ANSTO :: Browsing by Author "Greguric, I"

Browsing by Author "Greguric, I"

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[18F]Fluorination optimisation and the fully automated production of [18F]MEL050 using a microfluidic system
(CSIRO Publishing, 2014-06-06) Matesic, L; Kallinen, A; Wyatt, NA; Pham, TQ; Greguric, I; Pascali, G
The [18F]radiolabelling of the melanin-targeting positron-emission tomography radiotracer [18F]MEL050 was rapidly optimised using a commercial continuous-flow microfluidic system. The optimal [18F]fluorination incorporation conditions were then translated to production-scale experiments (35–150 GBq) suitable for preclinical imaging, complete with automated HPLC–solid phase extraction purification and formulation. [18F]MEL050 was obtained in 43 ± 10 % radiochemical yield in ~50 min. © 2015 CSIRO Publishing.
Alternative approaches for PET radiotracer development in Alzheimer's disease: imaging beyond plaque
(Wiley, 2013-12-11) Holland, JP; Liang, SH; Rotstein, BH; Collier, TL; Stephenson, NA; Greguric, I; Vasdev, N
Alzheimer's disease (AD) and related dementias show increasing clinical prevalence, yet our understanding of the etiology and pathobiology of disease-related neurodegeneration remains limited. In this regard, noninvasive imaging with radiotracers for positron emission tomography (PET) presents a unique tool for quantifying spatial and temporal changes in characteristic biological markers of brain disease and for assessing potential drug efficacy. PET radiotracers targeting different protein markers are being developed to address questions pertaining to the molecular and/or genetic heterogeneity of AD and related dementias. For example, radiotracers including [11C]-PiB and [18F]-AV-45 (Florbetapir) are being used to measure the density of Aβ-plaques in AD patients and to interrogate the biological mechanisms of disease initiation and progression. Our focus is on the development of novel PET imaging agents, targeting proteins beyond Aβ-plaques, which can be used to investigate the broader mechanism of AD pathogenesis. Here, we present the chemical basis of various radiotracers which show promise in preclinical or clinical studies for use in evaluating the phenotypic or biochemical characteristics of AD. Radiotracers for PET imaging neuroinflammation, metal ion association with Aβ-plaques, tau protein, cholinergic and cannabinoid receptors, and enzymes including glycogen-synthase kinase-3β and monoamine oxidase B amongst others, and their connection to AD are highlighted. Copyright © 2013 John Wiley & Sons, Ltd.
Alternative chromatographic processes for no-carrier added 177Lu radioisotope separation Part I. Multi-column chromatographic process for clinically applicable
(Springer, 2008-09-01) Le, VS; Morcos, N; Zaw, M; Pellegrini, PA; Greguric, I
The conventional multi-column solid phase extraction (SPE) chromatography technique using di-(2-ethylhexyl)orthophosphoric acid (HDEHP) impregnated OASIS-HLB sorbent based SPE resins (OASIS-HDEHP) was developed for the separation of no-carrier added (n.c.a) Lu-177 from the bulk quantity of ytterbium target. This technique exploited the large variation of lutetium metal ion distribution coefficients in the varying acidity of the HCl solution-OASIS-HDEHP resin systems for the consecutive loading-eluting cycles performed on different columns. The production batches of several hundred mCi n.c.a Lu-177 radioisotope separated from 50 mg Yb target activated in a nuclear reactor of medium neutron nux (Phi = 5 center dot 10(13) n.cm(-2).s(-1)) were successfully performed using the above mentioned separation technique. With the target irradiation in a reactor of thermal neutron flux Phi = 2 center dot 10(14) n.cm(-2).s(-1) or the parallel run of several separation units, many Ci-s of n.c.a Lu-177 can be profitably produced. The OASIS-HDEHP resin based multi-column SPE chromatography technique makes the separation process simple and economic and offers an automation capability for operation in highly radioactive hazardous environments. © 2008, Springer.
Alternative chromatographic processes for no-carrier added 177Lu radioisotope separation Part II. The conventional column chromatographic separation combined with HPLC for high purity
(Springer, 2008-09-01) Van So, L; Morcos, N; Zaw, M; Pellegrini, PA; Greguric, I; Nevissi, A
HPLC technique combined with the simple conventional column solid phase extraction (SPE) chromatography using di-(2- ethylhexyl)orthophosphoric acid (HDEHP) impregnated OASIS-HLB sorbent based SPE resins (OASIS-HDEHP ) was developed for the separation of no-carrier added (n.c.a) 177Lu from the bulk quantity of ytterbium target. This combination strategy was based on combining the advantages of the better resolution of HPLC separation of n.c.a 177Lu from the few milligram level Yb target with the high capacity of the OASISHDEHP column for the separation of 177Lu from the bulk Yb target. The production batches of several hundred mCi activity of n.c.a 177Lu radioisotope separated from 50 mg Yb target activated in a nuclear reactor of medium neutron flux (Φ = 5.1013 n.cm–2.s–1) were successfully performed using this combined separation technique. With the target irradiation in a reactor of higher thermal neutron flux or with the parallel run of several separation units, several Ci-s of n.c.a 177Lu can be profitably produced on a commercial production basis. © 2008, Springer.
Alternative method for Cu-64 radioisotope production
(Elsevier, 2008-05-12) Le, VS; Howse, J; Zaw, M; Pellegrini, PA; Katsifis, A; Greguric, I; Weiner, R
The method for 64Cu production based on a 64Ni target using an 18 MeV proton energy beam was developed. The studies on the optimisation of targetry for the 18 MeV proton bombardments were performed in terms of the cost-effective target utilisation and purity of the 64Cu product. The thickness-specific 64Cu yield (μCi/(μA×μm)) was introduced into the optimisation calculation with respect to cost-effective target utilisation. A maximum target utilisation efficacy factor (TUE) was found for the proton energy range of 2.5–13 MeV with corresponding target thickness of 36.2 μm. With the optimised target thickness and proton energy range, the 64Ni target thickness saving of 45.6% was achieved, while the overall 64Cu yield loss is only 23.9%, compared to the use of the whole effective proton energy range of 0–18 MeV with target thickness of 66.6 μm. This optimisation has the advantage of reducing the target amount to a reasonable level, and therefore the cost of the expensive 64Ni target material. The 64Ni target electroplated on the Au–Tl multi layer coated Cu-substrate was a new and competent design for an economic production of high quality 64Cu radioisotope using an 18 MeV proton energy cyclotron or a 30 MeV cyclotron with proton beam adjustable to 18 MeV. In this design, the Au coating layer plays a role of protection of “cold” Cu leakage from the Cu substrate and Tl serves to depress the proton beam energy (from 18 MeV to the energy optimised value 13 MeV). The ion exchange chromatographic technique with a gradient elution was applied to improve the 64Cu separation with respect to reducing the processing time and control of 64Cu product quality. © 2009, Elsevier Ltd.
Ascertaining the suitability of aryl sulfonyl fluorides for [18F]radiochemistry applications: a systematic investigation using microfluidics
(American Chemical Society, 2013-10-18) Matesic, L; Wyatt, NA; Fraser, BH; Roberts, MP; Pham, TQ; Greguric, I
Optimization of [18F]radiolabeling conditions and subsequent stability analysis in mobile phase, PBS buffer, and rat serum of 12 aryl sulfonyl chloride precursors with various substituents (electron-withdrawing groups, electron-donating groups, increased steric bulk, heterocyclic) were performed using an Advion NanoTek Microfluidic Synthesis System. A comparison of radiochemical yields and reaction times for a microfluidics device versus a conventional reaction vessel is reported. [18F]Radiolabeling of sulfonyl chlorides in the presence of competing nucleophiles, H-bond donors, and water was also assessed and demonstrated the versatility and potential utility of [18F]sulfonyl fluorides as synthons for indirect radiolabeling. © 2013 American Chemical Society
Clusterin facilitates in vivo clearance of extracellular misfolded proteins
(Springer Basel AG, 2011-12-01) Wyatt, AR; Yerbury, JJ; Berghofer, PJ; Greguric, I; Katsifis, A; Dobson, CM; Wilson, MR
The extracellular deposition of misfolded proteins is a characteristic of many debilitating age-related disorders. However, little is known about the specific mechanisms that act to suppress this process in vivo. Clusterin (CLU) is an extracellular chaperone that forms stable and soluble complexes with misfolded client proteins. Here we explore the fate of complexes formed between CLU and misfolded proteins both in vitro and in a living organism. We show that proteins injected into rats are cleared more rapidly from circulation when complexed with CLU as a result of their more efficient localization to the liver and that this clearance is delayed by pre-injection with the scavenger receptor inhibitor fucoidan. The CLU– client complexes were found to bind preferentially, in a fucoidan-inhibitable manner, to human peripheral blood monocytes and isolated rat hepatocytes and in the latter cell type were internalized and targeted to lysosomes for degradation. The data suggest, therefore, that CLU plays a key role in an extracellular proteostasis system that recognizes, keeps soluble, and then rapidly mediates the disposal of misfolded proteins. © 2012, Springer.
Comparative analysis of novel decynium-22 analogs to inhibit transport by the low-affinity, high-capacity monoamine transporters, organic cation transporters 2 and 3, and plasma membrane monoamine transporter
(Elsevier B. V., 2019-01) Fraser-Spears, R; Krause-Heuer, AM; Basiouny, M; Mayer, FP; Manishimwe, M; Wyatt, NA; Dobrowolski, JC; Roberts, MP; Greguric, I; Kumar, N; Koek, W; Sitte, HH; Callaghan, PD; Fraser, BH; Daws, LC
Growing evidence supports involvement of low-affinity/high-capacity organic cation transporters (OCTs) and plasma membrane monoamine transporter (PMAT) in regulating clearance of monoamines. Currently decynium-22 (D22) is the best pharmacological tool to study these transporters, however it does not readily discriminate among them, underscoring a need to develop compounds with greater selectivity for each of these transporters. We developed seven D22 analogs, and previously reported that some have lower affinity for α1-adrenoceptors than D22 and showed antidepressant-like activity in mice. Here, we extend these findings to determine the affinity of these analogs for OCT2, OCT3 and PMAT, as well as serotonin, norepinephrine and dopamine transporters (SERT, NET and DAT) using a combination of uptake competition with [3H]methyl-4-phenylpyridinium acetate in overexpressed HEK cells and [3H]citalopram, [3H]nisoxetine and [3H]WIN 35428 displacement binding in mouse hippocampal and striatal preparations. Like D22, all analogs showed greater binding affinities for OCT3 than OCT2 and PMAT. However, unlike D22, some analogs also showed modest affinity for SERT and DAT. Dual OCT3/SERT and/or OCT3/DAT actions of certain analogs may help explain their ability to produce antidepressant-like effects in mice and help account for our previous findings that D22 lacks antidepressant-like effects unless SERT function is either genetically or pharmacologically compromised. Though these analogs are not superior than D22 in discriminating among OCTs/PMAT, our findings point to development of compounds with combined ability to inhibit both low-affinity/high-capacity transporters, such as OCT3, and high-affinity/low-capacity transporters, such as SERT, as therapeutics with potentially improved efficacy for treatment of psychiatric disorders. © 2021 Elsevier B.V.
Comparison of cannabinoid CB1 receptor binding in adolescent and adult rats: a positron emission tomography study using [18F]MK-947
(Hindawi Publishing Corporation, 2011-01-01) Verdurand, M; Nguyen, VH; Stark, D; Zahra, D; Grégoire, MC; Greguric, I; Zavitsanou, K
Despite the important role of cannabinoid CB1 receptors (CB1R) in brain development, little is known about their status during adolescence, a critical period for both the development of psychosis and for initiation to substance abuse. In the present study, we assessed the ontogeny of CB1R in adolescent and adult rats in vivo using positron emission tomography with [18F]MK-9470. Analysis of covariance (ANCOVA) to control for body weight that would potentially influence [18F]MK-9470 values between the two groups revealed a main effect of age ( 𝐹 ( 1 , 1 0 9 ) = 5 . 0 , 𝑃 = 0 . 0 2 ) on [18F]MK-9470 absolute binding (calculated as percentage of injected dose) with adult estimated marginal means being higher compared to adolescents amongst 11 brain regions. This finding was confirmed using in vitro autoradiography with [3H]CP55,940 ( 𝐹 ( 1 0 , 9 9 ) = 1 4 0 . 1 , 𝑃 < 0 . 0 0 0 1 ). This ontogenetic pattern, suggesting increase of CB1R during the transition from adolescence to adulthood, is the opposite of most other neuroreceptor systems undergoing pruning during this period. Copyright © 2011 Mathieu Verdurand et al.
Comparison of in vivo binding properties of the 18-kDa translocator protein (TSPO) ligands [18F]PBR102 and [18F]PBR111 in a model of excitotoxin-induced neuroinflammation
(Springer Link, 2015-01) Callaghan, PD; Wimberley, CA; Rahardjo, GL; Berghofer, PJ; Pham, TQ; Jackson, TW; Zahra, D; Bourdier, T; Wyatt, N; Greguric, I; Howell, NR; Siegele, R; Pastuovic, Z; Mattner, F; Loc'h, C; Grégoire, MC; Katsifis, A
The in vivo binding parameters of the novel imidazopyridine TSPO ligand [18F]PBR102 were assessed and compared with those of [18F]PBR111 in a rodent model of neuroinflammation. The validity of the key assumptions of the simplified reference tissue model (SRTM) for estimation of binding potential (BP) was determined, with validation against a two-tissue compartment model (2TC). Methods Acute neuroinflammation was assessed 7 days after unilateral stereotaxic administration of (R,S)-α-amino-3-hydroxy-5-methyl-4-isoxazolopropionique (AMPA) in anaesthetized adult Wistar rats. Anaesthetized rats were implanted with a femoral arterial cannula then injected with a low mass of [18F]PBR102 or [18F]PBR111 and dynamic images were acquired over 60 min using an INVEON PET/CT camera. Another population of rats underwent the same PET protocol after pretreatment with a presaturating mass of the same unlabelled tracer (1 mg/kg) to assess the validity of the reference region for SRTM analysis. Arterial blood was sampled during imaging, allowing pharmacokinetic determination of radiotracer concentrations. Plasma activity concentration–time curves were corrected for unchanged tracer based on metabolic characterization experiments in a separate cohort of Wistar rats. The stability of neuroinflammation in both imaging cohorts was assessed by [125I] CLINDE TSPO quantitative autoradiography, OX42/GFAP immunohistochemistry, Fluoro-Jade C histology, and elemental mapping using microparticle-induced x-ray emission spectroscopy. The BP of each ligand were assessed in the two cohorts of lesioned animals using both SRTM and a 2TC with arterial parent compound concentration, coupled with the results from the presaturation cohort for comparison and validation of the SRTM. Results The BPs of [18F]PBR102 [18F]PBR111 were equivalent, with improved signal-to-noise ratio and sensitivity compared with [11C]PK11195. The presaturation study showed differences in the volume of distribution between the ipsilateral striatum and the striatum contralateral to the injury (0.7) indicating that an assumption of the SRTM was not met. The modelling indicated that the BPs were consistent for both ligands. Between the SRTM and 2TC model, the BPs were highly correlated, but there was a bias in BP. Conclusion [18F]PBR102 and [18F]PBR111 have equivalent binding properties in vivo, displaying significantly greater BPs with lower signal-to-noise ratio than [11C]PK11195. While an assumption of the SRTM was not met, this modelling approach was validated against 2TC modelling for both ligands, facilitating future use in longitudinal PET imaging of neuroinflammation.© 2014, Springer Nature
Detection of apoptotic cell death in the thymus of dexamethasone treated rats using [123I]Annexin V and in situ oligonucleotide ligation
(Springer Nature, 2007-06-29) Zavitsanou, K; Nguyen, VH; Greguric, I; Chapman, J; Ballantyne, P; Katsifis, A
In the present study we aimed to establish an animal model of dexamethasone (DEX)-induced apoptosis in the thymus of rats. The degree of apoptosis was determined in the same animals at 6 and 11 h after a single administration of DEX (5 mg/kg, ip) by (a) in vivo biodistribution of the uptake of [123I]Annexin V, a biomarker of the early stages of apoptosis; (b) in vitro evaluation of the apoptotic index (percentage of number of apoptotic cells versus total number of cells) in the form of DNA fragmentation, on tissue sections using in situ oligo ligation (ISOL). ISOL demonstrated a 62- and 90-fold increase of apoptotic index at 6 and 11 h after DEX administration respectively, in the outer part of the thymic lobule (cortex) and a 25- and 54-fold increases in the inner part of the thymic lobule (medulla) in the corresponding treatment groups. In the biodistribution study, [123I]Annexin V uptake was significantly increased in the thymus of rats 11 h after DEX administration (by 1.3- to 1.4-fold) and significantly decreased at the 6-h time point. We conclude that the specificity of the apoptotic signal provided by isotopic methods in vivo would always require confirmation by complementary in vitro techniques that verify the assessment of ongoing apoptosis accurately. © 2007 Springer Science+Business Media B.V.
Developing national capability for the production and use of radiometal based radiopharmaceuticals
(John Wiley & Sons, Inc, 2013-04-11) Lengkeek, NA; Pellegrini, PA; Oehlke, E; Fraser, BH; Greguric, I
Radiometals remain key radioisotopes for radio-medicine; the mainstay diagnostic medical imaging isotope, 99mTc, and important radioisotopes for radiotherapy, 90Y and 153Sm. However, the worldwide growth in PET centres, driven by the wildly successful [18F]-FDG, has provided a yet to be seized opportunity to deliver radiometal-based radiopharmaceuticals with clinical relevance to researchers. An ever growing set of radiometals is becoming available in Australia covering a wide range of half-lives, nuclear and chemical properties, these include 64Cu, 68Ga, 86Y, 89Zr, and looking into the near future, 44Sc, 45Ti and 90Nb. ANSTO's Lifesciences division is working to provide the Australian academic and clinical communities access to the following key areas. Radioisotope supply Two in-house 68Ge/68Ga generators; one solely for research and a larger generator for pre-clinical trials, provide access to this key radioisotope. ANSTO has provided financial and technical support to numerous cyclotron centres around Australia for development of solid targetry facilities, ensuring supply of 124I, 64Cu, 86Y and 89Zr in the short-term and 44Sc, 45Ti and 90Nb later. In coming years the OPAL reactor will begin producing pharmaceutical grade 177Lu as part of the Australian government's investment in nuclear medicine. Radiometal Labelling Research and Development In addition to our programs developing new ligands, tracers and improved radiometal labelling procedures, Lifesciences is supporting the development of new biomolecule treatments by providing researchers access to laboratories, procedures and expertise tailored specifically for functionalising biomolecules for radiometal labelling. Preclinical Radiometal Tracer Development We are working to deliver novel radiometal-based diagnostic tracers to the Australian research and clinical community by accessing the pre-clinical programs of our key international partnerships at Memorial Sloan Kettering Cancer Centre and Massachusetts General Hospital. Our initial objective is to supply the antibodies, i.e. [ 89 Zr]-J591. As our supply radiometals is diversified we will expand into proteins, peptides and other biomolecules. © 2013 John Wiley & Sons, Inc.
Development and validation of competition binding assays for affinity to the extracellular matrix receptors, αvβ3 and αIIbβ3 integrin
(Elsevier B.V., 2012-04-01) Szabo, A; Howell, NR; Pellegrini, PA; Greguric, I; Katsifis, A
The RGD (Arg-Gly-Asp) binding integrins αvβ3 and αIIbβ3 are integral components of various pathological and physiological processes, including tumor angiogenesis, osteoclast function, and thrombus formation. Because of this, there is interest in identifying novel compounds and proteins binding to these receptors as well as investigating the mechanism of these interactions. In this article, we describe the development and validation of competition binding assays for determining the affinity of test compounds to αvβ3 and αIIbβ3 integrin. Assays were successfully developed for each receptor, and the affinity of known compounds was comparable to published results. However, the inability of binding between αIIbβ3 integrin and the labeled echistatin protein ligand to reach equilibrium resulted in an assay that did not meet the assumptions of the competition binding model. Nevertheless, there was good agreement between this assay and known literature values, and intra- and interassay variability was acceptable. Binding by conformation-specific antibodies provided evidence that solid-phase bound αIIbβ3 receptor was in an activated conformation. This study also demonstrated that current models and methods for determining receptor affinity are simplistic and fail to account for common receptor–ligand interactions such as nondissociable interactions and varying receptor activation states. © 2012 by Elsevier Inc.
Development of novel ligands for emerging radiometal isotopes
(John Wiley & Sons, Inc., 2013-04-11) Ashford, ME; Burgess, L; Cheah, WC; Krause-Heuer, AM; Fraser, BH; Greguric, I; Lengkeek, NA
Background: The use of radiometals (non-Tc, non-Re) in targeted diagnosis and radiotherapy of different disease states has increased significantly over the last 15 years. ANSTO LifeSciences radiometals program seeks to provide a suite of radiometal tools for use in PET imaging and therapeutic modalities to improve upon the existing technologies which are currently dominated by 99mTc. This will enable researchers and clinicians to study and diagnose diseases with a greater efficacy and efficiency. Method: Many of the ligands currently available for radiometals have numerous drawbacks , including unfavourable in vivo properties such as thermodynamic and kinetic stability and poor lipophilicity. We are developing new ligand systems to have improved radiometal specificity while including design flexibility allowing us to manipulate properties such as biodistribution patterns, excretion rates, pharmacokinetics, thermodynamics and in vivo stability. The synthesis should be straightforward and cost effective and have the potential for bioconjugation in the initial design. Complexation studies are performed in vitro to assess the ligands suitability. Results: We are developing ligand systems for 68Ga, 89Zr, 64Cu, 90Y and 177Lu. We have prepared a novel analogue of the ubiquitous ligand NOTA (Figure 1, b); our system replaces the biologically labile carboxylic acid with a corresponding, biologically inert isotere, a tetrazole. This manipulation should provide additional stability and increased complex lipophilicity. Our studies have shown that a tetrazole analogue of NOTA (Figure 1, b) forms stable cold-metal complexes with potential PET metals of interest such as Ga3+. Conclusion: ANSTO LifeSciences provides a complete synthetic ligand and metal complex program, complementing its broader Radiometals Program. The aim of which is to provide an array of clinically relevant ligands that can be used in multiple applications for specific metal radiopharmaceuticals for improved patient outcomes. © 2013 John Wiley & Sons
Development of PET and SPECT radiopharmaceuticals to study multi-drug resistance (MDR)
(Australian Nuclear Science and Technology Organisation, 2002-04-29) Katsifis, A; Guilloteau, D; Dikic, B; Garrigos, M; Emond, P; Greguric, I; Knott, RB; Marvel, S; Mattner, F
Cellular resistance or Multidrug Resistance (MDR) to cytotoxic agents is the major cause of treatment failure in many human cancers. P-glycoprotein (Pgp), a Mr 17,0000 transmembrane protein and Multi Resistance Protein (MRP) are two proteins that are over expressed and confer resistance to a large number of chemotherapeutic agents by enhancing their extracellular transport. P-glycoprotein is expressed at a relative high level in treated and untreated human malignant tumours, including renal, colonic, adrenal, hepatocellular carcinoma and a considerable percentage of breast carcinomas. 99mTc-Sestamibi, a lipophilic cationic complex is a transport substrate for Pgp. In clinical studies of human neoplasms it was found that tumour uptake and clearance of this tracer correlate with Pgp expression and may be used for the phenotypic assessment of MDR. However, new tracers with better substrate specificity for Pgp and other drug transporters would greatly assist in optimising chemotherapeutic treatment and improving patient management by predicting tumour response to therapy and to assist in the development of antagonists, which may reverse or halt MDR. The aim of this project is therefore to develop PET and SPECT radiopharmaceuticals with improved affinity and selectivity for Pgp and MRP for the clinical evaluation of MDR in cancer patients. To optimise cellular transport characteristics, a number of chemical families that have been found to be substrates of Pgp and other drug efflux pumps, will be investigated. In the first instance, a series of drugs based on the flavonol natural product, Quercetin will be developed, screened for MDR and radiolabelled with PET and SPECT isotopes. Quercetin and related flavonol derivatives have been selected for this project because of their moderate to good affinity for Pgp. With the assistance of molecular modeling and in vitro studies, structural modification will be undertaken to improve the specificity and affinity for PgP. This generic structure also offers the flexibility to prepare a wide range of molecules that are readily suitable for halogenation with either Iodine-123 or F-18 for radiopharmaceutical development. Finally these phenolic type of molecules based on Quercetin are relatively less toxic than equivalent drugs. In this proposal an extensive research program is required to develop specific drugs for the different efflux pumps present in the body, which represent multi drug resistance. A successful outcome is critically dependent on the initial synthesis of a large number of compounds for screening. The combined effort of the three institutions will boost resources significantly to a critical level required to competitively produce successful outcomes in the project. Optimisation studies on derivatives of these flavonols will be made in parallel with the assistance of in vitro studies by measuring the binding of compounds to the ATP sites of Pgp. An extensive in vitro screening program has been established in Paris, prior to radiolabelling and in vivo evaluation. Structural optimisation and attachment of radionuclides to promising molecular targets will be explored using molecular modelling. Initially computational chemistry using Sybyl will be undertaken to develop a pharmacophore and to assist with the incorporation of the radionuclide in the appropriate position. In vivo evaluation will be undertaken in specific animal models both at the University of Tours in France as well as at the Sydney Cancer Centre in Australia. PET functional imaging studies may be undertaken on successful candidates at the SHFJ in Orsay, France whilst SPECT imaging will be undertaken in both Tours and in Sydney. In addition to intellectual property and potential commercial product(s), specific PET or SPECT radiopharmaceuticals can provide valuable information on the assessment of MDR in cancer patients through functional, non-invasive, imaging and therefore make significant contributions to the understanding of MDR. Scientific and clinical researchers from both countries identified the use of PET and SPECT functional imaging of MDR as a priority area of research. Finally the clear benefits to cancer patients include choice of treatment, with minimisation of ineffective drug treatments at an earlier stage, hence reduced drug side effects and discomfort to patients and improvements in their quality of life. There are also reduced health costs by avoiding expensive and ineffective drug treatments,
Different radiolabelling methods alter the pharmacokinetic and biodistribution properties of Plasminogen Activator Inhibitor Type 2 (PAI-2) forms
(Elsevier B.V., 2012-08-01) Ranson, M; Berghofer, PJ; Vine, KL; Greguric, I; Shepherd, R; Katsifis, A
Introduction Tumour-associated urokinase plasminogen activator (uPA) is a critical marker of invasion and metastasis, and it is recognised as having strong prognostic relevance as well as being a therapeutic target. The specific uPA inhibitor plasminogen activator inhibitor type-2 (PAI-2, SerpinB2) specifically targets cell bound uPA and is internalised. Furthermore, preclinical studies have established the “proof-of-principle” of uPA-targeting by PAI-2-cytotoxin conjugates in human carcinoma models. However, these studies also suggest that PAI-2 is rapidly cleared via the renal system with low total dose reaching the tumour. In this study, a comparative single photon emission computed tomography (SPECT) and biodistribution (BD) analysis of different forms of PAI-2 labelled with the radioisotopes iodine-123 (123I) and technetium-99m (99mTc) was undertaken. Methods The pharmacokinetic (PK) properties and BD of wild-type, ΔCD-loop and PEGylated ΔCD-loop PAI-2 labelled with the commonly used diagnostic SPECT radioisotopes 99mTc or 123I were compared in mouse models of human prostate carcinoma. Whole body SPECT imaging was also performed. Results Both wild-type and the shorter but active ΔCD-loop form of PAI-2 123I-labelled indirectly via conjugation to free amine groups (termed 123I-Bn-PAI-2) exhibited low tumour uptake, rapid excretion and similar PK profiles. Preliminary studies with a short branched-chain PEGylated 123I-Bn-PAI-2 ΔCD-loop indicated an increase in blood retention time and tumour uptake. All 123I-Bn-labelled radiotracers were largely excreted through the kidneys. By comparison, both wild-type 123I-PAI-2 (labelled directly via tyrosine residues) and 99mTc-PAI-2 displayed different PK/BD patterns compared to 123I-Bn-PAI-2, suggesting greater liver based catabolism and thus slower elimination. SPECT imaging mimicked the BD results of all radiotracers. © 2020 Elsevier B.V. Conclusion The different labelling methods gave distinct PAI-2 BD and tumour uptake profiles, with radioiodination resulting in the best non-tumour organ clearance profiles. Preliminary analyses with short branched-chain PEGylated 123I-Bn-PAI-2 ΔCD-loop suggest that further investigations with other PEGylation reagents are required to optimise this approach for tumour imaging. These findings impact on the use of PAI-2 for drug delivery and/or diagnostic development. © 2012 Published by Elsevier Inc.
Differential activity of decynium-22 analogs: novel targets for probing low-affinity/high-capacity biogenic amine transporters
(Federation of American Societies for Experimental Biology (FASEB), 2015-04-01) Fraser, R; Owens, A; Wyatt, NA; Krause-Heuer, AM; Greguric, I; Callaghan, PD; Fraser, BH; Daws, LC
We study neurotransmitter clearance by low-affinity, high-capacity uptake-2 transporters. This family includes plasma membrane monoamine transporter (PMAT) and three organic cation transporter isoforms (OCT1-3). We have shown uptake-2 transporters limit the effectiveness of the selective serotonin reuptake inhibitor (SSRI) fluvoxamine. Discerning the transporter type(s) involved is restricted by the lack of highly selective ligands. This project examines the pharmacological characteristics of novel uptake-2 compounds. Activity of ANSTO analogs, structurally based on the non-selective inhibitor decynium 22 (D22), was tested in human OCT3-HEK cells. Ligand competitions of [3H]MPP+ uptake were measured in whole, attached cells. Compared to D22, dose-responses of ANSTO compounds shifted 1- or 2 log-rightward, indicating reduced potency to inhibit OCT3 mediated [3H]MPP+ uptake. ANSTO analogs displayed similar potencies to corticosterone and may have higher selectivity at alternate uptake 2 subtypes. © 2015 Federation of American Societies for Experimental Biology (FASEB)
Discovery of [F-18]N-(2-(Diethylamino)ethyl)-6-fluoronicotinamide: a melanoma positron emission tomography imaging radiotracer with high tumor to body contrast ratio and rapid renal clearance
(American Chemical Society, 2009-09-10) Greguric, I; Taylor, SR; Denoyer, D; Ballantyne, P; Berghofer, PJ; Roselt, P; Pham, TQ; Mattner, F; Bourdier, T; Neels, OC; Dorow, DS; Loc'h, C; Hicks, RJ; Katsifis, A
The high melanoma uptake and rapid body clearance displayed by our series of [123I]iodonicotinamides prompted the development of [18F]N-(2-(diethylamino)ethyl)-6-fluoronicotinamide ([18F]2), a novel radiotracer for PET melanoma imaging. Significantly, unlike fluorobenzoates, [18F]fluorine incorporation on the nicotinamide ring is one step, facile, and high yielding. [18F]2 displayed high tumor uptake, rapid body clearance via predominantly renal excretion, and is currently being evaluated in preclinical studies for progression into clinical trials to assess the responsiveness of therapeutic agents. © 2009, American Chemical Society
Evaluation of radioisotope quality aspects for preparation of high specific activity [Ga-68]-NOTA-AnnexinA1.
(Elsevier, 2010-09-08) Fuchs, A; Greguric, I; Roe, G
The bi-functional chelator NOTA-(p-Bn)-NCS permits radio-labelling heat sensitive proteins with Ga-68 (t1/2=68 min) for positron emission tomography. Complexation at room temperature (RT) completes within minutes and in vivo transmetalation is negligible. Here, influences of trace metal cations, on radiochemical yield (RCY) and specific radioactivity (SRA) are assessed. Conjugation of NOTA-(p-Bn)-NCS to AnnexinA1 was performed at varying stoichiometries and conjugates purified by size exclusion high-performance liquid chromatography. Available complexation sites per protein were identified by colorimetric assay and titration with carrier added Ga-67. The complexation of Ga-68 by NOTA-AnnexinA1 at RT and 37°C was systematically studied with and without addition of competing trace metal cations. Ga-67 was used for confirmation of observed trends. Integrity of the radio-conjugate was assessed by addition of up to 104 fold excess of metal cations or apo-transferin and exposure to human serum at 37°C. Gallium-68 gave RCY of N99% within minutes at RT whereas, Ga-67 yielded max 85% dropping as the stock decayed. Presence of Fe(III) showed significant influence on RCY. Once formed, the radio-conjugate showed negligible loss of radioactivity under even the most extreme conditions investigated. SRA and RCY of the radio-conjugate depend significantly on absence of particularly Fe(III) during complexation. © 2020 Elsevier B.V
Evaluation of the antidepressant therapeutic potential of isocyanine and pseudoisocyanine analogues of the organic cation decynium-22
(Elsevier B. V., 2017-09-08) Krause-Heuer, AM; Fraser-Spears, R; Dobrowolski, JC; Ashford, ME; Wyatt, NA; Roberts, MP; Gould, GG; Cheah, WC; Ng, CKL; Bhadbhade, MM; Zhang, B; Greguric, I; Wheate, NJ; Kumar, N; Koek, W; Callaghan, PD; Daws, LC; Fraser, BH
Antidepressant-like activity Herein we describe the synthesis and evaluation of antidepressant properties of seven analogues (1–7) of the low affinity/high capacity transporter blocker decynium-22 (D-22). All analogues (1–7) were synthesized via base promoted coupling reactions between N-alkylated-2-methylquinolinium iodides or N-alkylated-4-methylquinolinium iodides and electrophilic N-alkylated-2-iodoquinolinium iodides. All final compounds were purified by re-crystallization or preparative HPLC and initial evaluation studies included; 1) screening for in vitro α1-adrenoceptor activity (a property that can lead to unwanted side-effects), 2) measuring antidepressant-like activity in a mouse tail suspension test (TST), and 3) measuring effects upon mouse locomotion. The results showed some analogues have lower affinities at α1-adrenoceptors compared to D-22 and showed antidepressant-like activity without the need for co-administration of SSRIs. Additionally, many analogues did not affect mouse locomotion to the same extent as D-22. Plans for additional evaluations of these promising analogues, including measurement of antidepressant-like activity with co-administration of selective serotonin re-uptake inhibitors (SSRIs), are outlined. © 2017 Elsevier B.V.

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