Design and Investigation of a [¹⁸F]-Labeled Benzamide Derivative as a High Affinity Dual Sigma Receptor Subtype Radioligand for Prostate Tumor Imaging

High overexpression of sigma (σ) receptors (σ₁ and σ₂ subtypes) in a variety of human solid tumors has prompted the development of σ receptor-targeting radioligands, as imaging agents for tumor detection. A majority of these radioligands to date target the σ₂ receptor, a potential marker of tumor proliferative status. The identification of approximately equal proportions of both σ receptor subtypes in prostate tumors suggests that a high affinity, dual σ receptor-targeting radioligand could potentially provide enhanced tumor targeting efficacy in prostate cancer. To accomplish this goal, we designed a series of ligands which bind to both σ receptor subtypes with high affinity. Ligand <b>3a</b> in this series, displaying optimal dual σ receptor subtype affinity (σ₁, 6.3 nM; σ₂, 10.2 nM) was radiolabeled with fluorine-18 (¹⁸F) to give [¹⁸F]<b>3a</b> and evaluated as a σ receptor-targeting radioligand in the mouse PC-3 prostate tumor model. Cellular assays with PC-3 cells demonstrated that a major proportion of [¹⁸F]<b>3a</b> was localized to cell surface σ receptors, while ∼10% of [¹⁸F]<b>3a</b> was internalized within cells after incubation for 3.5 h. Serial PET imaging in mice bearing PC-3 tumors revealed that uptake of [¹⁸F]<b>3a</b> was 1.6 ± 0.8, 4.4 ± 0.3, and 3.6 ± 0.6% ID/g (% injection dose per gram) in σ receptor-positive prostate tumors at 15 min, 1.5 h, and 3.5 h postinjection, respectively (<i>n</i> = 3) resulting in clear tumor visualization. Blocking studies conducted with haloperidol (a nonselective inhibitor for both σ receptor subtypes) confirmed that the uptake of [¹⁸F]<b>3a</b> was σ receptor-mediated. Histology analysis confirmed similar expression of σ₁ and σ₂ in PC-3 tumors which was significantly greater than its expression in normal organs/tissues such as liver, kidney, and muscle. Metabolite studies revealed that >50% of radioactivity in PC-3 tumors at 30 min postinjection represented intact [¹⁸F]<b>3a</b>. Prominent σ receptor-specific uptake of [¹⁸F]<b>3a</b> in prostate tumors and its subsequent clear visualization with PET imaging indicate potential utility for the diagnosis of prostate carcinoma

Discovery of Bifunctional Oncogenic Target Inhibitors against Allosteric Mitogen-Activated Protein Kinase (MEK1) and Phosphatidylinositol 3‑Kinase (PI3K)

The synthesis of a series of single entity, bifunctional MEK1/PI3K inhibitors achieved by covalent linking of structural analogs of the ATP-competitive PI3K inhibitor ZSTK474 and the ATP-noncompetitive MEK inhibitor PD0325901 is described. Inhibitors displayed potent in vitro inhibition of MEK1 (0.015 < IC₅₀ (nM) < 56.7) and PI3K (54 < IC₅₀ (nM) < 341) in enzymatic inhibition assays. Concurrent MEK1 and PI3K inhibition was demonstrated with inhibitors <b>9</b> and <b>14</b> in two tumor cell lines (A549, D54). Inhibitors produced dose-dependent decreased cell viability similar to the combined administration of equivalent doses of ZSTK474 and PD0325901. In vivo efficacy of <b>14</b> following oral administration was demonstrated in D54 glioma and A549 lung tumor bearing mice. Compound <b>14</b> showed a 95% and 67% inhibition of tumor ERK1/2 and Akt phosphorylation, respectively, at 2 h postadministration by Western blot analysis, confirming the bioavailability and efficacy of this bifunctional inhibitor strategy toward combined MEK1/PI3K inhibition

<i>In Vivo</i> Targeting and Positron Emission Tomography Imaging of Tumor with Intrinsically Radioactive Metal–Organic Frameworks Nanomaterials

Nanoscale metal–organic frameworks (nMOF) materials represent an attractive tool for various biomedical applications. Due to the chemical versatility, enormous porosity, and tunable degradability of nMOFs, they have been adopted as carriers for delivery of imaging and/or therapeutic cargos. However, the relatively low stability of most nMOFs has limited practical <i>in vivo</i> applications. Here we report the production and characterization of an intrinsically radioactive UiO-66 nMOF (⁸⁹Zr-UiO-66) with incorporation of positron-emitting isotope zirconium-89 (⁸⁹Zr). ⁸⁹Zr-UiO-66 was further functionalized with pyrene-derived polyethylene glycol (Py–PGA-PEG) and conjugated with a peptide ligand (F3) to nucleolin for targeting of triple-negative breast tumors. Doxorubicin (DOX) was loaded onto UiO-66 with a relatively high loading capacity (1 mg DOX/mg UiO-66) and served as both a therapeutic cargo and a fluorescence visualizer in this study. Functionalized ⁸⁹Zr-UiO-66 demonstrated strong radiochemical and material stability in different biological media. Based on the findings from cellular targeting and <i>in vivo</i> positron emission tomography (PET) imaging, we can conclude that ⁸⁹Zr-UiO-66/Py–PGA-PEG-F3 can serve as an image-guidable, tumor-selective cargo delivery nanoplatform. In addition, toxicity evaluation confirmed that properly PEGylated UiO-66 did not impose acute or chronic toxicity to the test subjects. With selective targeting of nucleolin on both tumor vasculature and tumor cells, this intrinsically radioactive nMOF can find broad application in cancer theranostics

Structure-Guided Design and Initial Studies of a Bifunctional MEK/PI3K Inhibitor (ST-168)

The structure-based design of a new single entity, MEK/PI3K bifunctional inhibitor (<b>7</b>, <b>ST-168</b>), which displays improved MEK1 and PI3K isoform inhibition, is described. <b>ST-168</b> demonstrated a 2.2-fold improvement in MEK1 inhibition and a 2.8-, 2.7-, 23-, and 2.5-fold improved inhibition toward the PI3Kα, PI3Kβ, PI3Kδ, and PI3Kγ isoforms, respectively, as compared to a previous lead compound (<b>4</b>; <b>ST-162</b>) in <i>in vitro</i> enzymatic inhibition assays. <b>ST-168</b> demonstrated superior tumoricidal efficacy over <b>ST-162</b> in an A375 melanoma spheroid tumor model. <b>ST-168</b> was comparatively more effective than <b>ST-162</b> in promoting tumor control when administrated orally in a tumor therapy study conducted in an A375 melanoma mouse model confirming its bioavailability and efficacy toward combined <i>in vivo</i> MEK1/PI3K inhibition

<i>In vivo</i> imaging of therapeutic response using contrast enhanced diffusion weighted MRI.

<p>(A) MR gadolinium (Gd) contrast-enhanced T1-weighted images and ADC color overlay maps demonstrate effects of <i>in vivo</i> effects of treatment with obtained from representative control, perifosine, CCI-779 and combination (perifosine + CCI-779) treated glioma mice at 5-7 days post-initiation of therapy. (B) Plot of mean MRI-determined tumor volumes based upon Gd-contrast enhanced regions versus time post-treatment initiation for control, perifosine, CCI-779 and combination (perifosine + CCI-779) treated mice. (Error bars ± SEM). (C) Plot of percent change of mean ADC values versus time post-treatment initiation for control, perifosine, CCI-779 and combination (perifosine + CCI-779) treated mice. (Error bars ± SEM).</p

Plots of the percent change in tumor volume and normalized ADC (ADC) for each of the treatment groups in <i>Study 2</i>.

<p>Presented are treatment groups (A) Control, (B) irradiation (IR), (C) gemcitabine (GEM) and (D) combination gemcitabine and irradiation (GEM+IR). Data is presented over the first week of the study as the mean ± SEM.</p

MR, histological images and western blots are presented from representative animals in <i>Study 1</i> treatment groups.

<p>(A) MRI data consists of anatomical contrast-enhancing T1-weighted images and ADC maps. Histological stains provide information on tumor cellularity (H&E) and apoptosis (cleaved Caspase-3). All data were acquired at day 7 post-treatment initiation. (B) Representative western blot for the detection of cleaved Caspase 3 in tumor tissue from all treatment groups. B-Actin was used as a loading control to ensure proper loading of the protein samples. The tumor tissue from all groups was acquired at day 2 post-treatment initiation.</p

Treatment schedule and Kaplan-Meier survival plots are presented for each therapy in <i>Study 2</i>.

<p>(A) Treatment schedule schematic for <i>Study 2</i>. Animals were randomized into four groups: control, IR, GEM and GEM+IR. Animals of the control group received vehicle 2 days a week for 2 weeks. Animals in the IR group received 1 Gy for 5 days as week with a two day break between treatment blocks for 2 weeks. The GEM group received 10 mg/kg GEM in saline i.p., and GEM+IR received GEM i.p. followed by 1 Gy with a 3 hour lag time between treatments. Control vehicle and GEM administration occurred every third day for a total of four doses. Arrows indicate the day of treatment. (B) Treatment groups are Controls, irradiation (IR), gemcitabine (GEM) and combination gemcitabine and irradiation (GEM+IR).</p

MR and histological images and western blots are presented from representative animals in <i>Study 2</i> treatment groups.

<p>(A) MRI data consists of anatomical contrast-enhancing T1-weighted images and ADC maps. Histological stains provide information on tumor cellularity (H&E) and apoptosis (caspase-3). All data were acquired at day 7 post-treatment initiation. (B) Tumor tissue from animals left untreated or treated with GEM, IR and GEM+IR at day two post-treatment initiation was assessed for cleaved Caspase 3. Western blot of representative animal tissue is shown and proper loading of protein samples was ensured by probing for Gapdh.</p

PDGF-B-driven glimoas treated with the combination of perifosine and CCI-779 undergo cell death and have decreased proliferation.

<p>Images of H&E, IHC with anti-p-S6RP, anti-PCNA, anti-Ki67, and TUNEL of (A) PTEN +/+ and (B) PTEN -/- GBMs after glioma-bearing mice were treated <i>in vivo</i> for 5 days with either vehicle, 30 mg/Kg perifosine, 40 mg/Kg CCI-779, or a combination of 30 mg/Kg perifosine with 40 mg/Kg CCI-779. PCNA and Ki67 images are 200x with the black bar indicating 100 microns. The graph on the right shows quantification of Ki67 and TUNEL staining for 3-4 independent tumors. *,**, and *** represent significance determined by ANOVA analysis for p>0.05, p>0.01, and p>0.001.</p