Radiolabeled Form Research Articles

Abstract PO4-19-03: FES-PET/MRI for Tailored treatment of luminal A and lobular breast cancer: a prospective cohort study

Abstract Background: Breast cancer (BC) staging is essential to planning the most appropriate treatment pathway. Currently, BC management has become more tailored to the tumor’s and patient’s characteristics. However, BC imaging shows varying performance according to tumor subtypes. In particular, routine imaging may pose some challenges when evaluating luminal A BC (LumA) and lobular BC (Lob). LumA, due to low grade and proliferation index (< 20%) shows lower sensitivity on axillary US, reduced MRI enhancement, and low FDG-avidity on PET. Similarly, Lob, due to its peculiar single-cell growth pattern, exhibits inferior sensitivity on axillary MRI and FDG-avidity. LumA and Lob account for >50% of all BCs. Despite their favourable prognosis, metastases and recurrences still occur; this translates into a higher absolute number of events than in other subtypes. As a result, a concrete risk of disease underestimation and under-treatment exists. Two ongoing studies on FDG-PET/MRI imaging in our institution have already demonstrated that its sensitivity also decreases in LumA and Lob. Based on these premises, our hypothesis states that by combining the advantages of hybrid PET/MRI with the high accuracy of 16α -18F-fluoro-17-beta-estradiol(FES), a radiolabeled form of estrogen binding to functionally active ER, we could obtain a reliable, non-invasive, operator-independent, one-stage imaging method for staging LumA and ER-positive Lob. Trial design: This is a prospective cohort study where patients with LumA and ER-positive Lob will be enrolled in four cohorts undergoing: A) primary surgery; B) induction endocrine therapy; C) neoadjuvant chemotherapy, and D) systemic therapy for metastatic disease. FES PET/MRI examinations will be performed at baseline for local and systemic staging in all cohorts and a second exam after systemic therapy in cohorts C-D. Correlations between the FES PET/MRI parameters and pathology, gene expression, and FDG PET parameters, when available, will be investigated. Study aims will evaluate: 1) the performance of the FES PET/MRI in axillary staging compared with axillary surgery; 2) potential correlations between changes in FES uptake and changes in proliferation index after three weeks of endocrine therapy (ET); 3) the performance of the FES PET/MRI in systemic staging of patients undergoing systemic therapy in comparison with standard imaging; 4) biological determinants of tumor heterogeneity on pathological, imaging, and genomic levels. Statistics: The primary analysis in cohort A will test the sensitivity of the FES PET/MRI in detecting macrometastatic axillary nodes. Assuming that the probability that a patient has positive nodes is 33%, 119 patients (10% drop-out included) will provide 80% power to reject the null hypothesis about sensitivity at a 5% significance level if the true sensitivity is at least 50%. Results from the FES PET/MRI will be also compared with standard imaging in terms of sensitivity and number of lesions detected using the Mc-Nemar test for paired proportions and the Wilcoxon signed-rank test for paired data. The primary analysis in Cohort B will test the association between the FES-SUV change from pre-ET to post-ET and the Ki-67 change from core biopsy to post-ET in ER positive BC patients with a Ki-67 >10%. The association between these changes will be measured by the Pearson correlation index ρ. Α total of 52 patients (10% drop-out included) from Cohort B will provide 80% power to reject the null hypothesis about ρ at a 5% significance level if the true ρ is at least 0.4. No formal sample size was calculated for cohort C and D: results will be considered descriptive in nature, informing sample size considerations for future trials. We intent to recruit 20 and 30 patients, respectively. Accrual has not started yet. Project funded by AIRC (Associazione Italiana Ricerca sul Cancro) Next Gen Clinician Scientist ID 28378 Contact: Dr. Rosa Di Micco, dimicco.rosa@hsr.it Citation Format: Rosa Di Micco, Carla Canevari, Francesca Gallivanone, Nicole Rotmensz, Veronica Zuber, Sara Baleri, Giovanni Cisternino, Mario Rampa, Stefania Zambelli, Giulia Viale, Patrizia Zucchinelli, Manuela Morgante, Paola Scifo, Elena Venturini, Paola Magnani, Isabella Sassi, Vincenzo Bagnardi, Pietro Panizza, Giampaolo Bianchini, Arturo Chiti, Oreste Davide Gentilini. FES-PET/MRI for Tailored treatment of luminal A and lobular breast cancer: a prospective cohort study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-19-03.

Potential improvement in the diagnostic accuracy in detecting the estrogen receptor status in metastatic and recurrent breast cancer using 18F-FES PET/CT in the US.

3063 Background: ER status helps classify BC for the most appropriate treatment pathway. Biopsy (Bx) and immunohistochemical (IHC) are established procedures to determine the ER status in metastatic BC (mBC, i.e., first occurrence of metastases) and recurrent BC (rBC, i.e., progression of mBC). In some cases, the accuracy of Bx/IHC to inform ER status may be challenged (e.g. difficult to access lesions; ER discordance within/across lesions and due to temporal heterogeneity; and when a Bx fails to obtain representative tissue). 16α-18F-fluoro-17β-fluoroestradiol (18F-FES) is a radiolabeled form of estrogen that binds to ER. For use with positron emission tomography /computed tomography imaging (PET/CT), it allows an assessment of whole-body ER+ status. In prospective trials and meta-analyses, 18F-FES PET/CT provided high diagnostic accuracy (DA) of ER+ disease. This study evaluated the potential increase in accuracy in determining ER+ status when 18F-FES PET/CT was added to IHC in mBC and rBC. Methods: An Excel based decision model was developed to estimate the impact on DA for ER+/HER2- US mBC and rBC patients over a five-year period by introducing 18F-FES PET/CT. The model assumed that 18F-FES PET/CT was added to Bx/IHC to classify ER status in three cases: (i) for mBC patients when Bx failed or was inconclusive, (ii) for mBC patients when Bx was not possible, or (iii) for all rBC patients. The DA between two scenarios was compared: scenario A, DA of Bx/IHC only and scenario B, 18F-FES PET/CT + Bx/IHC. Results: Scenario B, adding 18F-FES PET/CT, may lead to an increase in the initiation of appropriate endocrine- and chemotherapies, as defined by clinical practice guidelines, in all comparisons. The largest increase in DA was in rBC and, when BX is not possible, in mBC patients. Also, results indicate that introducing 18F-FES PET/CT decreases the number of re-biopsies performed except when Bx was not possible (ii). Conclusions: Adding 18F-FES PET/CT to Bx/IHC is expected to improve the DA of a patients’ ER status thereby increasing the information available to inform treatment, thus reducing the need for re-biopsies in mBC and rBC patients. By far the largest increase in DA of ER status using 18F-FES PET/CT was seen among patients with rBC (iii). [Table: see text]

Summary: Appropriate Use Criteria for Estrogen Receptor-Targeted PET Imaging with 16α-18F-Fluoro-17β-Fluoroestradiol.

PET imaging with 16α-18F-fluoro-17β-fluoroestradiol (18F-FES), a radiolabeled form of estradiol, allows whole-body, noninvasive evaluation of estrogen receptor (ER). 18F-FES is approved by the U.S. Food and Drug Administration as a diagnostic agent "for the detection of ER-positive lesions as an adjunct to biopsy in patients with recurrent or metastatic breast cancer." The Society of Nuclear Medicine and Molecular Imaging (SNMMI) convened an expert work group to comprehensively review the published literature for 18F-FES PET in patients with ER-positive breast cancer and to establish appropriate use criteria (AUC). The findings and discussions of the SNMMI 18F-FES work group, including example clinical scenarios, were published in full in 2022 and are available at https://www.snmmi.org/auc Of the clinical scenarios evaluated, the work group concluded that the most appropriate uses of 18F-FES PET are to assess ER functionality when endocrine therapy is considered either at initial diagnosis of metastatic breast cancer or after progression of disease on endocrine therapy, the ER status of lesions that are difficult or dangerous to biopsy, and the ER status of lesions when other tests are inconclusive. These AUC are intended to enable appropriate clinical use of 18F-FES PET, more efficient approval of FES use by payers, and promotion of investigation into areas requiring further research. This summary includes the rationale, methodology, and main findings of the work group and refers the reader to the complete AUC document.

Development of a PET radioligand for α2δ-1 subunit of calcium channels for imaging neuropathic pain

Neuropathic pain affects 7–10% of the adult population. Being able to accurately monitor biological changes underlying neuropathic pain will improve our understanding of neuropathic pain mechanisms and facilitate the development of novel therapeutics. Positron emission tomography (PET) is a noninvasive molecular imaging technique that can provide quantitative information of biochemical changes at the whole-body level by using radiolabeled ligands. One important biological change underlying the development of neuropathic pain is the overexpression of α2δ-1 subunit of voltage-dependent calcium channels (the target of gabapentin). Thus, we hypothesized that a radiolabeled form of gabapentin may allow imaging changes in α2δ-1 for monitoring the underlying pathophysiology of neuropathic pain. Here, we report the development of two 18F-labeled derivatives of gabapentin (trans-4-[18F]fluorogabapentin and cis-4-[18F]fluorogabapentin) and their evaluation in healthy rats and a rat model of neuropathic pain (spinal nerve ligation model). Both isomers were found to selectively bind to the α2δ-1 receptor with trans-4-[18F]fluorogabapentin having higher affinity. Both tracers displayed around 1.5- to 2-fold increased uptake in injured nerves over the contralateral uninjured nerves when measured by gamma counting ex vivo. Although the small size of the nerves and the signal from surrounding muscle prevented visualizing these changes using PET, this work demonstrates that fluorinated derivatives of gabapentin retain binding to α2δ-1 and that their radiolabeled forms can be used to detect pathological changes in vitro and ex vivo. Furthermore, this work confirms that α2δ-1 is a promising target for imaging specific features of neuropathic pain.

In Vivo Measurement of Granzyme Proteolysis from Activated Immune Cells with PET.

The biology of human granzymes remains enigmatic in part due to our inability to probe their functions outside of in vitro assays or animal models with divergent granzyme species. We hypothesize that the biology of human granzymes could be better elaborated with a translational imaging technology to reveal the contexts in which granzymes are secreted and biochemically active in vivo. Here, we advance toward this goal by engineering a Granzyme targeting Restricted Interaction Peptide specific to family member B (GRIP B) to measure secreted granzyme B (GZMB) biochemistry with positron emission tomography. A proteolytic cleavage of 64Cu-labeled GRIP B liberates a radiolabeled form of Temporin L, which sequesters the radioisotope by binding to adjacent phospholipid bilayers. Thus, at extended time points postinjection (i.e., hours, not seconds), tissue biodistribution of the radioisotope in vivo reflects relative units of the GZMB activity. As a proof of concept, we show in three syngeneic mouse cancer models that 64Cu-GRIP B detects GZMB from T cells activated with immune checkpoint inhibitors (CPI). Remarkably, the radiotracer detects the proteolysis within tumors but also in lymphoid tissue, where immune cells are activated by a systemic CPI. Control experiments with an uncleavable analogue of 64Cu-GRIP B and tumor imaging studies in germline GZMB knockout mice were applied to show that 64Cu-GRIP B is specific for GZMB proteolysis. Furthermore, we explored a potential noncytotoxic function for GZMB by applying 64Cu-GRIP B to a model of pulmonary inflammation. In summary, we demonstrate that granzyme biochemistry can be assessed in vivo using an imaging modality that can be scaled vertically into human subjects.

Labeling the 5' Termini of DNA with Bacteriophage T4 Polynucleotide Kinase.

The removal of 5' phosphates from nucleic acids with phosphatases and their readdition in radiolabeled form by bacteriophage T4 polynucleotide kinase is a widely used technique for generating 32P-labeled probes. When the reaction is performed efficiently, 40%-50% of the protruding 5' termini in the reaction becomes radiolabeled. However, the specific activity of the resulting probes is not as high as that obtained by other radiolabeling methods because only one radioactive atom is introduced per DNA molecule. Nevertheless, the availability of [γ-32P]ATP with specific activities in the 3000-7000 Ci/mmol range allows the synthesis of probes suitable for many purposes. This protocol includes procedures for labeling the 5' ends of dsDNA and oligonucleotides.

Automated synthesis of 18F radiolabelled indole containing Oncrasin-like molecules; a comparison of iodonium salts and boronic ester chemistry

BackgroundOncrasin-1 is a small molecule which was identified from a screen of KRAS mutant cancer cells and has shown specificity for KRAS mutant cell killing. We aimed to develop a radiolabelled form of Oncrasin-1 to enable in-vivo imaging of mutant KRAS expression in malignant tumours. This work outlines the synthesis of 3 fluorinated derivatives and development of iodonium salt and boronic ester precursors for radiolabelling with the 18F isotope.ResultsIn our hands, synthesis of iodonium salts were not easily accessible due to the 3-carbaldehyde indole structure being preferentially oxidized by conditions required for iodonium salt formation, rather than benzyl iodide. Synthesis and radiolabelling of boronic acid pinacol ester precursors were successful, with the products being obtained in yields of 10.76% ± 0.96% (n = 5), 14.7% ±8.58% (n = 3) and 14.92% ±3.9% (n = 3) for 18F KAM001, 18F KAM002 and 18F KAM003 respectively, with radiochemical purity of greater than 99%.ConclusionsThe successful synthesis of these tracers has been undertaken utilizing boronic ester radio-fluorination methods and will allow for investigation of Oncrasin based molecules as potential diagnostics for cancers expressing mutant KRAS protein.

Theranostic Targeting of CUB Domain Containing Protein 1 (CDCP1) in Pancreatic Cancer.

The recent emergence of radioligand therapies for cancer treatment has increased enthusiasm for developing new theranostic strategies coupling both imaging and cytotoxicity in the same entity. In this study, we evaluated whether CUB domain containing protein 1 (CDCP1), a single-pass transmembrane protein highly overexpressed in diverse human cancers, might be a target for cancer theranostics. The ectodomain of CDCP1 was targeted using radiolabeled forms of 4A06, a potent and specific recombinant human antibody that we developed. Imaging and antitumor assessment studies were performed in animal models of pancreatic cancer, including two patient-derived xenograft models we developed for this study. For antitumor assessment studies, the endpoints were death due to tumor volume >3,000 mm3 or ≥20% loss in body weight. Specific tracer binding or antitumor effects were assessed with an unpaired, two-tailed Student t test and survival advantages were assessed with a log rank (Mantel-Cox) test. Differences at the 95% confidence level were interpreted to be significant. 89Zr-4A06 detected a broad dynamic range of full length or cleaved CDCP1 expression on seven human pancreatic cancer tumors (n = 4/tumor). Treating mice with single or fractionated doses of 177Lu-4A06 significantly reduced pancreatic cancer tumor volume compared with mice receiving vehicle or unlabeled 4A06 (n = 8; P < 0.01). A single dose of 225Ac-4A06 also inhibited tumor growth, although the effect was less profound compared with 177Lu-4A06 (n = 8; P < 0.01). A significant survival advantage was imparted by 225Ac-4A06 (HR = 2.56; P < 0.05). These data establish that CDCP1 can be exploited for theranostics, a finding with widespread implications given its breadth of overexpression in cancer.

Cultivation at high osmotic pressure confers ubiquinone 8–independent protection of respiration on Escherichia coli

Cultivation at high osmotic pressure confers ubiquinone 8–independent protection of respiration on Escherichia coli

Tumor Imaging Using Radiolabeled Matrix Metalloproteinase-Activated Anthrax Proteins.

Increased activity of matrix metalloproteinases (MMPs) is associated with worse prognosis in different cancer types. The wild-type protective antigen (PA-WT) of the binary anthrax lethal toxin was modified to form a pore in cell membranes only when cleaved by MMPs (to form PA-L1). Anthrax lethal factor (LF) is then able to translocate through these pores. Here, we used a 111In-radiolabeled form of LF with the PA/LF system for noninvasive in vivo imaging of MMP activity in tumor tissue by SPECT. Methods: MMP-mediated activation of PA-L1 was correlated to anthrax receptor expression and MMP activity in a panel of cancer cells (HT1080, MDA-MB-231, B8484, and MCF7). Uptake of 111In-radiolabeled PA-L1, 111In-PA-WTK563C, or 111In-LFE687A (a catalytically inactive LF mutant) in tumor and normal tissues was measured using SPECT/CT imaging in vivo. Results: Activation of PA-L1 in vitro correlated with anthrax receptor expression and MMP activity (HT1080 > MDA-MB-231 > B8484 > MCF7). PA-L1–mediated delivery of 111In-LFE687A was demonstrated and was corroborated using confocal microscopy with fluorescently labeled LFE687A. Uptake was blocked by the broad-spectrum MMP inhibitor GM6001. In vivo imaging showed selective accumulation of 111In-PA-L1 in MDA-MB-231 tumor xenografts (5.7 ± 0.9 percentage injected dose [%ID]/g) at 3 h after intravenous administration. 111In-LFE687A was selectively delivered to MMP-positive MDA-MB-231 tumor tissue by MMP-activatable PA-L1 (5.98 ± 0.62 %ID/g) but not by furin-cleavable PA-WT (1.05 ± 0.21 %ID/g) or a noncleavable PA variant control, PA-U7 (2.74 ± 0.24 %ID/g). Conclusion: Taken together, our results indicate that radiolabeled forms of mutated anthrax lethal toxin hold promise for noninvasive imaging of MMP activity in tumor tissue.

ANNIVERSARY REVIEW: Octreotide, 40 years later.

Octreotide remains 40 years after its development a drug, which is commonly used in the treatment of acromegaly and GEP-NETs. Very little innovation that competes with this drug occurred over this period. This review discusses several aspects of 40 years of clinical use of octreotide, including the application of radiolabeled forms of the peptide.

Three classes of ligands each bind to distinct sites on the orphan G protein-coupled receptor GPR84

Medium chain fatty acids can activate the pro-inflammatory receptor GPR84 but so also can molecules related to 3,3′-diindolylmethane. 3,3′-Diindolylmethane and decanoic acid acted as strong positive allosteric modulators of the function of each other and analysis showed the affinity of 3,3′-diindolylmethane to be at least 100 fold higher. Methyl decanoate was not an agonist at GPR84. This implies a key role in binding for the carboxylic acid of the fatty acid. Via homology modelling we predicted and confirmed an integral role of arginine172, located in the 2nd extracellular loop, in the action of decanoic acid but not of 3,3′-diindolylmethane. Exemplars from a patented series of GPR84 antagonists were able to block agonist actions of both decanoic acid and 3,3′-diindolylmethane at GPR84. However, although a radiolabelled form of a related antagonist, [3H]G9543, was able to bind with high affinity to GPR84, this was not competed for by increasing concentrations of either decanoic acid or 3,3′-diindolylmethane and was not affected adversely by mutation of arginine172. These studies identify three separable ligand binding sites within GPR84 and suggest that if medium chain fatty acids are true endogenous regulators then co-binding with a positive allosteric modulator would greatly enhance their function in physiological settings.

Isoconcentration principle – an effective tool of (radio) analysis in environmental protection

The paper brings two determination methods for non-radioactive analytes based on isoconcentration principle. The first method represents a determination procedure for a non-radioactive substance by use of its radio-labelled form of known concentration by help of a (non-quantitative, but selective) separation method. The main advantage of this method consists in the fact, that the determination is executable only by the detection of radioactivity. The second described procedure enables the determination of the chosen analyte without requirement of radioactive form of the substance to be determinate.

A generic &lt;sup&gt;89&lt;/sup&gt;Zr labeling method to quantify the in vivo pharmacokinetics of liposomal nanoparticles with positron emission tomography

Liposomal nanoparticles are versatile drug delivery vehicles that show great promise in cancer therapy. In an effort to quantitatively measure their in vivo pharmacokinetics, we developed a highly efficient 89Zr liposome-labeling method based on a rapid ligand exchange reaction between the membrane-permeable 89Zr(8-hydroxyquinolinate)4 complex and the hydrophilic liposomal cavity-encapsulated deferoxamine (DFO). This novel 89Zr-labeling strategy allowed us to prepare radiolabeled forms of a folic acid (FA)-decorated active targeting 89Zr-FA-DFO-liposome, a thermosensitive 89Zr-DFO-liposome, and a renal avid 89Zr-PEG-DFO-liposome at room temperature with near-quantitative isolated radiochemical yields of 98%±1% (n=6), 98%±2% (n=5), and 97%±1% (n=3), respectively. These 89Zr-labeled liposomal nanoparticles showed remarkable stability in phosphate-buffered saline and serum at 37°C without leakage of radioactivity for 48 h. The uptake of 89Zr-FA-DFO-liposome by the folate receptor-overexpressing KB cells was almost 15-fold higher than the 89Zr-DFO-liposome in vitro. Positron emission tomography imaging and ex vivo biodistribution studies enabled us to observe the heterogeneous distribution of the 89Zr-FA-DFO-liposome and 89Zr-DFO-liposome in the KB tumor xenografts, the extensive kidney accumulation of the 89Zr-FA-DFO-liposome and 89Zr-PEG-DFO-liposome, and the different metabolic fate of the free and liposome-encapsulated 89Zr-DFO. It also unveiled the poor resistance of all three liposomes against endothelial uptake resulting in their catabolism and high uptake of free 89Zr in the skeleton. Thus, this technically simple 89Zr-labeling method would find widespread use to guide the development and clinical applications of novel liposomal nanomedicines.

Synthesis and Radiosynthesis of Prospective 2-Nitroimidazole Hypoxia­ PET Tracers via Thiazolidine Ligation with 5-Fluorodeoxyribose (FDR)

The first prospective fluorinated PET tracers for imaging hypoxia obtained via thiazolidine-ligation are reported. Three 1,2-thiol-amine linkers were combined with four different 2-nitroimidazole spacers via amide or urea bond formation. The resulting compounds were submitted to thiazolidine-ring-forming ligation reaction with the fluorinated carbohydrate l-5-fluoro-5-deoxy-ribose (FDR), affording the desired candidate PET tracers in variable yields. The same ligation reactions performed on l-ribose – a by-product of [18F]FDR radiosynthesis – under conditions mimicking a radiochemical production showed that the fluorinated adducts can be efficiently purified and isolated by HPLC. Finally, one of the prospective hypoxia tracers was successfully produced in radiolabelled form in 29.2% radiochemical yield from [18F]FDR.

Abstract LB-231: An optide (optimized knottin-peptide) that inhibits tumor cell growth In vitro and accumulates in sarcoma flank tumors in vivo

Abstract Antibody-drug conjugates (ADCs) have been FDA approved for the targeted delivery of chemotherapy to cancer. However, ADCs are limited for solid tumors and brain cancer by their poor penetration and inability to cross the blood-brain-barrier. A number of groups have shown that cystine-knot peptides (knottins) can be modified in ways that allow them to target cancer cells. Our lab demonstrated that an optide (optimized knottin-peptides) conjugate such as chlorotoxin-Cy5.5 are able to accumulate throughout tumors and cross an intact blood-brain barrier. However, chlorotoxin-Cy5.5 accumulates in mouse liver - a potential liability if used as a peptide-drug conjugate. We sought to identify a novel peptide that is capable of delivering chemotherapy selectively to tumor cells in vitro and in vivo. Our group has developed a mammalian protein expression system that is able to produce &amp;gt;20mg scale, endotoxin-free knottins and can rapidly generate variants of those peptides. We went in vivo to investigate if there was selective accumulation of our novel peptide-dye conjugates in sarcoma flank xenografts. Mice were injected intravenously with conjugate and accumulation was quantified in a number of tissues using IVIS imaging. We identified a novel peptide that accumulated in sarcoma flank tumors &amp;gt;10-fold relative to liver. We tested the ability of this peptide to deliver cytotoxic chemotherapy in vitro as a peptide-drug conjugate. We identified a novel conjugate capable of efficiently inhibiting the growth of a number of tumor cell lines in vitro. We tested the ability of various endocytosis inhibitors to prevent the uptake of this peptide and showed that inhibitors of GPI-anchored protein endocytosis prevented this accumulation. To verify tumor accumulation in preparation for efficacy studies we tracked the distribution of a radiolabeled form of the peptide in a whole body autoradiography model and observed sustained tumor uptake. Our novel peptide-conjugate is able to potently inhibit the growth of a number of tumor cell lines in vitro and the endocytosis of GPI-anchored proteins is involved in this accumulation. Additionally, this peptide accumulates in tumor tissue in vivo. Optide-drug conjugates may offer enhanced tumor penetration over antibody-drug conjugates and therefore increase the therapeutic index in delivering cytotoxic chemotherapy. Citation Format: Theo Sottero, Emily J. Girard, Colin Correnti, Mark R. Stroud, Brandon L. Kier, Andrew J. Mhyre, James Olson. An optide (optimized knottin-peptide) that inhibits tumor cell growth In vitro and accumulates in sarcoma flank tumors in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-231.

Investigation of the Brain Biodistribution of the Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Inhibitor [18F]GSK2647544 in Healthy Male Subjects

PurposeGSK2647544 is a potent and specific inhibitor of lipoprotein-associated phospholipase A2 (Lp-PLA2), which was in development as a potential treatment for Alzheimer’s disease (AD). In order to refine therapeutic dose predictions and confirm brain penetration, a radiolabelled form of the inhibitor, [18F]GSK2647544, was manufactured for use in a positron emission tomography (PET) biodistribution study.Procedures[18F]GSK2647544 was produced using a novel, copper iodide (Cu(I)) mediated, [18F]trifluoromethylation methodology. 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 (average injected activity and mass were 106 ± 47 MBq and 179 ± 55 μg, respectively) followed by dynamic PET scans for 120 min. Defined regions of interest (ROI) throughout the brain were used to obtain regional time-activity curves (TACs) and compartmental modelling analysis used to estimate the primary outcome measure, whole brain volume of distribution (VT). Secondary PK and safety endpoints were also recorded.ResultsPET dynamic data were successfully obtained from all four subjects and there were no clinically significant variations of the safety endpoints. Inspection of the TACs indicated a relatively homogenous uptake of [18F]GSK2647544 across all the ROIs examined. The mean whole brain VT was 0.56 (95 % CI, 0.41–0.72). Secondary PK parameters, Cmax (geometric mean) and Tmax (median), were 354 ng/ml and 1.4 h, respectively. Metabolism of GSK2647544 was relatively consistent across subjects, with 20–40 % of the parent compound [18F]GSK2647544 present after 120 min.ConclusionsThe study provides evidence that GSK2647544 is able to cross the blood brain barrier in healthy male subjects leading to a measurable brain exposure. The administered doses of GSK2647544 were well tolerated. Exploratory modelling suggested that a twice-daily dose of 102 mg, at steady state, would provide ~80 % trough inhibition of brain Lp-PLA2 activity.Trial RegistrationClintrials.gov: NCT01924858.

Targeting of radiolabeled J591 antibody to PSMA-expressing tumors: optimization of imaging and therapy based on non-linear compartmental modeling.

BackgroundWe applied a non-linear immunokinetic model to quantitatively compare absolute antibody uptake and turnover in subcutaneous LNCaP human prostate cancer (PCa) xenografts of two radiolabeled forms of the humanized anti-prostate-specific membrane antigen (PSMA) monoclonal antibody J591 (124I-J591 and 89Zr-J591). Using the model, we examined the impact of dose on the tumor and plasma positron emission tomography (PET)-derived time-activity curves. We also sought to predict the optimal targeting index (ratio of integrated-tumor-to-integrated-plasma activity concentrations) for radioimmunotherapy.MethodsThe equilibrium rates of antibody internalization and turnover in the tumors were derived from PET images up to 96 h post-injection using compartmental modeling with a non-linear transfer rate. In addition, we serially imaged groups of LNCaP tumor-bearing mice injected with 89Zr-J591 antibody doses ranging from antigen subsaturating to saturating to examine the suitability of using a non-linear approach and derived the time-integrated concentration (in μM∙hours) of administered tracer in tumor as a function of the administered dose of antibody.ResultsThe comparison of 124I-J591 and 89Zr-J591 yielded similar model-derived values of the total antigen concentration and internalization rate. The association equilibrium constant (ka) was twofold higher for 124I, but there was a ~tenfold greater tumoral efflux rate of 124I from tumor compared to that of 89Zr. Plots of surface-bound and internalized radiotracers indicate similar behavior up to 24 h p.i. for both 124I-J591 and 89Zr-J591, with the effect of differential clearance rates becoming apparent after about 35 h p.i. Estimates of J591/PSMA complex turnover were 3.9–90.5 × 1012 (for doses from 60 to 240 μg) molecules per hour per gram of tumor (20 % of receptors internalized per hour).ConclusionsUsing quantitative compartmental model methods, surface binding and internalization rates were shown to be similar for both 124I-J591 and 89Zr-J591 forms, as expected. The large difference in clearance rates of the radioactivity from the tumor is likely due to differential trapping of residualizing zirconium versus non-residualizing iodine. Our non-linear model was found to be superior to a conventional linear model. This finding and the calculated activity persistence time in tumor have important implications for radioimmunotherapy and other antibody-based therapies in patients.

Pharmacological Characterization of the αvβ6 Integrin Binding and Internalization Kinetics of the Foot-and-Mouth Disease Virus Derived Peptide A20FMDV2

A20FMDV2 is a peptide derived from the foot-and-mouth disease virus with a high affinity and selectivity for the alpha-v beta-6 (αvβ6) arginyl-glycinyl-aspartic acid (RGD)-binding integrin. It has been shown to be an informative tool ligand in pre-clinical imaging studies for selective labelling of the αvβ6 integrin in a number of disease models. In a radioligand binding assay using a radiolabelled form of the peptide ([³H]A20FMDV2), its high affinity (K_D: 0.22 nmol/l) and selectivity (at least 85-fold) for αvβ6 over the other members of the RGD integrin family was confirmed. [³H]A20FMDV2 αvβ6 binding could be fully reversed only in the presence of EDTA, whereas a partial reversal was observed in the presence of excess concentrations of an RGD-mimetic small molecule (SC-68448) or unlabelled A20FMDV2. Using flow cytometry on bronchial epithelial cells, the ligand-induced internalization of αvβ6 by A20FMDV2 and latency-associated peptide-1 was shown to be fast (t_1/2: 1.5 and 3.1 min, respectively), concentration-dependent (EC₅₀: values 1.1 and 3.6 nmol/l, respectively) and was followed by a moderately slow return of integrin to the surface. The results of the radioligand binding studies suggest that the binding of A20FMDV2 to the RGD-binding site on αvβ6 is required to maintain its engagement with the hypothesised A20FMDV2 synergy site on the integrin. In addition, there is evidence from flow cytometric studies that the RGD-ligand engagement of αvβ6 post-internalization plays a role in delaying recycling of the integrin to the cell surface. This mechanism may act as a homeostatic control of membrane αvβ6 following RGD ligand engagement.

Choline on the Move: Perspectives on the Molecular Physiology and Pharmacology of the Presynaptic Choline Transporter.

Genetic, biochemical, physiological, and pharmacological approaches have advanced our understanding of cholinergic biology for over 100 years. High-affinity choline uptake (HACU) was one of the last features of cholinergic signaling to be defined at a molecular level, achieved through the cloning of the choline transporter (CHT, SLC5A7). In retrospect, the molecular era of CHT studies initiated with the identification of hemicholinium-3 (HC-3), a potent, competitive CHT antagonist, though it would take another 30 years before HC-3, in radiolabeled form, was used by Joseph Coyle's laboratory to identify and monitor the dynamics of CHT proteins. Though HC-3 studies provided important insights into CHT distribution and regulation, another 15 years would pass before the structure of CHT genes and proteins were identified, a full decade after the cloning of most other neurotransmitter-associated transporters. The availability of CHT gene and protein probes propelled the development of cell and animal models as well as efforts to gain insights into how human CHT gene variation affects the risk for brain and neuromuscular disorders. Most recently, our group has pursued a broadening of CHT pharmacology, elucidating novel chemical structures that may serve to advance cholinergic diagnostics and medication development. Here we provide a short review of the transformation that has occurred in HACU research and how such advances may promote the development of novel therapeutics.