LXR[1]

Cancer immunotherapy is restricted to immune resistance caused by immunosuppressive tumor microenvironment. Pyroptosis involved in antitumor immunotherapy as a new schedule is prospective to reverse immunosuppression. Herein, acidic tumor microenvironment (TME)-evoked MRC nanoparticles (MRC NPs) co-delivering immune agonist RGX-104 and photosensitizer chlorine e6 (Ce6) are reported for pyroptosis-mediated immunotherapy. RGX-104 remodels TME by transcriptional activation of ApoE to regress myeloid-derived suppressor cells' (MDSCs) activity, which neatly creates foreshadowing for intensifying pyroptosis. Considering Ce6-triggered photodynamic therapy (PDT) can strengthen oxidative stress and organelles destruction to increase immunogenicity, immunomodulatory-photodynamic MRC nanodrugs will implement an aforementioned two-pronged strategy to enhance gasdermin E (GSDME)-dependent pyroptosis. RNA-seq analysis of MRC at the cellular level is introduced to first elucidate the intimate relationship between RGX-104 acting on LXR/ApoE axis and pyroptosis, where RGX-104 provides the prerequisite for pyroptosis participating in antitumor therapy. Briefly, MRC with favorable biocompatibility tackles the obstacle of hydrophobic drugs delivery, and becomes a powerful pyroptosis inducer to reinforce immune efficacy. MRC-elicited pyroptosis in combination with anti-PD-1 blockade therapy boosts immune response in solid tumors, successfully arresting invasive metastasis and extending survival based on remarkable antitumor immunity. MRC may initiate a new window for immuno-photo pyroptosis stimulators augmenting pyroptosis-based immunotherapy.[2] Adv Healthc Mater . 2022 Nov;11(21):e2201233.

When given orally to mice with visible tumors, RGX-104 (100 mg/kg daily) effectively suppressed the growth of several cancer types. The combination of RGX-104 and anti-PD-1 was found to be more effective than either drug administered alone. Significantly, mice that received RGX-104 in addition to anti-PD-1 treatment did so with good tolerance and showed no overt damage [1].

MDSC in vitro Proliferation Assay [1]
Myeloid-derived suppressor cells were isolated as previously described from splenic tissue of tumorbearing mice. One hundred thousand cells were plated in quadruplicates in poly-L-lysine coated plates. After 3 hours of treatment with 1uM Abequolixron (RGX-104) or DMSO as vehicle, cells were fixed with 4% PFA for 15 minutes and wash 3 times with 1X PBS prior staining. Rabbit monoclonal anti-Ki67 antibody (1:400 dilution) was applied at 4C overnight. Cells were incubated with Alexa Fluor 488 secondary antibody (1:200 dilution, Invitrogen) for one hour at room temperature, counterstained with DAPI (1:1000 dilution) and mounted with Prolong Gold. For the analysis of the percentage of Ki67 positive cells, five fields from each replicate were imaged at 20x magnification using Zeiss Axio Imager fluorescence microscope. Image analysis was performed using CellProfiler software.

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MDSC Adhesion Assay [1]
Myeloid-derived suppressor cells were isolated as previously described from splenic tissue of tumor-bearing mice. One hundred thousand cells were plated in triplicates in poly-L-lysine coated plates. Cells were treated with 1uM Abequolixron (RGX-104) or DMSO as vehicle for 2 hours and shaken at 300 rpm for 30 minutes. After this, cells were fixed with 4% PFA for 15 minutes, wash 3 times with 1X PBS, counterstained with DAPI and mounted using Prolong Gold. For the analysis, ten fields from each replicate were imaged at 20x magnification using Zeiss Axio Imager fluorescence microscope. The number of remaining cells was determined using CellProfiler software.

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In Vitro MDSC Apoptosis Assay [1]
Mouse spleens were isolated from either WT, LXRαβ−/−, ApoE−/− or LRP8−/− mice and homogenized to create a single cell suspension. The cells were treated with 1X ACK Lysing Buffer to lyse and remove erythrocytes. MDSCs were isolated from the resulting cell suspension using the Myeloid-Derived Suppressor Cell Isolation Kit. Isolated MDSCs were plated onto slides and treated with either Abequolixron (RGX-104) or murine recombinant ApoE, at the indicated concentrations and times. The samples were then stained with an antibody against Cleaved Caspase-3. RGX-104 was administered either through formulated drug chow at 100mg/kg/day or 50mg/kg/day or delivered via intraperitoneal injection (80mg/kg/day) in a vehicle suspension consisting of corn oil and ethanol (2.5% by volume) as indicated in each figure. Control cohorts were treated with either normal chow (Purina 5001) or with vehicle consisting of corn oil and ethanol (2.5% by volume), respectively. Tumor measurements were taken on the days indicted throughout the course of the experiment with calipers. For survival analysis, mice were euthanized when total tumor burden approached IACUC guidelines with a tumor burden exceeding 1,500 mm3 in volume. For the relevant experiments, anti-PD-1 mAb (clone RMP1-14) or a control isotype-matched antibody was administered at 10mg/kg intraperitoneally on days 3, 6, and 9 post-tumor injection. Gvax was generated as previously described and administered at high frequency (every 3 days) during the experiments as indicated.[1]

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Bone marrow cells are cultured with B16F10 melanoma cells and GM-CSF for 6 days. On day 3, RGX-104 (2 μM) is added to the culture. The mean number of Gr-1high CD11b+ cells per 50 mL of culture solution is assessed by flow cytometry on day 6[1].

Mice[1] B16F10 cancer cells are subcutaneously injected into C57BL/6 mice. Following tumor growth to 5-10 mm3 in volume, mice are fed either control chow, chow supplemented with GW3965 (100 mg/kg), or chow supplemented with RGX-104 (100 mg/kg)[1].

Animal/Disease Models: NOD SCID or RAG mice injected with 1×106 SKOV3 ovarian cancer cells[1].
Doses: 100 mg/kg.
Route of Administration: Oral administration daily for about 60 days.
Experimental Results: Robustly suppressed tumor growth and progression.

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Mice[1] B16F10 cancer cells are subcutaneously injected into C57BL/6 mice. Following tumor growth to 5-10 mm3 in volume, mice are fed either control chow, chow supplemented with GW3965 (100 mg/kg), or chow supplemented with RGX-104 (100 mg/kg)[1].

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RGX-104 was administered either through formulated drug chow at 100mg/kg/day or 50mg/kg/day or delivered via intraperitoneal injection (80mg/kg/day) in a vehicle suspension consisting of corn oil and ethanol (2.5% by volume) as indicated in each figure. Control cohorts were treated with either normal chow (Purina 5001) or with vehicle consisting of corn oil and ethanol (2.5% by volume), respectively. Tumor measurements were taken on the days indicted throughout the course of the experiment with calipers. For survival analysis, mice were euthanized when total tumor burden approached IACUC guidelines with a tumor burden exceeding 1,500 mm3 in volume. For the relevant experiments, anti-PD-1 mAb (clone RMP1-14) or a control isotype-matched antibody was administered at 10mg/kg intraperitoneally on days 3, 6, and 9 post-tumor injection. Gvax was generated as previously described and administered at high frequency (every 3 days) during the experiments as indicated.[1]

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[1]. LXR/ApoE Activation Restricts Innate Immune Suppression in Cancer. Cell. 2018 Feb 8;172(4):825-840.e18.

Abequolixron is an orally bioavailable agonist of the nuclear receptor liver X receptor beta (LXRbeta; NR1H2; LXR-b), with potential immunomodulating and antineoplastic activities. Upon oral administration, abequolixron selectively targets and binds to LXRbeta, thereby activating LXRbeta-mediated signaling, leading to the transcription of certain tumor suppressor genes and the downregulation of certain tumor promoter genes. This particularly activates the expression of apolipoprotein E (ApoE), a tumor suppressor protein, in tumor cells and certain immune cells. This activates the innate immune system, resulting in depletion of immunosuppressive myeloid-derived suppressor cells (MDSCs), tumor cells and endothelial cells in the tumor microenvironment. This reverses immune evasion, enhances anti-tumor immune responses and inhibits proliferation of tumor cells. LXRbeta, a member of the oxysterol receptor family, which is in the nuclear receptor family of transcription factors, plays a key role in cholesterol transport, glucose metabolism and the modulation of inflammatory responses; activation of LXRbeta suppresses tumor cell invasion, angiogenesis, tumor progression, and metastasis in a variety of tumor cell types. The expression of the ApoE protein becomes silenced in human cancers as they grow, become invasive, and metastasize; ApoE silencing is related to reduced survival in cancer patients. The LXR-ApoE pathway regulates the ability of cancers to evade the immune system and recruit blood vessels.

C34H33CLF3NO3

596.078939199448

595.21

C, 68.51; H, 5.58; Cl, 5.95; F, 9.56; N, 2.35; O, 8.05

610318-54-2

RGX-104 hydrochloride;610318-03-1

10218693

White to off-white solid powder

6.3

1

7

13

42

783

1

C[C@H](CCOC1=CC=CC(=C1)CC(=O)O)N(CC2=C(C(=CC=C2)C(F)(F)F)Cl)CC(C3=CC=CC=C3)C4=CC=CC=C4

ZLJZDYOBXVOTSA-XMMPIXPASA-N

InChI=1S/C34H33ClF3NO3/c1-24(18-19-42-29-16-8-10-25(20-29)21-32(40)41)39(22-28-15-9-17-31(33(28)35)34(36,37)38)23-30(26-11-4-2-5-12-26)27-13-6-3-7-14-27/h2-17,20,24,30H,18-19,21-23H2,1H3,(H,40,41)/t24-/m1/s1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.17 mg/mL (3.64 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.08 mg/mL (3.49 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 0.83 mg/mL (1.39 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)