PET in Drug Development

PET in Drug Development
SHREYA GUPTA

Flow of Presentation
• Introduction
• Molecular Imaging Techniques
• MICAD
• Roles of Molecular Imaging Techniques in Drug Development
• PET: Principle & Basics
• Applications & Techniques of PET
• PET in Preclinical drug development
• PET in Clinical development
• Recent Advances & Way Forward

Introduction
• Drug development process is a lengthy, high risk and costly endeavour
• Although the specifics and duration of each step depend on the target indication
as well as the drug class, in general, clinical development (from IND filing to
regulatory approval) takes about 10 years
• Although over the past two decades an increasing number of druggable targets
have been identified as a result of rapid advancements in molecular biology, the
number of approved drugs has not similarly increased

Introduction
• New strategies that aid early selection of promising candidates to move to
pivotal trials, or terminate candidates that are unlikely to be successful, could
significantly improve overall drug development process
• Molecular and functional imaging applied in the initial stages of drug
development can provide evidence of biological activity, confirm on-target drug
effects and identify patients who are more likely to benefit
• Considerable expectations & potential that investments in molecular imaging
technology will enhance drug development

Willmann JK, Van Bruggen N, Dinkelborg LM, Gambhir SS. Molecular imaging in drug development. Nature reviews Drug discovery. 2008 Jul;7(7):591-
607.

Characteristics of Molecular Imaging
• Molecular imaging attempts to characterize and quantify biological processes
at the cellular and subcellular level in intact living subjects
• Usually exploits specific molecular probes as well as intrinsic tissue
characteristics as the source of image contrast, and provides the potential for
understanding of integrative biology, earlier detection and characterization of
disease, and evaluation of treatment
Willmann JK, Van Bruggen N, Dinkelborg LM, Gambhir SS. Molecular imaging in drug development. Nature reviews Drug discovery. 2008 Jul;7(7):591-
607.

Advantages of Molecular Imaging
• Performed in the intact organism with sufficient spatial and temporal resolution
for studying biological processes in vivo
• Allows a repetitive, non-invasive, uniform and relatively automated study of
the same living subject using identical or alternative biological imaging assays at
different time points, thus harnessing the statistical power of longitudinal
studies, and reducing the number of animals required and cost

Basic Principles Of Imaging Modalities
Types of Imaging Techniques
Primarily
Morphological/Anatomical
Primarily Molecular Imaging

Primarily Anatomical
Imaging Technologies
• CT, MRI (with contrast agents injected at
millimolar blood concentrations) and
ultrasound
• Characterized by high spatial resolution
• Limitation: Unable to detect diseases until
tissue structural changes (for example,
growth of a tumour) are large enough to
be detected by the imaging modality
• Optical imaging, PET and SPECT (with
radiotracers injected at nanomolar blood
concentrations)
• Potential to detect molecular and cellular
changes of diseases (for example, before the
tumour is large enough to cause structural
changes)
• Limitation: poor spatial resolution
Primarily Molecular
Imaging Modalities

Molecular Imaging and Contrast Agent Database (MICAD)
• Large library of imaging probes that are directed against a large number of
known targets
• Database provides freely accessible, current, online scientific information
regarding molecular imaging (MI) probes and contrast agents (CA) used for PET,
SPECT, MRI, x-ray/CT, optical imaging and ultrasound imaging
• Available from MICAD: www.micad.nlm.nih.gov

Roles of Molecular
Imaging Techniques
in Drug
Development

Role in Early Phase Development
• Molecule biodistribution studies confirming molecule reaches the target tissue and does not
accumulate in non-target sites of potential toxicity
• Target PK (dose–target occupancy) measurements guiding dose selection
• Pharmacodynamic biomarkers for ‘proof of pharmacology’
• Translational preclinical imaging to identify or validate new imaging biomarkers and or
provide early differentiation between candidates based on target PK or PD responses
• In vivo measures for monitoring safety or toxicity

Role in Early
Phase
Development

Role In Late Phase Development
• Surrogate markers of response more sensitive than clinical measures
• Stratification of patients based on potential for treatment efficacy
• Pharmacological differentiation of asset from marketed drugs or new competitor
compounds

Role In Late Phase Development

Role in Drug Marketing
• Differentiation between available treatments
• Earlier detection of disease or associated pathology
• Improved disease classification/diagnosis
• Diagnosis of pre-symptomatic or minimally symptomatic disease
• Improved identification of chronic disease exacerbation/recurrence
• Patient stratification based on disease sub-phenotype or early treatment response

Positron Emission Tomography (PET)

Principles of PET
• An emitted positron collides with a local
electron, resulting in a mutual annihilation
and the production of a pair of photons that
travel at 180° to each other
• This coincidence event is detected by a
detector ring, which converts γ-rays into
visible light and finally into electrons and
allows localization and quantification of the
radiolabelled compound in the living subject
• Based on the use of short-lived positron
emitting radioisotopes such as carbon-1 1,
oxygen-15 and fluorine-18

Principles of PET
• PET radioligands are administered intravenously
• Distributed according to its characteristics such as BBB permeability, affinity
for target molecule, metabolism, and excretion from body
• Consequently, the degree of targeted molecule of each PET radioligand can be
evaluated quantitatively
• For example, [18F]fluorodeoxyglucose ([18F]FDG), which can measure the
glucose metabolism, accumulates in tissues of high glucose consumption such as
tumor and inflammation, and produces the large amount of gamma radiations
there

Procedure of PET imaging
Isotope production:
positron-emitting isotopes
are produced from
precursor molecules in a
cyclotron
Cyclotron accelerates
charged particles,
following which the
particles collide with
target precursor
molecules and transform
the appropriate atom in
them into a positron-
emitting isotope.
Labelling/tracer
synthesis: positron
emitting isotopes are
incorporated into the
target molecules such as
glucose, water, ammonia,
and drug candidates
using chemical reactions
done in a “hot” lab that
can handle
radiochemistry

Procedure of PET imaging
Tracer transportation:
labelled compound is
transported to a PET
facility
Tracer administration:
labeled compound is
administered to a
subject, usually via
injection.
Data Acquisition &
Analysis: data acquired
using a PET scanner,
processed and
interpreted

PET in Drug Development
• Applications of PET
• Techniques of PET
• Preclinical drug development
• Clinical development

Applications of PET
• Small animal PET/MicroPET
• Receptor occupancy quantification
• Drug biodistribution
• Pharmacokinetic (PK) analyses
• Validation of target engagement
• Treatment monitoring
• Measurement of neurotransmitter concentrations

Ghosh KK, Padmanabhan P, Yang CT, Ng DC, Palanivel M, Mishra S, Halldin C, Gulyás B. Positron emission tomographic imaging in drug discovery. Drug
discovery today. 2021 Jul 28.

Drug Development
Pre-
clinical
Small
animal PET
Target
expression
Clinical
Micro-
dosing
Pharmaco
-Kinetics
Pharmaco-
Dynamics
Drug
delivery

Strategies for using PET imaging in Drug Development and Research
Salvadori PA. Radiopharmaceuticals, drug development and pharmaceutical regulations in Europe. Current Radiopharmaceuticals. 2008 Jan 1;1(1):7-11.

Strategies for
using PET imaging
in Drug Development
and Research
Ghosh KK, Padmanabhan P, Yang CT, Ng DC, Palanivel M, Mishra S,
Halldin C, Gulyás B. Positron emission tomographic imaging in drug
discovery. Drug discovery today. 2021 Jul 28.

1. Target Expression
• After selection of a specific target, establishing that target is present under
pathological conditions is needed
• For example, for a drug intended to inhibit angiogenesis in cancer, there is a
need to assess whether and to what extent a specific target is expressed in new
blood vessels supplying different tumours
• Molecular imaging allows detection of specific targets in vivo, including
assessment of the presence of the targets, as well as the quantification of their
spatial and temporal distribution

STUDY RATIONALE: Overall response rate of VEGFR-targeted drugs therapeutics is highly variable,
most likely due to the temporal/spatial heterogeneity of the VEGFR expression levels amongst
individual patients. Therefore, methods for the non-invasive detection and quantification of VEGFR-1/2
expression
PET Imaging of VEGFR with a Novel 64Cu-Labeled Peptide. Kuan Hu, Jingjie Shang, Lin Xie, Masayuki Hanyu, Yiding Zhang, Zhimin Yang, et al.ACS
Omega 2020 5 (15), 8508-8514

• Imaging probe directly interacts
with the target(s) under
consideration
• Tracer predominantly bound to
VEGFR-2, as the tracer’s uptake in
the tumor is associated with the
expression level of VEGFR-2.
PET Imaging of VEGFR with a Novel 64Cu-Labeled Peptide. Kuan Hu, Jingjie Shang, Lin Xie, Masayuki Hanyu, Yiding Zhang, Zhimin Yang, et al.ACS
Omega 2020 5 (15), 8508-8514

2. Small animal PET
• Refers to imaging of animals such as rats and mice using a small, high-resolution
PET scanner designed specifically for this purpose
• Smaller detector ring ; saves detector cost and also improves the geometric
detection efficiency of the system
• Also known as ‘MicroPET’
• Allows the entire dynamic biodistribution of a labelled compound to be
measured in the same subject in a single scan, enables a single animal to be
studied multiple times over the course of the evaluation
Yao R, Lecomte R, Crawford ES. Small-animal PET: what is it, and why do we need it?. Journal of nuclear medicine technology. 2012 Sep 1;40(3):157-65.

2. Small animal PET
Yao R, Lecomte R, Crawford ES. Small-animal PET: what is it, and why do we need it?. Journal of nuclear medicine technology. 2012 Sep 1;40(3):157-65.

Applications of MicroPET
• Highly useful in neurology, oncology, and cardiology
• Clinical uses of micro-PET include estimating enzyme reactions, interactions
between ligand and receptor, cell proliferation, and cellular metabolism.
• Advanced applications of micro-PET such as in Alzheimer’s disease
pathophysiology or therapeutics are still at an early stage. The technique is currently
being used to enhance in vivo Alzheimer’s disease diagnosis, monitoring
propagation of the disease, and advancing clinical trials of the disease.
• The technique is being used for accelerating radiopharmaceuticals development
https://www.news-medical.net/life-sciences/Micro-PET-Principles-Strengths-and-Weaknesses.aspx

3-D PET/CT in situ detection of gastric tumors in mouse stomachs. Signals from 18F-FDG uptake in
fasudil-treated (4 weeks) and control mice presented as graded color code with standard uptake
value (SUVbw) range of 0.5-10. (Tu = tumor, bl =bladder).
Hinsenkamp I, Schulz S, Roscher M, Suhr A, Meyer B, Munteanu B, Fuchser J, Schoenberg SO, Ebert MPA, Wängler B, Hopf C and Burgermeister
E (2016) Inhibition of Rho-Associated Kinase 1/2 Attenuates Tumor Growth in Murine Gastric Cancer, Neoplasia, 18;500-511.

In vivo PET imaging and BLI of apoptosis after
Dox treatment
a) Micro-PET images of mice bearing cTK266-22B-
Fluc tumors at 2 h postinjection of 18F-FHBG
after treatment with 10 mg/kg of Dox for 72 h (n
= 6 per group). The white arrows point to
tumors
b) Quantification of the PET signal over the tumor
region, represented as a mean percentage
injected dose per gram (%ID/g)
c) Representative BLI images of the same groups
of mice after Dox treatment.*P < 0.05
d) Quantification of tumor BLI signal,P < 0.05
Wang F, Wang Z, Hida N, Kiesewetter DO, Ma Y, Yang K, Rong P, Liang J, Tian J, Niu G, Chen X. A cyclic HSV1-TK reporter for real-time PET imaging of
apoptosis. Proceedings of the National Academy of Sciences. 2014 Apr 8;111(14):5165-70.

Limitations of MicroPET
• Volumetric differences between tissues in small animals and humans
• Half-lives of radioisotopes used in this technique are very short and hence
cyclotrons may need to be present with the experimental apparatus for constant
generation of these isotopes
• Use of radiation can be harmful to small animals

Limitations of MicroPET
• Radiation also alters the size of the tumor in cancer research studies and thus
additional control groups may be required
• Spatial resolution offered by micro-PET is not very good
• It often needs to be combined with other tools such as micro-MRI or CT to
achieve a well rounded study involving both anatomical and molecular imaging
• This increases the cost as well as the need for specialized facilities.

1. PET in Microdosing studies
• In a microdosing (Phase 0) study, approximately 1% of the estimated therapeutic
dose of a drug (not exceeding 100 μg; at this low dose, toxic side effects are
typically not expected) is administered to healthy human volunteers or patients.
• Subsequently, the pharmacokinetic profiles of the drug such as the rate (Cmax)
and extent of drug absorption as well as the half-life can be measured by serial
PET scans (after direct radiolabelling of the drug) to obtain important
properties of a drug
Wagner CC, Langer O. Approaches using molecular imaging technology -- use of PET in clinical microdose studies. Adv Drug Deliv Rev. 2011 Jun
19;63(7):539-46. doi: 10.1016/j.addr.2010.09.011. Epub 2010 Sep 29. PMID: 20887762; PMCID: PMC3691790.

Advantages of PET in microdosing studies
• Uses minute amounts of radiolabelled drug tracers and thereby meets the
criteria for clinical microdose studies
• Determine the distribution of a radiolabelled drug microdose to different
organs and tissue over time, including the tissue(s) targeted for therapeutic drug
treatment
• Can give first evidence of the drug’s interaction with its pharmacological target
• PET imaging with radiolabelled tracers of CNS agents can be used to determine
BBB penetration in vivo, which preclinical models often fails to predict

Limitations of PET in microdosing studies
• Parent drug cannot be distinguished from radiolabelled metabolites in tissue,
because both give the same signal
• Consequently, for drugs which are extensively metabolized in vivo, the interpretation
of drug tissue PK may be confounded by the presence of radiolabelled metabolites
• For providing a quantitative description of PET data, such as the rate constants for
transfer of radiolabelled drug between plasma and different tissue compartments,
the concentration-time profile of the unmetabolized radiolabelled drug in arterial
plasma is required

2. Receptor Occupancy by PET
Receptor occupancy has been calculated to be the percentage of the receptor
population that is engaged by an unlabelled drug. Provides information about:
• Reachability of the drug to its intended targets
• Evaluation of the relationship between the target occupancy level and the
plasma kinetics of the drug
• Optimal occupancy level required to attain therapeutic effects (occupancy
threshold), by aiding the identification of the dose
• Occupancy levels above which adverse effects occur

Radioligands for PET determination of D2 and Di dopamine
receptor occupancy were [11C]raclopride and [11C]SCH23390,
respectively. Patients with acute extrapyramidal syndromes had
a higher D2 occupancy than those without side effects. This
finding indicates that neuroleptic-induced extrapyramidal
syndromes are related to the degree of central D2 occupancy
induced in the basal ganglia.

2. Receptor Occupancy by PET
Arakawa R, Takano A, Halldin C. PET technology for drug development in psychiatry. Neuropsychopharmacology Reports. 2020 Jun;40(2):114-21.

3. PET in Drug Biodistribution Studies
• Biodistribution studies are essential during initial-phase drug discovery because
they confirm whether the drug can reach the target tissue, and whether it has a
propensity to accumulate in nontarget sites or have a negative effect on target
sites, which could suggest possible toxicity
• Drug molecule is radiolabelled using isotopic replacement to avoid any
alteration to its biochemical properties
• Radioligand is then injected into healthy subject and the distribution of the
radioligand is traced over time using a dynamic PET scan
• During the dynamic PET measurement, blood samples are collected at different
time intervals from arterial cannulation

GSK2647544, specific inhibitor of lipoprotein-associated phospholipase A2 (Lp-PLA2), in development
as a potential treatment for AD. Healthy male subjects (n = 4, age range 34–42) received an oral dose of
unlabelled GSK2647544 (100 mg) and after 2 h an intravenous (iv) injection of [18F]GSK2647544
followed by dynamic PET scans for 120 min

4. Validation of Target Engagement by PET imaging
• When a PET radiotracer and a drug molecule share the same target, the PET
technique can be applied to check any interaction of candidate drug with its
target
• [11C]AZ10419369 was recently used as a suitable radioligand for the
quantification of in vivo 5-HT1B receptor binding. Currently, this radiotracer is
being used as a candidate to quantify the receptor occupancy effected by
compounds that target the 5-HT1B receptor

In vivo examination of the occupancy of AZD3783, a novel 5-HT1B receptor antagonist, at central 5-
HT1B receptors in a PET study with [11C]AZ10419369 in a human subject at baseline, and after oral
administration of 2 mg, 10 mg, and 40. AZD3783 decreased regional [11C]AZ10419369 binding in a
saturable and dose-dependent manner

5. Drug Treatment Monitoring by PET imaging
• One of the most important translations of PET imaging in drug discovery is to track
the progression of diseases, which alters the treatment course of therapeutic
candidates, such as the drug development process for Alzheimer’s disease (AD).
• To develop clinically relevant anti-amyloid therapeutic candidates that target the
amyloid cascade, PET imaging using beta amyloid binding radiotracers could be
applied using two different approaches:
1. Identify patients for studies who have pathological accumulation of beta amyloid
2. To track changes in its content with time, (i.e., to test the therapeutic capability of
the drug candidates)

PET using [11C]PIB to track the treatment effect of bapineuzumab, an anti-amyloid monoclonal
antibody. At 78 weeks of drug therapy, a profound decline in beta amyloid load was observed in the
brains of 26 patients with AD

6. Quantification Of Neurotransmitter Concentration by
PET
• Quantify the changes in endogenous neurotransmitter concentrations seen by
decrease in radiotracer binding to a neuroreceptor after an increase in the
neurotransmitter concentration
• [11C]AZ10419369 is one of the most sensitive radiotracers for 5-HT1B receptors,
used in psychiatric studies

Examined the effect of 3
serotonin concentration
enhancers on the binding of
[11C]AZ10419369 to 5-HT1B
receptors in the non-human
primate brain.
PET imaging data indicated
that [11C]AZ10419369 binding
was sensitive to alterations in
the levels of 5-HT triggered
by amphetamine, MDMA, or
5-HTP.
Yang KC, Takano A, Halldin C, Farde L, Finnema SJ. Serotonin concentration enhancers at clinically relevant doses reduce [11C]AZ10419369 binding to the 5-
HT1B receptors in the nonhuman primate brain. Transl Psychiatry. 2018 Jul 16;8(1):132. doi: 10.1038/s41398-018-0178-7.

Advantages & Limitations of PET in PK studies

PET in Oncology
• PET imaging is vital in the drug discovery
for oncological diseases
• PET with 2-[18F]fluoro-2-deoxyglucose
(FDG-PET) is often used for imaging
• Non-FDG PET imaging also serves a
beneficial role in cancer imaging and aids in
its drug development process, especially to
differentiate cancer phenotype from an
inflammatory pathology
Molecular imaging of tumour characteristics

Molecular and functional imaging strategies

PET in Cardiology
Areas of research:
• Perfusion imaging
• Metabolic imaging
• Receptor binding imaging
• Gene expression imaging

• Aim of this study was to develop and characterize a specific small-molecule tracer for PET
imaging that binds with high affinity to GPIIb/IIIa receptors.
• 18F-GP1, is a small molecule radiotracer that holds great promise in thrombus imaging
• Small arterial, venous thrombi, thrombotic depositions on damaged endothelial surface,
and small cerebral emboli were detected in vivo by PET imaging.

PET in Drug Delivery
• Potential to revolutionize patient care by in vivo assessment of drug
biodistribution and accumulation at the target site and real-time monitoring of
the therapeutic outcome
• Major goal to use molecular imaging to maximize effective therapy in diseased
tissues and to minimize systemic drug exposure in order to reduce toxicities
• Concept of “theranostic agent” ; Various drug carrier systems have been
radiolabelled with different positron emitter radionuclides for image-guided
drug delivery,

• PET image-guided tumor targeting using liposome based carrier.
• PET imaging of brain tumor using APRPG-modified liposomes, labeled with 1-[18F]fluoro-
3,6-dioxatetracosane.
Oku N.; Yamashita M.; Katayama Y.; Urakami T.; Hatanaka K.; Shimizu K.; Asai T.; Tsukada H.; Akai S.; Kanazawa H. PET imaging of brain
cancer with positron emitter-labeled liposomes. Int. J. Pharm. 2011, 403, 170–7

ImmunoPET and Personalized Medicine
• Combines Molecular imaging with radiolabelled antibodies
• Can provide quantitative information about antibody uptake at a whole-body
level
• Shows potential for the assessment of biomarker expression status and/or
prediction of clinical response, with a growing number of antibodies being
radiolabelled for immuno-PET
• ImmunoPET probes, such as 89Zr-Df-pertuzumab and 89Zr-atezolizumab, have
been successfully translated for clinical use

Scope of ImmunoPET
• Assessment of target expression
• Evaluation of the behaviour of the drug in relation to its intrinsic properties, and
optimization of drug design
• Optimization of dose, route and schedule of administration
• Prediction of efficacy and toxicity of drug treatment by performing target
occupancy studies
• Selection of patients with the highest chance of benefit from drug treatment

Concluding Remarks
• Use of PET is mostly centered in the fields of CNS and oncology-based
therapeutics development and, to a smaller extent, in the cardiac disorders
• The potential to be used in drug discovery and development, including
investigating drug biodistribution, plasma binding, absorption, distribution,
metabolism, metabolic clearance, and effective dosing
• An information-rich PET scan can help significantly condense and reduce the
length of clinical trials in their early phases
• Based on PET imaging studies, further advanced clinical trials can be cancelled
for unsuitable drug candidates

PET in Gene & Cell Therapy
• Positron emission tomography (PET) imaging reporter genes (IRGs) and PET
reporter probes (PRPs) are amongst the most valuable tools for gene and cell
therapy
• PET imaging probes reveal the presence of their target by accumulating on the
surface or inside the cells containing the target while clearing from other cells
• PET reporter probes (PRPs) image the expression of their PET IRGs in the same
manner, by accumulating on the surface or inside the cells expressing the PET
IRGs

Reporter-gene imaging, k/a PET imaging reporter genes (IRGS) and PET
reporter probes (PRPS)
• Amongst the most valuable tools for gene and cell therapy
• Used to non-invasively monitor all aspects of the kinetics of therapeutic
transgenes and cells in all types of living mammals.; Indirect more
generalizable approach
• Involves the simultaneous co-expression of the therapeutic target gene and a
reporter gene with both often driven by identical promoters
• The reporter gene encodes a protein that can interact with an imaging probe.
So, if the therapeutic target is present in or on a cell, this cell can be indirectly
visualized by trapping the imaging probe. By changing different components,
• Reporter gene can provide information on the regulation of DNA by upstream
promoters and the efficiency of vector transfection of cells.

PET in Drug Development

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