Innate immune activation plays a critical role in the onset and progression of many diseases. While positron emission tomography (PET) imaging provides a non-invasive means to visualize and quantify such immune responses, most available tracers are not specific for innate immune cells. To address this need, we developed [18F]OP-801 by radiolabeling a novel hydroxyl dendrimer that is selectively taken up by reactive macrophages/microglia and evaluated its ability to detect innate immune activation in mice following lipopolysaccharide (LPS) challenge. OP-801 was radiolabeled in two steps: [18F]fluorination of a tosyl precursor to yield [18F]3-fluoropropylazide, followed by a copper-catalyzed click reaction. After purification and stability testing, [18F]OP-801 (150-250 μCi) was intravenously injected into female C57BL/6 mice 24 h after intraperitoneal administration of LPS (10 mg/kg, n=14) or saline (n=6). Upon completing dynamic PET/CT imaging, mice were perfused, and radioactivity was measured in tissues of interest via gamma counting or autoradiography. [18F]OP-801 was produced with >95% radiochemical purity, 12-52 μCi/μg specific activity, and 4.3±1.5% decay-corrected yield. Ex vivo metabolite analysis of plasma samples (n=4) demonstrated high stability in mice (97±3% intact tracer >120 min post-injection). PET/CT images of mice following LPS challenge revealed higher signal in organs known to be inflamed in this context, including the liver, lung, and spleen. Gamma counting confirmed PET findings, showing significantly elevated signal in the same tissues compared to saline-injected mice: the liver (p=0.009), lung (p=0.030), and spleen (p=0.004). Brain PET/CT images (summed 50-60 min) revealed linearly increasing [18F]OP-801 uptake in the whole brain that significantly correlated with murine sepsis score (r=0.85, p<0.0001). Specifically, tracer uptake was significantly higher in the brain stem, cortex, olfactory bulb, white matter, and ventricles of LPS-treated mice compared to saline-treated mice (p<0.05). [18F]OP-801 is a promising new PET tracer for sensitive and specific detection of activated macrophages and microglia that warrants further investigation.

Abstract Purpose Innate immune activation plays a critical role in the onset and progression of many diseases. While positron emission tomography (PET) imaging provides a non-invasive means to visualize and quantify such immune responses, most available tracers are not specific for innate immune cells. To address this need, we developed [18F]OP-801 by radiolabeling a novel hydroxyl dendrimer that is selectively taken up by reactive macrophages/microglia and evaluated its ability to detect innate immune activation in mice following lipopolysaccharide (LPS) challenge. Procedures: OP-801 was radiolabeled in two steps: [18F]fluorination of a tosyl precursor to yield [18F]3-fluoropropyl azide, followed by a copper-catalyzed click reaction. After purification and stability testing, [18F]OP-801 (150–250 µCi) was intravenously injected into female C57BL/6 mice 24 hours after intraperitoneal administration of LPS (10 mg/kg, n = 14) or saline (n = 6). Upon completing dynamic PET/CT imaging, mice were perfused and radioactivity was measured in tissues of interest via gamma counting or autoradiography. Results [18F]OP-801 was produced with &gt; 95% radiochemical purity, 12–52 µCi/µg specific activity, and 4.3 ± 1.5% decay-corrected yield. Ex vivo metabolite analysis of plasma samples (n = 4) demonstrated high stability in mice (97 ± 3% intact tracer &gt; 120 min post-injection). PET/CT images of mice following LPS challenge revealed higher signal in organs known to be inflamed in this context, including liver, lung, and spleen. Gamma counting confirmed PET findings, showing significantly elevated signal in the same tissues compared to saline-injected mice: liver (p = 0.009), lung (p = 0.030), and spleen (p = 0.004). Brain PET/CT images (summed 50–60 min) revealed linearly increasing [18F]OP-801 uptake in whole brain that significantly correlated with murine sepsis score (r = 0.85, p &lt; 0.0001). Specifically, tracer uptake was significantly higher in the brain stem, cortex, olfactory bulb, white matter, and ventricles of LPS-treated mice compared to saline-treated mice (p &lt; 0.05). Conclusion [18F]OP-801 is a promising new PET tracer for sensitive and specific detection of activated macrophages and microglia that warrants further investigation.

Positron emission tomography (PET) is a powerful tool for studying neuroinflammatory diseases; however, current PET biomarkers of neuroinflammation possess significant limitations. We recently reported a promising dendrimer PET tracer ([18F]OP-801), which is selectively taken up by reactive microglia and macrophages. Here, we describe further important characterization of [18F]OP-801 in addition to optimization and validation of a two-step clinical radiosynthesis. [18F]OP-801 was found to be stable in human plasma for 90 min post incubation, and human dose estimates were calculated for 24 organs of interest; kidneys and urinary bladder wall without bladder voiding were identified as receiving the highest absorbed dose. Following optimization detailed herein, automated radiosynthesis and quality control (QC) analyses of [18F]OP-801 were performed in triplicate in suitable radiochemical yield (6.89 ± 2.23% decay corrected), specific activity (37.49 ± 15.49 GBq/mg), and radiochemical purity for clinical imaging. Importantly, imaging mice with tracer (prepared using optimized methods) 24 h following the intraperitoneal injection of liposaccharide resulted in the robust brain PET signal. Cumulatively, these data enable clinical translation of [18F]OP-801 for imaging reactive microglia and macrophages in humans. Data from three validation runs of the clinical manufacturing and QC were submitted to the Food and Drug Administration (FDA) as part of a Drug Master File (DMF). Subsequent FDA approval to proceed was obtained, and a phase 1/2 clinical trial (NCT05395624) for first-in-human imaging in healthy controls and patients with amyotrophic lateral sclerosis is underway.

Abstract Tumor-associated macrophages (TAMs) play a role in cancer progression and are associated with Sorafenib resistance in hepatocellular carcinoma (HCC)1. D-4559 is a new potent macrophage switching nanomedicine technology that selectively inhibits VEGF receptor tyrosine kinases (VEGFR1, 2, 3) in TAMs leading to a functional reprogramming of TAMs toward a pro-inflammatory activated phenotype. Here, we evaluate the effect of D-4559 on the M1 and M2 polarization of TAMs and its anti-tumor efficacy in a murine HCC tumor model. In vivo efficacy of D-4559 was examined in the subcutaneous Hepa 1-6 liver tumor model in C57BL/6 mice. Animals (n=15/group) were treated with D-4559 (i.p., 200 mg/kg daily) and free drug Sorafenib (p.o., 40 mg/kg daily) as a positive control for 4 weeks. The treatment started when the mean tumor size reached approximately 100 mm3, then the animals were randomly allocated into study groups. The day of randomization and treatment was denoted as day 0. Tumor sizes were measured using a caliper for 27 days, M1/M2 macrophage polarization was examined by flow cytometry at day 16, and cytokine biomarkers were evaluated with MSD Cytokine Multiplex Assay at day 16 and 27.D-4559 significantly reduced Hepa 1-6 tumor growth (**p&amp;lt;0.01 vs. vehicle). D-4559 and Sorafenib efficacy were similar with 40% of mice with a tumor volume less than 500 mm3 after 27 days of treatment. At day 16, D-4559 significantly increased M1/M2 ratio by inducing M1 macrophage infiltration and reprogramming of TAMs into M1 macrophages compared to vehicle treatment groups (* p&amp;lt;0.05). In contrast, Sorafenib did not switch TAM polarization from M2 to M1 phenotype. Moreover, D-4559 significantly increased TNF-a and IL-8 cytokines in the tumor microenvironment at days 16 and 27, indicative of a M1 signature. Increased levels of TNF-a and IL-8 have also been correlated with better survival in HCC patients2,3. D-4559 significantly increased M1 infiltration, shifted TAM polarization from M2 to M1 phenotype and favored pro-inflammatory cytokines suggesting that D-4559 creates an immunopermissive tumor microenvironment leading to a reduction in tumor growth. This preclinical study supports the development of the potent macrophage switching drug D-4559 as a safe and effective agent that can be used systemically for the treatment of HCC or in combination therapies to improve anti-tumor immune response.

2022 completes the first complete year of open-access (OA) publishing for SLAS Discovery. The change to OA has been well-received by the scientific community, allowing for more immediate and free access and sharing of cutting-edge science. We continue to publish intriguing and provocative research in the form of Reviews, Perspectives, Original Research Articles and Technical Briefs. In 2022, we also added a “Protocols” manuscript type to enable the publication of detailed methods. The goal of SLAS Discovery is, and continues to be, to inform, share, educate, and encourage collaboration.

AbstractBackgroundChronic activation of macrophages/microglia plays a critical role in the onset and progression of neurological diseases, including Alzheimer’s. While PET imaging could enable non‐invasive visualization and quantification of activated macrophages/microglia in vivo, most available PET tracers are nonspecific for macrophages/microglia (Lambert, 2009). To address this need, we developed [18F]OP‐801, a synthetic hydroxyl dendrimer‐based PET tracer that is selectively (&gt;95%) taken up by reactive macrophages/microglia across the blood‐brain barrier (Alnasser, 2018). Here, we evaluated the ability of [18F]OP‐801 to detect activated macrophages/microglia in the 5XFAD murine model of Alzheimer’s compared to an established neuroinflammation imaging approach (translocator protein 18 kDa [TSPO]‐PET, using [18F]GE180).Method[18F]OP‐801 (150‐250 μCi) was injected intravenously into female 5XFAD (TG) mice (n=12) and age/sex‐matched wild types (WT, n=7) at 3.75 and 5 months old. Sensitivity of [18F]OP‐801 was compared to [18F]GE180 by assessing brain signal‐to‐background ratios. [18F]GE180 (150‐250 μCi) was administered to a subset of TG (n=5) and WT (n=4) 3.75‐months‐old mice. Static 10‐minute PET/CT images were acquired at 50‐60 minutes post‐injection for both tracers. VivoQuant brain atlas was fit to CT images and fused to PET to quantify uptake in specific brain regions.ResultIn this model, macrophage/microglial activation is associated with amyloid plaque formation and can be detected using immunohistochemistry between 2‐4 months. Image quantification revealed 3‐fold higher PET signal in 3.75‐months‐old TG compared to WT mice using [18F]OP‐801, whereas [18F]GE180 signal provided no significant difference in brain regions known to contain activated microglia: cortex and hippocampus (Figure 1, Table 1). Significant differences in [18F]OP‐801 uptake were observed between 5‐months‐old TG and WT mice in cortex (p=0.005) (TG: 0.26±0.095%ID/g, WT: 0.11±0.041%ID/g), hippocampus (p=0.017) (TG: 0.18±0.065%ID/g, WT: 0.10±0.026%ID/g) and whole brain (p=0.004) (TG: 0.20±0.082%ID/g, WT: 0.10±0.039%ID/g). TG had almost 5‐fold higher [18F]OP‐801 signal compared to WT mice (Table 2).ConclusionThese results suggest that [18F]OP‐801 can detect early stage neuroinflammation with higher sensitivity than TSPO‐PET. We are currently replicating this study in a larger cohort of 5XFAD mice to correlate PET image findings with immunohistochemistry. [18F]OP‐801 is shows promise for visualizing the progression of neuroinflammation with high specificity and sensitivity, warranting further preclinical investigation.

BackgroundRecessive dystrophic epidermolysis bullosa (RDEB) is a rare, devastating blistering genodermatosis caused by mutations in the COL7A1 gene, which encodes for type VII collagen and is necessary for dermal-epidermal adhesion and integrity. Disease manifestations include severe and debilitating wounds, aggressive squamous cell carcinomas, and premature death; however, there are currently no approved therapies. This Phase 1/2a, open-label study evaluated the long-term efficacy and safety of gene-corrected autologous keratinocyte grafts (EB-101) for chronic RDEB wounds.MethodsAutologous keratinocytes were harvested from participants with severe RDEB, transduced with a retrovirus containing the full-length COL7A1 gene, and grown into 5 × 7 cm (35 cm2) sheets. Gene-corrected keratinocyte sheets were then transplanted onto chronic RDEB wounds present for ≥ 12 weeks.ResultsSeven adult participants with severe RDEB were grafted with six sheets each (42 total sheets) onto wounds and followed for a mean of 5.9 years (range 4–8 years). Long-term improvements in wound healing and symptoms were observed. At year five, 70% (21/30) of treated sites demonstrated ≥ 50% wound healing compared to baseline by investigator global assessment. No sites with ≥ 50% wound healing were painful or pruritic, compared to 67% (6/9) of sites with < 50% wound healing (p < 0.001) at year five. Grafts were well-tolerated throughout long-term follow-up. No serious adverse events related to treatment were reported over a mean of 5.9 years of follow-up. No persistent systemic autoimmunity against type VII collagen or replication-competent retrovirus infections were identified, and no participants developed squamous cell carcinomas related to treatment during long-term follow-up.ConclusionsTreatment with EB-101 appears safe and efficacious, and produces long-term improvements in wound healing, pain, and itch for RDEB patients. Results from the Phase 3 randomized controlled trial are forthcoming.Trial registrationClinicalTrials.gov, NCT01263379. Registered December 15, 2010. https://clinicaltrials.gov/ct2/show/NCT01263379

RDEB is a rare, severe dermatosis caused by mutations in type VII collagen (C7), a major component of structural attachments between the basement membrane and dermis. Manifestations include large, chronic wounds and blisters, increased squamous cell carcinoma (SCC) and early death. Currently, there are no approved therapies. We report long-term outcomes of a Phase 1/2a trial to treat large, chronic RDEB wounds with 35cm2 autologous skin sheets (EB-101) gene-corrected with a retroviral vector (RV).

Hyperinflammation triggered by SARS-CoV-2 is a major cause of disease severity, with activated macrophages implicated in this response. OP-101, a hydroxyl-polyamidoamine dendrimer-N-acetylcysteine conjugate that specifically targets activated macrophages, improves outcomes in preclinical models of systemic inflammation and neuroinflammation. In this multicenter, randomized, double-blind, placebo-controlled, adaptive phase 2a trial, we evaluated safety and preliminary efficacy of OP-101 in patients with severe COVID-19. Twenty-four patients classified as having severe COVID-19 with a baseline World Health Organization seven-point ordinal scale of ≥5 were randomized to receive a single intravenous dose of placebo (n = 7 patients) or OP-101 at 2 (n = 6), 4 (n = 6), or 8 mg/kg (n = 5 patients). All study participants received standard of care, including corticosteroids. OP-101 at 4 mg/kg was better than placebo at decreasing inflammatory markers; OP-101 at 4 and 8 mg/kg was better than placebo at reducing neurological injury markers, (neurofilament light chain and glial fibrillary acidic protein). Risk for the composite outcome of mechanical ventilation or death at 30 and 60 days after treatment was 71% (95% CI: 29%, 96%) for placebo and 18% (95% CI: 4%, 43%; P = 0.021) for the pooled OP-101 treatment arms. At 60 days, 3 of 7 patients given placebo and 14 of 17 OP-101-treated patients were surviving. No drug-related adverse events were reported. These data show that OP-101 was well tolerated and may have potential to treat systemic inflammation and neuronal injury, reducing morbidity and mortality in hospitalized patients with severe COVID-19.