A generator-produced gallium-68 radiopharmaceutical for PET imaging of myocardial perfusion

Lipophilic cationic technetium-99m-complexes are widely used for myocardial perfusion imaging (MPI). However, inherent uncertainties in the supply chain of molybdenum-99, the parent isotope required for manufacturing 99Mo/99mTc generators, intensifies the need for discovery of novel MPI agents incorporating alternative radionuclides. Recently, germanium/gallium (Ge/Ga) generators capable of producing high quality 68Ga, an isotope with excellent emission characteristics for clinical PET imaging, have emerged. Herein, we report a novel 68Ga-complex identified through mechanism-based cell screening that holds promise as a generator-produced radiopharmaceutical for PET MPI

A blood-based diagnostic test incorporating plasma Aβ42/40 ratio, ApoE proteotype, and age accurately identifies brain amyloid status: Findings from a multi cohort validity analysis

BACKGROUND: The development of blood-based biomarker tests that are accurate and robust for Alzheimer\u27s disease (AD) pathology have the potential to aid clinical diagnosis and facilitate enrollment in AD drug trials. We developed a high-resolution mass spectrometry (MS)-based test that quantifies plasma Aβ42 and Aβ40 concentrations and identifies the ApoE proteotype. We evaluated robustness, clinical performance, and commercial viability of this MS biomarker assay for distinguishing brain amyloid status. METHODS: We used the novel MS assay to analyze 414 plasma samples that were collected, processed, and stored using site-specific protocols, from six independent US cohorts. We used receiver operating characteristic curve (ROC) analyses to assess assay performance and accuracy for predicting amyloid status (positive, negative, and standard uptake value ratio; SUVR). After plasma analysis, sites shared brain amyloid status, defined using diverse, site-specific methods and cutoff values; amyloid PET imaging using various tracers or CSF Aβ42/40 ratio. RESULTS: Plasma Aβ42/40 ratio was significantly (p \u3c 0.001) lower in the amyloid positive vs. negative participants in each cohort. The area under the ROC curve (AUC-ROC) was 0.81 (95% CI = 0.77-0.85) and the percent agreement between plasma Aβ42/40 and amyloid positivity was 75% at the optimal (Youden index) cutoff value. The AUC-ROC (0.86; 95% CI = 0.82-0.90) and accuracy (81%) for the plasma Aβ42/40 ratio improved after controlling for cohort heterogeneity. The AUC-ROC (0.90; 95% CI = 0.87-0.93) and accuracy (86%) improved further when Aβ42/40, ApoE4 copy number and participant age were included in the model. CONCLUSIONS: This mass spectrometry-based plasma biomarker test: has strong diagnostic performance; can accurately distinguish brain amyloid positive from amyloid negative individuals; may aid in the diagnostic evaluation process for Alzheimer\u27s disease; and may enhance the efficiency of enrolling participants into Alzheimer\u27s disease drug trials

Second-Generation Triple Reporter for Bioluminescence, Micro–Positron Emission Tomography, and Fluorescence Imaging

Bioluminescence, positron emission tomography (PET), and fluorescence modalities are currently available for noninvasive imaging in vivo, each with its own merits. To exploit the combined strengths of each and facilitate multimodality imaging, we engineered a dual-reporter construct in which firefly luciferase (FLuc) and a 12–amino acid nonstructural linker were fused in frame to the N-terminus of a mutant herpes simplex virus thymidine kinase (mNLS-SR39TK) kinetically enhanced for positron emission tomography (PET). Furthermore, a triple-reporter construct was developed in which monster green fluorescent protein (MGFP), a recently available enhanced fluorescent protein, was introduced into the fusion vector downstream of an internal ribosome entry site (IRES) to allow analysis by fluorescence microscopy or flow cytometry without compromising the specific activities of the upstream fusion components. FLuc bioluminescence was measured with a cooled charge-coupled device camera and mNLS-SR39TK activity by 9-[4-[18F]fluoro-3-(hydroxymethyl) butyl guanine (18F-FHBG) microPET or 3H-penciclovir net accumulation. Importantly, HeLa cells transiently transfected with the FLuc-mNLS-SR39TK-IRES-MGFP triple reporter retained the same specific activities of the FLuc-mNLS-SR39TK heteroenzyme and the individual unfused enzymes with no change in protein half-lives. The presence of the IRES-MGFP modestly decreased upstream heteroprotein expression. In living mice, somatic gene transfer of a ubiquitin promoter-driven FLuc-mNLS-SR39TK-IRES-MGFP plasmid showed a > 1,000-fold increase in liver photon flux and a > 2-fold increase in liver retention of 18F-FHBG by microPET compared with mice treated with control plasmid. Multifocal hepatocellular fluorescence was readily observed by standard confocal microscopy. This second-generation triple reporter incorporating enhanced components enables bioluminescence, PET, and fluorescence imaging of cells and living animals

A New Nucleoside Analogue with Potent Activity against Mutant sr39 Herpes Simplex Virus-1 (HSV-1) Thymidine Kinase (TK)

Nucleoside analogues, such as penciclovir, ganciclovir, acyclovir, and their fluoro-substituted derivatives, have wide utility as antivirals. Among these analogues, FHBG (¹⁸F-Fluorohydroxybutylguanine) is a well-validated PET (positron emission tomography) probe for monitoring reporter gene expression. To evaluate whether or not imposing rigidity into the flexible side chain of FHBG 4 could also impact its interaction, with amino acid residues within the binding site of HSV1-TK (Herpes Simplex Virus-1 Thymidine Kinase), thus influencing its cytotoxic activity. Herein, the synthesis of a new fluorinated nucleoside analogue <b>6</b> (conceived via ligand-docking studies) is reported. Agent <b>6</b> demonstrates selective activity against HeLa cells stably transfected with mutant HSV1-sr39TK and is also 47-fold more potent than FHBG

Characterization of ⁶⁷Ga-labeled 5a <i>in cellulo</i>.

<p><b>A.</b> Cellular accumulation of <b>5a</b> in human MCF-7 cells (Pgp-) and stably transfected MCF-7/<i>MDR1</i> (Pgp+) cells. <i>Inset</i>: Immunodetectable levels of Pgp in plasma membrane preparations using mAb C219; MCF-7 cells (lane 1) and MCF-7/<i>MDR1</i> cells (lane 2). Arrows demarcate Pgp (170 kDa) and γ-tubulin (γ-t) loading control. <b>B.</b> Cellular accumulation of <b>5a</b> in KB-3–1 cells (Pgp-) and KB-8–5 (Pgp+). Shown are net uptakes at 90 min (fmol (mg protein)⁻¹ (nM₀)⁻¹) using control buffer in the absence (open column) or presence (solid column) of the MDR modulator LY335979 (1 µM). Each column represents the mean of 4 determinations; bar denotes ±SEM.</p

Pharmacokinetic data of 5a in WT and <i>mdr1a/1b^(−/−)</i> mice (n = 3).

<p>In WT mice, <b>5a</b> clears from the liver and does not penetrate significantly into the brain, but shows high and stable retention in heart tissue (a Pgp negative organ) in both WT and <i>mdr1a/1b^(−/−)</i> mice. Values represent the mean of 3 determinations; bar denotes ±SEM.</p

Representative metabolite analysis of ⁶⁷Ga-labeled 5a at 60 min post injection into a Sprague-Dawley rat (a time point compatible with ⁶⁸Ga imaging).

<p>Peaks are offset for clarity. Note the presence of a parental compound in all tissues.</p

Biodistribution Data (%ID/g) for ⁶⁷Ga-Complex <b>5a</b> in <i>mdr1a/1b</i>^(−/−) mice (n = 3).

<p>Biodistribution Data (%ID/g) for ⁶⁷Ga-Complex <b>5a</b> in <i>mdr1a/1b</i>^(−/−) mice (n = 3).</p

Representative MicroPET/CT Images.

<p>Sprague-Dawley rats (n = 3) were injected intravenously with HPLC-purified ⁶⁸Ga-labeled <b>5b</b> (600 µCi) and images were acquired 60 min post injection. <b>Top Panel</b>, microPET images co-registered with CT; <b>Lower Panel</b>, microPET images alone. Note retention of <b>5b</b> in the heart (white arrow) and rapid clearance of the tracer from the liver (white arrow head) into the intestines, properties ideal for a myocardial perfusion imaging agent.</p

Projection view of the cationic gallium(III) complex Ga-[3-isopropoxy-ENBDMPI]⁺ (4), with the iodide (I⁻) counteranion, showing the crystallographic numbering scheme.

<p>Projection view of the cationic gallium(III) complex Ga-[3-isopropoxy-ENBDMPI]⁺ (4), with the iodide (I⁻) counteranion, showing the crystallographic numbering scheme.</p