Noninvasive assessment of the lung inflammation-fibrosis axis by targeted imaging of CMKLR1.

Precision management of fibrotic lung diseases is challenging due to their diverse clinical trajectories and lack of reliable biomarkers for risk stratification and therapeutic monitoring. Here, we validated the accuracy of CMKLR1 as an imaging biomarker of the lung inflammation-fibrosis axis. By analyzing single-cell RNA sequencing datasets, we demonstrated CMKLR1 expression as a transient signature of monocyte-derived macrophages (MDMφ) enriched in patients with idiopathic pulmonary fibrosis (IPF). Consistently, we identified MDMφ as the major driver of the uptake of CMKLR1-targeting peptides in a murine model of bleomycin-induced lung fibrosis. Furthermore, CMKLR1-targeted positron emission tomography in the murine model enabled quantification and spatial mapping of inflamed lung regions infiltrated by CMKLR1-expressing macrophages and emerged as a robust predictor of subsequent lung fibrosis. Last, high CMKLR1 expression by bronchoalveolar lavage cells identified an inflammatory endotype of IPF with poor survival. Our investigation supports the potential of CMKLR1 as an imaging biomarker for endotyping and risk stratification of fibrotic lung diseases.

Dietary intake and glutamine-serine metabolism control pathologic vascular stiffness.

Perivascular collagen deposition by activated fibroblasts promotes vascular stiffening and drives cardiovascular diseases such as pulmonary hypertension (PH). Whether and how vascular fibroblasts rewire their metabolism to sustain collagen biosynthesis remains unknown. Here, we found that inflammation, hypoxia, and mechanical stress converge on activating the transcriptional coactivators YAP and TAZ (WWTR1) in pulmonary arterial adventitial fibroblasts (PAAFs). Consequently, YAP and TAZ drive glutamine and serine catabolism to sustain proline and glycine anabolism and promote collagen biosynthesis. Pharmacologic or dietary intervention on proline and glycine anabolic demand decreases vascular stiffening and improves cardiovascular function in PH rodent models. By identifying the limiting metabolic pathways for vascular collagen biosynthesis, our findings provide guidance for incorporating metabolic and dietary interventions for treating cardiopulmonary vascular disease.

ImmunoPET imaging of TIGIT in the glioma microenvironment.

Glioblastoma (GBM) is the most common primary malignant brain tumor. Currently, there are few effective treatment options for GBM beyond surgery and chemo-radiation, and even with these interventions, median patient survival remains poor. While immune checkpoint inhibitors (ICIs) have demonstrated therapeutic efficacy against non-central nervous system cancers, ICI trials for GBM have typically had poor outcomes. TIGIT is an immune checkpoint receptor that is expressed on activated T-cells and has a role in the suppression of T-cell and Natural Killer (NK) cell function. As TIGIT expression is reported as both prognostic and a biomarker for anti-TIGIT therapy, we constructed a molecular imaging agent, [89Zr]Zr-DFO-anti-TIGIT (89Zr-αTIGIT), to visualize TIGIT in preclinical GBM by immunoPET imaging. PET imaging and biodistribution analysis of 89Zr-αTIGIT demonstrated uptake in the tumor microenvironment of GBM-bearing mice. Blocking antibody and irrelevant antibody tracer studies demonstrated specificity of 89Zr-αTIGIT with significance at a late time point post-tracer injection. However, the magnitude of 89Zr-αTIGIT uptake in tumor, relative to the IgG tracer was minimal. These findings highlight the features and limitations of using 89Zr-αTIGIT to visualize TIGIT in the GBM microenvironment.

Evaluation of the pharmacokinetics, dosimetry, and therapeutic efficacy for the α-particle-emitting transarterial radioembolization (αTARE) agent [225Ac]Ac-DOTA-TDA-Lipiodol® against hepatic tumors.

BACKGROUND: The liver is a common site for metastatic disease for a variety of cancers, including colorectal cancer. Both primary and secondary liver tumors are supplied through the hepatic artery while the healthy liver is supplied by the portal vein. Transarterial radioembolization (TARE) using yttrium-90 glass or resin microspheres have shown promising results with reduced side-effects but have similar survival benefits as chemoembolization in patients with hepatocellular carcinoma (HCC). This highlights the need for new novel agents against HCC. Targeted alpha therapy (TAT) is highly potent treatment due to the short range (sparing adjacent normal tissue), and densely ionizing track (high linear energy transfer) of the emitted α-particles. The incorporation of α-particle-emitting radioisotopes into treatment of HCC has been extremely limited, with our recent publication pioneering the field of α-particle-emitting TARE (αTARE). This study focuses on an in-depth evaluation of the αTARE-agent [225Ac]Ac-DOTA-TDA-Lipiodol® as an effective therapeutic agent against HCC regarding pharmacokinetics, dosimetry, stability, and therapeutic efficacy. RESULTS: [225Ac]Ac-DOTA-TDA was shown to be a highly stable with bench-top stability at ≥ 95% radiochemical purity (RCP) over a 3-day period and serum stability was ≥ 90% RCP over 5-days. The pharmacokinetic data showed retention in the tumor of [225Ac]Ac-DOTA-TDA-Lipiodol® and clearance through the normal organs. In addition, the tumor and liver acted as suppliers of the free daughters, which accumulated in the kidneys supplied via the blood. The dose limiting organ was the liver, and the estimated maximum tolerable activity based on the rodents whole-body weight: 728-3641 Bq/g (male rat), 396-1982 Bq/g (male mouse), and 453-2263 Bq/g (female mouse), depending on an RBE-value (range 1-5). Furthermore, [225Ac]Ac-DOTA-TDA-Lipiodol® showed significant improvement in survival for both the male and female mice (median survival 47-days) compared with controls (26-days untreated, and 33-35-days Lipiodol® alone). CONCLUSIONS: This study shows that [225Ac]Ac-DOTA-TDA-Lipiodol® is a stable compound allowing for centralized manufacturing and distribution world-wide. Furthermore, the result of this study support the continue development of evaluation of the αTARE-agent [225Ac]Ac-DOTA-TDA-Lipiodol® as a potential treatment option for treating hepatic tumors.

Immuno-PET Imaging of CD69 Visualizes T-Cell Activation and Predicts Survival Following Immunotherapy in Murine Glioblastoma.

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. Immunotherapy may be promising for the treatment of some patients with GBM; however, there is a need for noninvasive neuroimaging techniques to predict immunotherapeutic responses. The effectiveness of most immunotherapeutic strategies requires T-cell activation. Therefore, we aimed to evaluate an early marker of T-cell activation, CD69, for its use as an imaging biomarker of response to immunotherapy for GBM. Herein, we performed CD69 immunostaining on human and mouse T cells following in vitro activation and post immune checkpoint inhibitors (ICI) in an orthotopic syngeneic mouse glioma model. CD69 expression on tumor-infiltrating leukocytes was assessed using single-cell RNA sequencing (scRNA-seq) data from patients with recurrent GBM receiving ICI. Radiolabeled CD69 Ab PET/CT imaging (CD69 immuno-PET) was performed on GBM-bearing mice longitudinally to quantify CD69 and its association with survival following immunotherapy. We show CD69 expression is upregulated upon T-cell activation and on tumor-infiltrating lymphocytes (TIL) in response to immunotherapy. Similarly, scRNA-seq data demonstrated elevated CD69 on TILs from patients with ICI-treated recurrent GBM as compared with TILs from control cohorts. CD69 immuno-PET studies showed a significantly higher tracer uptake in the tumors of ICI-treated mice compared with controls. Importantly, we observed a positive correlation between survival and CD69 immuno-PET signals in immunotherapy-treated animals and established a trajectory of T-cell activation by virtue of CD69-immuno-PET measurements. Our study supports the potential use of CD69 immuno-PET as an immunotherapy response assessment imaging tool for patients with GBM. Significance: Immunotherapy may hold promise for the treatment of some patients with GBM. There is a need to assess therapy responsiveness to allow the continuation of effective treatment in responders and to avoid ineffective treatment with potential adverse effects in the nonresponders. We demonstrate that noninvasive PET/CT imaging of CD69 may allow early detection of immunotherapy responsiveness in patients with GBM.

Targeted imaging of very late antigen-4 for noninvasive assessment of lung inflammation-fibrosis axis.

BACKGROUND: The lack of noninvasive methods for assessment of dysregulated inflammation as a major driver of fibrosis (i.e., inflammation-fibrosis axis) has been a major challenge to precision management of fibrotic lung diseases. Here, we determined the potential of very late antigen-4 (VLA-4)-targeted positron emission tomography (PET) to detect inflammation in a mouse model of bleomycin-induced fibrotic lung injury. METHOD: Single time-point and longitudinal VLA-4-targeted PET was performed using a high-affinity peptidomimetic radiotracer, 64Cu-LLP2A, at weeks 1, 2, and 4 after bleomycin-induced (2.5 units/kg) lung injury in C57BL/6J mice. The severity of fibrosis was determined by measuring the hydroxyproline content of the lungs and expression of markers of extracellular matrix remodeling. Flow cytometry and histology was performed to determine VLA-4 expression across different leukocyte subsets and their spatial distribution. RESULTS: Lung uptake of 64Cu-LLP2A was significantly elevated throughout different stages of the progression of bleomycin-induced injury. High lung uptake of 64Cu-LLP2A at week-1 post-bleomycin was a predictor of poor survival over the 4-week follow up, supporting the prognostic potential of 64Cu-LLP2A PET during the early stage of the disease. Additionally, the progressive increase in 64Cu-LLP2A uptake from week-1 to week-4 post-bleomycin correlated with the ultimate extent of lung fibrosis and ECM remodeling. Flow cytometry revealed that LLP2A binding was restricted to leukocytes. A combination of increased expression of VLA-4 by alveolar macrophages and accumulation of VLA-4-expressing interstitial and monocyte-derived macrophages as well as dendritic cells was noted in bleomycin-injured, compared to control, lungs. Histology confirmed the increased expression of VLA-4 in bleomycin-injured lungs, particularly in inflamed and fibrotic regions. CONCLUSIONS: VLA-4-targeted PET allows for assessment of the inflammation-fibrosis axis and prediction of disease progression in a murine model. The potential of 64Cu-LLP2A PET for assessment of the inflammation-fibrosis axis in human fibrotic lung diseases needs to be further investigated.

2-deoxy-2-[18F]fluoro-D-glucose Positron Emission Tomography to Monitor Lung Inflammation and Therapeutic Response to Dexamethasone in a Murine Model of Acute Lung Injury.

PURPOSE: To image inflammation and monitor therapeutic response to anti-inflammatory intervention using 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) positron emission tomography (PET) in a preclinical model of acute lung injury (ALI). PROCEDURES: Mice were intratracheally administered lipopolysaccharide (LPS, 2.5 mg/kg) to induce ALI or phosphate-buffered saline as the vehicle control. A subset of mice in the ALI group received two intraperitoneal doses of dexamethasone 1 and 24 h after LPS. [18F]FDG PET/CT was performed 2 days after the induction of ALI. [18F]FDG uptake in the lungs was quantified by PET (%ID/mLmean and standardized uptake value (SUVmean)) and ex vivo γ-counting (%ID/g). The severity of lung inflammation was determined by quantifying the protein level of inflammatory cytokines/chemokines and the activity of neutrophil elastase and glycolytic enzymes. In separate groups of mice, flow cytometry was performed to estimate the contribution of individual immune cell types to the total pulmonary inflammatory cell burden under different treatment conditions. RESULTS: Lung uptake of [18F]FDG was significantly increased during LPS-induced ALI, and a decreased [18F]FDG uptake was observed following dexamethasone treatment to an intermediate level between that of LPS-treated and control mice. Protein expression of inflammatory biomarkers and the activity of neutrophil elastase and glycolytic enzymes were increased in the lungs of LPS-treated mice versus those of control mice, and correlated with [18F]FDG uptake. Furthermore, dexamethasone-induced decreases in cytokine/chemokine protein levels and enzyme activities correlated with [18F]FDG uptake. Neutrophils were the most abundant cells in LPS-induced ALI, and the pattern of total cell burden during ALI with or without dexamethasone therapy mirrored that of [18F]FDG uptake. CONCLUSIONS: [18F]FDG PET noninvasively detects lung inflammation in ALI and its response to anti-inflammatory therapy in a preclinical model. However, high [18F]FDG uptake by bone, brown fat, and myocardium remains a technical limitation for quantification of [18F]FDG in the lungs.

Molecular imaging of chemokine-like receptor 1 (CMKLR1) in experimental acute lung injury.

The lack of techniques for noninvasive imaging of inflammation has challenged precision medicine management of acute respiratory distress syndrome (ARDS). Here, we determined the potential of positron emission tomography (PET) of chemokine-like receptor-1 (CMKLR1) to monitor lung inflammation in a murine model of lipopolysaccharide-induced injury. Lung uptake of a CMKLR1-targeting radiotracer, [64Cu]NODAGA-CG34, was significantly increased in lipopolysaccharide-induced injury, correlated with the expression of multiple inflammatory markers, and reduced by dexamethasone treatment. Monocyte-derived macrophages, followed by interstitial macrophages and monocytes were the major CMKLR1-expressing leukocytes contributing to the increased tracer uptake throughout the first week of lipopolysaccharide-induced injury. The clinical relevance of CMKLR1 as a biomarker of lung inflammation in ARDS was confirmed using single-nuclei RNA-sequencing datasets which showed significant increases in CMKLR1 expression among transcriptionally distinct subsets of lung monocytes and macrophages in COVID-19 patients vs. controls. CMKLR1-targeted PET is a promising strategy to monitor the dynamics of lung inflammation and response to anti-inflammatory treatment in ARDS.

Modular Smart Molecules for PSMA-Targeted Chemotherapy.

New targeted chemotherapeutics are urgently needed to minimize off-target toxicity and reduce the high-mortality rate associated with metastatic prostate cancer. Herein, we report on the modular synthesis, pharmacokinetics, and efficacy of two small-molecule-drug conjugates (SMDC) targeted to prostate-specific membrane antigen (PSMA) incorporating either: (i) a cathepsin-B-cleavable valine-citrulline (Val-Cit), or (ii) an acid-cleavable phosphoramidate linker. Crucial components used in the design of the conjugates include: (i) CTT1298, a nanomolar affinity ligand that binds irreversibly to PSMA and has proven in past studies to rapidly internalize and shuttle payloads into PSMA-expressing prostate cancer cells, (ii) MMAE, a known potent cytotoxic payload, and (iii) an albumin-binder, proven to improve residence time of drug conjugates. At dose of 0.8 mg/kg (∼250 nmol/kg), the two SMDCs showed significant efficacy in a PSMA(+) PC3-PIP mouse model of human prostate cancer compared with controls, without inducing systemic toxicity. Though localization of the SMDCs was observed in tissues apart from the tumor, release of MMAE was observed predominantly in tumor tissue, at levels that were 2-3 orders of magnitude higher than non-target tissues. Furthermore, SMDC2, which incorporated a novel pH-responsive phosporamidate linker, demonstrated significantly improved efficacy over SMDC1 that has a Val-Cit linker, with a 100% survival over 90 days and 4 out of 8 mice showing complete tumor growth inhibition after 6 weekly doses of 0.8 mg/kg (244 nmol/kg). Our findings demonstrate the potential of irreversible PSMA inhibitors combined with pH-responsive linkers as a way to specifically deliver chemotherapeutic drugs to prostate cancer tumors with minimal toxicity.

PET Imaging of VLA-4 in a New BRAFV600E Mouse Model of Melanoma.

PURPOSE: Despite unprecedented responses to immune checkpoint inhibitors and targeted therapy in melanoma, a major subset of patients progresses and have few effective salvage options. We have previously demonstrated robust, selective uptake of the peptidomimetic LLP2A labeled with Cu-64 ([64Cu]-LLP2A) for positron emission tomography (PET) imaging in subcutaneous and metastatic models of B16F10 murine melanoma. LLP2A binds with high affinity to very late antigen-4 (VLA-4, integrin α4ß1), a transmembrane protein overexpressed in melanoma and other cancers that facilitates tumor growth and metastasis. Yet B16F10 fails to faithfully reflect human melanoma biology, as it lacks certain oncogenic driver mutations, including BRAF mutations found in ≥ 50 % of clinical specimens. Here, we evaluated the PET tracer [64Cu]-CB-TE1A1P-PEG4-LLP2A ([64Cu]-LLP2A) in novel, translational BRAFV600E mutant melanoma models differing in VLA-4 expression-BPR (VLA-4-) and BPRα (VLA-4+). PROCEDURES: BPR cells were transduced with α4 (CD49d) to overexpress intact cell surface VLA-4 (BPRα). The binding affinity of [64Cu]-LLP2A to BPR and BPRα cells was determined by saturation binding assays. [64Cu]-LLP2A internalization into B16F10, BPR, and BPRα cells was quantified via a plate-based assay. Tracer biodistribution and PET/CT imaging were evaluated in mice bearing subcutaneous BPR and BPRα tumors. RESULTS: [64Cu]-LLP2A demonstrated high binding affinity to BPRα (Kd = 1.4 nM) but indeterminate binding to BPR cells. VLA-4+ BPRα and B16F10 displayed comparable time-dependent [64Cu]-LLP2A internalization, whereas BPR internalization was undetectable. PET/CT showed increased tracer uptake in BPRα tumors vs. BPR tumors in vivo, which was validated by significantly greater (p < 0.0001) BPRα tumor uptake in biodistribution analyses. CONCLUSIONS: [64Cu]-LLP2A discriminates BPRα (VLA-4+) vs. BPR (VLA-4-) melanomas in vivo, supporting translation of these BRAF-mutated melanoma models via prospective imaging and theranostic studies. These results extend the utility of LLP2A to selectively target clinically relevant and therapy-resistant tumor variants toward its use for therapeutic patient care.

Novel theranostic agent for PET imaging and targeted radiopharmaceutical therapy of tumour-infiltrating immune cells in glioma.

BACKGROUND: Malignant gliomas are deadly tumours with few therapeutic options. Although immunotherapy may be a promising therapeutic strategy for treating gliomas, a significant barrier is the CD11b+ tumour-associated myeloid cells (TAMCs), a heterogeneous glioma infiltrate comprising up to 40% of a glioma's cellular mass that inhibits anti-tumour T-cell function and promotes tumour progression. A theranostic approach uses a single molecule for targeted radiopharmaceutical therapy (TRT) and diagnostic imaging; however, there are few reports of theranostics targeting the tumour microenvironment. METHODS: Utilizing a newly developed bifunctional chelator, Lumi804, an anti-CD11b antibody (αCD11b) was readily labelled with either Zr-89 or Lu-177, yielding functional radiolabelled conjugates for PET, SPECT, and TRT. FINDINGS: 89Zr/177Lu-labeled Lumi804-αCD11b enabled non-invasive imaging of TAMCs in murine gliomas. Additionally, 177Lu-Lumi804-αCD11b treatment reduced TAMC populations in the spleen and tumour and improved the efficacy of checkpoint immunotherapy. INTERPRETATION: 89Zr- and 177Lu-labeled Lumi804-αCD11b may be a promising theranostic pair for monitoring and reducing TAMCs in gliomas to improve immunotherapy responses. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Molecular Imaging of Very Late Antigen-4 in Acute Lung Injury.

Inflammation plays a central role in the pathogenesis of acute lung injury (ALI) during both the acute pneumonitis stage and progression into the chronic fibroproliferative phase, leading to pulmonary fibrosis. Currently, there is an unmet clinical and research need for noninvasive ways to monitor lung inflammation through targeting of immunoregulatory pathways contributing to ALI pathogenesis. In this study, we evaluated the role of targeted imaging of very late antigen-4 (VLA-4), as a key integrin mediating the adhesion and recruitment of immune cells to inflamed tissues, in quantifying lung inflammation in a mouse model of lipopolysaccharide-induced ALI. Methods: ALI was induced by a single intratracheal administration of lipopolysaccharide (10, 20, or 40 µg per mouse) in C57BL/6J mice. Control mice were intratracheally instilled with sterile phosphate-buffered saline. VLA-4-targeted PET/CT was performed 24 h after intravenous injection of a 64Cu-labeled high-affinity peptidomimetic ligand referred to as 64Cu-LLP2A, which is conjugated with the chelator (1,4,8,11-tetraazacyclotetradecane-1-(methane phosphonic acid)-8-(methane carboxylic acid) and a polyethylene glycol 4 linker, at day 2 after the induction of ALI. Ex vivo biodistribution of 64Cu-LLP2A was determined by γ-counting of harvested organs. The severity of lung inflammation was assessed histologically and by measuring the expression of inflammatory markers in the lung tissue lysates using reverse transcription quantitative polymerase chain reaction. Results: Intratracheal lipopolysaccharide instillation led to an acute inflammatory response in the lungs, characterized by increased expression of multiple inflammatory markers and infiltration of myeloid cells, along with a significant and specific increase in 64Cu-LLP2A uptake, predominantly in a peribronchial distribution. There was a strong correlation between the lipopolysaccharide dose and 64Cu-LLP2A uptake, as quantified by in vivo PET (R = 0.69, P < 0.01). Expression levels of both subunits of VLA-4, that is, integrins α4 and ß1, significantly correlated with the expression of multiple inflammatory markers, including tumor necrosis factor-α, interleukin-1ß, and nitric oxide synthase-2, highlighting the potential of VLA-4 as a surrogate marker of acute lung inflammation. Notably, in vivo 64Cu-LLP2A uptake significantly correlated with the expression of multiple inflammatory markers and VLA-4. Conclusion: Our study demonstrates the feasibility of molecular imaging of VLA-4, as a mechanistically relevant target in ALI, and the accuracy of VLA-4-targeted PET in quantification of ongoing lung inflammation in a murine model.

Integrin VLA-4 as a PET imaging biomarker of hyper-adhesion in transgenic sickle mice.

In sickle cell disease (SCD), very late antigen-4 (VLA-4 or integrin α4ß1) mediates the adhesion of reticulocytes to inflamed, proinflammatory endothelium, a key process in promoting vaso-occlusive episodes (VOEs). We hypothesized that a radionuclide tracer targeting VLA-4 could be harnessed as a positron emission tomography (PET) imaging biomarker of VOEs. We tested the VLA-4 peptidomimetic PET tracer 64Cu-CB-TE1A1P-PEG4-LLP2A (64Cu-LLP2A) for imaging hyper-adhesion-associated VOEs in the SCD Townes mouse model. With lipopolysaccharide (LPS)-induced VOEs, 64Cu-LLP2A uptake was increased in the bone marrow of the humeri and femurs, common sites of VOEs in SCD mice compared with non-SCD mice. Treatment with a proven inhibitor of VOEs (the anti-mouse anti-P-selectin monoclonal antibody [mAb] RB40.34) during LPS stimulation led to a reduction in the uptake of 64Cu-LLP2A in the humeri and femurs to baseline levels, implying blockade of VOE hyper-adhesion. Flow cytometry with Cy3-LLP2A demonstrated an increased percentage of VLA-4-positive reticulocytes in SCD vs non-SCD mice in the bone and peripheral blood after treatment with LPS, which was abrogated by anti-P-selectin mAb treatment. These data, for the first time, show in vivo imaging of VLA-4-mediated hyper-adhesion, primarily of SCD reticulocytes, during VOEs. PET imaging with 64Cu-LLP2A may serve as a valuable, noninvasive method for identifying sites of vaso-occlusion and may provide an objective biomarker of disease severity and anti-P-selectin treatment efficacy in patients with SCD.

Validation of Ultrasound Super-Resolution Imaging of Vasa Vasorum in Rabbit Atherosclerotic Plaques.

Acute coronary syndromes and strokes are mainly caused by atherosclerotic plaque (AP) rupture. Abnormal increase of vasa vasorum (VV) is reported as a key evidence of plaque progression and vulnerability. However, due to their tiny size, it is still challenging to noninvasively identify VV near the major vessels. Ultrasound super resolution (USR), a technique that provides high spatial resolution beyond the acoustic diffraction limit, demonstrated an adequate spatial resolution for VV detection in early studies. However, a thorough validation of this technology in the plaque model is particularly needed in order to continue further extended preclinical studies. In this letter, we present an experiment protocol that verifies the USR technology for VV identification with subsequent histology and ex vivo micro-computed tomography ( µ CT). Deconvolution-based USR imaging was applied on two rabbits to identify the VV near the AP in the femoral artery. Histology and ex vivo µ CT imaging were performed on excised femoral tissue to validate the USR technique both pathologically and morphologically. This established validation protocol could facilitate future extended preclinical studies toward the clinical translation of USR imaging for VV identification.

Preclinical Dosimetry, Imaging, and Targeted Radionuclide Therapy Studies of Lu-177-Labeled Albumin-Binding, PSMA-Targeted CTT1403.

PURPOSE: Prostate-specific membrane antigen (PSMA) continues to be the hallmark biomarker for prostate cancer as it is expressed on nearly all prostatic tumors. In addition, increased PSMA expression correlates with castration resistance and progression to the metastatic stage of the disease. Recently, we combined both an albumin-binding motif and an irreversible PSMA inhibitor to develop the novel PSMA-targeted radiotherapeutic agent, CTT1403. This molecule was novel in the field of PSMA-targeted agents as its key motifs resulted in extended blood circulation time and tumor uptake, rapid and extensive internalization into PSMA+ cells, and promising therapeutic efficacy. The objective of this study was to perform IND-enabling translational studies on CTT1403 in rodent models. PROCEDURES: A dose optimization study was performed in PC3-PIP (PSMA+) tumor-bearing mice. Treatment groups were randomly selected to receive one to three 14-MBq injections of CTT1403. Control groups included (1) saline, (2) non-radioactive [175Lu]CTT1403, or (3) two injections of 14 MBq CTT1751, a Lu-177-labeled non-targeted albumin-binding moiety. Tumor growth was monitored up to 120 days. Small-animal single photon emission tomography/X-ray computed tomography imaging was performed with CTT1403 and CTT1751 in PC3-PIP tumor-bearing mice to visualize infiltration of the Lu-177-labeled agent into the tumor. In preparation for a first-in-human study, human absorbed doses were estimated based on rat biodistribution out to 5 weeks to determine a safe CTT1403 therapy dose in humans. RESULTS: Two to 3 injections of 14 MBq CTT1403 yielded significant tumor growth inhibition and increased survival compared with all control groups and mice receiving 1 injection of 14 MBq CTT1403. Five of 12 mice receiving 2 or 3 injections of CTT1403 survived to the 120-day post-treatment study endpoint. Dosimetry identified the kidneys as the dose-limiting organ, with an equivalent dose of 5.18 mSv/MBq, resulting in a planned maximum dose of 4.4 GBq for phase 1 clinical trials. CONCLUSIONS: The preclinical efficacy and dosimetry of CTT1403 suggest that this agent has significant potential to be safe and effective in humans.

A Novel Sulforaphane-Regulated Gene Network in Suppression of Breast Cancer-Induced Osteolytic Bone Resorption.

Bone is the most preferred site for colonization of metastatic breast cancer cells for each subtype of the disease. The standard of therapeutic care for breast cancer patients with bone metastasis includes bisphosphonates (e.g., zoledronic acid), which have poor oral bioavailability, and a humanized antibody (denosumab). However, these therapies are palliative, and a subset of patients still develop new bone lesions and/or experience serious adverse effects. Therefore, a safe and orally bioavailable intervention for therapy of osteolytic bone resorption is still a clinically unmet need. This study demonstrates suppression of breast cancer-induced bone resorption by a small molecule (sulforaphane, SFN) that is safe clinically and orally bioavailable. In vitro osteoclast differentiation was inhibited in a dose-dependent manner upon addition of conditioned media from SFN-treated breast cancer cells representative of different subtypes. Targeted microarrays coupled with interrogation of The Cancer Genome Atlas data set revealed a novel SFN-regulated gene signature involving cross-regulation of runt-related transcription factor 2 (RUNX2) and nuclear factor-κB and their downstream effectors. Both RUNX2 and p65/p50 expression were higher in human breast cancer tissues compared with normal mammary tissues. RUNX2 was recruited at the promotor of NFKB1 Inhibition of osteoclast differentiation by SFN was augmented by doxycycline-inducible stable knockdown of RUNX2. Oral SFN administration significantly increased the percentage of bone volume/total volume of affected bones in the intracardiac MDA-MB-231-Luc model indicating in vivo suppression of osteolytic bone resorption by SFN. These results indicate that SFN is a novel inhibitor of breast cancer-induced osteolytic bone resorption in vitro and in vivo.

Prevention of breast cancer-induced osteolytic bone resorption by benzyl isothiocyanate.

Osteolytic bone resorption is the primary cause of pain and suffering (e.g. pathological bone fracture) in women with metastatic breast cancer. The current standard of care for patients with bone metastasis for reducing the incidence of skeletal complications includes bisphosphonates and a humanized antibody (denosumab). However, a subset of patients on these therapies still develops new bone metastasis or experiences adverse effects. Moreover, some bisphosphonates have poor oral bioavailability. Therefore, orally-bioavailable and non-toxic inhibitors of breast cancer-induced osteolytic bone resorption are still clinically desirable. We have shown previously that benzyl isothiocyanate (BITC) decreases the incidence of breast cancer in a transgenic mouse model without any side effects. The present study provides in vivo evidence for inhibition of breast cancer-induced osteolytic bone resorption by BITC. Plasma achievable doses of BITC (0.5 and 1 µM) inhibited in vitro osteoclast differentiation induced by co-culture of osteoclast precursor cells (RAW264.7) and breast cancer cells representative of different subtypes. This effect was accompanied by downregulation of key mediators of osteoclast differentiation, including receptor activator of nuclear factor-κB ligand and runt-related transcription factor 2 (RUNX2), in BITC-treated breast cancer cells. Doxycycline-inducible knockdown of RUNX2 augmented BITC-mediated inhibition of osteoclast differentiation. Oral administration of 10 mg BITC/kg body weight, 5 times per week, inhibited MDA-MB-231-induced skeletal metastasis multiplicity by ~81% when compared with control (P = 0.04). The present study indicates that BITC has the ability to inhibit breast cancer-induced osteolytic bone resorption in vivo.

177Lu-Labeled Phosphoramidate-Based PSMA Inhibitors: The Effect of an Albumin Binder on Biodistribution and Therapeutic Efficacy in Prostate Tumor-Bearing Mice.

Prostate-specific membrane antigen (PSMA) continues to be an active biomarker for small-molecule PSMA-targeted imaging and therapeutic agents for prostate cancer and various non-prostatic tumors that are characterized by PSMA expression on their neovasculature. One of the challenges for small-molecule PSMA inhibitors with respect to delivering therapeutic payloads is their rapid renal clearance. In order to overcome this pharmacokinetic challenge, we outfitted a 177Lu-labeled phosphoramidate-based PSMA inhibitor (CTT1298) with an albumin-binding motif (CTT1403) and compared its in vivo performance with that of an analogous compound lacking the albumin-binding motif (CTT1401). The radiolabeling of CTT1401 and CTT1403 was achieved using click chemistry to connect 177Lu-DOTA-N3 to the dibenzocyclooctyne (DBCO)-bearing CTT1298 inhibitor cores. A direct comparison in vitro and in vivo performance was made for CTT1401 and CTT1403; the specificity and efficacy by means of cellular uptake and internalization, biodistribution, and therapeutic efficacy were determined for both compounds. While both compounds displayed excellent uptake and rapid internalization in PSMA+ PC3-PIP cells, the albumin binding moiety in CTT1403 conferred clear advantages to the PSMA-inhibitor scaffold including increased circulating half-life and prostate tumor uptake that continued to increase up to 168 h post-injection. This increased tumor uptake translated into superior therapeutic efficacy of CTT1403 in PSMA+ PC3-PIP human xenograft tumors.

Secreted Phosphoprotein 1 and Sex-Specific Differences in Silica-Induced Pulmonary Fibrosis in Mice.

BACKGROUND: Fibrotic lung diseases occur predominantly in males, and reports describe better survival in affected females. Male mice are more sensitive to silica-induced lung fibrosis than silica-treated female mice. Secreted phosphoprotein 1 (SPP1, also known as osteopontin) increases in pulmonary fibrosis, and Spp1 transcription may be regulated by estrogen or estrogen receptor-related receptors. OBJECTIVE: We determined whether differences in silica-induced SPP1 levels contribute to sex differences in lung fibrosis. METHODS: Male and female mice were treated with 0.2 g/kg intratracheal silica, and lung injury was assessed 1, 3, or 14 days post-exposure. Gene-targeted (Spp1-/-) mice, control Spp1+/+ (C57BL/6J) mice, ovariectomized (OVX) female mice, and estrogen-treated male mice were treated with silica, and lung injury was assessed. RESULTS: Silica-induced SPP1 in lung tissue, bronchoalveolar lavage, and serum increased more in male than in female mice. Following silica treatment, bronchoalveolar lavage cell infiltrates decreased in female Spp1-/- mice compared with female Spp1+/+ mice, and lung hydroxyproline decreased in male Spp1-/- mice compared with male Spp1+/+ mice. OVX female mice had increased lung SPP1 expression in response to silica compared with silica-treated sham female mice. Silica-induced lung collagen and hydroxyproline (markers of fibrosis), and SPP1 levels decreased in estrogen-treated males compared with untreated males. CONCLUSION: These findings suggest that sex-specific differences in SPP1 levels contribute to the differential sensitivity of male and female mice to the development of silica-induced fibrosis. CITATION: Latoche JD, Ufelle AC, Fazzi F, Ganguly K, Leikauf GD, Fattman CL. 2016. Secreted phosphoprotein 1 and sex-specific differences in silica-induced pulmonary fibrosis in mice. Environ Health Perspect 124:1199-1207; http://dx.doi.org/10.1289/ehp.1510335.