Favipiravir, umifenovir and camostat mesylate: a comparative study against SARS-CoV-2

Since the first cases the coronavirus disease caused by SARS-CoV-2 (COVID-19) reported in December 2019, worldwide continuous efforts have been placed both for the prevention and treatment of this infectious disease. As new variants of the virus emerge, the need for an effective antiviral treatment continues. The concept of preventing SARS-CoV-2 on both pre-entry and post-entry stages has not been much studied. Therefore, we compared the antiviral activities of three antiviral drugs which have been currently used in the clinic. In silico docking analyses and in vitro viral infection in Vero E6 cells were performed to delineate their antiviral effectivity when used alone or in combination. Both in silico and in vitro results suggest that the combinatorial treatment by favipiravir and umifenovir or camostat mesylate has more antiviral activity against SARS-CoV-2 rather than single drug treatment. These results suggest that inhibiting both viral entry and viral replication at the same time is much more effective for the antiviral treatment of SARS-CoV-2.

HA/PEI-coated acridine orange-loaded gold-core silica shell nanorods for cancer-targeted photothermal and chemotherapy

Aims: To develop a tumor-targeted chemo-photothermal nanomedicine through the functionalization of acridine orange (AO)-loaded gold-core mesoporous silica shell (AuMSS) nanorods with polyethylenimine (PEI) and hyaluronic acid (HA). Methods: Functionalization of the AuMSS nanorods was achieved through the chemical linkage of PEI followed by electrostatic adsorption of HA. Results: HA functionalization improved AuMSS' cytocompatibility by decreasing blood hemolysis, and PEI-HA inclusion promoted a controlled and sustained AO release. In vitro assays revealed that HA functionalization increased the internalization of nanoparticles by human negroid cervix epithelioid carcinoma cancer (HeLa) cells, and the combinatorial treatment mediated by AuMSS/PEI/HA_AO nanorods presented an enhanced effect, with >95% of cellular death. Conclusion: AuMSS/PEI/HA_AO formulations can act as tumor-targeted chemo-photothermal nanomedicines for the combinatorial therapy of cervical cancer.

Co-Targeting E-Selectin/CXCR4 with GMI-1359 Facilitates AML Stem Cell Mobilization and Protects BM Niches from Anti-Leukemia Therapy

Acute myeloid leukemia (AML) is characterized by the heterogeneous clonal expansion of undifferentiated myeloid cells in the bone marrow (BM). AML cells compete with normal hematopoietic cells and rewire the BM microenvironment into niches that selectively support leukemia stem cells (LSC). The leukemic niche produces soluble factors that facilitate the retention of LSC and provide protection from cytotoxic and targeted agents. The vascular adhesion molecule, E-selectin is expressed on endothelial cells (EC) in the perivascular niche where therapy-resistant AML cells have an increased affinity to E-selectin compared to normal hematopoietic stem cells (HSC) (Winkler et al., 2020). We previously demonstrated (Chang et al., ASH 2020) that E-selectin blockade by the pharmacological antagonist, GMI-1271 (uproleselan; GlycoMimetics, Inc) sensitized therapy-resistant LSC to Bcl-2 targeted therapy. Efficacious eradication of LSC in the BM however requires blocking multiple receptors and/or associated signaling pathways. A more optimal dislodgement of LSC from the BM could be attained by combining an E-selectin antagonism with blockade of the CXCR4/SDF-1α axis. The dual antagonist of E-selectin and CXCR4, GMI-1359 (GlycoMimetics, Inc.), has been tested in a phase 1 clinical trial (NCT02931214). Previously, we showed that GMI-1359 in combination with a FLT3-ITD inhibitor, improved survival in a xenograft model of FLT3-ITD + AML (Zhang et al., 2016). Hence, we hypothesized that co-targeting E-selectin/CXCR4 more efficiently mobilizes AML cells from BM niches and synergizes with the anti-leukemia activity of venetoclax/hypomethylating agent (Ven/HMA). Intra-vital 2-photon imaging and tracking of individual leukemia cells in triple reporter mice (Blood: dextran-TRITC; Host T-cells: DsRed; Host myeloid CD11 cells: EYFP) injected with AML cells carrying a turquoise fluorescent protein reporter gene suggested that dual inhibition of E-selectin/CXCR4 with GMI-1359 significantly enhanced AML cell motility (Fig 1. from 2.2 um/min to 5.4 um/min, p<0.001). Individual cells were dislodged from the niche and traveled long-distance. The combined inhibition of E-selectin and CXCR4 depleted BM leukemia cells in vascular niche areas. In a patient-derived primary AML xenograft (PDX) model (harboring mutations in JAK2 and c-Kit), combinatorial treatment of GMI-1359 with Ven/HMA significantly reduced BM retention of LSC compared to control cohorts or to Ven/HMA alone (p = 0.02 and p=0.003, respectively). In order to better understand how the augmented AML mobilization improves the efficacy of AML therapy, BM cells from PDX mice treated for 2 weeks with GMI-1271, GMI-1359, Ven/HMA, and their combinations were analyzed by single-cell proteomics (CyTOF). Blockade of E-selectin alone or dual E-selectin/CXCR4 inhibition in combination with Ven/HMA diminished levels of E-selectin ligand, mTOR, pFAK, pRb, cMyc, while increasing p21 and cleaved caspase3, which was associated with significant reduction of BM-resident LSC compared to Ven/HMA alone (CD45+34+CD38-CD123+, p= 0.03). AML blasts from the BM of the combinatorial treatment groups showed altered signaling including decreased Ki67, pRb, pNFkB, pPI3K, and E-selectin ligand, and increased levels of cleaved caspase 3. We further found that Ven/HMA significantly diminished CD31+ EC in the BM compared to control cohorts (p= 0.009). However, pharmacological antagonists of E-selectin or E-selectin/CXCR4 protected EC from Ven/HMA-induced detrimental insults through upregulation of survival signaling cascades including pAKT, pERK, pMAPK and decreased eNOS expression in EC compared to Ven/HMA treatment alone. Both EC and MSC were protected by dual inhibition of E-selectin/CXCR4 with GMI-1359. We also observed upregulated pro-survival signaling pathways such as phosphorylation of AKT-MAPK-ERK along with increased Bcl-xL, Bcl-2, and Idu expression in MSC from the GMI-1359 + Ven/HMA treated PDX mice compared to Ven/HMA single treatment cohorts. Collectively, our results provide strong evidence that co-targeting E-selectin/CXCR4 with GMI-1359 profoundly reduces BM retention of LSC as well as protects BM niche component cells from apoptosis induced by targeted therapy, resulting in improving the anti-leukemia activity of Ven/HMA therapy in AML. Figure 1 Figure 1. Disclosures Fogler: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Carter: Ascentage: Research Funding; Syndax: Research Funding. Andreeff: Oxford Biomedica UK: Research Funding; ONO Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Karyopharm: Research Funding; Breast Cancer Research Foundation: Research Funding; Syndax: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Aptose: Consultancy; Glycomimetics: Consultancy; Medicxi: Consultancy; Senti-Bio: Consultancy.

607 Regional delivery of a TLR9 agonist to boost checkpoint inhibitor responsiveness in liver metastases

BackgroundClass C TLR9 agonists, CpG oligodeoxynucleotides (ODNs) enhance responsiveness to anti-PD1 therapy in solid tumors through favorable modulation of the tumor microenvironment (TME) [1]. Recently, we reported that regional delivery of a TLR9 agonist eliminated myeloid derived suppressor cells (MDSC) and promoted pro-inflammatory/anti-tumorigenic M1 macrophage programming in the TME of liver metastases (LM) [2]. Further, we found enhanced TLR9 activation in LM following regional TLR9 agonist infusion compared to the systemic treatment. We hypothesize that regional delivery of a TLR9A into LM will enhance the responsiveness to systemically infused anti-PD1 therapy.MethodsIn this study, we treated mice with established MC38-CEA-Luc LM with ODN-2395 (30µg/mouse) regionally with or without anti PD-1 antibody (250µg/mouse) intraperitoneally.ResultsControl of LM growth (Figure 1) was significantly higher with combinatorial treatment as compared to anti-PD1 (p<0.01) or PBS treatments (p<0.05). To study the impact of TLR9 activation on human MDSC, we treated healthy donor PBMCs with ODN-2395 or SD101. We found that both reduced the hu-MDSC (CD11b+CD33+HLADR-) population in a dose-dependent manner with an increase in PD-L1 expression as determined by flow cytometry (FC) analysis (Figure 2). Moreover, by using Luminex, demonstrated that ODN-2395 and SD101 enhanced expression of IL 29, IFNα, and NFκB, along with downstream cytokines IL 6 and IL 10. To investigate the effect of SD101 in modulating the differentiation of huMDSC from huPBMC, we treated huPBMC with IL6+GM-CSF in the presence or absence of SD101. By FC analysis, we found that SD-101 blocked huMDSC development induced by IL6+GM-CSF, preferentially limited the more immunosuppressive monocytic MDSC subtype, and drove M1 macrophage polarization. Treatment of SD101 only once for 48hrs was sufficient to inhibit huMDSC differentiation for two weeks.Abstract 607 Figure 1Combinatorial treatment of CPI and ODN’s reduces tPV = portal vein; IP = intraperitoneal.Eight to twelve weeks old male C57/BL6 mice were challenged intra-splenic with 0.5e6 MC38-CEA-Luc cells for a week. Bioluminescence value was determined by IVIS on D0, and mice were randomized accordingly and treated with 30 µg/mouse ODN2395 via PV with or without 250 µg/mouse anti-PD1 antibody via IP on D0, D+3 and D+6. PBS served as the vehicle (Veh) control and administered via PV. Fold change of the tumor burden was calculated based on D0 baseline bioluminescence. Tumor progression was analyzed unpaired t test among groups. (*p <0.05).Abstract 607 Figure 2Human PBMC treated with ODN2395 and SD101 reducesCtrl = control; MDSC = ODN = oligodeoxynucleotide’ PBMC = peripheral blood monocytes.Human PBMC were isolated from the Leukoreduction system chamber. 1e6/ml PBMCs were treated with increasing concentrations (0.04–10 µM) SD101, ODN2395 along with ctrl ODN5328 (1µM) for 48 hours. Panels A and B: MDSC population and their corresponding PD–L1 expression were evaluated (n=12). Four donors with three replicates were used. Data represented as mean ± SEM.ConclusionsBoth the in vitro and in vivo findings suggest that regional TLR9 stimulation in a model of LM improves responsiveness to systemic anti-PD-1 therapy through elimination of MDSC, and the effect on huMDSC was confirmed in vitro. Increased PDL-1 expression in response to TLR9 stimulation among MDSC may further enhance the anti-PD-1 effect. Therefore, combing regional infusions of a TLR9 agonist with systemic anti-PD-1 agents may be a promising approach for liver tumor treatment.ReferencesWang, S., et al., Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells. Proc Natl Acad Sci U S A, 2016. 113(46): p. E7240-E7249.Ghosh CC, H.K., O’Connell K, Laporte J, Guha P, Cox B, Jaroch D, Katz SC, Regional administration of class C CpG Oligodeoxynucleotides results in superior intrahepatic TLR9 activation and immunomodulation compared to systemic infusion, Abstract: AACR Annual Meeting. 2021.

Synergistic Cytotoxicity between Elephantopus scaber and Tamoxifen on MCF-7-Derived Multicellular Tumor Spheroid

Elephantopus scaber Linn, a traditional herb, exhibited anticancer properties, and it was cytotoxic against the monolayer estrogen receptor-positive breast cancer cell line, MCF-7, in the previous study. In order to determine the potential of E. scaber as a complementary medicine for breast cancer, this study aimed to evaluate the synergism between E. scaber and tamoxifen in cytotoxicity using MCF-7 in the form of 3-dimensional multicellular tumor spheroid (MCTS) cultures. MCTS represents a more reliable model for studying drug penetration as compared to monolayer cells due to its greater resemblance to solid tumor. Combination of E. scaber ethanol extract and tamoxifen, which were used in concentrations lower than their respective IC50 values, had successfully induced apoptosis on MCTS in this study. The combinatorial treatment showed >58% increase of lactate dehydrogenase release in cell media, cell cycle arrest at the S phase, and 1.3 fold increase in depolarization of mitochondrial membrane potential. The treated MCTS also experienced DNA fragmentation; this had been quantified by TUNEL-positive assay, which showed >64% increase in DNA damaged cells. Higher externalization of phospatidylserine and distorted and disintegrated spheroids stained by acridine orange/propidium iodide showed that the cell death was mainly due to apoptosis. Further exploration showed that the combinatorial treatment elevated caspases-8 and 9 activities involving both extrinsic and intrinsic pathways of apoptosis. The treatment also upregulated the expression of proapoptotic gene HSP 105 and downregulated the expression of prosurvival genes such as c-Jun, ICAM1, and VEGF. In conclusion, these results suggested that the coupling of E. scaber to low concentration of tamoxifen showed synergism in cytotoxicity and reducing drug resistance in estrogen receptor-positive breast cancer.

Challenges and Future Perspectives of Immunotherapy in Pancreatic Cancer

To date, extensive efforts to harness immunotherapeutic strategies for the treatment of pancreatic ductal adenocarcinoma (PDAC) have yielded disappointing results in clinical trials. These strategies mainly focused on cancer vaccines and immune checkpoint inhibitors alone or in combination with chemotherapeutic or targeted agents. However, the growing preclinical and clinical data sets from these efforts have established valuable insights into the immunological characteristics of PDAC biology. Most notable are the immunosuppressive role of the tumour microenvironment (TME) and PDAC’s characteristically poor immunogenicity resulting from tumour intrinsic features. Moreover, PDAC tumour heterogeneity has been increasingly well characterized and may additionally limit a “one-fits-all” immunotherapeutic strategy. In this review, we first outline mechanisms of immunosuppression and immune evasion in PDAC. Secondly, we summarize recently published data on preclinical and clinical efforts to establish immunotherapeutic strategies for the treatment of PDAC including diverse combinatorial treatment approaches aiming at overcoming this resistance towards immunotherapeutic strategies. Particularly, these combinatorial treatment approaches seek to concomitantly increase PDAC antigenicity, boost PDAC directed T-cell responses, and impair the immunosuppressive character of the TME in order to allow immunotherapeutic agents to unleash their full potential. Eventually, the thorough understanding of the currently available data on immunotherapeutic treatment strategies of PDAC will enable researchers and clinicians to develop improved treatment regimens and to design innovative clinical trials to overcome the pronounced immunosuppression of PDAC.

Bioinformatics Analysis Confirms the Target Protein Underlying Mitotic Catastrophe of 4T1 Cells under Combinatorial Treatment of PGV-1 and Galangin

Pentagamavunon-1 (PGV-1), a potential chemopreventive agent with a strong cytotoxic effect, modulates prometaphase arrest. Improvement to get higher effectiveness of PGV-1 is a new challenge. A previous study reported that the natural compound, galangin, has antiproliferative activity against cancer cells with a lower cytotoxicity effect. This study aims to develop a combinatorial treatment of PGV-1 and galangin as an anticancer agent with higher effectiveness than a single agent. In this study, 4T1, a TNBC model cell, was treated with a combination of PGV-1 and galangin. As a result, PGV-1 and galangin showed a cytotoxic effect with IC50 values of 8 and 120 µM, respectively. Combining those chemicals has a synergistic impact, as shown by the combination index (CI) value of 1. Staining with the May Grunwald-Giemsa reagent indicated mitotic catastrophe evidence, characterized by micronuclear and multinucleated morphology. Moreover, the senescence percentage was higher than the single treatment. Furthermore, bioinformatics investigations showed that PGV-1 and galangin target CDK1, PLK1, and AURKB, overexpression proteins in TNBC that are essential in regulating cell cycle arrest. In conclusion, the combination of PGV-1 and galangin exhibit a synergistic effect and potential to be a chemotherapeutic drug by the mechanism of mitotic catastrophe and senescence induction.

Dynamicity in Host Metabolic Adaptation Is Influenced by the Synergistic Effect of Eugenol Oleate and Amphotericin B During Leishmania donovani Infection In Vitro

Immune metabolic adaptation in macrophages by intracellular parasites is recognized to play a crucial role during Leishmania infection. However, there is little accessible information about changes in a metabolic switch in L. donovani infected macrophages. In previous studies, we have reported on the anti-leishmanial synergic effect of eugenol oleate with amphotericin B. In the present study, we demonstrated that glycolytic enzymes were highly expressed in infected macrophages during combinatorial treatment of eugenol oleate (2.5 µM) and amphotericin B (0.3125 µM). Additionally, we found that the biphasic role in arachidonic acid metabolite, PGE2, and LTB4, is released during this treatment. In vitro data showed that COX-2 mediated PGE2 synthesis increased significantly (p&lt;0.01) in infected macrophages. Not only was the level of prostaglandin synthesis decreased 4.38 fold in infected macrophages after treatment with eugenol oleate with amphotericin B. The mRNA expression of PTGES, MPGES, and PTGER4 were also moderately expressed in infected macrophages, and found to be decreased in combinatorial treatment. In addition, NOS2 expression was activated by the phosphorylation of p38MAPK when combination-treated macrophages were promoted to kill intracellular parasites. The findings of the present study indicate that the synergism between eugenol oleate and amphotericin B could play an important role in immune metabolism adaptation with a concomitant increase in host immune response against the intracellular pathogen, L. donovani.