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⁶⁷Ga-metalloprobes: monitoring the impact of geometrical isomers on accumulation profiles in rat cardiomyoblasts and human breast carcinoma cells.

ABSTRACT: Geometrically similar monocationic gallium(iii) complexes and their radiolabeled SPECT counterparts were obtained from Schiff base precursor ligands using ligand exchange reactions to evaluate the impact of cis and trans-isomers on their cellular accumulation profiles in rat cardiomyoblasts (H9c2(2-1)) and human breast carcinoma (MCF-7neo) cells. ⁶⁷Ga-metalloprobes comprising trans-phenolates showing an overall octahedral geometry and exhibiting uniform spatial distribution of positive charges on their molecular surface show steady-state accumulation in H9c2(2-1) and MCF-7neo cells, and localize in the mitochondria of the cells. Importantly, the surrogate geometrically similar and monocationic metalloprobe counterparts possessing the cis arrangement of phenolates do not show cellular uptake in H9c2(2-1) and MCF-7neo cells. Exploiting their modest fluorescent traits, live cell imaging indicates that trans-isomers of metalloprobes localize within the mitochondria of cells following their penetration, thereby indicating the excellent correlation of radiotracer data and live-cell microscopy results. Overall, these results indicate that the cell uptake profiles of metalloprobes within this class are mediated by the spatial distribution of charges over their molecular surface and hydrophobicity.

SUBMITTER: Sivapackiam J

PROVIDER: S-EPMC6071725 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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67Ga-metalloprobes: monitoring the impact of geometrical isomers on accumulation profiles in rat cardiomyoblasts and human breast carcinoma cells.

Sivapackiam Jothilingam J Harpstrite Scott E SE Rath Nigam P NP Sharma Vijay V

MedChemComm 20161109 1

Geometrically similar monocationic gallium(iii) complexes and their radiolabeled SPECT counterparts were obtained from Schiff base precursor ligands using ligand exchange reactions to evaluate the impact of cis and trans-isomers on their cellular accumulation profiles in rat cardiomyoblasts (H9c2(2-1)) and human breast carcinoma (MCF-7neo) cells. 67Ga-metalloprobes comprising trans-phenolates showing an overall octahedral geometry and exhibiting uniform spatial di ...[more]

PMID: 30108701

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Project description:IntroductionMolecular radiotherapy exploiting short-range Auger electron-emitting radionuclides has potential for targeted cancer treatment and, in particular, is an attractive option for managing micrometastatic disease. Here, an approach using chelator-trastuzumab conjugates to target radioactivity to breast cancer cells was evaluated as a proof-of-concept to assess the suitability of 67Ga as a therapeutic radionuclide.MethodsTHP-trastuzumab and DOTA-trastuzumab were synthesised and radiolabelled with Auger electron-emitters 67Ga and 111In, respectively. Radiopharmaceuticals were tested for HER2-specific binding and internalisation, and their effects on viability (dye exclusion) and clonogenicity of HER2-positive HCC1954 and HER2-negative MDA-MB-231 cell lines was measured. Labelled cell populations were studied by microautoradiography.ResultsLabelling efficiencies for [67Ga]Ga-THP-trastuzumab and [111In]In-DOTA-trastuzumab were 90% and 98%, respectively, giving specific activities 0.52 ± 0.16 and 0.61 ± 0.11 MBq/μg (78-92 GBq/μmol). At 4 nM total antibody concentration and 200 × 103 cells/mL, [67Ga]Ga-THP-trastuzumab showed higher percentage of cell association (10.7 ± 1.3%) than [111In]In-DOTA-trastuzumab (6.2 ± 1.6%; p = 0.01). The proportion of bound activity that was internalised did not differ significantly for the two tracers (62.1 ± 1.4% and 60.8 ± 15.5%, respectively). At 100 nM, percentage cell binding of both radiopharmaceuticals was greatly reduced compared to 4 nM and did not differ significantly between the two (1.2 ± 1.0% [67Ga]Ga-THP-trastuzumab and 0.8 ± 0.9% for [111In]In-DOTA-trastuzumab). Viability and clonogenicity of HER2-positive cells decreased when each radionuclide was incorporated into cells by conjugation with trastuzumab, but not when the same level of radioactivity was confined to the medium by omitting the antibody conjugation, suggesting that 67Ga needs to be cell-bound or internalised for a therapeutic effect. Microautoradiography showed that radioactivity bound to individual cells varied considerably within the population.Conclusions[67Ga]Ga-THP-trastuzumab reduced cell viability and clonogenicity only when cell-bound, suggesting 67Ga holds promise as a therapeutic radionuclide as part of a targeted radiopharmaceutical. The causes and consequences of non-homogeneous uptake among the cell population should be explored.

Project description:Introduction: Radiohybrid (rh) ligands, a novel class of prostate-specific membrane antigen (PSMA)-targeted radiopharmaceuticals, can be labeled either with [18F]fluorine via isotopic exchange or with radiometals (such as [68Ga]Gallium, [177Lu]Lutetium, [225Ac]Actinium). Among these, [18F, natGa]rhPSMA-7 has recently entered clinical assessment.Aim: Since [18F, natGa]rhPSMA-7 is composed of four stereoisomers ([18F, natGa]rhPSMA-7.1, -7.2, -7.3 and -7.4), we initiated a preclinical selection process to identify the isomer with the most favorable pharmacokinetics for further clinical investigation.Methods: A synthetic protocol for enantiopure [19F, natGa]rhPSMA-7 isomers has been developed. The comparative evaluation of the four isomers comprised human serum albumin binding, lipophilicity, IC50, internalization and classical biodistribution studies and competition experiments in LNCaP tumor-bearing CB-17 SCID mice. In addition, a radio high-performance liquid chromatography-based method was developed allowing quantitative, intraindividual comparison of [18F, natGa]rhPSMA-7.1 to -7.4 in LNCaP tumor-bearing mice.Results: Cell studies revealed high PSMA affinity and internalization for [18/19F, natGa]rhPSMA-7.2, -7.3 and -7.4, whereas [18/19F, natGa]rhPSMA-7.1 showed approximately twofold lower values. Although the biodistribution profile obtained was typical of PSMA inhibitors, it did not allow for selection of a lead candidate for clinical studies. Thus, an intraindividual comparison of all four isomers in LNCaP tumor-bearing mice was carried out by injection of a diastereomeric mixture, followed by analysis of the differential uptake and excretion pattern of each isomer. Based on its high tumor accumulation and low uptake in blood, liver and kidneys, [18F, natGa]rhPSMA-7.3 was identified as the preferred isomer and transferred into clinical studies.Conclusion: [18F, natGa]rhPSMA-7.3 has been selected as a lead compound for clinical development of a [18F]rhPSMA-based candidate. The intraindividual differential uptake and excretion analysis in vivo allowed for an accurate comparison and assessment of radiopharmaceuticals.

Project description:PurposeProstate-specific membrane antigen (PSMA) agents, such as [68Ga]Ga-PSMA-11, have an unprecedented accuracy in staging prostate cancer (PCa) and detecting disease recurrence. PSMA PET/CT may also be used for response monitoring by displaying molecular changes, instead of morphological changes alone. However, there are still limited data available on the variability in biodistribution and intra-prostatic uptake of PSMA targeting radiotracers. Therefore, the aim of this study was to assess the repeatability of [68Ga]Ga-PSMA-11 uptake in primary PCa patients in a 4-week interval.MethodsTwenty-four primary PCa patients were prospectively included, who already were scheduled for [68Ga]Ga-PSMA-11 PET/CT scan on clinical indication (??cT3, Gleason score???7 or PSA???20 ng/mL). These patients received two [68Ga]Ga-PSMA-11 PET/CT scans with a 4-week interval. No treatment was started in between the scans. Semiquantitative measurements (SULmax, SULmean, and SULpeak) were determined in the prostate tumor, normal tissues, and blood pool. The repeatability coefficient of every region was determined. All scans were visually analyzed by two nuclear medicine physicians.ResultsWithin-subject coefficient of variation of [68Ga]Ga-PSMA-11 uptake between the two scans was on average 10% in the prostate tumor, normal tissues (liver, kidney, parotid), and blood pool. The repeatability coefficient of the prostate tumor was 18% for SULpeak and 22% for SULmax. Lesion uptake was visually different in 5 patients, though not clinically relevant.ConclusionResults of test-retest [68Ga]Ga-PSMA-11 PET/CT scans in a 4-week interval show that [68Ga]Ga-PSMA-11 uptake is repeatable, with a clinical irrelevant variation in tumor and physiological distribution. Based on the presented repeatable uptake, [68Ga]Ga-PSMA-11 PET/CT scans can potentially be used for disease surveillance and therapy response monitoring. Changes in uptake larger than the RC are therefore likely to reflect actual biological changes in PSMA expression. Trial registration NL8263 at Trialregister.nl retrospectively registered on 03-01-2020. https://www.trialregister.nl/trial/8263.

Project description:PurposeThe widespread use of 68Ga for positron emission tomography (PET) relies on the development of radiopharmaceutical precursors that can be radiolabelled and dispensed in a simple, quick, and convenient manner. The DATA (6-amino-1,4-diazapine-triacetate) scaffold represents a novel hybrid chelator architecture possessing both cyclic and acyclic character that may allow for facile access to 68Ga-labelled tracers in the clinic. We report the first bifunctional DATA chelator conjugated to [Tyr3]octreotide (TOC), a somatostatin subtype 2 receptor (SST2)-targeting vector for imaging and functional characterisation of SSTR2 expressing tumours.MethodsThe radiopharmaceutical precursor, DATA-TOC, was synthesised as previously described and used to complex natGa(III) and 68Ga(III). Competition binding assays of [natGa]Ga-DATA-TOC or [natGa]Ga-DOTA-TOC against [125I-Tyr25]LTT-SS28 were conducted in membranes of HEK293 cells transfected to stably express one of the hSST2,3,5 receptor subtypes (HEK293-hSST2/3/5 cells). First in vivo studies were performed in female NMRI-nude mice bearing SST2-positive mouse phaeochromocytoma mCherry (MPC-mCherry) tumours to compare the in vivo SST2-specific tumour-targeting of [68Ga]Ga-DATA-TOC and its overall pharmacokinetics versus the [68Ga]Ga-DOTA-TOC reference. A direct comparison of [68Ga]Ga-DATA-TOC with the well-established PET radiotracer [68Ga]Ga-DOTA-TOC was additionally performed in a 46-year-old male patient with a well-differentiated NET (neuroendocrine tumour), representing the first in human administration of [68Ga]Ga-DATA-TOC.ResultsDATA-TOC was labelled with 68Ga with a radiolabelling efficiency of > 95% in less than 10 min at ambient temperature. A molar activity up to 35 MBq/nmol was achieved. The hSST2-affinities of [natGa]Ga-DATA-TOC and [natGa]Ga-DOTA-TOC were found similar with only sub-nanomolar differences in the respective IC50 values. In mice, [68Ga]Ga-DATA-TOC was able to visualise the tumour lesions, showing standardised uptake values (SUVs) similar to [68Ga]Ga-DOTA-TOC. Direct comparison of the two PET tracers in a NET patient revealed very similar tumour uptake for the two 68Ga-radiotracers, but with a higher tumour-to-liver contrast for [68Ga]Ga-DATA-TOC.Conclusion[68Ga]Ga-DATA-TOC was prepared, to a quality appropriate for in vivo use, following a highly efficient kit type process. Furthermore, the novel radiopharmaceutical was comparable or better than [68Ga]Ga-DOTA-TOC in all preclinical tests, achieving a higher tumour-to-liver contrast in a NET-patient. The results illustrate the potential of the DATA-chelator to facilitate the access to and preparation of 68Ga-radiotracers in a routine clinical radiopharmacy setting.

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⁶⁷Ga-metalloprobes: monitoring the impact of geometrical isomers on accumulation profiles in rat cardiomyoblasts and human breast carcinoma cells.

Publications

<sup>67</sup>Ga-metalloprobes: monitoring the impact of geometrical isomers on accumulation profiles in rat cardiomyoblasts and human breast carcinoma cells.

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