Arginase (specifically arginase 1). The compound is a potent inhibitor of this enzyme with an IC50 of 89 nM .
CB-1158 analog targets the enzyme arginase, which is secreted by myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. Arginase depletes L-arginine, an amino acid essential for T-cell proliferation and function, thereby suppressing anti-tumor immune responses. By inhibiting arginase, the compound restores L-arginine levels and enhances T-cell-mediated antitumor immunity.

In vitro activity: CB-1158 (previously known as INCB01158) is a potent and orally-bioavailable small-molecule inhibitor of the arginase. Myeloid cells are an abundant leukocyte in many types of tumors and contribute to immune evasion. Expression of the enzyme arginase 1 (Arg1) is a defining feature of immunosuppressive myeloid cells and leads to depletion of L-arginine, a nutrient required for T cell and natural killer (NK) cell proliferation. CB-1158 has shown high anti-cancer activity in renal cell cancer, breast cancer, non-small cell lung cancer, acute myeloid leukemia, and other tumor types where arginase-secreting MDSCs are known to play an immunosuppressive role. Arginase blockade by CB-1158 may be an effective therapy in multiple types of cancer, also the combination of CB-1158 with standard-of-care chemotherapy or other immunotherapies that target T-cell activation, such as CTLA-4 and PD-1 antibodies may yield improved clinical responses.
Kinase Assay: Recombinant human arginase 2 (Arg2) comprising amino acids 23–254 was purchased from US Biological (Salem, MA). Activity assays using 2 nM Arg1 or 4 nM Arg2 were performed in reaction buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, 0.005% Triton X-100, 0.5 mM DTT, 0.5 mM MgCl2, 0.1 mM CaCl2, and 160 μM or 20 mM L-arginine, pH 7.4) at 37 °C for 30 min with a dose-titration of CB-1158. Activity was determined by a spectrophotometric assay using the QuantiChrom Urea Assay Kit (BioAssay Systems, Hayward, CA) or by quantification of the generation of 13C(5)-L-ornithine from 13C(6)-L-arginine using a SCIEX API4000 mass spectrometer. Urea produced or 13C(5)-L-ornithine peak areas were plotted and fitted to a four-parameter equation using GraphPad Prism software (San Diego, CA) to determine IC50 values.
Cell Assay: T cells or NK cells were purified from healthy donor human blood or from murine splenocytes using a negative selection kit for the appropriate cell type and species from Stemcell Technologies. Isolated T cells or NK cells were loaded with carboxyfluorescein succinimidyl ester (CFSE, Thermo Fisher) and stimulated for 72–96 h in complete growth medium containing a minimum of either 50 μM L-arginine (NK cells) or 100 μM L-arginine (T cells). For T cell stimulation, a solution of 10 μg/mL anti-CD3 (human clones UCHT1 or OKT3; murine clone 145-2C11) was used to coat the wells of a 96-well plate and then T cells were stimulated on immobilized anti-CD3 in the presence of 2 μg/mL soluble anti-CD28 (human clone CD28.2; murine clone 37.51). NK cells were stimulated with recombinant IL-2. Proliferation was quantified by analyzing CFSE dilution by flow cytometry.

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In vitro, CB-1158 analog inhibits arginase activity, thereby preventing the depletion of L-arginine in the tumor microenvironment. This restores T-cell proliferation and function that are suppressed by arginase-secreting MDSCs. Detailed in vitro activity data are not provided in the available references.

Female wild-type C57BL/6 and Balb/c mice (5–6 weeks old) were purchased from Charles River Laboratories (Hollister, CA). Severe combined immune deficient (SCID, B6.CB17-PrkdcSCID/SzJ) and Pmel-1 TCR transgenic (B6.Cg-Thy1a/Cy Tg(TcraTcrb)8Rest/J) mice (5–6 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were housed and treated in accordance with Institutional Animal Care and Use Committee guidelines. For the 4T1 tumor model, 105 cells were injected orthotopically into the mammary fat pad; for all other tumor models, 106 cells were injected subcutaneously (s.c.) in the right flank. For all studies, CB-1158 was administered by oral gavage twice per day at 100 mg/kg starting on study day 1 (1 day after tumor implant). Control groups received vehicle (water) twice daily by gavage. Tumor volume measured by digital caliper (length × width × width/2) and body weight were recorded three times per week. Animals were euthanized when tumors necrotized or volumes reached 2000 mm3. For the CT26 model, anti-PD-L1 antibody (5 mg/kg, clone 10F.9G2, BioXCell, West Lebanon, NH) was injected intraperitoneally (i.p.) on days 5, 7, 9, 11, 13, and 15. For the 4T1 model, anti-CTLA-4 antibody (5 mg/kg, clone 9H10, BioXCell) was injected i.p. on days 2, 5, and 8; anti-PD-1 antibody (5 mg/kg, clone RMP1–14, BioXCell) was injected i.p. on days 3, 6, and 9. 4T1 tumors were harvested on study day 25 into Fekete’s solution and tumor nodules were enumerated visually. Gemcitabine was dosed 50 mg/kg i.p. on days 10 and 16 for the CT26 model, 60 mg/kg i.p. on days 6 and 10 for the LLC model, or 30 mg/kg i.p. on day 5 for the 4T1 model. With these regimens, gemcitabine modestly reduces tumor growth and spares most tumor-infiltrating immune cells, allowing for the evaluation of combination activity with CB-1158. For CD8+ cell depletion, mice were injected i.p. with anti-CD8 antibody (25 mg/kg, clone 2.43, BioXCell) on days −1, 0, + 5, and +10. For NK cell depletion, mice were injected i.p. with anti-NK1.1 antibody (25 mg/kg, clone PK136, BioXCell) in the LLC and B16 models or with anti-Asialo GM1 sera (20 μL) in the CT26 model, per the same schedule as anti-CD8.

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In vivo, CB-1158 has shown high anti-cancer activity in renal cell cancer, breast cancer, non-small cell lung cancer, acute myeloid leukemia, and other tumor types where arginase-secreting MDSCs are known to play an immunosuppressive role. As an orally bioavailable compound, it is administered orally in preclinical models.

The compound's inhibition of arginase is measured using recombinant human arginase enzyme in a biochemical assay. The assay determines the concentration of compound required to inhibit the enzyme's activity by 50% (IC50). The specific reported IC50 value for arginase inhibition is 89 nM .

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Arginase inhibitory activity is determined using cell-free enzyme assays with recombinant arginase enzyme and its substrate L-arginine. The conversion of L-arginine to L-ornithine and urea is measured, and IC50 values are calculated from dose-response curves.

To assess the reversal of immune suppression, T-cells are co-cultured with immunosuppressive cells like MDSCs or PMNs in the presence of the compound. T-cell proliferation and function are then measured to determine the compound's ability to overcome arginase-mediated suppression .

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Cell-based assays for CB-1158 analog would use co-culture systems of T cells and MDSCs. T-cell proliferation and function (e.g., cytokine production) are measured in the presence of arginase-secreting MDSCs with or without the compound. Restoration of T-cell activity by arginase inhibition is assessed.

CB-1158 was administered by oral gavage twice per day at 100 mg/kg starting on study day 1
Female wild-type C57BL/6 and Balb/c mice (5–6 weeks old)

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In vivo animal studies are conducted in syngeneic mouse tumor models where arginase-secreting MDSCs are known to play an immunosuppressive role, including renal cell cancer, breast cancer, non-small cell lung cancer, and AML models. CB-1158 analog is administered orally, and tumor growth inhibition and immune cell infiltration are assessed.

In a first-in-human Phase 1 study, the drug was rapidly absorbed following oral administration, reaching peak plasma concentration (Tmax) at approximately 4 hours. The elimination half-life was approximately 6 hours. At doses of 50 mg and 100 mg twice daily, the steady-state plasma trough levels were 1.6 µM and 4.5 µM, respectively, which were sufficient to achieve >90% inhibition of arginase activity. This target engagement led to a 2.4-fold and 4-fold increase in plasma arginine levels, respectively .

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CB-1158 analog has the molecular formula C₁₈H₂₇BCl₂N₂O₄ with a molecular weight of 417.14. It is orally bioavailable. It is a small-molecule arginase inhibitor. Detailed pharmacokinetic parameters such as half-life and bioavailability are not disclosed.

In the ongoing Phase 1 clinical trial (NCT02903914), Numidargistat has been reported to be generally well-tolerated. As of the data cut-off, no dose-limiting toxicities (DLTs) or drug-related Grade 3 adverse events had been observed .

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No specific toxicity data are detailed in the available references. As an arginase inhibitor, potential toxicities would relate to the role of arginase in normal physiology, including wound healing and immune regulation. The compound's selectivity for arginase would be important for minimizing off-target effects.

[1]. J Immunother Cancer.2017 Dec 19;5(1):101;
[2]. WO 2017075363 A1.

CB-1158 analog (also known as Numidargistat analog) is a research compound not yet approved for clinical use. It is a potent, orally bioavailable small-molecule arginase inhibitor with potential applications in cancer immunotherapy. By targeting arginase-secreting MDSCs, it aims to restore anti-tumor T-cell immunity in various cancers.

C18H27BCL2N2O4

417.13

416.144

C, 51.83; H, 6.52; B, 2.59; Cl, 17.00; N, 6.72; O, 15.34

1345810-21-0

2095732-06-0 ;1345810-21-0;

66832679

Typically exists as solid at room temperature

4

6

9

27

480

0

B(CCCCC(C1CCN(CC1)CC2=C(C=C(C=C2)Cl)Cl)(C(=O)O)N)(O)O

ISYBHIALKUAHGV-UHFFFAOYSA-N

InChI=1S/C18H27BCl2N2O4/c20-15-4-3-13(16(21)11-15)12-23-9-5-14(6-10-23)18(22,17(24)25)7-1-2-8-19(26)27/h3-4,11,14,26-27H,1-2,5-10,12,22H2,(H,24,25)

CB-1158 analog; CHEMBL5276302; CB1158 analog; 2-amino-6-borono-2-[1-[(2,4-dichlorophenyl)methyl]piperidin-4-yl]hexanoic acid; Arginase inhibitor 2; ...INCB01158,CB-1158,INCB 01158,CB 1158,INCB-01158; CB1158

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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