Drug Discovery/Development & Delivery
Insect Models for Drug Discovery
Currently, it takes from $800 m to $1
bn and 10-16 years to bring a new
drug to market. It is estimated that only
four in 5,000 - 10,000 compounds that
begin preclinical testing will progress to
human testing, and only one of those
will be approved for human use. About
40-60% of the compounds fail due to
absorption, distribution, metabolic,
elimination and toxicological (ADMET)
properties in clinical development where
the cost per drug candidate is the most
expensive1. Thus, it is essential for the
pharmaceutical companies to reduce
the costs and the time to market for
new drugs. To succeed in doing this
more and more complex in vitro models
are developed and applied as filters to
select the most suitable compounds for
the in vivo models. However, the gap
between in vitro and in vivo models is still
large and this may introduce problems
when linking in vitro findings to in vivo
readouts. Hence, there is demand for
intermediate models that give a better
prediction of in vivo ADMET parameters
than in vitro models, and at the same
time are faster and cheaper than
traditional vertebrate in vivo models.
There are strong indications that insect
models may fill the gap between in vitro
and in vivo.
Certain invertebrates have served
as useful models for understanding
many different biological processes.
In particular, the fruit fly, Drosophila
melanogaster (Dm) is a well-recognised
model research organism, which has
already made significant contributions
to the understanding of, e.g., genetics,
neurobiology, and molecular biology2.
Generally, insects and vertebrates have
many physiological features in common.
They are all multi-cell organisms, but
the anatomy of insects is relatively
simple and the physiology is wellstudied. Moreover, there are remarkable
molecular and functional similarities
with mammals, and this suggests that
insects may be relevant as models in
drug discovery research.
Transgenic models of Dm have been
developed to imitate neurodegenerative
human diseases. Some of these
models have demonstrated their
22 INTERNATIONAL PHARMACEUTICAL INDUSTRY
efficiency for testing relevant drugs,
and concordance of drug efficacy in
flies and mammals is seen for diseases
like Huntington’s, Parkinson’s and
Alzheimer’s3,4. In addition, Dm has been
used to provide model systems for
research on drug abuse5. In fact, such
studies led to identification of genes
required for cocaine sensitisation in
mammals. Hence, insects have proved
their potential as experimental models
in drug discovery. The homologies
between insects and vertebrates also
allow studies of ADMET properties of
chemicals. Thus, insects are potential
new model organisms for studying
blood brain barrier (BBB) permeability,
pharmacokinetics
(PK),
intestinal
absorption and transporter mediated
efflux/influx of chemicals.
Insect Physiology
In contrast to vertebrates, insects do not
have a blood vessel-based circulatory
system. Instead the body fluid (i.e. the
hemolymph) circulates (by aid of a dorsal
beating tubular heart) throughout the
insect body. In the hemolymph nutrients
are transported to the various organs while
waste products are transported to the
excretory organ, i.e. Malpighian tubule. An
optimal internal environment is obtained
by regulation of hemolymph osmotic
pressure. This regulation is executed by
the Malpighian tubule, long thin blindly
ended tubes arising from the posterior end
of the midgut, exhibiting a most efficient
capacity to exchange material with the
hemolymph and maintain a proper internal
environment (Figure 1).
The principal cells of the Malpighian
tubule are enriched with closely packed
microvillus, and the cells contain
numerous mitochondria indicating
metabolically very active cells. The
principles for water and molecular entry
to the Malpighian lumen are the same
as that for primary urine formation in
vertebrates. In vertebrates the liver is very
important for a first pass metabolism of
orally-administered drugs. Metabolisms
in insects occur in a number of organs,
mainly the intestinal mucosa, the fat
body (a specialised tissue consisting
of cells with enormously increased
outer membrane surface area and high
capacity for uptake and metabolism
similar to that of vertebrate hepatocytes)
and the Malpighian tubule. In Dm it has
been shown that there is a remarkable
enrichment of detoxification genes (e.g.
cytochrome P450 (CYP450) enzymes
and
glutathione-S-transferases)
in
the Malpighian tubule6. Furthermore,
microarray studies have shown that
nearly every subclass of the huge ABC
transporter gene family, as well as the
OAT, OCT, OATP, sugar and amino
acid transporter families, are highly
expressed in the Malpighian tubule7.
Pharmacokinetics
To make a successful drug, displaying
a therapeutic effect, the drug must
pass all pharmacokinetic hurdles in
order to reach the target organ and
exert its pharmacological action. Thus,
it is important to investigate to what
extent compounds permeate cellular
membranes, how they are distributed,
the rate and extent of metabolism, etc.
The metabolism is usually addressed
in vivo by measuring the compound
concentration in the blood over time.
It is known that insects are able to
metabolise xenobiotic compounds,
e.g. pesticides, in a similar manner to
mammals. This is the basis for adding
compounds like piperonyl butoxide to
Figure 1. The Malpighian organ in insects
consists of long blindly ending tubular
structures that arise at the junction between the
midgut and the hindgut, and functions as an
excretory organ maintaining the homeostasis
in the hemolymph. The functional similarities
between human and insect primary urine
formation and its neuroendocrine control
combined with the metabolic capacity of the
Mapighian organ makes insects excellent
models for basic and applied pharmacology
research in drug metabolism and clearance.
Volume 3 Issue 2
Drug Discovery/Development & Delivery
insecticide mixtures, as these potentiate
the effect of the toxic pesticides through
inhibition of CYP450s in the insects. In
a recent study we injected Quinidine
into six grasshoppers and hemolymph
samples were extracted from each
insect at five different time-points. From
this study it was seen that Quinidine is
metabolised by insects over time as
shown in Figure 2.
Intestinal Absorption
The vertebrate intestine is known for
its absorptive function because of
the presence of large numbers of
microvilli and the expression of several
physiological relevant uptake carriers
for nutrients. In addition there is a high
expression of both phase I and phase
II metabolising enzymes contributing
to the so-called first pass metabolism.
Furthermore, the high abundance of the
efflux transporters like P-glycoprotein
(Pgp), breast cancer resistance protein
(BCRP), and multidrug resistance protein
2 (MRP2) severely limit the absorption
of numerous drugs. Much attention has
been given to the dynamic interaction
of metabolism, secretion and the role
of transporters on drug absorption.
Because of the significant role of the
intestine in the first pass metabolism and
permeation of drugs a number of models
have been established from cellular in
vitro models (e.g. CaCo-2) via intestinal
tissue preparation (Ussing chamber)
to in vivo techniques. However, factors
affecting drug absorption include drug
permeability, metabolism, transporters
and interaction between these factors,
and this is difficult to model in vitro8.
On the other hand from traditional
vertebrate in vivo ADMET models it is
difficult to distinguish the effect related
to the actual intestinal absorption from
the metabolising effects in the liver.
In insects the alimentary midgut cells
are tall, columnar and morphologically
very similar to vertebrate enterocytes
and exhibit huge numbers of microvilli
towards the luminal side. Also similar
to the vertebrate intestine there are
proliferative zones with the same stem
cell principles of reproduction and
differentiation as in vertebrates. The
presence of the CYP450 system in the
insect midgut cells has been shown,
but there is sparse information on the
presence of transporters, though an
24 INTERNATIONAL PHARMACEUTICAL INDUSTRY
unexpected similarity between intestinal
sugar absorption in vertebrates and
insects has been reported9.
BBB Permeability
The vertebrate BBB represents the
physiologic barrier between the brain
tissue and blood vessels, which restricts
the exchange of solutes and regulates
absorption of exogenic agents (e.g.
drugs) from the blood into the brain.
Penetration of the BBB is one of the
major hurdles in the development of
successful CNS drugs. The insect
BBB has largely been ignored as a
model system for the mammalian BBB,
because it was for a long time assumed
that epithelial barrier junctions in insects
and vertebrates were analogous rather
than homologous structures. However,
identification of homologous proteins,
i.e. claudines, at the epithelial junctions
of both septate junctions in flies and
tight junctions in mammals10 has led to
a re-evaluation of this view11.
The insect brain is bathed in
hemolymph. This is in contrast to the
vertebrate brain which contains a huge
number of blood vessels. Consequently,
the insect brain surface and thereby
the exposure to the hemolymph is thus
expected to be much lower than the
mammalian brain surface. However, as
in vertebrates, insects have complex
compartmentalised nervous systems
for specialised functions like vision,
olfaction, learning, and memory (Figure
3)3.
Transmembrane permeability in
the vertebrate BBB is limited by the
presence of efflux transporters that
actively block the passage of drugs
or endogenous compounds from the
blood to the brain. The most important
efflux transporters expressed in the
membrane of endothelial cells are Pgp,
BCRP, and MRP2.
In a recent study using the
grasshopper (Locusta migratoria) it was
found that a number of CNS active test
compounds crossed the locus brain
barrier, whereas peripherally acting nonCNS compounds were excluded from
the brain. Furthermore, it was found in
an ex vivo locust model that treatment
with a human Pgp inhibitor facilitated
the uptake of a typical Pgp substrate12.
Transporters
During the last decades, numbers
of in vitro and animal studies have
shown
that
multidrug-transporting
proteins (MDTP) are important for drug
absorption and excretion. Moreover,
these proteins affect drug disposition
and pharmacological activity at the
cellular target site. Most MDTP research
has so far been focusing on the efflux
transporters e.g. Pgp and other
multidrug resistance proteins (MRPs).
During recent years the importance of
other cellular membrane transporters
has been investigated. Of main interest
have been the studies of the organic
anion and cation transporters (OATs
and OCTs) but also the organic anion
transporting polypeptides (OATPs).
The major interest in these studies has
Figure 2. The utility of insects in pharmacokinetic studies is illustrated by
the time course concentration of Quinidine in the hemolymph of the locust
after a single injection of the compound into the hemolymph.
Volume 3 Issue 2
Drug Discovery/Development & Delivery
been focusing on transporters in the
BBB, intestine, liver and kidney tissues
and cells since this is important in drug
absorption, metabolism and excretion
and consequently the disposition,
therapeutic efficacy and adverse
effects. Transporter proteins may also
cause safety issues related to drug-drug
interactions. Thus, the identification
of membrane transporters influencing
the disposition and safety of drugs is a
new challenge in drug discovery. Of the
numerous drug transporters identified so
far, their importance in drug absorption
(e.g. intestinal Pgp and BCRP), in drug
distribution (e.g. Pgp at the BBB and
OATPs in hepatocyte uptake) and in
drug excretion (OATs and OCTs for renal
elimination) have been considered.
It has been shown that insects have
OATPs and Pgp in the intestinal wall,
while the Pgp function has been
shown in both Dm and locusts12. This
indicates that insects may be useful as
in vivo systems in the early drug ADMET
characterisation.
Thus, taken together, there are
many reasons to believe that the insect
hemolymph-fat
body-Malpighianmetabolising-excretion system could
be a relevant model for highly efficient
documentation of some key ADMET
parameters in the early drug discovery
phase.
Future
Insect models could potentially be
used as a filter between in vitro and in
vivo models, filling the gap between the
‘quick’ in vitro models and the ‘slow,
expensive but more reliable’ in vivo
models. In vitro models are used for
high- or medium-throughput screening,
but it may be difficult to judge which
compounds have the highest probability
of successfully passing mouse studies.
In contrast to in vitro models, the insect
models are based on more complex
biological organisms that comprise
many parameters not present in in vitro
models. Moreover, many homologies on
molecular as well as functional levels
are reported, and the data generated
from using insects have so far been
very promising. This, in combination
with the fact that insect models have
been shown to be both cost- and timeefficient, imply that insect models may
be useful as intermediate screening
models in the drug discovery phase.
26 INTERNATIONAL PHARMACEUTICAL INDUSTRY
Figure 3. Main structure of the locust brain
consisting of three fused ganglionic masses,
the protocerebrum, the deutocerebrum and the
tritocerebrum. The protocerebrum is a major
integrative centre receiving information from
many sensory sources like the compound
eye and the ocelli. This part is also believed
to be involved in olfactory learning. The
deutocerebrum receives input from mechanoand chemosensory receptors on the antenna
and the tritocerebrum integrates the signals that
are sent to various muscles in the insect body.
Another interesting feature is the fact
that insect models only require tiny
amounts of compound material; existing
DMSO stock solutions are suitable, i.e.
re-synthesis is not needed.
It is obvious that insects are not
substituting in vitro or in vivo models,
but used in the right way insect models
could potentially enhance the drug
discovery phase and reduce the number
of traditional animal studies. So far, the
research performed on insects indicates
that insects could be a new model
species in ADMET profiling of chemical
compounds n
References
1 Charles River Laboratories, Annual Repot
(2009)
2 Gullan, P.J. & Cranston, P.S. The insects.
An outline of entomology. Blackwell
Science Ltd. (2000)
3 Marsh, J.L., & Thompson, L.M. Can flies
help humans treat neurodegenerative
diseases? Bioessays 26, 485–496 (2004)
4 Marsh, J.L. & Thompson, L.M. Drosophila
in the Study of Neurodegenerative
Disease. Neuron 52, 169-178 (2006)
5 Wolf, F.W., & Heberlein, U. Invertebrate
Models of Drug Abuse. J. Neurosci, 54,
161-78 (2003)
6 Dow, J.A.T. Insights into the Malpighian
tubule from functional genomics. J. Exp.
Biol. 212, 435-445 (2009)
7 Wang, J., Kean, L., Yang, J., Allan, A.K.,
Davies, S.A., Herzyk, P., & Dow, J.A.T.
Function-informed transcriptome analysis
of Drosophila renal tubule. Genome
Biology, 5, R69.1-R69.21 (2004)
8P
ang, K.S. Modeling of Intestinal Drug
Absorption: Roles of Transporters and
Metabolic enzymes (For the Gillette
Review Series). Drug Metab. Disp. 31,
1507-1519 (2003)
9C
accia, S., Casartelli, M., Grimaldi, A.,
Losa, E., de Eguileor, M., Pennacchio,
F. & Giordana, B. Unexpected similarity
of intestinal sugar absorption by SGLT1
and apical GLUT2 in an insect (Aphidius
ervi, Hymenoptera) and mammals. Am. J.
Physiol. Regul. Integr. Comp. Physiol. 292,
R2284-R2291 (2007)
10 W
u, V.M., Schulte, J., Hirschi, A., Tepass,
U. & Beitel, G.J. Sinuous is a Drosophila
claudin required for septate junction
organization and epithelial tube size
control. The J. Cell Biol. 164, (2), 313–
323 (2004)
11 D
aneman, R. & Barres, B.A. The BloodBrain Barrier—Lessons from Moody
Flies. Cell 123, 9-12 (2005)
12 N
ielsen, P.Aa., Andersson, O., Hansen,
S.H., Simonsen, K.B. & Andersson, G.
Models for predicting blood-brain barrier
permeation. Drug Discov. Today (in
press)
Peter
Aadal
Nielsen is CEO at
EntomoPharm.
He
received
his
PhD
in
Computational
Chemistry
from University of Copenhagen. He
has worked as senior scientist at
AstraZeneca R&D and 7TM Pharma.
During his work he has been focusing
on developing computational models
for prediction of ADMET properties.
Email: [email protected]
G u n n a r
Andersson
is
CSO
at
EntomoPharm.
He received his
PhD in Animal
Physiology from
University of Lund. He has worked as
Head of Department of Experimental
Animal Pharmacology at various
pharmaceutical companies. He has
been Adj. Professor at University
of Lund with a main focus on cell
proliferation and differentiation.
Email: [email protected]
Volume 3 Issue 2