The Efficacy of Nanoparticle Delivery to Hypoxic Solid Tumors by ciRGD Co-Administration Depends on Neuropilin-1 and Neutrophil Levels.

The ability to improve nanoparticle delivery to solid tumors is an actively studied domain, where various mechanisms are looked into. In previous work, the authors have looked into nanoparticle size, tumor vessel normalization, and disintegration, and here it is aimed to continue this work by performing an in-depth mechanistic study on the use of ciRGD peptide co-administration. Using a multiparametric approach, it is observed that ciRGD can improve nanoparticle delivery to the tumor itself, but also to tumor cells specifically better than vessel normalization strategies. The effect depends on the level of tumor perfusion, hypoxia, neutrophil levels, and vessel permeability. This work shows that upon characterizing tumors for these parameters, conditions can be selected that can optimally benefit from ciRGD co-administration as a means to improve NP delivery to solid tumors.

Targeting of PDGF-C/NRP-1 autocrine loop as a new strategy for counteracting the invasiveness of melanoma resistant to braf inhibitors.

Melanoma resistance to BRAF inhibitors (BRAFi) is often accompanied by a switch from a proliferative to an invasive phenotype. Therefore, the identification of signaling molecules involved in the development of metastatic properties by resistant melanoma cells is of primary importance. We have previously demonstrated that activation of neuropilin-1 (NRP-1) by platelet-derived growth factor (PDGF)-C confers melanoma cells with an invasive behavior similar to that of BRAFi resistant tumors. Aims of the present study were to evaluate the role of PDGF-C/NRP-1 autocrine loop in the acquisition of an invasive and BRAFi-resistant phenotype by melanoma cells and the effect of its inhibition on drug resistance and extracellular matrix (ECM) invasion. Furthermore, we investigated whether PDGF-C serum levels were differentially modulated by drug treatment in metastatic melanoma patients responsive or refractory to BRAFi as single agents or in combination with MEK inhibitors (MEKi). The results indicated that human melanoma cells resistant to BRAFi express higher levels of PDGF-C and NRP-1 as compared to their susceptible counterparts. Overexpression occurs early during development of drug resistance and contributes to the invasive properties of resistant cells. Accordingly, silencing of NRP-1 or PDGF-C reduces tumor cell invasiveness. Analysis of PDGF-C in the serum collected from patients treated with BRAFi or BRAFi+MEKi, showed that in responders PDGF-C levels decrease after treatment and raise again at tumor progression. Conversely, in non-responders treatment does not affect PDGF-C serum levels. Thus, blockade of NRP-1 activation by PDGF-C might represent a new therapeutic approach to counteract the invasiveness of BRAFi-resistant melanoma.

Targeting the vascular endothelial growth factor A/neuropilin 1 axis for relief of neuropathic pain.

ABSTRACT: Vascular endothelial growth factor A (VEGF-A) is a pronociceptive factor that causes neuronal sensitization and pain. We reported that blocking the interaction between the membrane receptor neuropilin 1 (NRP1) and VEGF-A-blocked VEGF-A-mediated sensory neuron hyperexcitability and reduced mechanical hypersensitivity in a rodent chronic neuropathic pain model. These findings identified the NRP1-VEGF-A signaling axis for therapeutic targeting of chronic pain. In an in-silico screening of approximately 480 K small molecules binding to the extracellular b1b2 pocket of NRP1, we identified 9 chemical series, with 6 compounds disrupting VEGF-A binding to NRP1. The small molecule with greatest efficacy, 4'-methyl-2'-morpholino-2-(phenylamino)-[4,5'-bipyrimidin]-6(1H)-one, designated NRP1-4, was selected for further evaluation. In cultured primary sensory neurons, VEGF-A enhanced excitability and decreased firing threshold, which was blocked by NRP1-4. In addition, NaV1.7 and CaV2.2 currents and membrane expression were potentiated by treatment with VEGF-A, and this potentiation was blocked by NRP1-4 cotreatment. Neuropilin 1-4 reduced VEGF-A-mediated increases in the frequency and amplitude of spontaneous excitatory postsynaptic currents in dorsal horn of the spinal cord. Neuropilin 1-4 did not bind to more than 300 G-protein-coupled receptors and receptors including human opioids receptors, indicating a favorable safety profile. In rats with spared nerve injury-induced neuropathic pain, intrathecal administration of NRP1-4 significantly attenuated mechanical allodynia. Intravenous treatment with NRP1-4 reversed both mechanical allodynia and thermal hyperalgesia in rats with L5/L6 spinal nerve ligation-induced neuropathic pain. Collectively, our findings show that NRP1-4 is a first-in-class compound targeting the NRP1-VEGF-A signaling axis to control voltage-gated ion channel function, neuronal excitability, and synaptic activity that curb chronic pain.

Inhibition of neuropilin-1 enhances the therapeutic effects of lenvatinib in suppressing cholangiocarcinoma cells via the c-Met pathway.

Neuropilin-1 (NRP-1) participates in the progression of cholangiocarcinoma (CCA) and lenvatinib is an approved tyrosine kinase inhibitor treating several other cancers. Our exploratory study reveals that the inhibitory activities of lenvatinib largely rely on the expression levels of NRP-1 in CCA cells, leading to the present study that aims to investigate whether inhibition of NRP-1 could enhance the effects of lenvatinib in suppressing CCA. By using stable transfected CCA cells depleted of NRP-1 and EG00229, a specific NRP-1 inhibitor, we examined cell proliferation, cell cycle distribution and apoptosis, and detected the expression of key molecules and the involvement of the c-Met pathway. Xenograft mouse models were employed to verify the in vitro results. NRP-1 depletion and EG00229 strengthened lenvatinib in inhibiting the proliferation and promoting the apoptosis of CCA cells, and their additive or synergistic effects were confirmed in animal models. Mechanistically, lenvatinib induced the activation of the c-Met pathway, while either NRP-1 depletion or EG00229 inhibited this activation, which could be stimulated by its ligand hepatocyte growth factor. NRP-1 inhibition resulted in a significant alteration in the expression/activation of downstream pathways and molecules, which are key factors regulating cell proliferation and apoptosis. In conclusion, the present results indicate that the inhibition of NRP-1 enhances the efficacy of lenvatinib via the c-Met pathway, and warrant further studies on the pharmacological utility of EG00229, particularly, in the combination of lenvatinib as a promising adjunct therapeutic strategy for combating CCA.

Dual αV-integrin and neuropilin-1 targeting peptide CEND-1 plus nab-paclitaxel and gemcitabine for the treatment of metastatic pancreatic ductal adenocarcinoma: a first-in-human, open-label, multicentre, phase 1 study.

BACKGROUND: CEND-1 is a novel cyclic peptide that targets αV integrins and neuropilin-1 and enhances tumour delivery of co-administered anticancer drugs. We investigated the safety, tolerability, and biological activity of CEND-1 in patients with metastatic pancreatic ductal adenocarcinoma in combination with nab-paclitaxel and gemcitabine. METHODS: This open-label, multicentre, phase 1 study, conducted at three hospitals in Australia, enrolled participants aged 18 years or older with histologically confirmed metastatic pancreatic ductal adenocarcinoma who had one or more lesions measurable on MRI or CT, an Eastern Cooperative Oncology Group performance status score of 0 or 1, and a life expectancy of at least 3 months. Exclusion criteria included previous chemotherapy and brain metastases or other malignancy (unless receiving curative intent). There was no randomisation or masking. CEND-1 monotherapy was given as an intravenous fluid bolus on day 1 of a run-in phase of 7 days (0·2-3·2 mg/kg) followed by CEND-1 plus intravenous gemcitabine (1000 mg/m2) and nab-paclitaxel (125 mg/m2) on days 1, 8, and 15 of 28-day treatment cycles until disease progression. The primary safety endpoints were incidence, severity, and duration of treatment-emergent and treatment-related adverse events; overall survival; and clinical laboratory results, which were all assessed in the safety population. This study is registered with ClinicalTrials.gov, NCT03517176, and the Australian New Zealand Clinical Trials Registry, ACTRN12618000804280. FINDINGS: Between Aug 13, 2018, and Nov 30, 2019, 31 patients were enrolled (eight in the dose-escalation phase [cohort 1a] and 23 in the expansion phase [cohort 1b]). Two patients were excluded from the efficacy population. No CEND-1 dose-limiting toxicities were observed in the safety population (n=31). The most common grade 3 or 4 events were neutropenia (17 [55%] patients), anaemia (eight [26%]), leukopenia (five [16%]), and pulmonary embolism (four [13%]). Serious adverse events occurred in 22 (71%) patients, mostly related to disease progression. Ten deaths occurred during the study due to progression of metastatic pancreatic cancer (n=9) and a left middle cerebral artery stroke (n=1). In the efficacy population (n=29), 17 (59%) patients had an objective response, including one complete response and 16 partial responses. After a median follow-up of 26 months (IQR 24-30), median overall survival was 13·2 months (95% CI 9·7-22·5). INTERPRETATION: CEND-1 with nab-paclitaxel and gemcitabine has an acceptable safety profile, with no dose-limiting toxicities and encouraging activity. Adverse events were generally consistent with those seen with nab-paclitaxel and gemcitabine. Further randomised trials to determine the efficacy of CEND-1 are warranted. FUNDING: DrugCendR Australia Pty.

Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation.

BACKGROUND: Triple-negative breast cancers (TNBC) have a relatively poor prognosis and responses to targeted therapies. Between 25 and 39% of TNBCs are claudin-low, a poorly differentiated subtype enriched for mesenchymal, stem cell and mitogen-activated signaling pathways. We investigated the role of the cell-surface co-receptor NRP1 in the biology of claudin-low TNBC. METHODS: The clinical prognostic value of NRP1 was determined by Kaplan-Meier analysis. GSVA analysis of METABRIC and Oslo2 transcriptomics datasets was used to correlate NRP1 expression with claudin-low gene signature scores. NRP1 siRNA knockdown was performed in MDA-MB-231, BT-549, SUM159 and Hs578T claudin-low cells and proliferation and viability measured by live cell imaging and DNA quantification. In SUM159 orthotopic xenograft models using NSG mice, NRP1 was suppressed by shRNA knockdown or systemic treatment with the NRP1-targeted monoclonal antibody Vesencumab. NRP1-mediated signaling pathways were interrogated by protein array and Western blotting. RESULTS: High NRP1 expression was associated with shorter relapse- and metastasis-free survival specifically in ER-negative BrCa cohorts. NRP1 was over-expressed specifically in claudin-low clinical samples and cell lines, and NRP1 knockdown reduced proliferation of claudin-low cells and prolonged survival in a claudin-low orthotopic xenograft model. NRP1 inhibition suppressed expression of the mesenchymal and stem cell markers ZEB1 and ITGA6, respectively, compromised spheroid-initiating capacity and exerted potent anti-tumor effects on claudin-low orthotopic xenografts (12.8-fold reduction in endpoint tumor volume). NRP1 was required to maintain maximal RAS/MAPK signaling via EGFR and PDGFR, a hallmark of claudin-low tumors. CONCLUSIONS: These data implicate NRP1 in the aggressive phenotype of claudin-low breast cancer and offer a novel targeted therapeutic approach to this poor prognosis subtype.

Modeling Neoadjuvant chemotherapy resistance in vitro increased NRP-1 and HER2 expression and converted MCF7 breast cancer subtype.

BACKGROUND AND PURPOSE: Patients with locally advanced breast cancer usually receive third-generation neoadjuvant chemotherapy (NAC). Although NAC treatment improved the overall survival, patients' response varies, some acquire resistance and others exhibit a conversion in their breast cancer molecular subtype. We aimed to identify the molecular changes involved in NAC resistance attempting to find new therapeutic targets in different breast cancer subtypes. EXPERIMENTAL APPROACH: We modelled NAC treatments used in clinical practice and generated resistant cell lines in vitro. The resistant cells were generated by consecutive treatment with four cycles of doxorubicin (adriamycin)/cyclophosphamide (4xAC) followed by an additional four cycles of paclitaxel (4xAC + 4xPAC). KEY RESULTS: Our data revealed distinct mechanisms of resistance depending on breast cancer subtype and drugs used. MDA-MB-231 cells resistant to 4xAC + 4xPAC activated neuropilin-1/TNC/integrin ß3/FAK/NF-κBp65 axis and displayed a decrease in breast cancer resistance protein (BCRP/ABCB2). However, MCF7 cells resistant to 4xAC treatments induced HER2 expression, which converted MCF7 subtype from luminal A to luminal B HER2 type, up-regulated neuropilin-1, oestrogen receptor-α, and EGFR, and activated PI3K/Akt/NF-κBp65 axis. However, MCF7 cells resistant to 4xAC + 4xPAC exhibited down-regulation of the survival axis and up-regulated BCRP/ABCG2. Co-immunoprecipitation demonstrated a novel interaction between HER2 and neuropilin-1 driving the resistance features. CONCLUSIONS AND IMPLICATIONS: The concurrent increase in neuropilin-1 and HER2 upon resistance and the inverse relationship between neuropilin-1 and BCRP/ABCG2 suggest that, in addition to HER2, neuropilin-1 status should be assessed in patients undergoing NAC, and as a potential drug target for refractory breast cancer.

Synthesis, 3D-structure and stability analyses of NRPa-308, a new promising anti-cancer agent.

We report herein the synthesis of a newly described anti-cancer agent, NRPa-308. This compound antagonizes Neuropilin-1, a multi-partners transmembrane receptor overexpressed in numerous tumors, and thereby validated as promising target in oncology. The preparation of NRPa-308 proved challenging because of the orthogonality of the amide and sulphonamide bonds formation. Nevertheless, we succeeded a gram scale synthesis, according to an expeditious three steps route, without intermediate purification. This latter point is of utmost interest in reducing the ecologic impact and production costs in the perspective of further scale-up processes. The purity of NRPa-308 has been attested by means of conventional structural analyses and its crystallisation allowed a structural assessment by X-Ray diffraction. We also reported the remarkable chemical stability of this molecule in acidic, neutral and basic aqueous media. Eventually, we observed for the first time the accumulation of NRPa-308 in two types of human breast cancer cells MDA-MB231 and BT549.

Neuropilin 1 ameliorates electrical remodeling at infarct border zones in rats after myocardial infarction.

BACKGROUND: Electrical remodeling at infarct border zone (IBZ) has been shown to contribute to the occurrence of ventricular arrhythmias after myocardial infarction (MI). Sema3A has been demonstrated to reduce the inducibility of ventricular arrhythmias. Neuropilin 1 (NRP1) is the receptor of Sema3A. In the present study, we investigated whether treatment with NRP1 can ameliorate electrical remodeling at IBZ after MI. METHODS AND RESULTS: Wistar rats underwent sham operation (n = 20), the ligation of left coronary artery (MI group, n = 30), MI with control adenovirus (Ad group, n = 30), and MI with NRP1 adenovirus (NRP1 group, n = 30). Eight weeks after treatment, electrophysiological properties including heart rate variability (HRV), monophasic action potential duration (MAPD), effective refractory period (ERP) and the inducibility of ventricular arrhythmias and the expression of arrhythmia-related ion channel proteins including Kv4.2, Kv4.3, KChIP2 and Kir2.1 at the IBZ of the left ventricle were examined. Compared with the MI or Ad group, NRP1 significantly increased HRV and shortened MAPD and ERP (all P < 0.05). Inducibility of VT by electrophysiological study was significantly lower in the NRP1 group than in the MI or Ad group (P < 0.05). The expression levels of Kv4.2, Kv4.3, KChIP2 and Kir2.1 proteins were significantly decreased in MI group and Ad group. In contrast, the expression levels of these proteins were restored in NRP1 group, which may represent the molecular basis of the NRP1-mediated inhibition of electrical remodeling. CONCLUSIONS: NRP1 can ameliorate electrical remodeling at IBZ after MI.

Multifunctional ultrasmall nanoplatforms for vascular-targeted interstitial photodynamic therapy of brain tumors guided by real-time MRI.

Photodynamic therapy (PDT) for brain tumors appears to be complementary to conventional treatments. A number of studies show the major role of the vascular effect in the tumor eradication by PDT. For interstitial PDT (iPDT) of brain tumors guided by real-time imaging, multifunctional nanoparticles consisting of a surface-localized tumor vasculature targeting neuropilin-1 (NRP-1) peptide and encapsulated photosensitizer and magnetic resonance imaging (MRI) contrast agents, have been designed. Nanoplatforms confer photosensitivity to cells and demonstrate a molecular affinity to NRP-1. Intravenous injection into rats bearing intracranial glioma exhibited a dynamic contrast-enhanced MRI for angiogenic endothelial cells lining the neovessels mainly located in the peripheral tumor. By using MRI completed by NRP-1 protein expression of the tumor and brain adjacent to tumor tissues, we checked the selectivity of the nanoparticles. This study represents the first in vivo proof of concept of closed-head iPDT guided by real-time MRI using targeted ultrasmall nanoplatforms. From the clinical editor: The authors constructed tumor vascular peptide targeting multifunctional silica-based nanoparticles, with encapsulated gadolinium oxide as MRI contrast agent and chlorin as a photosensitizer, as a proof of concept novel treatment for glioblastoma in an animal model.

A Phase Ib study evaluating MNRP1685A, a fully human anti-NRP1 monoclonal antibody, in combination with bevacizumab and paclitaxel in patients with advanced solid tumors.

PURPOSE: MNRP1685A is a human monoclonal antibody that blocks binding of vascular endothelial growth factor (VEGF), VEGF-B, and placental growth factor 2 to neuropilin-1 resulting in vessel immaturity and VEGF dependency. The safety of combining MNRP1685A with bevacizumab, with or without paclitaxel, was examined. METHODS: Patients with advanced solid tumors received escalating doses of MNRP1685A (7.5, 15, 24, and 36 mg/kg) with bevacizumab 15 mg/kg every 3 weeks in Arm A (n = 14). Arm B (n = 10) dosing consisted of MNRP1685A (12 and 16 mg/kg) with bevacizumab 10 mg/kg (every 2 weeks) and paclitaxel 90 mg/m(2) (weekly, 3 of 4 weeks). Objectives were to determine safety, pharmacokinetics, pharmacodynamics, and the maximum tolerated dose of MNRP1685A. RESULTS: Infusion reactions (88 %) and transient thrombocytopenia (67 %) represent the most frequent study drug-related adverse events (AEs). Drug-related Grade 2 or 3 proteinuria occurred in 13 patients (54 %). Additional study drug-related AEs occurring in >20 % of patients included neutropenia, alopecia, dysphonia, fatigue, and nausea. Neutropenia occurred only in Arm B. Grade ≥3 study drug-related AEs in ≥3 patients included neutropenia (Arm B), proteinuria, and thrombocytopenia. Two confirmed and three unconfirmed partial responses were observed. CONCLUSIONS: The safety profiles were consistent with the single-agent profiles of all study drugs. However, a higher than expected rate of clinically significant proteinuria was observed that does not support further testing of MNRP1685A in combination with bevacizumab.