV.
State S1. Mild illness of limited duration.
AIDS. State S1 is not shared despite the presence of shared, relevant properties.
b. Immune system. Many commonalities. Constructed on generally the same plan.
c. White blood cells present and function similarly.
d. Receptors on white blood cells also present and function similarly.
e. Shared intracellular components of white blood cells.
Correspondingly, Giere, Bickle, and Mauldin
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note that some question the use of causal models in the study of humans because humans are complex systems whereas casual models assume a deterministic system: an outcome in a simple system is fixed by the variables. The problems of determining causation are further explored by Bunge
61
in his neodeterminism explanation alluded to above and his analysis is highly relevant to this discussion. While we will attempt to contrast the traditional deterministic view of causality in light of complexity science, this article will not do justice the current thinking on causation and we refer the reader to Bunge
61
for a fuller explanation.Giere, Bickle, and Mauldin suggest a probabilistic relationship instead of a 100% causal relationship for the model: “C is a positive causal factor (probabilistic) for E in an individual, I, characterized by residual state, S, if in I the probability of E given C is greater than the probability of E given Not-C.” Likewise, LaFollette and Shanks raise the question as to whether animal models can be weak CAMs: “Begin with two systems Ssub 1 and S2. S1 has causal mechanisms {a,b,c,d,e}, S2 has mechanisms {a,b,c,x,y}. When we stimulate sub-system {a,b,c} of S1 with stimuli sf response rf regularly occurs. We can therefore infer that were we to stimulate sub-systems {a,b,c}of S2 with sf rf would probably occur” [
58
p141]. LaFollette and Shanks then explain that this outcome will be highly probable if and only if {a,b,c} are causally independent of {d,e} and {x,y}.Again we anticipate problems in using animal models as weak CAMs, even in the traditional deterministic-causation view, because, as we shall discuss, various properties of complex systems will likely give rise to difficulties in isolating subsystems, which would be required for an animal model to be a weak CAM. These problems have been referred to as causal/functional asymmetry and mandates caution in extrapolating data between species. Kirschner and Gerhart give an example of this:"The case of the octopus and the human camera eye has been looked into, and the lessons are clear. Underneath the gross anatomical similarities are many differences. The eye derives from different tissues by different developmental means. Although both structures use the same pigment (rhodopsin) for photoreception, and both send electrical signals to the brain, we now know that the intervening circuitry is completely different [
1
p240-01]."Independent evolution has also produced spindle neurons in species as diverse as humans and cetaceans. Spindle neurons connect parts of the brain involved in higher cognition and were thought to only occur in primates but have recently been discovered in cetaceans, such as humpback whales and fin whales, as well as elephants
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. Convergent evolution, the acquisition of the same trait in different lineages, is also important when considering the role of animal models.
Evolved complex systems
Reductionism is a method of study that seeks to break a system down into its component parts, study each part individually, and then reach a conclusion about the system as a whole or at least the role of the individual part. Descartes introduced the concept and it has proven effective for ascertaining many facts about the material universe. Conversely, the clockwork universe of Descartes has not held up to scrutiny on all levels. Quantum mechanics, relativity, chaos, and complexity have revealed the stochastic nature of the supposedly clockwork, deterministic universe. Regrettably, while physicists recognized the limitations of reductionism, biologists were uncritically embracing it. Francis Crick extended reductionism to all aspects of biology when he stated: “The ultimate aim of the modern movement in biology is to explain all biology in terms of physics and chemistry”
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. Biological reductionism arguably reached its zenith in the Human Genome Project (HGP)
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and, ironically, the consequences of the HGP—that humans have a relatively small number of genes—have, in large part, been responsible for a re-examination of the role of reductionism in biology. This has been especially true for human pathophysiology where animals are used as models for humans.Systems can be categorized as simple or complex. The world of Newton and Descartes was largely confined to simple systems hence reductionism functioned well for discovery. At some levels, the components of a complex system can be simple systems and thus are subject to study by reductionism while at other levels these simple systems combine to make complex systems thus necessitating study of the intact whole. Mazzocchi points out that when reductionism takes a component out of its natural environment it has consequences for extrapolating the results back to the organism as a whole: “But this extrapolation is at best debatable and at worst misleading or even hazardous. The failure of many promising drug candidates in clinical research shows that it is not always possible to transfer results from mice or even primates to humans”
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.While evolution is defined as a change in allele frequency over time, complexity science can be defined as “the study of the behaviour of large collections of simple, interacting units, endowed with the potential to evolve with time”
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. Living organisms are complex systems that have highly variable evolutionary histories and as such are best modeled using nonlinear differential equations. The difficulty with this approach is that the values for many of the factors are unknown; hence solving the equation is impossible
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.Animals and humans are examples of living complex adaptive systems and as such exhibit the following properties
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:1. Complex systems are composed of many components. Some of these components may be simple systems, but many are complex systems. These components exist on many scales and interact extensively with each other. A complex system is a “system of systems.”2. The components can be grouped as modules. For example, the following could be considered as modules: the cell; the various processes in a cell; gene networks; gene-gene interactions; gene-protein interactions; protein-protein interactions; organs; and all the factors that influence the natural history of a disease. However, failure in one module does not necessarily spread demise to the system as a whole as redundancy and robustness (see #s 5 and 6 below) also exist and the various modules also communicate with each other.3. The different components of a complex system are linked to and affect one another in a synergistic manner. There is positive and negative feedback in a complex system
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.4. A complex system demonstrates hierarchal levels of organization
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. These levels range from the subatomic to the molecular to the whole individual to collections of individuals
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. Emergence (see # 13 below) occurs at each level; therefore, even a complete understanding of the lower level is insufficient for explaining the upper level. The various levels interact such that there is both upward causation and downward causation. In order to understand a particular level, one must alternate between looking at the components and looking at the whole while taking into account the connections between each
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. Moreover, the various levels may respond differently to the same perturbation.The various levels of organization are important when considering which responses to specific perturbations can be extrapolated among species. Living complex systems have numerous properties that can be studied without consideration of the fact that the whole, intact organism is a complex system. Some systems or components follow only the laws of physics, or even simple geometry, while others are best described by their physicochemical properties or just by chemistry. Some properties of complex systems can be described simply by math formulas. Growth, for example, can be described as geometrical in some cases and exponential in others. The surface area of a body increases by the square of the linear dimensions while the volume increases by the cube. This is a consequence of geometry and is important in physiology, in part, because heat loss is proportional to surface area while heat production is proportional to volume. Haldane stated: “Comparative anatomy is largely the story of the struggle to increase surface in proportion to volumes”
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. For example, chewing increases the surface area of food, the rate the small bowel absorbs nutrients and other chemicals depends in part on the surface area of the small bowel, and air sacs in the lungs rely on surface area for gas exchange, as do capillaries.Allometry is the study of the relationship of body size to shape. Examples of allometric laws include Kleiber’s law: q
0 ~ M
¾ where q
0 is metabolic rate and is proportional to M, body mass, raised to the ¾ power. The rate t, of breathing and heart contractions are proportional to M, body mass, raised to ¼ power: t ~ M
¼. Further, many physiological functions affect or depend on surface area.Levels of organization can also be described based on whether they are primarily chemical reactions and hence subject to analysis by chemistry. Reactions or perturbations that involve the denaturation of proteins should affect all systems, be they simple or complex, similarly because at this level of organization other factors do not come into play. Exactly what effects sulfuric acid would have in a person over an extended period of time are irrelevant as it denatures protein more or less immediately. Perhaps species differences would manifest if small amounts of H2SO4 were infused over long periods of time, but the immediate effects are the same across species because of the chemical properties of the acid.Animals can be successfully used for numerous purposes in science (see 3–9 in Table 1). One of the purposes for which animals can be successfully used is to evaluate phenomena that can be described by the physicochemical properties of the organism. The same applies to basic physiologic functions. There are physiological parameters that can be applied across species lines by the use of conversion factors based on the weight or surface area of the organism. There are also properties of organisms that can be anticipated by the physical or chemical properties of the substance acting on the organism. All of these are instances of successfully treating a complex system as if it were a simple system. Problems arise however, as the level of organization under study increases. Allometric scaling based on body surface area (BSA), for example, does not include differences that manifest at higher levels of organization for example in the elimination or metabolism of drugs. Different levels of organization can be acted on by single factors or many factors but perturbations of simple systems, or systems that can be described as simple on the level or organization being affected, should produce similar results.5. Complex systems are robust, meaning they have the capacity to resist change. This can be illustrated by the fact that knocking out a gene in one strain of mouse may produce negligible effects while being lethal to another strain. Gene pleiotropy is an additional example
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.6. Complex systems exhibit redundancy. For example, living systems exhibit redundancy of some genes and proteins
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.7. Complex systems are dynamic. They communicate with, and are acted on by, their environment.8. Complex systems exhibit self-organization, which allows adaptation to the environment
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. The intact cell is a prime example of this property.9. Complex systems are dependent on initial conditions. The well-known example of the butterfly flapping its wings and causing a weather catastrophe on the other side of the earth—the butterfly effect—is an example of dependence on initial conditions. An example in living complex systems would be that very small differences in genetic makeup between two systems could result in dramatic differences in response to the same perturbation. For example, monozygotic twins raised in the same environment may have different predispositions to diseases such as multiple sclerosis and schizophrenia
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. Additionally, the above-mentioned observation that knocking out a gene results in different outcomes in two stains of mice illustrates the concept that small differences in initial conditions—genetic makeup—can mean the difference between life and death
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.10. The initial conditions of a complex living system are determined, in part, by evolution. Various species have different evolutionary histories and thus are differently organized complex systems. Initial conditions can be different, despite the exact same genes, secondary to modifier genes, differences in regulation or expression of genes, epigenetics, and mutations among others factors. For example, small epigenetic changes probably account for the dissimilarities between monozygotic twins in terms of disease susceptibility
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.11. Perturbations to complex systems result in effects that are nonlinear
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. Large disturbances may result in no change to the system while minor perturbations may cause havoc
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. Efforts to describe complex systems in terms of linear cause and effect relationships are prone to failure
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. Extrapolating among complex systems is even more problematic because of nonlinearity, along with the other factors described.12. The whole of a complex system is greater than the sum of the parts; hence, some processes and or perturbations are not amenable to study by reductionism.13. Complex systems have emergent properties that cannot be predicted even in light of full knowledge of the component parts.Animal models have historically been utilized for the prediction of human responses to drugs and disease and this use has also been the justification for animal use in research in general
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. But because various levels of organization and different modules can be acted on by the same perturbation, in order to evaluate whether an animal model can be used as a predictive modality, one needs to understand the levels affected by the perturbation, what rules are being followed at those levels, and whether the system is simple or complex at the respective levels. Empirical evidence, explained and placed in context by theory developed from complexity science and evolutionary biology, suggests animal models cannot predict human responses to drugs and disease
14
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, despite the presence of shared physicochemical properties and conserved processes.
Conserved processes
Theodosius Dobzhansky famously stated: “Nothing in biology makes sense except in the light of evolution.” We want to examine the consequences that various characteristics of evolved complex systems, such as modules and different levels of organization, have on processes conserved by evolution in terms of determining the response of whole organisms to perturbations. Conserved processes and genes are the subject of much interest today
1
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. Kirschner and Gerhardt state: “all organisms are a mixture of conserved and nonconserved processes (said otherwise, or changing and unchanging processes)” [
1
p34-35]. Conserved processes are not reactions to the laws of physics or the determination of properties of an organism as they relate to chemistry or geometry. Nevertheless, conservation reaches across phyla and even kingdoms. Kirschner and Gerhardt have pointed out that processes conserved include those involved in cell function and organization, development, and metabolism and that these processes are similar in animals, yeast, and bacteria. They note that novelty has been the result of using the conserved processes in different ways rather than inventing completely new processes [
1
p34-35]. This has critical implications for what can be learned from interspecies study.Housekeeping genes in general perform the same function; make the same proteins, in mice, frogs or humans. The role of FOX transcription factors is conserved among species
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as is the role of Sarco(endo)plasmic reticulum (SER) Ca2+ ATPases (SERCA) pumps
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. Modules have also been conserved. The fin module of the modern fish for example, arose roughly 400 million years ago and has been conserved ever since [
1
p65].Conserved processes include core genes like those in the homeobox that are involved in the same developmental processes. Because these processes and genes are conserved among species, we could reasonably expect the same outcome from the same perturbation, regardless of the species containing these processes. But is this the case? In 1978 Lewis
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published his seminal work on the anterior-posterior layout of Drosophila. This was followed in 1984 by the discovery of the homeobox by McGinnis et al.
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. The field of evo devo developed in large part from this work. In the last decade, enormous strides have been made as a result of research in evo devo and the various genome projects. The results of such research have revealed an enormous genetic similarity among mammals. At the level of the genes centrally involved in development, e.g., the homeobox genes, bilaterians are virtually identical. The homeobox class of genes
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are conserved across species lines, functioning in early cellular organization and anterior-posterior body plan layout
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. There are important differences however. For example, there are nine Hox genes in flies but thirty-nine in mammals. Pertinently, we understand how modifications (gene duplications, deletions, changes in the regulatory processes and so forth) to these conserved processes have resulted in the evolution of different body types and indeed different species
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.MicroRNA (miRNA) has been found in essentially all species from Caenorhabditis elegans to humans and plays a large role in gene regulation
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. Apparently, over 50% of miRNAs are conserved across species lines in vertebrates
145
. An important consideration for drug development, however, is the fact that even though miRNA is conserved, up to 50% of miRNAs differs among vertebrates. This is important when considering the use of animals as predictive human models. Furthermore, miRNA expression levels change when tissues deteriorate from a healthy state to a diseased state
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. Thus the exact role of miRNA may differ intra-individually depending on age and disease. Hence, we see both inter-species and intra-individual differences with respect to this conserved process.It is well known that humans and nonhuman primates respond differently to infections. For example, untreated humans usually progress to AIDS when infected with HIV, are susceptible to malaria (except those with sickle cell anemia), have different reactions to hepatitis B and C than nonhuman primates and, appear more susceptible to many cancers and Alzheimer’s disease
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. Barreiro et al.
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studied gene expression levels in monocytes from humans, chimpanzees, and rhesus macaques and found that all three species demonstrated “the universal Toll-like receptor response” when stimulated with lipopolysaccharide (LPS). However they also discovered that only 58% of genes identified in the Toll-like receptor response “showed a conserved regulatory response to stimulation with LPS,” and only 31% of those genes demonstrated the same conserved regulatory response when exposed to viruses or bacteria. Barreiro et al. also discovered that 335 genes in humans are unique among the species in responding to LPS, with 273 genes responding only in chimpanzees, and 393 only in rhesus macaques
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. Even in conserved processes, there are going to be significant differences that influence the outcomes from disease perturbations. Significant differences in the details of conserved processes (also illustrated by Figure figr fid F1 1
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) mean that there are differences in the initial conditions of the complex system and this has major implications for inter-species extrapolation.
fig Figure 1Variation in sialic acid (Sia)-recognizing Ig-superfamily lectins among primates“Expression of CD33rSiglecs on human and great ape lymphocytestext
Variation in sialic acid (Sia)-recognizing Ig-superfamily lectins among primates. “Expression of CD33rSiglecs on human and great ape lymphocytes. (A) Percentage of positive lymphocytes for each Siglec Ab (staining above negative controls) for 16 chimpanzees, 5 bonobos, and 3 gorillas are shown, as well as data for 8 humans (the latter were tested on one or more occasions). Examples of flow cytometry histograms of human (B) and chimpanzee (C) lymphocytes using Abs recognizing Siglec-3, Siglec-5, Siglec-7, and Siglec-9 (y axis: normalized cell numbers expressed as percent of maximum cell number detected). In later samples examined, low levels of Siglec-11 staining (<5% positive) were occasionally detected on lymphocytes in both great apes and humans (data not shown)” 156.
graphic file 1742-4682-9-40-1 The implications of the various properties of complex systems also become apparent when scientists study processes such as preimplantation embryonic development (PED). PED is thought to be highly conserved among species which led Xie et al.
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to study gene expression profiles in embryos from humans, mice, and cows. They found that: “40.2% orthologous gene triplets exhibited different expression patterns among these species.” Differences in expression profiles have implications for drug and disease response.The Cdc14 gene was discovered in the yeast Saccharomyces cerevisiae and is classified as a dual-specificity phosphatase. It has since been found in many organisms including humans. Human Cdc14B fulfills the role, in yeast, of the yeast gene Cdc14. Because the yeast gene plays a role in regulating late mitosis, it was assumed the gene would have the same role in mammals. In actuality, neither Cdc14A nor Cdc14B are necessary for cell-cycle progression in humans
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. Thus, we have a conserved gene but not a conserved function.Pyrin proteins have been found to be ubiquitous in mammals. Pyrin-only protein 2 (POP2) was found in humans and thought to be important in inflammatory diseases. Atianand et al.
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studied mice but did not find POP2. They then discovered that POP2 was not in rodents or many other mammals but was present in chimpanzees (Pan troglodytes) and rhesus macaques (Macaca mulatta). Moreover, the chimpanzee POP2 was identical to humans POP2 at both the DNA and protein levels but the macaque POP2 was not.Conserved processes act, are affected by, or interact at multiple levels of organization. As Cairns-Smith points out, proteins, catalysts, nucleic acids, membranes, and lipids are interlocked and all are dependent on the others for their production. Cairns-Smith summarizes by stating: “Subsystems are highly interlocked The inter-locking is tight and critical. At the centre everything depends on everything” [
81
p39]. The same perturbation may result in different effects or outcomes for different levels of organization in the same intact system. This further complicates our ability to predict outcomes between two intact living complex systems. Thus it appears that a perturbation of complex system S1 containing conserved process P1 resulting in outcome O1 will not necessarily result in O1 in the very similar complex system S2 that also has P1.We will now examine in more detail the response of organisms to inhalational anesthetics and anti-neoplastic agents in order to illustrate what can and cannot be extrapolated between species knowing that species are acted on and affected by the fundamental principles of geometry, chemistry, and physics as well as shared conserved processes.
Conserved processes in anesthesia
General anesthesia by means of inhalational anesthetics (IAs) provides us with an excellent opportunity to examine where the effects of conserved processes can and cannot be extrapolated between species. We expect to see various effects at different levels of organization and in different modules. We also anticipate effects on emergent properties. Because IAs act on the system as a whole, we expect to see effects that cannot be predicted from reductionism. This has implications for what can be expected in terms of predicting human response by studying a different species or perhaps even a different individual. Therefore, both the primary effect of the anesthetic agent as well as the side effects may vary.Broadly speaking, general anesthesia in humans and animals is defined by amnesia, controlled insensitivity and consciousness, and immobility. It has been observed that most, if not all, extant vertebrate species exhibit an anesthetic-like response to a wide variety of chemicals that seemingly have little in common. This has been termed the universal response. Multiple mechanisms for the universal response have been postulated and this is an area of intense current research
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. There seems to be general agreement that ligand gated ion channel (LGIC) protein receptors are involved as well as possible effects on the cellular membrane. Regardless of the exact details, the conservation of mechanisms can be seen in that inhalational anesthetics (IAs) have observable effects on motor or motility responses in vertebrates and invertebrates
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, tactile plants
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and ciliated protists
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. (We note that this is probably an example of an exaptation, specifically a spandrel, rather than an adaptation
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.) Interestingly, effects have even been observed in S. cerevisiae (Baker’s yeast)
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, suggesting that crucial aspects of the universal response go beyond metazoans to include Eukaryotes. Moreover, IAs have been shown to have effects on membrane composition in prokaryote species
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e.g., A. laidlawii
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, Bacillus halodurans
175
and E. coli
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and the single-celled eukaryote tetrahymena
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(a ciliated protozoan). The universal response appears to date far back in evolutionary time and strongly suggests that the mechanism has been conserved among species.However, there are differences in outcomes with respect to IAs. Humphrey et al.
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studied genes in Caenorhabditis elegans and Drosophila melanogaster in order to assess the function of genes thought involved in the response to IAs. They found that a gene in C elegans, unc-79, and a gene in Drosophila, narrow abdomen (na), were related to each other and play a conserved role in response to anesthetics. However, mutations in each gene produced unique changes in sensitivity to IAs. The sensitivity to halothane, an IA, was increased but the sensitivity to enflurane, a different IA, was unchanged or perhaps even lowered. This is perplexing because one would have expected the two inhalational agents to be affected in a similar fashion by the mutation. The gene unc-79 appears to be a post-transcriptional regulator of na, thus the genes operate in the same pathway. Interestingly, both genes are also associated with similar phenotypes regarding locomotion: “fainting” in C. elegans and “hesitant walking” in Drosophila.Stimulation of the conserved processes controlling the universal response results in clinically significant variability among humans, even though the minimum alveolar concentration (MAC) for IAs for most species is approximately the same. MAC is the most often used metric to assess IA potency. However, the concept of MAC implies variability. MAC50, simply called MAC in anesthesiology, is the minimum alveolar concentration necessary to suppress movement in response to painful stimuli in 50% of subjects
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. MAC is significantly variable among humans depending on a number of factors including age and sex. Why is this the case?Sonner et al. reported, “one hundred forty-six statistically significant differences among the 15 strains [of mice] were found for the three inhaled anesthetics (isoflurane, desflurane, and halothane)”
164
. They concluded that multiple genes must be involved in anesthetic potency. Wang et al. developed two strains of mice that manifested different sensitivities to isoflurane
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. MAC is an example of a phenomenon controlled by quantitative trait loci
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, which may explain in part why, while one can obtain a rough approximation of MAC by studying other species, there will still be clinically significant differences.IAs also function at different levels of organization and on modules in addition to the one involved in the universal response. The side effects of the same chemical that produce an effect on the conserved receptors or other processes vary greatly from species to species and in some cases, even from person to person. A good example is the case of isoflurane and coronary steal. In the 1980s, there was heated controversy regarding the administration of the inhalation anesthetic isoflurane to patients with heart disease. The controversy centered on research using canines that indicated that the drug caused myocardial ischemia during certain situations in patients with coronary disease. The phenomenon appeared to result from isoflurane causing dilation of the normal coronary arteries, and thus blood being shunted away from the occluded coronary arteries; the arteries and tissues that most needed it. This was called coronary steal. Further, this situation was worsened by a decrease in blood pressure; a condition that often occurs during general anesthesia with IAs. This supposed danger, based almost entirely on studies in canines, was seized on by many in the anesthesiology community as dogma
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.This was an interesting reaction from clinicians for two reasons. First, experiments with other species had failed to demonstrate coronary steal
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and second, anesthesiologists had not noticed ischemic changes associated with isoflurane despite much use of the agent. The situation was also troublesome because isoflurane was a needed addition to an anesthesiologist’s armamentarium when initially approved for clinical practice. Six years after its introduction, it was the most frequently used IA, in part because of the favorable properties of the drug
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. Further studies continued to demonstrate varying effects intra- and inter-species
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. Ultimately, studies began to appear that suggested isoflurane was in fact cardio-protective. The mechanism for this protection was called “preconditioning and involves the opening of adenosine triphosphate-dependent potassium channels”
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. Isoflurane went from being contraindicated in patients with coronary artery disease to being the drug of choice in such patients. Studies from animals, specifically dogs, figured heavily in forming both, mutually exclusive, conclusions.Just as with the homeobox, miRNAs, and the response to inflammation, there are differences among species in how the conserved process known as the universal response to anesthesia manifests. Clinically, these differences are significant and limit the amount of information that can be extrapolated between species even when the underlying process is conserved. Inhalation anesthetics are also a good example of why, when the level of organization or module being examined changes, extrapolation breaks down. The same chemical that induces general anesthesia in a dog will probably result in the same effect in humans but the dose may vary in a clinically significant fashion and the side effects will most likely vary, as the conserved process does not dictate the side effects. Differences in outcomes from perturbations like the ones we have seen above have been explained by evolution-based species-specific differences, for example background genes, mutations, expression levels, and modifier genes
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.
Anti-neoplastic drugs acting on mitosis
As discussed, a relationship exists between BSA and many physiological parameters
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. For example, Reagan-Shaw, Nihal, and Ahmad state: “BSA correlates well across several mammalian species with several parameters of biology, including oxygen utilization, caloric expenditure, basal metabolism, blood volume, circulating plasma proteins, and renal function”
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. Dosing algorithms for first-in-man (FIM) trials are based on the assumption that there is a one-to-one dose scale between humans and animals when BSA is taken into account
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. The first study suggesting a relationship between dose and body surface area was performed by Pinkel in 1958
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involving anti-neoplastic agents, drugs where the effects and side effects are largely the same—cell death. Subsequently, Freireich et al.,
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studied 18 anti-neoplastic drugs in six animal species and concluded that the maximum tolerated dose (MTD) for humans was 1/12 of the dose in mice that resulted in the death of 10% of the mice (LD10). They also noted that the MTD was 1/7 of the LD10 in rats. These were also the ratios for converting from a mg/kg dose to a dose based on BSA. Fifty anti-neoplastic drugs were then studied using this formula and all were reportedly introduced into human trials without incident
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. The standard for FIM doses then became the 1/10th the LD10 for mice. Actually Freireich recommended a starting dose of 1/3rd the LD10 not 1/10th but that changed over time. The 1/3rd recommendation was found to be too large for FIM and was changed to 1/10th
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. More studies appeared to confirm the 1/10th value
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.The above makes a prima facie case that animal models can predict a starting dose for humans in clinical trials for anti-neoplastics. Further substantiating this is the fact that anti-neoplastics are not always metabolized by the liver
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, thus possibly eliminating a complex system from consideration. Cell division by mitosis is arguably the most conserved process one can find in biology and the traditional drugs for treating cancer act by interfering with mitosis. (Newer drugs act on targeted pathways as opposed to the cell cycle.) Anti-neoplastics kill the cells that are dividing most rapidly—the cancer cells. However, hair cells, cells in the bone marrow, and cells in the gut also divide at a similar rate such that anti-neoplastics can affect them. Thus, in part, the effects and side effects of anti-neoplastics are the same—cell death. The problem with traditional anti-neoplastics is that they do not discriminate adequately.Anti-neoplastic drugs are unique in medicine in that: 1) they are nonspecific; 2) long term toxicities are anticipated and accepted because the patient frequently does not have any other viable options; 3) the effects and side effects of the drugs are the same—cell death; and 4) they act on a universally conserved process—mitosis. This is why body surface area appears to be so effective for calculating FIM dose. Whereas, when one is examining effects and side effects of drugs based on interactions at the level of organization where complexity is relevant, for example metabolism
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, there are simply too many other factors to allow for the expectation of one-to-one correlations. Species-specific differences create perturbations in the complex system thus the differences among species outweigh the similarities
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.However, in the final analysis even the FIM dose of the anti-neoplastic agents cannot be reliably ascertained based on BSA. Most anti-neoplastics are effective only at doses near the maximum tolerated dose and the drugs are given in an escalating fashion during clinical trials. “Patients treated at the lower end of the dose escalation strategy are unlikely to receive even a potentially therapeutic dose since most cytotoxic drugs are only active at or near the MTD”
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. Differences among species in dose response for anti-neoplastics are due in part to differences in pharmacokinetics
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, which cannot be accounted for based on BSA. Brennan et al. state that: “While proper determination of drug doses can be complicated within the same species, it can be an incredible challenge and burden between species”
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. Brennan et al. continue by pointing out that metabolism and clearance differ among species and that “…the liver, kidneys and hematopoietic system between species may have significant differences in their sensitivity to chemotherapeutic agents. None of these factors are taken into account with the use of the species-specific dose calculations”
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. They recommend area under the curve (AUC) for calculating FIM dose but then concede: “However, there are numerous examples in which the species-specific conversion dose varies significantly from the AUC guided dose and/or far exceeds the animal’s maximum tolerated dose.” They then list examples from pediatrics where the recommended and actual doses differ significantly
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.Horstmann et al.
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reviewed 460 Phase I National Cancer Institute trials involving 11,935 adults that occurred between 1991 and 2002. Approximately 25% of the trials were FIM trials. Horstmann et al. found that serious nonfatal effects occurred in 15% of the patients undergoing single chemotherapy, with 58 deaths that were probably treatment-related
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. Concern has also been expressed that animal models have derailed anti-neoplastics that would have been successful in humans
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.FIM dose based on animal models is ineffective for predicting dose for other drug classes as well-TGN1412 being a recent notable example
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. An unnamed clinician, speaking of toxicity trials for new drugs in general in humans, was quoted in Science, stating, “If you were to look in [a big company’s] files for testing small-molecule drugs you’d find hundreds of deaths”
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. Chapman reinforced this stating: “. but other incidents of harm [besides TGN1412], even death, to participants in Phase I trials, some then known and other unpublicized, had taken place”
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. It is also important to note that the 1/3rd or 1/10th safety factor is fabricated. Perlstein et al. state: “Due to uncertainty in translating animal model findings to humans, particularly for unprecedented mechanisms, a wide dose range (1000-fold) is expected to cover the entire exposure–response curve”
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. Extrapolating from species to species should not require fudge factors if the process is truly science-based. In Phase I trials, where FIM or first in human (FIH) occurs, scientists want to characterize the drug’s PK properties and safety margins
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. Wexler and Bertelsen summarize the situation when they state: “Although allometric scaling techniques continue to provide poor predictive estimates for human pharmacokinetic parameters, FIH starting doses are selected with substantial safety factors applied to human equivalent dose, often in excess of regulatory guidelines. Approaches that could enhance the predictive nature of a compound’s disposition and adaptive nature of FIH studies could provide a tremendous benefit for drug development”
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. FIM for all classes of drug could be easily accomplished using microdosing
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with the first dose of one nanogram
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and increasing subsequent doses to the desired endpoint.Finally, one must recall that 95%
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of anti-neoplastic agents fail in clinical trials. Oncology drugs fail more frequently in clinical trials than most other categories
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and a higher percentage of anti-neoplastic drugs fail in Phase III trials than drugs from any other category
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. Reasons for the attrition include the fact that most of the effects and side effects, even of the anti-neoplastic agents, when placed into the context of a complex system, are not predicted from animal studies. Interfering in mitosis is a universal phenomenon but the degree and success of that interference varies. The FIM dose estimation is apparently successful because the level of organization in question is very basic and conserved and because the dose is lowered even further by fudge factors. Picking a starting dose based on the most toxic substances in nature
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would be more scientific. The apparent success also breaks down because the types of cancers in humans differ from those in animals, the genetic background of humans varies from that in animals, and because the reality of a complex system—the interactions of all the other systems (for example how the drugs are eventually metabolized and eliminated and how those metabolites interact with other systems and so on)—eventually appear. These are the problems that cannot be solved by animal models and are why the attrition rate is 95%. Weinberg stated: “it’s been well known for more than a decade, maybe two decades, that many of these preclinical human cancer models have very little predictive power in terms of how actual human beings—actual human tumors inside patients—will respond preclinical models of human cancer, in large part, stink hundreds of millions of dollars are being wasted every year by drug companies using these [animal] models”
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. Others have also pointed out the inadequacy of animal models of cancer, including genetically modified animal models
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.
Conserved processes in light of systems biology
As the level of organization in a complex system increases, we expect to see an increase in the number of emergent properties as well as more overall interactions. A gene or process that has been conserved will interact with the intact whole organism yielding new processes and states. Perturbations of these conserved genes or processes will thus likely result in new states not seen in other organisms that share the conserved processes; perhaps not even in organisms of the same lineage (clade) or species.The lack of appreciation for the differences between levels of organization and other properties of complex systems is apparent in the following from Kardong [
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p2], writing in his textbook of comparative vertebrate anatomy: “For example, by testing a few vertebrate muscles, we may demonstrate that they produce a force of 15 N (newtons) per square centimeter of muscle fiber cross section. Rather than testing all vertebrate muscles, a time-consuming process, we usually assume that other muscles of similar cross section produce a similar force (other things being equal). The discovery of force production in some muscles is extrapolated to others. In medicine, the comparative effects of drugs on rabbits or mice are extrapolated to tentative use in humans.” At the level of organization where one studies the force generated by muscle fibers, no doubt inter-species extrapolation is useful, but that is an entirely different level from where drug actions occur.Indeed the successes from using animal models have been examples of perturbations occurring at subsystems that can be described as simple systems and or outcomes or characteristics that apply on the gross level of examination. For example, the Germ Theory of Disease applies to humans and animals. The immune system reacts to foreign entities in a manner that is grossly similar across species lines. The details of immunity are clinically very different, for example HIV infection leads to AIDS in humans but not chimpanzees
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. Nevertheless, grossly, inflammation, white blood cells, and antibodies are identifying characteristics of the immune system in the phylum Chordata. Likewise, while the heart functions to circulate the blood in mammals, the diseases various mammalian hearts are subject to differ considerably
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. The failures of animal models have occurred when attempting to extrapolate data from higher levels of organization, levels where complexity is an important component in the system or subsystem under consideration. For example, a drug that has passed animal tests and is in Phase I human clinical trials has only an 8% chance of making it to market
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. Over 1,000 drugs have been shown to improve outcomes in cerebral ischemia in animal models but none, save aspirin and thrombolysis, which were not animal-based discoveries, have been successful in humans
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. The animal model for polio, monkeys, revealed a pathophysiology that was very different from that of humans
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. Extracranial-intracranial bypass for inoperable carotid artery disease was successful in animals but results in net harm for humans
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.Most diseases are multifactorial hence it should come as no surprise that conserved processes play a small, although at times important role, in major diseases like heart disease, cancer and stroke. The field of systems biology was formed in part in an attempt to place the parts of molecular biology and genetics in the larger context of the human system; the system that actually responds to drugs and disease. An editorial in Nature asks: “What is the difference between a live cat and a dead one? One scientific answer is 'systems biology'. A dead cat is a collection of its component parts. A live cat is the emergent behaviour of the system incorporating those parts”
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. According to the Department of Systems Biology at Harvard Medical School: “Systems biology is the study of systems of biological components, which may be molecules, cells, organisms or entire species. Living systems are dynamic and complex, and their behavior may be hard to predict from the properties of individual parts”
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. Systems biology
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takes a top-down approach as opposed to reductionism, which evaluates organisms from the bottom-up. Systems biology is concerned more with networks than individual components, although both are studied. It also recognizes the importance of emergent phenomena. (See Figure F2 2
79
). Such top-down approaches are used by the fields commonly referred to as “Omics,” for example: interactomics, metabolomics, proteomics, transcriptomics, and even fractalomics
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.
Figure 2Reductionism versus systems biology
Reductionism versus systems biology.
1742-4682-9-40-2 Nobel laureate Sydney Brenner, in 1998, emphasized that the interactions of components was important in understanding an organism
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. Only by studying proteins and processes in the context of their systems can we expect to understand what happens to the intact organisms as a result of these processes and genes. Further, evolution uses old pathways and processes in different ways to create novelty
1
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. Everything is context dependent. Noble stresses that in order to predict how drugs will act, one must understand “how a protein behaves in context” at higher levels of organization
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.Heng
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, writing in JAMA states that, because of reductionism, biological scientists have sought individual components in a disease process so they could intervene. A linear cause and effect relationship was assumed to exist. Heng cites diabetes intervention in an attempt to control blood glucose and cancer therapies as examples. He points out that while this has worked well in many cases, very tight control of blood glucose was recently found to increase the risk of death
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. Along the same lines, chemotherapies for cancer have decreased the size of the tumors but at the expense of an increase in frequency of secondary tumors and a very adversely affected lifestyle. Furthermore, most chemotherapy does not prolong life or result in a longer, high quality life
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. Instead of focusing on small modules or components of a system, complexity theory mandates that biomedical science look at the system as a whole.Closely related to systems biology are the concepts of personalized medicine and pharmacogenomics
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. It has long been appreciated that humans respond differently to drugs and have different susceptibilities to disease. Based on studies of twins, there appears to be a genetic component to susceptibility to leprosy, poliomyelitis and hepatitis B, as well as response to opioids
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. Other infectious diseases that appear to have a genetic component to susceptibility include HIV, Hepatitis C, malaria, dengue, meningococcal disease, variant Creutzfeldt–Jakob disease and perhaps tuberculosis among others
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. Differences in drug and disease response are manifest among ethnic groups
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and sexes
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. Even monozygotic twins manifest differences in response to such perturbations
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. Rashmi R Shah, previous Senior Clinical Assessor, Medicines and Healthcare products Regulatory Agency, London stated in 2005: “During the clinical use of a drug at present, a prescribing physician has no means of predicting the response of an individual patient to a given drug. Invariably, some patients fail to respond beneficially as expected whereas others experience adverse drug reactions (ADRs)”
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.Similarly, Allen Roses, then-worldwide vice-president of genetics at GlaxoSmithKline (GSK), said fewer than half of the patients prescribed some of the most expensive drugs derived any benefit from them: “The vast majority of drugs more than 90% only work in 30 or 50% of the people.” Most drugs had an efficacy rate of 50% or lower
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. Because of differences in genes, like SNPs, all children may not currently be protected by the same vaccine
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. It is estimated that “between 5 and 20 per cent of people vaccinated against hepatitis B, and between 2 and 10 per cent of those vaccinated against measles, will not be protected if they ever encounter these viruses”
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. In the future such children may be able to receive a personalized shot. Currently, numerous drugs have been linked to genetic mutations and alleles. See Table T5 5
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and Table T6 6
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. The number of personalized medicine products has increased from 13 in 2006 to 72 as of 2012
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.
Table 5
Examples of drugs with genetic information in thier labels
Drug
Sponsor
Indication
Gene or genotype
Effect of genotype
Clinical directive on label
*All drug labels were accessed through Drugs @FDA at www.accessdata.fda.gov/scripts/cder/drugsatfda. HIV-1 denotes human immunodeficiency virus type 1, TPMT thiopurine methyltransferase, UGT1A1 UDP glucuronosyltransferanse 1 family polypeptide A1, and VKORC1 vitamins K epoxide reductase complex subunit 1
Abacavir (Ziagen)
GlaxoSmithKline
HIV-1
HLA-B*5701
Hypersensitivity
Black-box warning. "Prior to initiating therapy with abacavir, screening for the HLA-B*5701 allele is recommended." "Your doctor can determine with a blood test if you have this gene variation."
Azathioprine (Imuran)
Prometheus
Renal allograft transplantation, rheumatoid
TPT*2TPT*3Aand TPMT*3C
Severe myeloxicity
"TPT genotyping or phenotyping can help identify patients who are at an increased risk for developing Imuran toxicity." "Phenotyping and genotyping methods are commercially available."
Carbamazepine (Tegretol)
Novartis
Epilepsy, trigeminal neuralgia
HLA-B*1502
Stevens-Johnson syndrome or toxic epidermal necrolysis
Black-box warning: "Patients with ancestry in genetically at-risk populations should be screened for the presence of HLA-B*1502 prior to initiating treatment with Tegretol. Patients testing positive for the allele should not be treated with Tegretol." "For genetically at-risk patients, high-resolution HLA-B*1502 typing is recommended."
Cetuximab (Erbitux)
Imclone
Metastatic colorectal cancer
KRAS mutations
Efficacy
"Retrospective subset analyses of metastatic or advanced colerectal cancer trials have not shown a treatment benefit for Erbitux in patients whose tumors had KRAS mutations in codon 12 or 13. Use of Erbitux is not recommended for the treatment of colorectal cancer with mutations."
Clopidogrel (Plavix)
Bristol-Myer Squibb
Anticoagulation
CYP2C19*2*3
Efficacy
"Tests are available to identify a patient’s CYP2C19 genotype; these tests can be used as an aid in determining therapeutic strategy. Consider alternative treatment or treatment strategies in patienrs identified as CYP2C19 poor metabolizer."
Irinotecan (Camptosar)
Pfizer
Metastatic colorectal cancer
UGT1A1*28
Diarrhea neutropenia
"A reduction in the starting dose by at least one level of Camptosar should be consider for patients knows to be homozygous for the UGT1A1*28 allele. "A laboratory test is available to determine the UGT1A1 status of patients."
Pantumumab (Vectibix)
Amgen
Metastatic colorectal cancer
KRAS mutations
Efficacy
"Retrospective subset analyses of metastatic colorectal cancer trials have not shown a treatment benefit for Vectibix in patients whose tumors had KRAS mutations in codon 12 or 13. Use of Vectibix is not recommended for the treatment of colorectal cancer with these mutations."
Transtuzumab (Herceptin)
Genetech
HER2-positive breastcancer
HER2 expression
Efficacy
"Detection of HER2 protein overexpression is necessary for selection of patients appropriate for Herceptin therapy because these are the only patients studied and for whom benefit has shown." "Several FDA-approved commercial assays are available to aid in the selection of breast cancer and metastatic cancer patients for Herptin therapy."
Wafarin (Coumadin)
Bristol-Myer Squibb
Venous thrombosis
CYP2C9*2*3 and VKORC1 variants
Bleeding complications
Includes the following table: Range of Expected Therapeutic Warfarin Doses Based on CYP2CP and VKORC1 Genotypes.
Table 6
The most significant genetic predictors of drug response
Organ or system involved
Associated gene/allele
Drug/drug response phenotype
Blood
Red blood cells
G6PD
Primaquine and others
Neutrophils
TMPT*2
Azathioprine/6MP-induced neutropenia
UGT1A1*28
Irintotecan-induced neutropenia
Plates
CYP2C19*2
Stent thrombusis
Coagulation
CY2C9*2, *3, VKORC1
Warfarin dose-requirement
Brain and peripheral nervous system
CNS depression
CYP2D6*N
Codeine-related sedation and respiratory depression
Anaesthesia
Butyrylcholinesterase
Prolonged apnoea
Peripheral nerves
NAT-2
Isoniazid-induced peripheral neuropathy
Drug hypersesitivity
HLA-B*5701
Abacavir hypersensitivity
HLA-B*1502
Carbamazepine-induced Steve Johnson syndrome (in some Asian groups )
HLA-A*3101
Carbamazepine-induced hypersensitivity in Causians and Japanese
HLA-B*5801
Allopurinol-induced serious cutaneous reactions
Drug-induced liver injury
HLA-B*5701
Flucloxacillin
HLA-DR81*1501-DQ81*0602
Co-amoxiclav
HLA-DR81*1501-DQ81*0602
Lumiracoxib
HLA-BR81*07-DOA1*02
Ximelagatran
HLA-DQA1*0201
Lapatinib
Infection
HIV-1 infection
CCRS
Maraviroc efficacy
Hepatitis C infection
IL288
Interferon-alpha efficacy
Malignancy
Breast cancer
CYP2D^
Response to tamoxifen
Chronic myeloid leukaemia
BCR-ABL
Imatinib and other tyrosine kinase inhibitors
Colon cancer
KRAS
Cetuximab efficacy
GI stromal tumours
c-kit
Imatinib efficacy
Lung cancer
EGFR
Gefinib efficacy
EML4-ALK
Crizotinib efficacy
Malignant melanoma
BRAF V600E
Vemurafenib efficacy
When animals were being used as models in the 19th century, many of the scientists who were using them had not accepted evolution and believed that animal parts were interchangeable with their human counterparts
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. Given that we now understand that intra-human variation results in such markedly different responses to drugs and disease, attempting to predict human response from animal models, even for perturbations acting on conserved processes, seems unwarranted. Yet, despite the implications of personalized medicine
22
, some scientists continue to commit the fallacy described by Burggren and Bemis: “Yet the use of ‘cockroach as insect,’ ‘frog as amphibian,’ or ‘the turtle as reptile’ persists, in spite of clear evidence of the dangers of this approach. Not surprisingly, this type of comparative physiology has neither contributed much to evolutionary theories nor drawn upon them to formulate and test hypotheses in evolutionary physiology” [
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p206]. Comparative research will yield a nice comparison of the trait or process among species or phyla. However, one simply cannot assume that the outcome from a specific perturbation in, say the cockroach, will be seen in insects in general and this concept becomes even more important when relying on animal models for medical interventions in humans.
Conclusion
A perturbation of living complex system S1 containing conserved process P1 resulting in outcome O1 will not result in O1 in the very similar living complex system S2 that also has P1 often enough to qualify S1 as a predictive modality for S2 when the trait or response being studied is located at higher levels of organization, is in a different module, or is influenced by other modules. However, when the examination of the conserved process occurs at the same or lower level of organization or in the same module, and hence is subject to study solely by reductionism, then extrapolation is possible. We believe this is a valuable principle.Our current understanding of evo devo, evolution in general, complexity science, and genetics allows us to generalize regarding trans-species extrapolation, even when conserved processes are involved. Shanks and Greek:"Living complex systems belonging to different species, largely as a result of the operation of evolutionary mechanisms over long periods of time, manifest different responses to the same stimuli due to: (1) differences with respect to genes present; (2) differences with respect to mutations in the same gene (where one species has an ortholog of a gene found in another); (3) differences with respect to proteins and protein activity; (4) differences with respect to gene regulation; (5) differences in gene expression; (6) differences in protein-protein interactions; (7) differences in genetic networks; (8) differences with respect to organismal organization (humans and rats may be intact systems, but may be differently intact); (9) differences in environmental exposures; and last but not least; (10) differences with respect to evolutionary histories. These are some of the important reasons why members of one species often respond differently to drugs and toxins, and experience different diseases. Immense empirical evidence supports this position (
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p358)."The failures of animal models as a predictive modality for human response to disease and drugs, even when such perturbations are acting on conserved processes, can be explained in the context of evolved complex systems. One does not need to study every such perturbation in every species in order to conclude that the animal model will not be a predictive modality for humans when perturbations occur at higher levels of organization or involve different modules or affect the system as a whole. This is not to deny that animal models, as characterized by 3–9 in Table 1, have contributed and will continue to contribute to scientific advancements.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
The authors contributed equally to this paper.
Authors’ information
Ray Greek, MD has been on faculty in the Department of Anesthesiology at the University of Wisconsin-Madison and at Thomas Jefferson University in Philadelphia. He is currently president of the not-for-profit Americans For Medical Advancement (http://www.AFMA-curedisease.org).Mark Rice, MD is currently on faculty at the University of Florida (UF). He is chief of the liver transplant division at UF Department of Anesthesiology, has seven US patents, and reviews for several major journals.
bm
ack
Acknowledgements
None.
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