Cellular Efficacy Research Articles

Enhanced sucrose-mediated cryoprotection of siRNA-loaded poly (lactic-co-glycolic acid) nanoparticles

The present study aimed to determine the effects of sucrose on the physical stability, cellular entry pathways and functional efficacy of poly(lactic-co-glycolic acid) nanoparticles (PLGA-NPs). PLGA-NPs were synthesized in the absence or presence of 10 % sucrose, using HEI-101, an unmodified small interfering RNA (siRNA), as a drug model. The newly synthesized HEI-101-loaded PLGA-NPs (HEI-101-NPs) were exposed to repeated freeze-thaw cycles and iteratively tested over a six-month evaluation period. The effect of sucrose stabilization on HEI-101-NPs was independently tested in vitro for biocompatibility and cellular uptake in IMO-2B1 cells. Data analyses suggest that, without sucrose, freeze-thaw cycles of HEI-101-NPs resulted in increased particle diameter, increased polydispersity index, and reduced zeta potential. In contrast, a substantial improvement in the physical stability of HEI-101-NPs was observed in the presence of 10 % sucrose. The data revealed that the release of HEI-101 from the PLGA-NPs was governed by polymer erosion and drug diffusion. Data from cellular uptake study in IMO-2B1 cells demonstrated that, 10 % sucrose significantly reduced the inhibitory effect of nocodazole on the microtubule-dependent uptake of PLGA-NPs. In addition, the presence of 10 % sucrose seemed to lessen the inhibitory effect of sodium azide on the energy-dependent uptake of PLGA-NPs. Overall, the current data suggest that the cellular internalization of PLGA-NPs occurred through the polymerization of actin filaments under the control of the microtubules. Our findings reveal cryoprotective effect of 10 % sucrose on HEI-101-NPs that confers marked improvements in the stability, cellular uptake and efficiency for the delivery of biomolecules to inner ear cells.

IL-2K35C-moFA, a Long-Acting Engineered Cytokine with Decreased Interleukin 2 Receptor α Binding, Improved the Cellular Selectivity Profile and Antitumor Efficacy in a Mouse Tumor Model.

Simple SummaryThe application of IL-2 for treating cancer is limited owing to its toxicity and short half-life. Its high binding ability to IL-2 receptor α expands immunosuppressive Treg cells, which represents an undesirable toxicity in cancer immunotherapy. Moreover, its small molecular size is the reason for its short half-life. We solved these problems by using a covalent modification strategy of IL-2 variant IL-2K35C with fatty acid by maleimide chemistry, namely, IL-2K35C-moFA. The experiments performed in vitro and in vivo proved that IL-2K35C-moFA is a novel immunotherapeutic agent with the potential to selectively stimulate CD8+ T cells and NK cells. Compared to IL-2WT, IL-2K35C-moFA showed a specifically reduced potency for the stimulation of Treg cells. Our results also showed that fatty acid conjugation appears to be effective in half-life extension. The combination of selective lymphocyte expansion and its long half-life means IL-2K35C-moFA should be evaluated as a potential human immunotherapeutic in the future.Human interleukin 2 (IL-2) has shown impressive results as a therapeutic agent for cancer. However, IL-2-based cancer therapy is limited by strong Treg amplification owing to its high binding affinity to IL-2 receptor α (IL-2Rα) and its short half-life owing to its small molecular size. In this study, we solved these problems using a covalent modification strategy of the IL-2 variant, i.e., substituting cysteine (C) for lysine (K) at position 35, using octadecanedicarboxylic acid through maleimide chemistry, creating IL-2K35C-moFA. IL-2K35C-moFA was equipotent to human IL-2 wild type (IL-2WT) in activating tumor-killing CD8+ memory effector T cells (CD8+ T) and NK cells bearing the intermediate affinity IL-2 receptors, and less potent than IL-2WT on CTLL-2 cells bearing the high-affinity IL-2 receptors. Moreover, it was shown to support the preferential activation of IL-2 receptor β (IL-2Rβ) over IL-2Rα because of the mutation and fatty acid conjugation. In a B16F10 murine tumor model, IL-2K35C-moFA showed efficacy as a single dose and provided durable immunity for 1 week. Our results support the further evaluation of IL-2K35C-moFA as a novel cancer immunotherapy.

Triggered amplification of gene theranostics with high accuracy and efficacy using metallo-nanoassemblies

RNA-cleaving DNAzymes hold great promise for gene regulation, yet the low in vivo catalytic efficacy and the unavoidable nonspecific response in normal tissues hinder their practical applications. Herein, we develop a DNA prodrug-laden metallo-nanoassembly for sensitive orthogonal activation of DNAzymes to regulate disease gene with high precision and efficacy. With rich positive charges, metallo-nanoassemblies efficiently load DNA prodrugs (functional DNAzyme subunits), and thus improve drug stability and cellular uptake efficacy. Under tumor microenvironment, the cancer-specific biomarker triggers the amplified assembly of DNA prodrugs into active DNAzymes to efficiently cleave tumor-involved mRNA. Meanwhile, these glutathione-responsive metallo-nanoassemblies decompose and release metal ions as self-sufficient DNAzyme cofactors, which are accommodated into the afore-assembled DNAzymes to enhance the gene cleavage activity. Moreover, upon laser stimulus, these photothermal metallo-nanoassemblies can also remarkably promote the catalytic reaction of DNAzymes via the elevated local temperature. Such a smart delivery approach allows disease diagnosis via sensitive fluorescence and photothermal imaging, and achieves the precise and robust gene regulation with photothermal ablation as an auxiliary therapy. These results provide a new route to design programmable and activatable prodrugs for precision nanomedicine.

Lenvatinib Synergistically Promotes Radiation Therapy in Hepatocellular Carcinoma by Inhibiting Src/STAT3/NF-κB-Mediated Epithelial–Mesenchymal Transition and Metastasis

This study suggested that lenvatinib may incapacitate hepatocellular carcinoma (HCC) to radiation treatment by abrogating radiation-induced Src/signal transducer and the activator of transcription 3 signaling (STAT3)/nuclear factor-κB (NF-κB) to escalate radiation-induced extrinsic and intrinsic apoptosis. These findings uncover the role of targeting Src and its arbitrating epithelial-mesenchymal transition (EMT), which could increase the anti-HCC efficacy of radiation therapy (RT). Lenvatinib and sorafenib are multikinase inhibitors used to treat HCC. Lenvatinib is noninferior to sorafenib in the therapeutic response in HCC. However, whether lenvatinib intensifies the anti-HCC efficacy of RT is ambiguous. Several oncogenic kinases and transcription factors, such as Src, STAT3, and NF-κB, enhance the radiosensitivity of cancers. Therefore, we aimed to investigate the roles of the Src/STAT3/NF-κB axis in HCC after RT treatment and assessed whether targeting Src by lenvatinib may enhance the effectiveness of RT. Hep3B, Huh7, HepG2, and SK-Hep1 HCC cells and 2 types of animal models were used to identify the efficacy of RT combined with lenvatinib. Cellular toxicity, apoptosis, DNA damage, EMT/metastasis regulation, and treatment efficacy were validated by colony formation, flow cytometry, Western blotting, and in vivo experiments, respectively. Knockdown of Src by siRNA was also used to validate the role of Src in RT treatment. Silencing Src reduced STAT3/NF-κB signaling and sensitized HCC to radiation. Lenvatinib reversed radiation-elicited Src/STAT3/NF-κB signaling while enhancing the anti-HCC efficacy of radiation. Both lenvatinib and siSrc promoted the radiation effect of cell proliferation on suppression, inhibition of the invasion ability, and induction of apoptosis in HCC. Lenvatinib also alleviated radiation-triggered oncogenic and EMT-related protein expression. Our findings uncovered the role of the Src/STAT3/NF-κB regulatory axis in response to radiation-induced toxicity and confirmed Src as the key regulatory molecule for radiosensitization of HCC evoked by lenvatinib.

Monitoring of Both Humoral and Cellular Immunities Could Early Predict COVID-19 Vaccine Efficacy Against the Different SARS-CoV2 Variants.

Reliable immunoassays are essential to early predict and monitor vaccine efficacy against SARS-CoV-2. The performance of an Interferon Gamma Release Assay (IGRA, QuantiFERON® SARS-CoV-2), and a current anti-spike serological test, compared to a plaque reduction neutralization test (PRNT) taken as gold standard were compared. Eighty vaccinated individuals, whose 16% had a previous history of COVID-19, were included in a longitudinal prospective study and sampled before and two to four weeks after each dose of vaccine. In non-infected patients, 2 doses were required for obtaining both positive IGRA and PRNT assays, while serology was positive after one dose. Each dose of vaccine significantly increased the humoral and cellular response. By contrast, convalescent subjects needed a single dose of vaccine to be positive on all 3 tests. Both IGRA and current serology assay were found predictive of a positive titer of neutralizing antibodies that is correlated with vaccine protection. Patients over 65 or 80 years old had a significantly reduced response. The response tended to be better with the heterologous scheme (vs. homologous) and with the mRNA-1273 vaccine (vs. BNT162b2) in the homologous group, in patients under 55 and under 65 years old, respectively. Finally, decrease intensity or absence of IGRA response and to a less extent of anti-spike serology were also correlated to reinfection which has occurred during the follow up. In conclusion, both IGRA and current anti-spike serology assays could be used at defined thresholds to monitor the vaccine response against SARS-CoV-2 and to simply identify non-responding individuals after a complete vaccination scheme.Capsule summaryTwo available specific tests (IGRA and anti-spike antibodies) could early assess the vaccine-induced immunity against SARS-CoV-2 at the individual scale, to potentially adapt the vaccination scheme in non-responder patients.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10875-022-01354-x.

Replica-mold nanopatterned PHEMA hydrogel surfaces for ophthalmic applications

Biomimicking native tissues and organs require the development of advanced hydrogels. The patterning of hydrogel surfaces may enhance the cellular functionality and therapeutic efficacy of implants. For example, nanopatterning of the intraocular lens (IOL) surface can suppress the upregulation of cytoskeleton proteins (actin and actinin) within the cells in contact with the IOL surface and, hence, prevent secondary cataracts causing blurry or opaque vision. Here we introduce a fast and efficient method for fabricating arrays consisting of millions of individual nanostructures on the hydrogel surface. In particular, we have prepared the randomly distributed nanopillars on poly(2-hydroxyethyl methacrylate) hydrogel using replica molding and show that the number, shape, and arrangement of nanostructures are fully adjustable. Characterization by atomic force microscopy revealed that all nanopillars were of similar shape, narrow size distribution, and without significant defects. In imprint lithography, choosing the appropriate hydrogel composition is critical. As hydrogels with imprinted nanostructures mimic the natural cell environment, they can find applications in fundamental cell biology research, e.g., they can tune cell attachment and inhibit or promote cell clustering by a specific arrangement of protrusive nanostructures on the hydrogel surface.

Cellular antioxidant and viability efficacy of Harpephyllum caffrum peel and Syzygium guineense seed extracts

Crude phenolic extracts of indigenous sub-Saharan fruit byproducts have potent antioxidant activity. Purification of these extracts and evaluation of their antioxidant activity and viability using appropriate biological models, has, however, not yet been completed. The current study quantitatively evaluated the phenolic composition and in vitro antioxidant activity of crude and purified extracts (PE) of Harpephyllum caffrum peel (HCP) and Syzygium guineense seed (SGS), and cellular antioxidant and viability efficacy of the PE. Phenols were extracted by 60% ethanol (v/v) solvent system and purified using XAD7HP macroporous adsorption resin. The UPLC/Q-TOF-MS was used for the characterization and identification of phenols. Cellular antioxidant activity (CAA) assay was applied to assess the antioxidant efficacy and cytotoxicity of the PE. Analysis of variance of data was done using a generalized linear mixed model with fruit type, extract purity and their interaction as fixed effects. Purification increased (P ≤ 0.05) HCP total phenolic concentration (TPC) by over 200%, while SGS TPC decreased (P ≤ 0.05) by 20%. Regardless of fruit type, PE had higher (P ≤ 0.05) quantities of proanthocyanidins and anthocyanins than crude extracts. The HCP-PE had greater (P ≤ 0.05) in vitro antioxidant activity than the other extracts (P ≤ 0.05). Cellular antioxidant activity of HCP-PE was comparable to that of sodium metabisulphite (P > 0.05). Also, HCP-PE had lower (P ≤ 0.05) cytotoxic effects than SGS and sodium metabisulphite with increasing antioxidant concentration. It was concluded that HCP-PE is a potential source of safe natural antioxidants for nutraceutical applications.

Development of minimal physiologically-based pharmacokinetic-pharmacodynamic models for characterizing cellular kinetics of CAR T cells following local deliveries in mice

Chimeric antigen receptor (CAR) T cell therapies have revolutionized the treatment of hematologic malignancies and have potentials for solid tumor treatment. To overcome limited CAR T cell infiltration to solid tumors, local delivery of CAR T cells is a practical strategy that has shown promising therapeutic outcome and safety profile in the clinic. It is of great interest to understand the impact of dosing routes on CAR T cell distribution, subsequent proliferation and tumor killing in a quantitative manner to identify key factors that contribute to CAR T efficacy and safety. In this study, we established mouse minimal physiologically-based pharmacokinetic (mPBPK) models combined with pharmacodynamic (PD) components to delineate CAR T cell distribution, proliferation, tumor growth, and tumor cell killing in the cases of pleural and liver tumors. The pleural tumor model reasonably captured published CAR T cellular kinetic and tumor growth profiles in mice. The mPBPK-PD simulation of a liver tumor mouse model showed a substantial increase in initial tumor infiltration and earlier CAR T cell proliferation with local hepatic artery delivery compared to portal vein and intravenous (i.v.) injections whereas portal vein injection showed little difference from i.v. administration, suggesting the importance of having the injection site close to tumor for maximal effect of non-systemic administration. Blood flow rate in the liver tumor was found to be a sensitive parameter for cellular kinetics and efficacy, indicating a potential role of tumor vascularization in the efficacy of CAR T cell therapies.

Production of high loading insulin nanoparticles suitable for oral delivery by spray drying and freeze drying techniques

Insulin nanoparticles (NPs) with high loading content have found diverse applications in different dosage forms. This work aimed to evaluate the impact of freeze-drying and spray drying process on the structures of insulin-loaded chitosan nanoparticles, with or without mannitol as cryoprotectants. We also assessed the quality of these nanoparticles by redissolving them. Before dehydration, the chitosan/sodium tripolyphosphate/insulin crosslinked nanoparticles were optimized to 318 nm of particle size, 0.18 of PDI, 99.4% of entrapment efficiency, and 25.01% of loading content. After reconstitution, all nanoparticles, except the one produced by the freeze-drying method without using mannitol, maintained their spherical particle structure. The nanoparticles dehydrated by spray drying without mannitol also showed the smallest mean particle size (376 nm) and highest loading content (25.02%) with similar entrapment efficiency (98.7%) and PDI (0.20) compared to mannitol-containing nanoparticles dehydrated by either spray drying or freeze-drying techniques. The nanoparticles dried by spray drying without mannitol also resulted in the fastest release and highest cellular uptake efficacy of insulin. This work shows that spray drying can dehydrate insulin nanoparticles without the need for cryoprotectants, creating a significant advantage in terms of greater loading capacity with lower additive requirements and operating costs as compared to conventional freeze drying approaches.

Abstract LB090: Targeting GCN2 kinase-driven stress response inactivation to restore immunity in AML Cancers

Abstract Introduction/Purpose: The most common leukemia in adults is Acute myeloid leukemia (AML) and the 5-year overall survival (OS) in all AML patients is approximately 24%. The highest of AML deaths in the US alone is among older patients at ages ≥ 65 emphasizing the need for better therapeutics. The older patients have more limited curative options available due to their inability to tolerate aggressive chemotherapy. Identifying ways to boost immunotherapy responses could change the paradigm of AML, a disease still difficult to treat despite several approved targeted immunotherapeutic. The development of effective small molecules would be significant since small molecules not only target immunosuppressive mechanisms like mAbs but can stimulate intracellular pathways where antibodies are unable to permeate. In addition, small molecules can provide optimal pharmacokinetics and pharmacodynamics for oral administration, amenable clinical dosing, and can induce relatively acute antitumor efficacy to avoid systemic immunogenicity, thus providing a superior therapeutic potential. The highly proliferative nature of tumor cells, along with infiltration of myeloid cells into the AML leads to depletion of nutrients such as functional/natural amino acids. A key meditator of this nutrient stress pathway, the cytoplasmic GCN2 (EIF2AK4) kinase, switches on reduction of amino acids, and this activity results in T-cell inactivation, T-cell death, regulatory T-cell expansion, and the potentiation of myeloid-derived suppressor cells (MDSCs). We have discovered a series of novel small molecule immunotherapeutic agents (HCI-1046) that reversibly bind to GCN2 kinase, competitively block the ATP site, and elicit responses in AML patient samples and cell lines. Experimental Procedures: GCN2 cell-free kinase binding, EIF2α family selectivity, pGCN2 inhibition assays were performed and confirmed its on-target efficacy and potency of lead inhibitor HCI-1046. Additional experiments were conducted including CellTiterGlo, IncuCyte Live Cell Imaging, FACS Flow Cytometry, Cytotoxicity, Western Blot, ELISpot, and ELISA assays. Results and Summary: GCN2 cell-free kinase binding, kinome selectivity, pGCN2, pEIF2α, ATF4 inhibition data confirmed on-target activities of our lead GCN2 inhibitor HCI-1046. HCI-1046 demonstrated potent activity, with an IC50 of 36 nM in inhibiting GCN2 kinase, and exhibited cellular efficacy with an IC50 of 0.1 to 0.5 μM range. Our preliminary results support the hypothesis that the inhibition of GCN2 reinstates anti-tumor immunity and blocks tumor progression in the AML cancer model in vivo. Our in vivo PK studies on HCI-1046 in rodent species showed excellent PK properties with 55% oral bioavailability, low clearance, and >5.0-hour half-life. Thus, HCI-1046 is nominated as a pre-clinical agent. Additional data regarding the evaluation of the effects of HCI-1046 on the MDSC-suppressive function on T-cells using ELISpot, ELISA assays in AML patient samples, and other mouse model efficacy studies results will be discussed. Citation Format: Zhaoliang Li, Kyle Medley, Dongqing Yan, Kimberly Coffman, Tony Pomicter, David Lum, David J. Bearss, Hariprasad Vankayalapati. Targeting GCN2 kinase-driven stress response inactivation to restore immunity in AML Cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB090.

Abstract LB137: Orally available, CDK9 selective, small molecule inhibitors shows promise in n-Myc-driven neuroendocrine prostate cancers (NEPC)

Abstract Introduction/Purpose: Amplification of N-MYC causes its overexpression in Neuroendocrine Prostate Cancer (NEPC) patients is prevalent, and it is a rare lethal subtype of cancer detected in 2% of all Prostate Cancers (PC)and over 10-17% of mCRPC (Metastatic Castration-Resistant PC) patients. N-MYC and activated CDK9 act as oncogenic drivers sufficient to transform human-derived prostate cancer cells to take on NEPC phenotypic changes resulting in a more aggressive disease identified in late-stage human PC patients. Additionally, N-MYC is required for tumor resistance, and its downregulation through CDK9 inhibitors lessens the tumor burden. Our efforts in this study have proven that the targeting of N-NYC as a driver of NEPC with CDK9 inhibitors such as BLX-3030 and its series could be a viable approach for therapeutic intervention of mCRPC and NEPCs. Experimental Procedures: Utilizing our FIELDS fragment-based design strategies, we have developed and synthesized a series of novel small molecule CDK9 agents that reversibly bind to CDK9, competitively block the ATP site, and elicit pharmacological responses in N-MYC and other prostate cancer cells in vitro. CDK9 cell-free kinase binding, CDKs selectivity, pCDK9 inhibition assays were performed and confirmed its on-target efficacy and potency of lead CDK9 inhibitor BLX-3030. Results and Summary: In these studies, our lead CDK9 kinase inhibitor BLX-3030 from over 60 plus analogs demonstrated potent activity with an IC50 of <5 nM in inhibiting CDK9. CDK9 and N-MYC expression has been detected in PC-3, DU-145, LNCaP, and in LASCPC-01, NCI-H660 and 22RV1 cell lines. HCI-3030 exhibited potent cellular efficacy with an IC50 of 22 to 590 nM in PC cell lines and in N-MYC expressed cells had an activity of 76, 470, and 132 nM respectively and reduced pCDK9, C-MYC, Mcl-1, N-MYC and its downstream RNA Pol II (Ser-2) and pAR (Ser-81) dose-dependently. Our preliminary results support the hypothesis that inhibition of CDK9 reinstates anti-tumor and blocks tumor progression in both mCRPC and NEPCs cellular models. BLX-3030 and a few of its analogs had excellent PK properties in mouse species in the range of 28-94% F, low clearance, and >3 hours half-life. BLX-3030 is nominated as a pre-clinical agent for additional 3D, FACS/apoptosis, cell migration, live-cell imaging including immunofluorescent studies along with in vivo prostate cancer mouse model efficacy studies as well and these results will be presented. Conclusions: In summary, a series of BLX-3030, its analogs as CDK9 inhibitors, display a range of potency in prostate cancer and NEPC cells with ideal PK parameters, were identified. Within compounds of this profile, BLX-3030 was selected as a candidate CDK9 inhibitor upon in-depth evaluation of the in vitro and in Vivo PK properties further supporting our conclusion that BLX-3030 had the appropriate qualities for development as a pre-clinical candidate. Citation Format: Kyle Medley, Zhaoliang Li, Saisha Vankayalapati, Sowmya Paritala, Dongqing Yan, Kimberly Coffman, Neeraj Agarwal, David J. Bearss. Orally available, CDK9 selective, small molecule inhibitors shows promise in n-Myc-driven neuroendocrine prostate cancers (NEPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB137.

Development of Irinotecan Liposome Armed with Dual-Target Anti-Epidermal Growth Factor Receptor and Anti-Fibroblast Activation Protein-Specific Antibody for Pancreatic Cancer Treatment.

Pancreatic cancer is one of the most common causes of death in Taiwan. Previous studies have shown that more than 90% of pancreatic cancer cells presented epidermal growth factor receptor (EGFR) cell marker, and this marker is thought to be important as it is related to activation of cancer cell proliferation, angiogenesis, and cancer progression. Moreover, tumor-associated fibroblasts were involved in tumor proliferation and progression. In this study, we fabricated an anti-EGFR and anti-fibroblast activation protein bispecific antibody-targeted liposomal irinotecan (BS−LipoIRI), which could specifically bind to pancreatic cancer cells and tumor-associated fibroblasts. The drug encapsulation efficiency of BS−LipoIRI was 80.95%, and the drug loading was 8.41%. We proved that both pancreatic cancer cells and fibroblasts could be targeted by BS−LipoIRI, which showed better cellular uptake efficacy compared to LipoIRI. Furthermore, an in vivo mouse tumor test indicated that BS−LipoIRI could inhibit pancreatic cancer growth up to 46.2% compared to phosphate-buffered saline control, suggesting that BS−LipoIRI could be useful in clinical cancer treatment.

Phytochemical profiling and cellular antioxidant efficacy of different rice varieties in colorectal adenocarcinoma cells exposed to oxidative stress

In the present study, white (Baegilmi), brown (hyunmi) and black (chalheugmi) Korean local rice varieties ethanol extracts were analyzed for in-vitro antioxidant assays (ABTS, FRAP and DPPH), cellular antioxidant activities (CAAs) and phenolic phytochemicals content. The highest antioxidant assays, phenolic, flavonoid and anthocyanins content were identified among the free fractions of black rice. Phenolic phytochemicals were detected and quantified using the ultra-high-performance liquid tandem chromatography quadrupole flight mass spectrometry (UHPLC-Q-TOF-MS2). Which indicated the richness of several phytochemicals like ascorbic acid, vanillic acid, p-coumaric acid, catechin, epigallocatechin and quercetin in black rice than in other rice samples. The cellular antioxidant activities (CAA) of black rice were found equivalent to that of ascorbic acid, the standard employed in the assay. The CAAs of free fractions were as follows: white rice < brown rice < black rice. These findings are significant for enhancing human health through increased consumption of black and brown rice in the development of functional food products.

Layered Double Hydroxides-Loaded Sorafenib Inhibit Hepatic Stellate Cells Proliferation and Activation In Vitro and Reduce Fibrosis In Vivo.

A core feature of liver fibrosis is the activation of hepatic stellate cells (HSCs), which are transformed into myofibroblasts and lead to the accumulation of extracellular matrix (ECM) proteins. In this study, we combined in vitro cellular efficacy with in vivo antifibrosis performance to evaluate the outcome of sorafenib (SRF) loaded layered double hydroxide (LDH) nanocomposite (LDH-SRF) on HSCs. The cellular uptake test has revealed that sorafenib encapsulated LDH nanoparticles were efficiently internalized by the HSC-T6 cells, synergistically inducing apoptosis of hepatic stellate cells. Moreover, the apoptosis rate and the migration inhibition rate induced by LDHs-SRF were 2.5 and 1.7 times that of SRF. Western Blot showed that the TGF-β1/Smad/EMT and AKT signaling pathway was significantly inhibited in HSC-T6 cells treated with LDHs-SRF. For the in vivo experiment, LDHs-SRF were administered to rat models of CCl4-induced liver fibrosis. H&E, masson and sirius red staining showed that LDHs-SRF could significantly reduce inflammatory infiltrate and collagen fiber deposition and immunohistochemical results found that LDHs-SRF treatment significantly inhibited the protein expressions of α-SMA in the liver, these results suggesting that LDHs-SRF exhibited better anti-fibrotic effect than SRF alone and significantly inhibited the proliferation and activation of rat hepatic stellate cells and collagen fiber synthesis.

MO040: Development of BBP-711, a small molecule inhibitor of glycolate oxidase for primary hyperoxaluria type 1 and recurrent kidney stone formers

Abstract BACKGROUND AND AIMS BBP-711 is an investigational small molecule inhibitor of glycolate oxidase (GO), currently in development for the treatment of primary hyperoxaluria type 1 (PH1) and recurrent nephrolithiasis. Selection of BBP-711 as a drug candidate followed structural activity optimization and a pharmacological testing strategy that included functional activity in enzyme assays, target engagement in cellular assays and efficacy studies in a rodent model (Agxt−/− mice) of primary hyperoxaluria type 1. METHOD BBP-711 was developed by iterative synthesis and study of the structure–activity relationship of more than 50 GO inhibitors. Lead molecules including BBP-711 were assessed for functional activity by determining inhibition of the conversion of glycolate to oxalate with purified human, mouse and rat HAOX1 enzymes. The ability of BBP-711 to bind directly to immobilized purified hGO (HAOX1 gene product) was evaluated by surface plasmon resonance. Selectivity of BBP-711 was assessed by determining its inhibition of several physiologically related gene products including that from HAOX2. In vitro efficacy of BBP-711 was tested via inhibition of oxalate production in primary hepatocytes cultured from hyperoxaluric Agxt−/− mice. In vivo efficacy of BBP-711 on urinary oxalate excretion was assessed at various doses given by oral gavage in Agxt −/− mice. Pharmacokinetics of BBP-711 was investigated to assess plasma and liver concentrations associated with efficacy. RESULTS BBP-711 is a potent inhibitor of human, rat and mouse GO (IC50 = 15.4 nM, 22.4 nM and 149 nM, respectively). BBP-711 demonstrated stronger direct binding to hGO1 (KD = 6.31 nM) than to rGO2 (KD = 12.8 nM), indicating its preference for GO1 as a target. BBP-711 specificity was demonstrated with &amp;lt;10% activity against off-target enzymes D-amino acid oxidase, dihydroorotate dehydrogenase and lactate dehydrogenase-A at 10 µM. BBP-711 demonstrated concentration-dependent inhibition of oxalate production by Agxt−/− hepatocytes (IC50 = 24.2 nM at 24 hours, IC50 = 42.9 nM at 48 hours). When orally dosed at 7 mg/kg for 5 days in Agxt−/− mice, BBP-711 produced a maximum reduction of urinary oxalate (60%) as well as maximum inhibition of GO activity (&amp;gt;88%). Higher doses did not result in greater reduction in urinary oxalate. Liver exposures (AUC0-24hr) were higher than plasma exposures at all doses tested. GO engagement by BBP-711 was also assessed in an ex vivo assay. Liver extracts from Agxt−/− and wild-type mice dosed with BBP-711 show a dose-dependent reduction in ex vivo GO activity at 1 and 4-hour post-dose. CONCLUSION Iterative and rational optimization of small molecule GO inhibitors resulted in BBP-711, a potent and specific inhibitor of GO with drug-like properties.

New ZW4864 Derivatives as Small-Molecule Inhibitors for the β-Catenin/BCL9 Protein-Protein Interaction.

A series of 1-(3-(2-amino-2-oxoethoxy)phenyl)piperidine-3-carboxamide derivatives was reported as new small-molecule β-catenin/B-cell lymphoma 9 (BCL9) protein-protein interaction (PPI) inhibitors. Compounds 17-21 were discovered to inhibit the β-catenin/BCL9 PPI with K i = 0.85-2.7 μM. The effects of 21 on the β-catenin/BCL9 PPI in cellular context were demonstrated by β-catenin/BCL9 pull-down inhibition and dose-dependent suppression of Wnt/β-catenin signal transactivation. Notably, compound 21 is more potent than ZW4864, a previously reported analogue, in modulating transcription and expression of β-catenin target genes and suppressing survival of β-catenin-dependent cancer cells. The cellular on-target efficacy of 21 was demonstrated by β-catenin rescue experiments. Compound 21 represents a promising starting point for further optimization of β-catenin/BCL9 PPI inhibitors.

NEUROPROTECTIVE EFFECT OF VANDA TESSELLATA AS “RASNA” SPECIES, ON ALUMINIUM CHLORIDE INDUCED ALZHEIMER’S IN RATS

The present study focused on evaluation of neuroprotective effect of hydromethanolic extracts of Vanda tessellate (VT), also considered as Rasna. Aluminum Chloride (AlCl3) induces neuroinflammation in rats and finally the development of AD. PASS online and molecular docking insilico studies were conducted with PPAR-γ for β-sitosterol and AChE for gigantiol. Total 36 trained Wistar rats were divided into VI groups 6 in each. Group I - normal control, Group II – Disease control, Group III – Rivastigmine (0.3mg/kg, p.o), Group IV and V – Hydromethanolic extract of VT (HMEVT, 150mg/kg, 300mg/kg, p.o) respectively, Group VI - Ayurvedic Formulation of Rasna (AFR) (1ml/kg, p.o). All the animals received Aluminum Chloride (AlCl3) (300mg/kg, p.o) except group I. The rats were treated for 20 days but mean time behavioural study, body weight changes were monitored on 0th, 7th, 14th&amp; 20th day. On 21th day, rats were sacrificed, brains were isolated, then antioxidant enzymes levels, protein content and neurotransmitters levels were determined. Histopathology of cortex and hippocampus parts of the brain were studied. Group II animals showed reduction in locomotor activity, increased in the number of entries as well as time spent in closed arm and time taken to climb the pole was increased but it was reversed in groups treated with 150mg/kg, 300mg/kg doses of HMEVT and AFR. Increased level of protein content, malondialdehyde, reduction in body weight and antioxidants enzymes like superoxide dismutase, catalase, glutathione were observed in disease control group and it was due to free radicals generation and were corrected and restored in groups treated with HMEVT and AFR. Moreover, the histopathological report also showed cellular level protection efficacy found with HMEVT and AFR. The neuroprotective action of HMEVT was due to the active constituents and was proved in insilico study. The order of neuroprotective efficacy was HMEVT &gt; AFR.

In Vivo Cellular Expansion of Lisocabtagene Maraleucel and Association With Efficacy and Safety in Relapsed/Refractory Large B-Cell Lymphoma.

Lisocabtagene maraleucel (liso‐cel) is an autologous, CD19‐directed, chimeric antigen receptor T‐cell product for the treatment of adult patients with relapsed or refractory large B‐cell lymphoma (LBCL) after 2 or more lines of systemic therapy. In vivo cellular expansion after single‐dose administration of liso‐cel has been characterized. In this article, in vivo liso‐cel expansion in the pivotal study TRANSCEND NHL 001 (ClinicalTrials.gov identifier, NCT02631044) was further characterized to assess the relationship between in vivo cellular expansion after single‐dose administration of liso‐cel and efficacy or safety after adjusting for key baseline characteristics. Two bioanalytical methods, quantitative polymerase chain reaction and flow cytometry, were used for the assessment of cellular kinetics of liso‐cel, which showed high concordance for in vivo cellular expansion. Multivariable logistic regression analyses demonstrated that higher in vivo cellular expansion of liso‐cel was associated with a higher overall response and complete response rate, and a higher incidence of cytokine release syndrome and neurological events in patients with relapsed or refractory LBCL. Age and tumor burden (by sum of the product of perpendicular diameters) were likely to confound the relationship between in vivo cellular expansion and efficacy, where the association became stronger after controlling for these factors. Repeat dosing of liso‐cel was tested in the study; however, in vivo cellular expansion of liso‐cel was lower after repeat dosing than after the initial dose. These findings should enable a comprehensive understanding of the in vivo cellular kinetics of liso‐cel and the association with outcomes in relapsed/refractory LBCL.

Effects of the Surface Charge of Graphene Oxide Derivatives on Ocular Compatibility.

The incorporation of functional groups endows graphene oxide (GO) with different surface charges, which plays important roles in biological interactions with cells. However, the effect of surface charge of GO derivatives on ocular biocompatibility has not been fully elucidated. Previously, we found that positively, negatively and neutrally charged PEGylated GO (PEG-GO) nanosheets exerted similar effect on the viability of ocular cells. In this work, we performed in vitro and in vivo studies to comprehensively study the effect of surface charge of PEG-GO on ocular compatibility. The in vitro results showed that the cellular uptake efficacy of negatively charged PEG-GO nanosheets was significantly decreased compared with positively charged and neutrally charged analogs. However, three kinds of PEG-GO nanosheets produced similar amounts of intracellular reactive oxygen species and showed similar influence on mitochondrial membrane potential. By analysis of global gene expression profiles, we found that the correlation coefficients between three kinds of PEG-GO-treated cells were more than 0.98. Furthermore, in vivo results showed that all these PEG-GO nanosheets had no significant toxicity to ocular structure and function. Taken together, our work suggested that surface charge of PEG-GO exerted negligible effect on its ocular compatibility, except for the cellular uptake. Our work is conducive to understanding the relationship between surface charge and biocompatibility of GO derivatives.

Discovery of HDAC6-Selective Inhibitor NN-390 with in Vitro Efficacy in Group 3 Medulloblastoma.

Histone deacetylase 6 (HDAC6) has been targeted in clinical studies for anticancer effects due to its role in oncogenic transformation and metastasis. Through a second-generation structure-activity relationship (SAR) study, the design, and biological evaluation of the selective HDAC6 inhibitor NN-390 is reported. With nanomolar HDAC6 potency, >200-550-fold selectivity for HDAC6 in analogous HDAC isoform functional assays, potent intracellular target engagement, and robust cellular efficacy in cancer cell lines, NN-390 is the first HDAC6-selective inhibitor to show therapeutic potential in metastatic Group 3 medulloblastoma (MB), an aggressive pediatric brain tumor often associated with leptomeningeal metastases and therapy resistance. MB stem cells contribute to these patients' poor clinical outcomes. NN-390 selectively targets this cell population with a 44.3-fold therapeutic margin between patient-derived Group 3 MB cells in comparison to healthy neural stem cells. NN-390 demonstrated a 45-fold increased potency over HDAC6-selective clinical candidate citarinostat. In summary, HDAC6-selective molecules demonstrated in vitro therapeutic potential against Group 3 MB.