Radiopharm Metal Iso
PEOPLE
MARIE CURIE ACTIONS
International Incoming Fellowships (IIF)
Call: FP7-People-2011-IIF
Final report:
Radiopharm Metal Iso
(299009)
Radiopharm Metal Iso
Publishable summary
(i) New bifunctional tris(hydroxypyridinone) chelators for rapid labelling with gallium-68: conjugates with SSTR2and v3-targeting peptides, and an immunoconjugate of the HER2-targeting trastuzumab
Objectives: To synthesise and evaluate labelling and biodistribution of conjugates of new isothiocyanatefunctionalised tris(hydroxypyridinone) chelators with tumour targeting peptides.
Methods: Two tris(hydroxypyridinones) each with a pendant isothiocyanate group were synthesised and
conjugated to amine groups of Tyr3-octreotate for SSTR2 targeting (1), monovalent and trivalent cyclic-(RGDfK)
derivatives (e.g. 2) for v3 targeting (Figure) and the monoclonal antibody trastuzumab. Eluate from an iThemba
Labs 1.8 GBq 68Ge/68Ga generator was processed on a cation-exchange cartridge, eluted in 90% ethanol/0.9 N HCl
(J Nucl Med 2014, 55:1023), and added to each bioconjugate before neutralising with ammonium acetate.
Biodistribution was assessed in balb/c nude mice bearing U87MG (v3) or AR42J (SSTR2) tumours, by PET at 1
and 2 h post-injection, after which animals were killed and tissues/organs harvested and counted. Co-injection of
unconjugated peptide (400 g per animal) with each radiotracer, followed by scanning and biodistribution 1 h postinjection assessed specificity of the radiotracers.
Results: All peptide conjugates were labelled with 68Ga3+ in  95% radiochemical yield and high specific activity
(80 - 100 MBq nmol-1) at 25 °C and formulated to pH 6-7 in < 5 min. Biodistribution showed tumour-specific
uptake and retention of target receptor affinity. Excretion was rapid and predominantly renal (Figure). Co-injection
of unconjugated peptide substantially reduced tumour uptake in all cases. A HER2-targeting mAb, trastuzumab,
was radiolabeled in < 5 min by direct addition of 68Ga3+ generator eluate to a solution of immunoconjugate at pH 6
– 7, with specific activities of 50 MBq nmol-1. The radiolabeled immunoconjugate accumulated specifically in
HER2-expressing cells in vitro.
Significance: The new bifunctional tris(hydroxypyridinone) chelators enable easy preparation of bioconjugates,
and rapid radiolabelling with 68Ga3+ under mild conditions without subsequent purification. The simplicity and
efficiency of labelling at very low concentrations under mild conditions brings, for the first time, the possibility of
kit-based 68Ga tracer production without complex automated synthesis typical of multistep PET radiochemistry.
This would greatly increase 68Ga PET access to hospitals, expanding the use of the 68Ga generator.
(ii) A tripodal tris(hyroxypyrdinone) ligand for immunoconjugate PET imaging with zirconium-89
Objectives: Due to its long half-life (78.4h) and decay properties (+, 897keV), 89Zr is an appealing radionuclide
for immunoPET imaging with antibodies. Desferrioxamine-B (DFO) is the most widely used bifunctional chelator
to coordinate 89Zr4+ because the radiolabelling of the resulting immunoconjugates is rapid under mild conditions.
89
Zr-DFO complexes are stable in vitro but recent data demonstrated the release and subsequent bone uptake of the
Radiopharm Metal Iso
radiometal in vivo. We aim to evaluate a tripodal tris(hydroxypyridinone) ligand CP256 (Berry DJ, et al. (2011)
ChemComm, 47, 7068-70) developed in our laboratory, as a 89Zr4+ chelator and compare it with DFO.
Methods: To compare the ability of CP256 to complex Zr4+ with that of DFO, decreasing concentrations of each
chelator were labelled with [89Zr(oxalate)4]. Competition experiments were also undertaken. The radiolabelled
products and radiochemical yields were evaluated by ITLC and reverse phase HPLC-MS coupled to a scintillation
detector. To assess the stability of DFO/CP256-immunoconjugates radiolabeled with 89Zr, maleimide derivatives of
the chelators were conjugated to the monoclonal antibody trastuzumab via reduced cysteine side chains. Serum
stability studies and in vivo biodistribution and microPET/CT studies with normal C57Bl/6j mice were undertaken
on the 89Zr-radiolabelled immunoconjugates.
Results: CP256 coordinates Zr4+ (natural abundance) and comparison of HPLC-MS/scintillator chromatograms
confirms that addition of [89Zr(oxalate)4] to CP256 results in formation of [89Zr(CP256)]+. DFO and CP256 can be
radiolabelled with [89Zr(oxalate)4] at ambient temperature in quantitative yield at pH 6-7 at millimolar
concentrations. Competition experiments demonstrate that 89Zr4+ does not substantially dissociate (<10%) from
[89Zr(CP256)]+ in the presence of a ten-fold greater concentration of DFO relative to CP256. However, 89Zr4+
dissociates from [89Zr(DFO)]2+ in the presence of one equivalent of CP256 (relative to DFO), resulting largely in
[89Zr(CP256)]+. The immunoconjugates, CP256-trastuzumab and DFO-trastuzumab, can be labelled with 89Zr in
>98% yield at specific activities of 55MBq mg-1 and 91MBq mg-1 respectively. Both 89Zr-labelled
immunoconjugates are stable in serum with respect to dissociation of the radiometal. MicroPET/CT and
biodistribution studies indicate that after one day, 89Zr4+ dissociates from CP256-trastuzumab with significant
amounts of activity associated with bones and joints (25.88±0.58% ID g-1 after one week). In contrast, <8% ID g-1
of 89Zr activity is associated with bone for animals administered 89Zr-DFO-trastuzumab over the course of one
week.
Significance: The tris(hydroxypyridinone) ligand, CP256 coordinates 89Zr4+ rapidly under mild conditions, but the
89
Zr-labelled immunoconjugate, 89Zr-CP256-trastuzumab, was observed to release significant amounts of 89Zr4+ in
vivo, demonstrating inferior stability when compared with 89Zr-DFO-trastuzumab.
Project objectives
The following aims were originally proposed:
1. Synthesise bifunctional/trifunctional chelators for 99mTc and 188Re that can be attached to targeting
molecules (MSH-based peptide and bisphosphonate), providing multimeric derivatives for imaging
studies. These derivatives will be further developed to provide molecular 99mTc and 188Re agents with
improved target affinity/tumour uptake.
2. Test the hypothesis that “multivalency” (rather than prolonged bioavailability of a radiotracer) is the main
contributor to enhanced tumour uptake.
3. Synthesise a multivalent chelator platform that can be attached to a targeting molecule for a “pre-targeting
approach” using MSH/MT1R as a model targeting system so that targeting molecules with slow
pharmacokinetics such as anti-melanoma antibodies can be used with short half-life radioisotopes and
hence reduce radiation dose to patients.
4. Optimise a selected design for development of each of the above towards clinical application.
The following milestones were identified as critical to achieving project objectives:
1. Synthesis of novel bifunctional chelators for generator-produced isotopes
2. Conjugation of bifunctional chelators to targeting peptides, and radiolabeling
3. In vitro uptake and in vivo biodistribution of labelled conjugates
Radiopharm Metal Iso
As described below (Deviations from Annex) the following milestone/objective was also added to the project:
4. Evaluation of tris(hydroxypyridinone) ligands for radiolabeling and imaging antibodies with 89Zr4+
Work progress and achievements
Progress towards objectives
Progress results and research outcomes critical to work progress and achievements are listed under project
milestones, and referenced to project aims (in parentheses).
Milestone 1: Synthesis of novel bifunctional chelators for generator-produced isotopes
 A dithiocarbamate (DTC) ligand derived from carbon disulfide and iminodiacetic acid has been synthesized
for coordination of the generator-produced SPECT isotope, 99mTc. The resulting ligand (DTC-IDA)) contains two
acetate groups that in future could be activated and conjugated to primary amines of biological targeting molecues.
Reaction of this ligand at room temperature with a solution containing (either) the [ 99mTcN]2+ or [99TcN]2+ core
results in formation of [99mTcN(DTC-IDA)2]2-, as determined by LC-MS coupled to a radioscintillation detector.
Furthermore, the ligand reacts with solutions containing Zn2+, forming [Zn(DTC-IDA)2]2-, which undergoes
transmetallation with [99mTcN]2+ to form [99mTcN(DTC-IDA)2]2-, providing a second radiochemical synthetic route.
The DTC-IDA ligand also reacts at room temperature with [ReNCl2(PPh3)2] to form [ReN(DTC-IDA)2]2-. (Aim 1)
 Two new bifunctional tris(hydroxypyridinone) (THP) chelators (THP-PhNCS and THP-NCS) designed
specifically for rapid labeling with 68Ga have been synthesized, each with pendant isothiocyanate groups and three
1,6-dimethyl-3-hydroxypyridin-4-one groups. (Aim 3)
 A bifunctional chelator containing three tris(hydroxypyridinone) groups (an enneakis(hydroxypyridinone)) for
coordination of up to three 68Ga3+ ions has been synthesised THP3-PhNCS. This compound also contains an
isothiocyanate group that reacts with primary amines. (Aim 3)
Milestone 2: Conjugation of bifunctional chelators to targeting peptides, and radiolabeling
 Both THP-PhNCS and THP-NCS have been conjugated with the primary amine group of a cyclic integrin
targeting peptide, cyclic(RGDfK), providing THP-PhNCS-RGD and THP-NCS-RGD. Each conjugate can be
radiolabeled and formulated for intravenous injection by treatment with generator-produced 68Ga3+ in over 95 %
radiochemical yield under ambient conditions in less than 5 min, with specific activities of 60 - 80 MBq nmol-1.
Additionally, a scaffold has been prepared from which three peptidic cyclic integrin targeting groups (RGD
peptide) for binding the v3 integrin receptor have been attached. Conjugation of the new bifunctional chelators
THP-PhNCS and THP3-PhNCS with the scaffold containing multiple peptide groups has yielded a trimeric
compound that contains three peptidic groups and one chelator group (THP-PhNCS-RGD3), and a dendritic
compound that contains three peptidic groups and three chelator groups (THP3-PhNCS-RGD3). (Aim 3)
 THP-NCS has been conjugated to the somatostatin receptor 2 (SSTR2) targeting peptide, Tyr 3-octreotate
(TATE) via a PEG linker to provide THP-NCS-TATE, for comparison with the clinically used conjugate, DOTATATE. THP-NCS-TATE can be radiolabeled with 68Ga3+ in over 95 % radiochemical yield in specific activities of
up to 100 MBq nmol-1. (Aim 3, 4)
 We are yet to conjugate DTC-IDA to a targeting peptide, but this project will contribute to a continuing
programme of research undertaken by the researcher.
Milestone 3: In vitro uptake and in vivo biodistribution of 68Ga-labeled conjugates
 In vitro measurements and in vivo PET scanning and ex vivo biodistribution in mice bearing v3-positive
U87MG tumours demonstrate that the new 68Ga3+-labeled THP peptide conjugates retain affinity for the v3integrin receptor, clear within 1 – 2 h from circulation predominantly via a renal route and undergo receptormediated tumor uptake in vivo. In vivo tumour uptake and retention of [68Ga(THP-PhNCS-RGD] is higher than that
of [68Ga(THP-NCS-RGD]. (Aim 2, 4)
Radiopharm Metal Iso
 The 68Ga-labelled trimeric conjugate, [68Ga(THP-PhNCS-RGD3)] demonstrates higher tumour accumulation
and a higher tumour to blood ratio at 1 and 2 h post-injection in vivo than monomeric [68Ga(THP-PhNCS-RGD)].
Indeed, blood activity at 1 h PI for both conjugates is the same (a measure of similar bioavailability), but tumor
uptake of the trimeric conjugate (4.28±0.53 %ID g-1) is significantly higher than that of the monomeric compound
(2.86±0.43 %ID g-1). (Aim 2, 4)
 Differences in accumulated radioactivity in tumours for trimeric [ 68Ga(THP-PhNCS-RGD3)] and dendritic
[(68Ga)n(THP3-PhNCS-RGD3)] (n=1-3) are not statistically significant. Notably, high liver uptake of dendritic
[(68Ga)n(THP3-PhNCS-RGD3)] renders this specific tracer entirely unsuitable for PET imaging, however in vivo
experiments demonstrate that attachment of a bulky enneakis(hydroxypyridinone) does not adversely decrease the
affinity of a peptide targeting group for tumour receptors in vivo. (Aim 2, 4)
 In vitro experiments have shown that the 68Ga-labeled radiotracer, [68Ga(THP-NCS-TATE)] specifically binds
AR42J receptors and is internalised rapidly, consistent with reported agonist behaviour of octreotate peptide
derivatives. In vivo PET scanning and ex vivo biodistribution studies demonstrate that tumour uptake of [68Ga(THPNCS-TATE)] is comparable to the clinically used radiopharmaceutical, [68Ga(DOTATATE)] in mice bearing
SSTR2-positive AR42J tumours. However, in comparison to [68Ga(DOTATATE)], [68Ga(THP-NCS-TATE)] has
prolonged kidney retention. (Aim 4)
Milestone 4: Evaluation of tris(hydroxypyridinone) ligands for radiolabeling and imaging antibodies with 89Zr4+
 The tris(hydroxypyridinone) chelator, H3CP256 and its bifunctional maleimide derivative, H3YM103, was
evaluated for coordination of Zr4+. The NMR spectra, and the 89Zr4+ radiolabeling, antibody conjugation, serum
stability and in vivo distribution of radiolabeled immunoconjugates was compared with those of the commercial
standard used for 89Zr4+ coordination, H3DFO and its analogs. H3CP256 coordinates 89Zr4+ at carrier-free
concentrations forming [89Zr(CP256)]+. Competition experiments demonstrate that 89Zr4+ dissociates from
[89Zr(DFO)]+ in the presence of one equivalent of H3CP256 (relative to H3DFO) at pH 6 – 7, resulting largely in
[89Zr(CP256)]+.
 To assess the stability of H3DFO and H3YM103 immunoconjugates radiolabeled with 89Zr, maleimide
derivatives of the chelators were conjugated to the monoclonal antibody trastuzumab via reduced cysteine side
chains. Both immunoconjugates were labeled with 89Zr4+ in >98 % yield at high specific activities and the labeled
immunoconjugates were stable in serum with respect to dissociation of the radiometal.
 In vivo studies in mice indicate that 89Zr4+ dissociates from YM103-trastuzumab with significant amounts of
activity becoming associated with bones and joints (25.88 ± 0.58 % ID g-1 7 days post-injection). In contrast, < 8 %
ID g-1 of 89Zr activity becomes associated with bone in animals administered 89Zr-DFO-trastuzumab over the course
of 7 days. The significantly lower in vivo stability of the tris(hydroxypyridinone) conjugate is likely to be a result
of lower kinetic stability of the Zr4+ tris(hydroxypyridinone complex) relative to that of DFO and its derivatives.
Transfer of knowledge activities
Conference and workshop presentations
Transfer of knowledge activities such as participation in conferences and workshops are detailed in the section
entitled Dissemination Activities, including oral presentations at three conferences. Additionally, the researcher was
invited to speak at the workshop entitled Radioisotopes in Cancer Imaging and Therapy (February 2015, London,
UK), hosted by the Institute of Cancer Research. Recently, the researcher also represented the Chemistry
Cluster/Scientific Programme at the CRUK’s KCL/UCL (King’s College London/University College London)
Comprehensive Cancer Imaging Centre annual meeting (February 2015, London, UK), attended by all programme
leads and members of the KCL/UCL CCIC.
Student supervision
The researcher has supervised four masters-level projects that included:
 Synthesis of a simple tris(hydroxypyridinone) ligand, and conjugation of the ligand to an antibody and
radiolabelling for the resulting conjugate.
 Radiosynthesis of simple 64Cu lipophilic compounds based on bis(phosphines).
 Radiolabelling of peptide-based ligands engineered to coordinate metal ions.
Radiopharm Metal Iso
In doing so, the fellow trained students to design and synthesis chelators for coordination of radiometals,
characterise the structures of relevant metal complexes spectroscopically, evaluate the radiolabeling, and
characterise the resulting radiolabelled species.
The researcher has also assisted in supervising and training PhD students within the department in areas of design
and synthesis of chelators and peptides, radiolabeling of conjugates and evaluation of new radiotracers.
Participation in Departmental scientific meetings
The researcher has regularly presented laboratory work updates and presentations to her host department, the
Department of Imaging Chemistry and Biology within the Division of Imaging Sciences and Bioengineering. In
addition to this, the fellow has also presented at Chemistry and Biology meetings where members of the
Department of Chemistry, School of Pharmacy and Department of Imaging Chemistry and Biology meet to discuss
their research outcomes. Lastly, the fellow has also participated in organising, planning and presenting at monthly
meetings of members of the KCL/UCL CCIC chemistry cluster/programme.
Collaborations with industry and clinicians
The host institute has licensed new tris(hydroxypyridinone) technology to industry partners, and the researcher has
liaised with industry partners, CheMatech, providing advice on synthetic procedures to produce commercial
tris(hydroxypyridinone) chelator products. These are now available commercially:
http://www.chematech-mdt.com/index.php?page=products&lvl=3&ids=-1_75_26_86&hc=0
The researcher was also awarded a Royal Society of Chemistry (RSC) mobility fellowship, allowing her to
undertake collaborative research at the Peter MacCallum Centre in Australia, assessing the biology of conjugates
synthesised at the host institute in a highly translational research centre/hospital. During this time, the researcher
collaborated with radiochemists, biologists and clinicians.
Significant results
Significant scientific results are outlined in the Publishable summary, and elaborated upon under Progress towards
objectives. In brief:

A tris(hydroxypyridinone) chelator, CP256, coordinates 89Zr4+ rapidly, and immunoconjugates can be
radiolabeled in comparable radiochemical yields to that of desferrioxamine immunoconjugates. However, in vivo,
89 4+
Zr complexes of this tris(hydroxypyridinone) are not stable, resulting in demetallation and accumulation of
89 4+
Zr in the skeleton.

Two new bifunctional tris(hydroxypyridinone) chelators designed specifically for rapid labeling with 68Ga
have been synthesized, each with pendant isothiocyanate groups and three 1,6-dimethyl-3-hydroxypyridin-4-one
groups. Both compounds have been conjugated with the primary amine group of a cyclic integrin targeting peptide,
cyclic(RGDfK). Each conjugate can be radiolabeled by treatment with generator-produced 68Ga3+ in high
radiochemical yield (> 95 %) under ambient conditions in < 5 min, with specific activities of 60 - 80 MBq nmol-1.
Biodistribution of an v 3-targeted conjugate showed tumor-specific uptake, retention of target receptor affinity
and rapid clearance. The ability to synthesise 68Ga-based radiopharmaceuticals under mild concentrations at very
low concentrations of conjugate brings about the possibility of kit-based 68Ga radiopharmaceutical production
similar to that utilised for 99mTc-based radiopharmaceuticals that are prevalent in clinical nuclear imaging. This
would greatly increase 68Ga PET access to hospitals, expanding the use of the 68Ga generator.

The novel 68Ga-labelled SSTR2-targeting conjugate, [68Ga(THP-NCS-TATE)] can be similarly labelled in
high radiochemical yield (> 95%) under ambient conditions in < 5 min, with specific activities of up to 100 MBq
nmol-1. The radiotracer demonstrates receptor-mediated uptake in AR42J tumours, comparable to that of the
clinically-used neuroendocrine molecular imaging agent, [68Ga(DOTATE)], although kidney retention is prolonged
in the former radiotracer.
 A novel trimeric scaffold for conjugation of three peptidic groups has been synthesised, alongside an
enneakis(hydroxypyridinone) bifunctional chelator. A novel trimeric v3-targeting cyclic(RGD) conjugate,
[68Ga(THP-PhNCS-RGD3)] of a tris(hydroxypyridinone) demonstrates higher tumour uptake and higher tumour to
background ratios than that of the monomeric conjugate. A trimeric v3-targeting cyclic(RGD) conjugate of the
enneakis(hydroxypyridinone) bifunctional chelator has also been prepared, providing the dendritic 68Ga-labelled
radiotracer, [68Gan(THP3-PhNCS-RGD3)] (n = 1-3), that can be radiolabeled in very high specific activities (180 –
Radiopharm Metal Iso
240 MBq nmol-1). The latter dendritic radiotracer demonstrates similar tumour uptake to that of the trimeric
radiotracer, although liver uptake substantially reduces the ability of tumours to be visualized clearly by PET scans.
Deviations from Annex
The researcher has not used the MT1R-targeting MSH peptide originally specified in the project proposal.
Instead, the researcher has substituted this peptide for an v3-targeting cyclic(RGDfK) peptide. This is because
(i) expertise and resources were readily available for in vitro and in vivo v3-expressing models, and (ii) it was
more propitious to undertake proof-of-concept studies for the well-understood v3 integrin receptor.
Additionally, although not originally described in the grant proposal, the researcher has also worked on a related
project. This related project uses tris(hydroxypyridinone) ligands (described above) to coordinate the positronemitting isotope, 89Zr4+. Since award of the fellowship, this isotope has recently (2012/2013) become commercially
available in the European area, and is a significant new technology for PET-based molecular imaging. It was
determined that research on this isotope in the context of tris(hydroxypyridinone) ligands would enhance the
impact of the research.
Use of resources
There has been no deviation from planned researcher months. The full budget allocated for laboratory
consumables/expenses and travel has been spent. The monies have been spent on laboratory consumables,
chemicals, instrument access fees, costs associated with in vivo work, conference registration and associated travel.
Dissemination activities
As indicated in the publishable summary, the research undertaken over the course of the last two years can be
categorised into two separate projects, and thus dissemination activities are reported accordingly.
(i) New bifunctional tris(hydroxypyridinone) chelators for rapid labelling with gallium-68: conjugates with SSTR2and v3-targeting peptides
Two research papers on the tris(hydroxypyridinone) work have been drafted, and the manuscripts are undergoing
final review by all authors prior to submission of the manuscript. A third manuscript is currently under preparation.
Copies of these drafts can be provided upon request.
The tris(hydroxypyridinone) work has recently been presented as an oral presentation at the following conferences:
o
New bifunctional tris(hydroxypyridinone) chelators for rapid labelling with gallium-68: conjugates with
SSTR2- and v3-targeting peptides, Third Theranostics World Congress on Ga-68 and PRRT, March 2015, Baltimore,
USA
o
Rapid 68Ga-Radiolabelling of Proteins in Mild Conditions Exploiting a Novel tris(hydroxypyridinone)
Bifunctional Chelator, Third Theranostics World Congress on Ga-68 and PRRT, March 2015, Baltimore, USA
o
One-step, kit-based biomolecule labelling and molecular imaging with gallium-68 tris(hydroxypyridinone)
chelators, Radioisotopes in Cancer Imaging and Therapy, February 2015, Institute of Cancer Research, London, UK –
invited presentation
The tris(hydroxypyridinone) work lead to the basis of two successful early career researcher awards:
o
STEM Early Career Award - NIHR Biomedical Research Centre at Guy’s and St Thomas’ NHS Foundation Trust and
King’s College London (£ 6,972, January – March 2015)
o
Royal Society of Chemistry Research Mobility Fellowship – (£ 5000, 2014)
(ii) A tripodal tris(hyroxypyrdinone) ligand for immunoconjugate PET imaging with zirconium-89
The 89Zr tris(hydroxypyridinone) work has been presented as an oral presentation at a Dalton Discussions meeting
(York, UK; September, 2014) and has recently been published:
o Michelle T. Ma*, Levente K. Meszaros, Brett M. Paterson, David J. Berry, Maggie S. Cooper, Yongmin Ma, Robert
C. Hider, Philip J. Blower, Tripodal tris(hydroxypyridinone) ligands for immunoconjugate PET imaging with
comparison with desferrioxamine-B, Dalton Transactions, 2015, 44, 4884-4900.
89
Zr4+:
Radiopharm Metal Iso
The work has also been presented as an oral presentation at the European Association of Nuclear Medicine
Congress, where it was shortlisted for the EANM Marie Curie Award:
o
Tripodal tris(hydroxypyridinone) ligands for immunoconjugate PET imaging with 89Zr4+: comparison with
desferrioxamine-B, European Association of Nuclear Medicine Congress, October 2013, Lyon, France
The work was also presented as a poster at the International Conference on BioInorganic Chemistry 16 (July 2013,
Grenoble, France).
Project Management
Project planning and management has been minimal. Monthly meetings between the scientist in charge (Prof.
Blower) and the research fellow (Michelle Ma) have taken place in order to discuss results and plan experiments
and reporting. There have been no major changes to planned milestones. Minor deviations from the project
proposal are described above, under "work progress and achievements during the period". There have been no
gender or ethical issues, and no changes to the legal status of any of the beneficiaries.