THERAPEUTICSTRATEGIES

DRUG DISCOVERY

TODAY

The use of small animal imaging inrespiratory disease drug discoveryK. Ask1,2, A. Moeller2, J. Gauldie2, T.H. Farncombe3, R. Labiris1, M.R.J. Kolb1,2,*1Department of Pathology and Molecular Medicine, Center for Gene Therapeutics, McMaster University, Hamilton, Ontario, Canada2Department of Medicine, Firestone Institute for Respiratory Health, McMaster University, Hamilton, Ontario, Canada3Department of Nuclear Medicine, Hamilton Health Sciences, McMaster University, Hamilton, Ontario, Canada

Drug Discovery Today: Therapeutic Strategies Vol. 5, No. 2 2008

Editors-in-Chief

Raymond Baker – formerly University of Southampton, UK and Merck Sharp & Dohme, UK

Eliot Ohlstein – GlaxoSmithKline, USA

Respiratory diseases

The expansion of scientific knowledge and technology

over the past decades did not result in an increased rate

of drug discoveries for respiratory diseases. Despite the

identification of numerous targets and lead com-

pounds, few new therapeutic molecules have reached

patient care. It is generally acknowledged that the large

cost of drug development is mainly because of the high

failure rate during clinical testing. Animal models of

respiratory disorders used for experimental proof-of-

principle and efficacy studies are often criticized

because of inherent limitations, one of them being

the tool-box used for the assessment of outcomes.

Noninvasive imaging is a novel approach which might

help to reverse this trend. Different imaging modalities

can be combined and molecular, functional and anato-

mical events can be assessed quantitatively. Com-

pounds with potential beneficial effects can be

radiolabeled and proof-of-principle experiments can

be addressed to ensure that the drug meets the area

with pathology at sufficient concentrations. This

method will also help to further characterize different

animal models of respiratory disease calling research-

ers attention to the inherent limitations of each respec-

tive model, thus preventing subsequent futile use.

*Corresponding author: M.R.J. Kolb (kolbm@mcmaster.ca)

1740-6773/$ � 2008 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddstr.2008.08.001

Furthermore, preclinical trials can be performed long-

itudinally in animals in which the pathology has been

quantitatively assessed before drug administration.

The obvious benefit from a drug discovery perspective

is that this approach is a vigorous test for any new drug

or indication and probably will facilitate more ‘earlier

and better failures’. This review is focusing on multiple

components of assessing respiratory diseases with non-

invasive small animal imaging tools and how they can

be adapted for drug discovery.

Section Editor:Martin Braddock – AstraZeneca R&D, Charnwood,Loughborough, UK

Introduction

The overall cost involved in the release of a New Chemical

Entity (NCE) to the market exceeds US$ 1.7 billion [1]. World-

wide, 25 NCEs enter the market every year and, on average,

represent 12 years of development [1]. Most of this cost is the

result of a high failure ratio. Drug discovery today involves

multiple aspects including identification of target candidates,

synthesis and characterization of lead compounds and multi-

ple assays to evaluate therapeutic efficacy. There is an unmet

need to develop methods for a better selection of therapeutic

candidates for patients, and to identify probable drug failures

at an earlier stage of development. The use of animal models

and in vivo imaging technologies currently available for small

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Drug Discovery Today: Therapeutic Strategies | Respiratory diseases Vol. 5, No. 2 2008

Table 1. Comparison of different imaging modalities

Spatial resolution (mm) Time of acquisition Sensitivity Analysis Cost of scan Clinical applicability

CT <1c c a b b b

MRI <1c a a b b c

PET >1a b c c c c

SPECT >1a a c c b c

Optical >3a c c a a a

Ultrasound <1c c b a a b

a Low.b Moderate.c High.

Table 2. Imaging modalities for drug discovery

Identification of molecular targets involved in disease

Functional imaging

Emission tomography (PET/SPECT)

Optical imaging (BLI)

Biodistribution of therapeutic compounds/gene products

Emission tomography (PET/SPECT)

Optical imaging (BLI)

Proof of efficacy

Anatomical imaging (CT/MRI)

Functional imaging (ventilation perfusion)

animals (Table 1) has the potential to positively impact these

shortcomings.

The process of drug discovery

Historically, some of the most important drug discoveries can

be classified as ‘serendipitous discoveries’ [2]. To rationalize

and streamline drug development, different paradigms have

emerged and the most widely used method today is target-

based drug discovery [3]. This process implicates identifica-

tion of a ‘drugable’ target which is causally involved in the

disease mechanism. This approach was favored following the

decoding of the human genome, which increased the knowl-

edge of molecular pathways implicated in a given disease

process, and also because of the availability of advanced

technology allowing high-throughput screening [4]. Despite

its promise, target-based drug discovery has not yet had the

success initially expected [5]. An older paradigm, physiology-

based drug discovery [5], is based on the goal to ameliorate a

disease phenotype as a primary readout, with mechanisms

and identification of drug targets being examined at later

stages of the development. With this approach, larger scale

screening of compounds is typically initiated in cell systems,

in lower organisms (worm, insect) or in animal models of

disease. Recently, a combination of both physiology- and

target-based drug discovery has been proposed, named target

deconvolution strategy [6]. This approach aims to first iden-

tify lead molecules that show phenotypic changes in model

systems, before the molecular pathways of this process are

identified. As a result of the steady decline in drugs reaching

patients, the NIH has developed a roadmap which states that

better scientific and technical methods are urgently needed

to improve both predictability and efficiency of drug devel-

opment from proof-of-concept to the commercial product

[1]. It has been suggested that this can be achieved by

enhancing translational research, with a particular focus

on the development and characterization of animal disease

models and preclinical drug efficacy studies. The develop-

ment of small animal models of lung disease and different

imaging modalities (Table 2) has the potential to allow

82 www.drugdiscoverytoday.com

researchers to identify compound failures both ‘earlier’ and

‘better’, implying that ‘early failure’ is less costly and that

‘better failure’ gives investigators the chance to learn from

their mistakes and implement this knowledge in subsequent

research [7].

Animal models of respiratory disease

Numerous animal models of the most common human lung

disorders have been developed to investigate pathological

mechanisms and to evaluate treatment options [8]. It is

possible to mimic certain features of pneumonia [9], asthma

[10], acute lung injury [11,12], chronic obstructive lung dis-

ease [13], pulmonary fibrosis [14,15], pulmonary hyperten-

sion [16] and lung cancer [17]. However, none of these animal

models truly reflects the complexity of the human disease.

Despite an incredibly large number of successful intervention

studies in animal models, there are still substantial short-

comings for the therapy of many human lung diseases,

particularly for chronic diseases like COPD or fibrosis. Several

possible explanations may account for these obvious drug

discovery failures. Most of the models have inherent limita-

tions such as anatomy, structure and function of the lungs

which is different between animal species, and even more so

between mice and men [8]. Another common criticism to

animal models, especially related to drug efficacy studies in

chronic disease, is the timing of drug therapy. In the clinical

Vol. 5, No. 2 2008 Drug Discovery Today: Therapeutic Strategies | Respiratory diseases

situation patients usually present with advanced disease,

which is in contrast to the experimental models where most

published intervention studies are of preventative nature.

These interventions might target different biological

mechanisms and it is questionable that they are relevant

for chronic disease in humans. Another area of dispute is

that assessment methods used to show efficacy in animal

models do not necessarily correlate with clinical assessments

and may be inappropriate to evaluate the usefulness of novel

compounds for human disease [18].

Molecular imaging

The understanding of the molecular pathways implicated in

initiation and progression of a given disease is the key to

identify therapeutic targets. Imaging technologies such as

emission tomography (PET or SPECT; reviewed in [19]) or

bioluminescence imaging (BLI) are suitable for performing in

vivo molecular imaging in small animals [20–22], and can

help investigate aspects of gene function and pharmacoge-

nomics [22,23]. The measurement of gene regulation can be

addressed utilizing appropriate in vivo radiolabeled reporter

genes which are coding for regulatory elements such as

promoters or enhancers; gene expression can be analyzed

in vivo if radiolabeled ligands, antibodies or substrates are

employed [23]. Once a clear association of gene products with

disease activity has been established, the imaging of these

molecules can be used as marker of disease progression or

therapeutic efficacy of a drug. Further, radiolabeling of drugs

and other therapeutic options allows us to perform distribu-

tion studies in vivo. One of the major advantages of this

technology is in its quantitative potential and its direct

translation from the research laboratory to clinical evalua-

tions in humans [24]. This augmented translational potential

suggests that preclinical data could be reproduced in patients

in the early stage of clinical trials and that proof-of-principle

could be re-evaluated in patients before larger, costlier trials

are initiated.

There are many examples of distinct phenotypes and

applicability of molecular imaging that are directly related

to respiratory disease models. Neutrophilic inflammatory

changes can be followed by PET imaging by using radi-

olabeled fluorine-18-fluorodeoxyglucose ([18F]-FDG) [25].

Alternatively, fluorine-18-proline ([18F]-proline) has been

used by our group in a rat model of pulmonary fibrosis as a

marker of collagen turnover. Very encouraging preliminary

data show that the development of fibrotic changes can be

followed over time using this technology [26]. PET tracers

are usually short-lived (hours) and imaging must be per-

formed soon after the administration of the radiolabeled

compounds. By contrast, SPECT technology utilizes tracers

with somewhat longer half-lives, which can be used to

follow molecules or cells over an extended period, from

one day (99mTc has a half-life of six hours) up to a few

months (125I has a 60-day half-life). It would be beyond the

scope of this review to list all possible applications of

molecular imaging, but the interested reader may be

referred to several excellent reviews which develop the pros

and cons of this technology [22–24,27,28]. One limitation

of molecular imaging is that it requires sophisticated tech-

nology and an experienced radiochemistry department to

synthesize radiotracers. To help the scientific community

with these issues, the NIH provides scientific information

about different in vivo molecular and imaging contrast

agents, which is freely available at http://micad.nih.gov

[29]. It is helpful to place the information about cellular

function as provided by molecular imaging into a struc-

tural, ideally three-dimensional framework, which can be

done using anatomical imaging methods. Combined sys-

tems such as SPECT/computed tomography (CT), PET/

SPECT/CT or PET/magnetic resonance imaging (MRI)

machines are available for small animal research, which

allows us to assess both anatomical and functional para-

meters in living animals [30]. The immense potential of

these modalities (and any imaginable combination) is in

the possibility of following biological changes during the

course of experimental disease, including initiation, pro-

gression and hopefully in therapeutic intervention studies,

reversal of pulmonary pathology.

Once a suitable disease pathway and/or target have been

determined, lead molecules or biological compounds are

identified by screening. Then, proof-of-concept experiments

are set up to validate the viability of the concept before

extensive pharmacokinetics and toxicity studies are initiated.

Small animal imaging with radiolabeled compounds brings

another advantage to this step of drug development, because

it can be applied to confirm the location of the novel com-

pounds and can give valuable information about biodistribu-

tion and pharmacokinetics.

Anatomical and functional imaging

Experimental drug research in pulmonary diseases typically

involves invasive and terminal procedures. For preclinical

drug studies, the value of following the same animal over

time with noninvasive methods is obvious and represents a

tremendous advantage over current invasive methods

(Fig. 1). The possibility to induce a disease process, identify

the extent of structural and/or functional damage, followed

by administration of treatments will increase the translation

to the clinical situation.

High-resolution X-ray CT (micro-CT) is extensively used in

clinic to provide information about lung density and volumes.

Current techniques can now make use of respiratory gating for

small animals [31,32] during imaging, thereby reducing

motion-based artifacts and providing additional functional

information regarding lung volume. An example of a respira-

tory disease model particularly adapted to micro-CT analysis is

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Drug Discovery Today: Therapeutic Strategies | Respiratory diseases Vol. 5, No. 2 2008

Figure 1. Role of small animal imaging procedures in the evaluation

of drug efficacy. Imaging can be used as a noninvasive assessment tools

and pathology can be quantitated in an animal before treatment is

administered (e.g. at time 2, with a pathology score of 50%).

Treatment efficacy can then be assessed in the same animal over time

and progression of disease can be monitored.

experimental emphysema [33]. Models inducing higher den-

sity parenchymal changes such as pulmonary fibrosis [18,34]

can also be assessed by CT-analysis, albeit the analysis is more

challenging.

MRI can also be applied to assess lung structure and func-

tion in small animal models of respiratory disease (reviewed

in [35]). So far, this technology has been less utilized com-

pared to X-ray CT, because of the inherent difficulties of low

signal content from lung tissue. However, respiratory disor-

Figure 2. Example of focal distal left lung fibrosis in rat. (a) Axial slice of CT-sca

area color-coded in green 28 days after AdTGF-b1 administration in the dista

84 www.drugdiscoverytoday.com

ders with increased influx of water-rich material or cells into

the lung tissue or alveolar/bronchial lumen can be imaged by

MRI, because MRI can easily detect the signal of proton

density of water. As such, MRI can be used to assess inflam-

mation [36], edema, fibrosis [37] and tumor burden in small

animals. A novel and exciting approach to bypass the pro-

blems associated with signal detection in MRI is to use con-

trast agents. Particularly, the use of hyperpolarized gas such as129Xe [38] or 3He [39] has been very helpful to generate

ventilation and perfusion images in various experimental

models of respiratory diseases.

Unilateral respiratory models

We are currently working on a new approach to reduce the

duration and cost of lung disease models: the establishment

of specific small animal models which pursue experimental

interventions in one lung only, thus providing the contral-

ateral lung as internal control. It has been shown that uni-

lateral administration of porcine elastase in dogs using a

bronchoscope causes extensive emphysema only in the

one lobe into which elastase has been administered [40]. It

is obviously more challenging to apply this technique to

smaller animals such as rats or mice. Single lung intubation

can be done blindly, but this method may not be a reliable

and reproducible approach. In our laboratory we have devel-

oped a rigid bronchoscope to administer compounds directly

into individual lung lobes of small animals. As an example,

we have shown that an adenovector over-expressing active

TGF-b1 deposited in the distal part of the left lung of rats via

this micro-bronchoscope induces only fibrosis in this area

(Fig. 2). One of the major advantages of this model system for

drug development is that specific therapeutic compounds

could be administered directly into the pathological area

n and its corresponding 3D reconstructed lungs (b) with a dense fibrotic

l left lung (blue color indicates lung tissue with normal lung density).

Vol. 5, No. 2 2008 Drug Discovery Today: Therapeutic Strategies | Respiratory diseases

securing proof-of-concept studies, especially when the tech-

nology is combined with small animal imaging.

Summary

Small animal imaging technology including functional and

anatomical assessment of lung pathology, lung function and

drug distribution in rodents has the potential to increase the

identification of therapeutic compounds that are useful in

patients. Despite relatively high equipment costs, the increas-

ingly high financial challenge of successful drug discovery

could be efficiently addressed by proper use of these tech-

nologies. It is expected that over the next few years, validated

imaging methods of different respiratory pathologies as well

as drug distribution studies in small animals will increase the

number of safe, therapeutic compounds that finally reach

patients not only in early phase clinical trials but also in

clinical practice.

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