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Abequolixron HCl (formerly RGX-104; SB-742881; SB742881), the hydrochloride salt of RGX104, is a novel potent and orally bioavailable liver-X nuclear hormone receptor-beta (LXR) agonist that modulates innate immunity via transcriptional activation of the ApoE gene. It has potential immunomodulating and antineoplastic activities. RGX-104 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.
Physicochemical Properties
| Molecular Formula | C34H34CL2F3NO3 | |
| Molecular Weight | 632.54 | |
| Exact Mass | 631.186 | |
| Elemental Analysis | C, 64.56; H, 5.42; Cl, 11.21; F, 9.01; N, 2.21; O, 7.59 | |
| CAS # | 610318-03-1 | |
| Related CAS # | RGX-104;610318-54-2; 610318-03-1 (HCl); 2648455-06-3 (zinc) | |
| PubChem CID | 68861574 | |
| Appearance | White to off-white solid powder | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 7 | |
| Rotatable Bond Count | 13 | |
| Heavy Atom Count | 43 | |
| Complexity | 783 | |
| Defined Atom Stereocenter Count | 1 | |
| SMILES | C1C=CC(C(C2=CC=CC=C2)CN(CC2C=CC=C(C=2Cl)C(F)(F)F)[C@H](C)CCOC2=CC(=CC=C2)CC(=O)O)=CC=1.Cl |
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| InChi Key | LCMIYQOJZLRHTO-GJFSDDNBSA-N | |
| InChi Code | InChI=1S/C34H33ClF3NO3.ClH/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);1H/t24-;/m1./s1 | |
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| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
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. |
| ln Vitro |
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. RGX-104 (1 μM) significantly reduced the survival of myeloid-derived suppressor cells (MDSCs) isolated from tumor-bearing mice after 3 hours of incubation in vitro. [1] RGX-104 (2 μM) significantly reduced the number of Gr-1high CD11b+ granulocytic MDSCs generated in a transwell co-culture system of bone marrow cells with B16F10 melanoma cells and GM-CSF. [1] RGX-104 (1 μM) significantly enhanced cleaved caspase-3 staining (apoptosis) in MDSCs isolated from wild-type tumor-bearing mice after a 6-hour culture, but this effect was abolished in MDSCs from LXRαβ−/− mice. [1] Direct in vitro treatment of isolated CD8+ T cells with RGX-104 did not modulate their activation or proliferation. [1] |
| ln Vivo |
When mice with visible tumors are given GW3965 or RGX-104 hydrochloride orally, the growth of many cancer types is considerably suppressed. Large tumor-bearing animals also exhibit strong inhibition of tumor growth. Sometimes the therapy results in either full or partial tumor regression. A broad range of cancers, including as lung cancer, melanoma, glioblastoma, ovarian, renal cell, triple-negative breast, and colon cancer, have responses[1]. Oral administration of RGX-104 (100 mg/kg/day via formulated chow) significantly suppressed the growth of large SKOV3 ovarian cancer xenograft tumors in RAG−/− mice, causing some complete regressions. [1] Oral RGX-104 (100 mg/kg/day) significantly suppressed tumor growth in immunocompetent C57BL/6 mice bearing established GL261 glioblastoma, LLC lung cancer, Renca renal cancer, B16F10 melanoma, or MC38 colon cancer tumors. [1] Oral RGX-104 (100 mg/kg/day) significantly reduced the growth of large U118 glioblastoma xenograft tumors in NOD SCID mice. [1] Intraperitoneal administration of RGX-104 (80 mg/kg/day) to C57BL/6 mice bearing B16F10 tumors significantly reduced the abundance of both granulocytic and monocytic MDSCs within the tumor, spleen, and peripheral blood. [1] This MDSC depletion was associated with a ~7-fold increase in tumor-infiltrating IFN-γ+ Granzyme B+ CD8+ cytotoxic T lymphocytes (CTLs) and a ~4-fold increase in IFN-γ+ TNF-α+ CD4+ T cells. [1] Combination of oral RGX-104 (50 mg/kg/day) with adoptive transfer of gp100-specific CD8+ T cells and vaccination enhanced anti-B16F10 tumor activity and increased mouse survival compared to adoptive cell therapy alone. [1] In a B16F10 model combining adoptive cell therapy, vaccination, and anti-PD-1, co-administration of oral RGX-104 (100 mg/kg/day) was superior to anti-PD-1 alone. [1] In the Lewis Lung Carcinoma (LLC) model, co-administration of oral RGX-104 (100 mg/kg/day) with anti-PD-1 yielded synergistic anti-tumor activity. [1] In a B16F10 model without vaccination, co-administration of oral RGX-104 (50 mg/kg/day) with anti-PD-1 significantly enhanced anti-tumor activity and augmented tumor-infiltrating CTL abundance compared to anti-PD-1 alone. [1] In a B16F10 model where Gvax increased MDSC accumulation and rendered anti-PD-1 ineffective, the addition of oral LXR agonist (GW3965, 100 mg/kg/day) to anti-PD-1 reduced tumoral MDSCs to baseline levels and significantly impaired tumor growth. [1] B16F10 tumors grown in LXRαβ−/− mice failed to exhibit significant growth inhibition upon RGX-104 treatment. [1] LXR treatment failed to significantly reduce tumoral MDSC levels or tumor volume in Apoe−/− mice bearing ApoE-depleted B16F10 tumor cells. [1] |
| Cell Assay |
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].
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. 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. 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. For the in vitro MDSC apoptosis assay, MDSCs were isolated from the spleens of tumor-bearing mice using a magnetic bead-based isolation kit. Isolated MDSCs were plated and treated with RGX-104 or vehicle for specified times. Cells were then fixed and stained with an antibody against cleaved caspase-3. The percentage of cleaved caspase-3 positive cells was quantified by fluorescence microscopy or flow cytometry. [1] For the MDSC in vitro proliferation assay, isolated MDSCs were plated in coated plates, treated with RGX-104 or vehicle, fixed, permeabilized, and stained with an anti-Ki67 antibody. The percentage of Ki67 positive cells was quantified by fluorescence microscopy. [1] For the MDSC adhesion assay, isolated MDSCs were plated in coated plates, treated with RGX-104 or vehicle, shaken, fixed, and stained. The number of remaining adherent cells was counted by fluorescence microscopy. [1] For the T cell suppression assay, CD8+ T cells were isolated from naive mice, labeled with a proliferation dye, and co-cultured with isolated MDSCs at various ratios in the presence of activator beads and IL-2. After incubation, T cell activation (IFN-γ production) and proliferation (dye dilution) were assessed by flow cytometry. [1] For the in vitro MDSC differentiation assay, bone marrow cells were cultured with B16F10 melanoma cells in transwell inserts and GM-CSF. After several days, with RGX-104 or vehicle added, cells were harvested and analyzed by flow cytometry for MDSC markers. [1] |
| Animal Protocol |
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].
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] For subcutaneous tumor models, cancer cells suspended in PBS were mixed 1:1 with Matrigel and injected into the flank of 6-8 week old mice. When tumors reached a specified volume, mice were randomly assigned to treatment groups. [1] RGX-104 was administered orally via formulated drug chow at doses of 50 or 100 mg/kg/day, or via intraperitoneal injection at 80 mg/kg/day in a vehicle consisting of corn oil with 2.5% ethanol by volume. Control groups received normal chow or the corn oil/ethanol vehicle. [1] For combination therapy with immune checkpoint inhibitors, an anti-PD-1 monoclonal antibody or an isotype control was administered intraperitoneally at 10 mg/kg on specified days post-tumor injection. [1] For adoptive T cell therapy experiments, CD8+ T cells isolated from transgenic mice were adoptively transferred into recipient tumor-bearing mice. A peptide vaccine was administered into footpads on specified days. [1] For MDSC adoptive transfer experiments, MDSCs were isolated from donor mice, labeled with a fluorescent tracker dye, and transferred into recipient mice, which were then treated with RGX-104 or control before analysis. [1] Tumor dimensions were measured regularly with calipers. Mice were euthanized when tumor burden exceeded guidelines. [1] |
| Toxicity/Toxicokinetics |
In the first six evaluable cancer patients treated with oral RGX-104 in a Phase 1 trial, the drug was well tolerated with no dose-limiting toxicities reported. [1] In mice, co-administration of RGX-104 with anti-PD-1 therapy was well tolerated with no overt signs of toxicity reported. [1] |
| References |
[1]. LXR/ApoE Activation Restricts Innate Immune Suppression in Cancer. Cell. 2018 Feb 8;172(4):825-840.e18. |
| Additional Infomation |
To the best of our knowledge, RGX-104 represents the first MDSC-targeting therapeutic that sufficiently curbs immune-suppression as a single-agent to elicit CTL activation in humans. These findings suggest that LXR activation may be effective at preventing metastasis formation and inhibiting progression of metastatic disease given its multi-mechanistic effects on curbing immune suppression, angiogenesis, and tumor invasion. Additionally, LXR therapy may augment anti-tumor responses when given in combination with checkpoint inhibitors or adoptive T cell therapies or may render patients who are refractory to these immunotherapies responsive.[1] RGX-104 is a potent LXRβ agonist under clinical development. [1] It is currently in an ongoing multicenter dose-escalation Phase 1 clinical trial (NCT02922764) in patients with metastatic solid cancers or lymphomas refractory to standard therapies, including immune checkpoint inhibitors. [1] The primary mechanism of action involves LXR activation leading to transcriptional upregulation of ApoE, which suppresses the survival of immunosuppressive myeloid-derived suppressor cells (MDSCs) via the LRP8 receptor. [1] Depletion of MDSCs reverses innate immune suppression, leading to enhanced activation of cytotoxic T lymphocytes (CTLs). [1] This supports its use as a combination agent to enhance the efficacy of other immunotherapies. [1] In the Phase 1 trial, treatment with RGX-104 significantly decreased circulating granulocytic MDSC levels (median 86% decrease) and increased the fraction of activated (GITR+) CD8+ T cells, particularly within the PD-1+ population, in cancer patients. [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.29 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 2: ≥ 1.5 mg/mL (2.37 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 15.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.5809 mL | 7.9046 mL | 15.8093 mL | |
| 5 mM | 0.3162 mL | 1.5809 mL | 3.1619 mL | |
| 10 mM | 0.1581 mL | 0.7905 mL | 1.5809 mL |