- Arginase 1 (Arg1) – IC50 = 86 nM (recombinant human Arg1, assayed with 160 μM L-arginine) [1]
- Arginase 2 (Arg2) – IC50 = 296 nM (recombinant human Arg2, assayed with 20 mM L-arginine) [1]
- No inhibition of NOS isoforms (endothelial NOS, neuronal NOS, inducible NOS) observed at 50 μM [1]
IC50: 86 nM (Arginase 1); 296 nM (Arginase 2)[1]

- CB-1158 inhibited native Arg1 in human granulocyte lysates with IC50 = 178 nM (±28); in human erythrocyte lysates with IC50 = 116 nM; in human hepatocyte lysates with IC50 = 158 nM (±23); in cancer patient plasma with IC50 = 122 nM (±32) [1]
- CB-1158 showed low potency against intracellular arginase in intact cells: IC50 = 32 μM (±5.6) in HepG2 cells, 139 μM (±8.8) in K-562 cells, and 210 μM in primary human hepatocytes (measured by urea production after 24 h), likely due to inefficient cell membrane penetration [1]
- In T cell proliferation assays, human and murine T cells required extracellular L-arginine for proliferation; no proliferation occurred in media lacking L-arginine [1]
- Isolated human granulocytes spontaneously activated in vitro and depleted L-arginine from the media; co-culture with autologous T cells inhibited T cell proliferation; CB-1158 blocked L-arginine depletion and restored T cell proliferation to 90% of control levels (granulocyte to T cell ratio 0.25:1) [1]
- Conditioned media from cancer patient-derived granulocytic MDSCs (lung cancer) or granulocytes (head and neck cancer) inhibited T cell proliferation and depleted L-arginine; CB-1158 dose-dependently restored L-arginine levels and T cell proliferation to 99% or 79% of control levels, respectively [1]
- CB-1158 restored secretion of interferon-γ and granzyme-B from T cells co-cultured with cancer patient granulocytes [1]
- CB-1158 showed no direct cytotoxicity against murine cancer cell lines (CT26, LLC, B16, 4T1) at concentrations up to 1 mM in 72 h CellTiterGlo assays [1]
- CB-1158 (50 μM) showed no inhibitory activity against endothelial NOS, neuronal NOS, or inducible NOS [1]

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Numidargistat is a strong and orally accessible arginase inhibitor with IC50 values of 86 and 296 nM for recombinant human arginase 1 and 2, respectively. Numidargistat inhibits native rginase 1 (Arg1) in human granulocyte, erythrocyte, and hepatocyte lysate at IC50s of 178 nM, 116 nM, and 158 nM, respectively, and inhibits Arg1 in cancer patient plasma (IC50, 122 nM). Numidargistat effectively inhibits arginase in human HepG2, K562 cell lines, and primary human hepatocytes with IC50 values of 32, 139, and 210 μM, respectively. Numidargistat has no effect on NOS. Furthermore, Numidargistat is not directly cytotoxic to murine cancer cell lines [1].

- In CT26, LLC, B16, and 4T1 syngeneic mouse tumor models, oral CB-1158 (100 mg/kg twice daily) significantly inhibited tumor growth [1]
- In LLC tumor-bearing mice with conditional myeloid-specific Arg1 deletion (Arg1^flox/flox; Tie2-Cre^+), tumor growth was reduced similar to CB-1158-treated wild-type mice; CB-1158 treatment of Arg1-deleted mice conferred no further tumor growth reduction, confirming on-target activity [1]
- CB-1158 efficacy was abrogated in SCID mice (LLC model), indicating requirement for intact immune system [1]
- Depletion of CD8⁺ T cells or NK cells in B16, CT26, and LLC tumor models blocked CB-1158 efficacy, demonstrating requirement for both cell types [1]
- In CT26 tumors, CB-1158 treatment increased tumor-infiltrating CD8⁺CD25⁺ T cells (day 14) [1]
- In B16 tumors, CB-1158 increased CD25⁺CD8⁺ T cells (day 9) [1]
- In LLC tumors, CB-1158 increased CD8⁺ T cells and decreased CD68⁺ myeloid cells (day 14) [1]
- In 4T1 tumors, CB-1158 increased T cells and NK cells, and decreased myeloid cells (day 10) [1]
- NanoString gene expression analysis of LLC tumors showed CB-1158 increased T cell and NK cell markers and interferon response genes [1]
- Luminex cytokine analysis of LLC tumors showed CB-1158 increased inflammatory cytokines and chemokines [1]
- CB-1158 combined with anti-PD-L1 antibody (CT26 model) or anti-CTLA-4/anti-PD-1 (4T1 model) showed enhanced tumor growth inhibition compared to either single agent [1]
- CB-1158 combined with adoptive T cell therapy (Pmel-1 T cells in B16 model) significantly reduced tumor growth compared to either single agent [1]
- CB-1158 combined with adoptive NK cell therapy (CT26 lung metastasis model) significantly reduced lung metastases [1]
- CB-1158 combined with gemcitabine (CT26, LLC, 4T1 models) showed enhanced tumor growth inhibition; combination increased NK cells and inflammatory (M1-type, CD80⁺) macrophages and decreased immunosuppressive (M2-type, CD206⁺) macrophages in CT26 tumors [1]
- CB-1158 raised L-arginine levels in plasma and tumors of tumor-bearing mice, indicating on-target pharmacodynamic effect [1]
- In human cancer patient samples, Arg1 protein was significantly elevated in plasma of 76 cancer patients across 13 histologies compared to 31 healthy volunteers; plasma L-arginine was significantly lower in 26 cancer patients across 7 histologies compared to 20 healthy volunteers [1]
- Immunohistochemistry of tumor tissue microarrays from 11 different tumor types (NSCLC, breast, gastric, colorectal, prostate, pancreatic, ovarian, bladder, renal cell, etc.) revealed abundant Arg1⁺ myeloid cells in tumors; immunofluorescence showed Arg1 more frequently associated with CD15⁺ granulocytes than with CD68⁺ macrophages [1]

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In mice, imidargistat (100 mg/kg, po, twice daily) reduces myeloid cells and increases the number of cytotoxic cells that infiltrate tumors. In mice containing CT26 cancer cells, the combination of fumarate plus gemcitabine or PD-L1 inhibition slows the formation of tumors[1].

- Recombinant arginase activity assay: Full-length human Arg1 (2 nM) or human Arg2 (4 nM, amino acids 23-254) was incubated 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, with either 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 spectrophotometric urea assay or by quantification of ¹³C(5)-L-ornithine generation from ¹³C(6)-L-arginine using mass spectrometry. IC50 values were calculated by fitting to a four-parameter equation [1]
- Native arginase activity in cell lysates: Human granulocyte, erythrocyte, or hepatocyte lysates were prepared by microtip sonication and clarification by centrifugation. Lysates were incubated with a dose-titration of CB-1158 in reaction buffer containing 160 μM ¹³C(6)-L-arginine at 37°C for 30 min. Arginase activity was determined by quantification of ¹³C(5)-L-ornithine generation by mass spectrometry [1]
- Native arginase activity in cancer patient plasma: Plasma samples from renal cell carcinoma patients were incubated with a dose-titration of CB-1158 in reaction buffer containing 160 μM ¹³C(6)-L-arginine at 37°C for 30 min. Arginase activity was determined by quantification of ¹³C(5)-L-ornithine generation by mass spectrometry [1]
- NOS activity assay: Activity of 50 μM CB-1158 against recombinant murine inducible NOS, recombinant bovine endothelial NOS, and native rat cerebellar neuronal NOS was determined by either quantitation of radiolabeled L-citrulline or spectrophotometric measurement of nitrite [1]

- Intact cell arginase activity assay: HepG2 cells (100,000 cells/well, seeded one day prior) or K-562 cells (200,000 cells/well) were treated with a dose-titration of CB-1158 in SILAC RPMI-1640 media containing 5% heat-inactivated dialyzed FBS, 10 mM L-arginine, 0.27 mM L-lysine, and 2 mM L-glutamine. Media were harvested after 24 h and urea was quantified using a urea assay kit. Background controls were wells containing media without cells [1]
- Primary human hepatocyte arginase assay: Frozen human hepatocytes were thawed, allowed to adhere onto collagen-coated wells for 4 h, then incubated for 48 h in SILAC-RPMI containing 10 mM L-ornithine, no L-arginine, and a dose-titration of CB-1158. Media were analyzed for urea [1]
- Cytotoxicity assay: Cells were seeded in fully-supplemented RPMI-1640 medium, treated with a dose-titration of CB-1158 in triplicate wells, and incubated for 72 h. Cytotoxicity was assayed by addition of CellTiterGlo reagent followed by fluorescence quantification [1]
- T cell and NK cell proliferation assay: T cells or NK cells were purified from healthy donor human blood or murine splenocytes using negative selection kits. Isolated cells were loaded with CFSE and stimulated for 72-96 h in complete growth medium containing minimum 50 μM (NK cells) or 100 μM (T cells) L-arginine. T cells were stimulated on immobilized anti-CD3 (10 μg/mL) with soluble anti-CD28 (2 μg/mL). NK cells were stimulated with recombinant IL-2. Proliferation was quantified by CFSE dilution flow cytometry [1]
- T cell/myeloid cell co-culture assay: Granulocytes were purified from healthy donor peripheral blood and incubated in SILAC-RPMI medium containing 10% charcoal-stripped FBS, 0.27 mM L-lysine, 20 μM MnCl₂, 100 μM L-arginine, pH 7.4, with a dose-titration of CB-1158 for 48 h. T cells from the same donor were loaded with CFSE and plated with immobilized anti-CD3 and soluble anti-CD28 in the presence of aged granulocytes at ratios of granulocytes to T cells as indicated (or fixed ratio of 4 T cells to 1 granulocyte). Co-cultures were incubated for 3-4 days. Media were analyzed for L-arginine and L-ornithine by mass spectrometry, and T cell proliferation was determined by flow cytometry [1]
- Cancer patient myeloid cell assay: G-MDSCs (from PBMC layer) or granulocytes (from RBC layer) were purified from cancer patient blood. Freshly isolated cells were incubated in co-culture medium containing 100 μM L-arginine for 48 h, then cells were removed and conditioned media were used to incubate healthy donor CFSE-loaded T cells on immobilized anti-CD3/soluble anti-CD28 for 3-4 days. Cytokines in co-culture media were quantified by Cytometric Bead Array [1]

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Intracellular arginase activity is determined for the arginase-expressing HepG2 and K-562 cell lines as follows. HepG2 cells are seeded at 100,000 cells per well one day prior to treatment with CB-1158. K-562 cells are seeded at 200,000 cells per well on the day of CB-1158 treatment. Cells are treated with a dose-titration of CB-1158 in SILAC RPMI-1640 media containing 5% heat-inactivated and dialyzed FBS, antibiotics/anti-mycotic, 10 mM L-arginine, 0.27 mM L-lysine, and 2 mM L-glutamine. The medium is harvested after 24 h and urea generated is determined. Wells containing media without cells are used as background controls. For assessing the effect of CB-1158 on Arg1 in primary hepatocytes, frozen human hepatocytes are thawed, allowed to adhere onto collagen-coated wells for 4 h, and then incubated for 48 h in SILAC-RPMI containing 10 mM L-ornithine, no L-arginine, and a dose-titration of CB-1158, at which time the media are analyzed for urea[1].

- General dosing: CB-1158 was dissolved in Milli-Q water and 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 [1]
- Tumor models: Female wild-type C57BL/6 and Balb/c mice (5-6 weeks old) were used. For 4T1 model, 1×10⁵ cells injected orthotopically into mammary fat pad; for all other models (CT26, LLC, B16), 1×10⁶ cells injected subcutaneously in right flank. Tumor volume measured by digital caliper (length × width × width/2) three times per week. Animals euthanized when tumors necrotized or volumes reached 2000 mm³ [1]
- Checkpoint blockade combination: In CT26 model, anti-PD-L1 antibody (5 mg/kg, clone 10F.9G2) injected i.p. on days 5, 7, 9, 11, 13, and 15. In 4T1 model, anti-CTLA-4 antibody (5 mg/kg, clone 9H10) injected i.p. on days 2, 5, and 8; anti-PD-1 antibody (5 mg/kg, clone RMP1-14) injected i.p. on days 3, 6, and 9 [1]
- Gemcitabine combination: Gemcitabine dosed at 50 mg/kg i.p. on days 10 and 16 (CT26), 60 mg/kg i.p. on days 6 and 10 (LLC), or 30 mg/kg i.p. on day 5 (4T1) [1]
- Immune cell depletion: For CD8⁺ cell depletion, anti-CD8 antibody (25 mg/kg, clone 2.43) injected i.p. on days -1, 0, +5, and +10. For NK cell depletion, anti-NK1.1 antibody (25 mg/kg, clone PK136) injected i.p. in LLC and B16 models, or anti-Asialo GM1 sera (20 μL) in CT26 model, per same schedule [1]
- SCID mouse study: B6.CB17-Prkdc^scid/SzJ mice implanted with LLC cells, dosed with CB-1158 100 mg/kg twice daily [1]
- Conditional Arg1 knockout mice: Arg1^flox/flox; Tie2-Cre^+ mice (myeloid-specific Arg1 deletion) and Cre-negative littermate controls. LLC cells (1×10⁶) injected s.c. in flank. Mice orally gavaged with 100 mg/kg CB-1158 or vehicle every 12 h for 14 days. Tumors excised and weighed [1]
- Adoptive T cell transfer: B16 tumor-bearing C57BL/6 mice. CB-1158 100 mg/kg BID oral gavage starting day 1. On day 7, lymphopenia induced by cyclophosphamide (250 mg/kg) and fludarabine (50 mg/kg) i.p. On day 9, 1×10⁶ Pmel-1 T cells (gp100-specific CD8⁺) administered i.v. IL-2 (200,000 IU/dose) i.p. twice daily for 3 days starting day of T cell transfer [1]
- Adoptive NK cell transfer: Balb/c mice inoculated i.v. with 1×10⁵ CT26 cells. On same day, 1×10⁶ NK cells (isolated from Balb/c spleens, incubated with IL-2 and IL-15 for 18 h) transferred. Mice treated with vehicle or CB-1158 for 14 days. Lungs harvested and tumor nodules counted [1]
- Tumor dissociation for flow cytometry: Excised tumors placed on ice in RPMI-1640 with 5% FBS, minced, dissociated with tumor dissociation enzymes on a tissue dissociator. Dissociated tumors strained through 70 μm nylon mesh, washed, blocked with anti-CD16/CD32, stained for cell surface antigens. For B16 and 4T1 tumors, dead cells removed using Dead Cell Removal MicroBeads prior to staining. For intracellular staining, cells fixed and permeabilized. Samples acquired on flow cytometer and analyzed with FlowJo software [1]
- Pharmacokinetic/pharmacodynamic study: LLC tumor-bearing mice (N=5 per group) administered single dose or 5 twice-daily doses of CB-1158. Samples collected 2 h after last dose. CB-1158 and L-arginine in plasma and tumor lysates measured by LC/MS [1]

- Following a single dose or twice-daily dosing for 5 days, dose-dependent exposure of CB-1158 was observed in both plasma and tumors (data shown in figures; specific PK parameters not reported in text) [1]
- Oral dosing of CB-1158 in tumor-bearing mice raised L-arginine levels in plasma and tumors, indicating on-target pharmacodynamic effect [1]

- CB-1158 was well-tolerated at doses of 100 mg/kg twice daily for 23 days, with no significant clinical observations or impact on body weight [1]
- Twice-daily oral dosing of CB-1158 was well-tolerated in mice for at least 40 days [1]
- No endotoxin contamination of CB-1158 preparations was observed [1]
- No LD50, hepatotoxicity, nephrotoxicity, or other detailed toxicity parameters were described in this paper [1]

[1]. Inhibition of arginase by CB-1158 blocks myeloid cell-mediated immune suppression in the tumor microenvironment. J Immunother Cancer. 2017 Dec 19;5(1):101.

- CB-1158 is a potent, selective, and orally-bioavailable small-molecule inhibitor of arginase [1]
- CB-1158 does not readily enter cells, exhibiting IC50 values for intracellular arginase two orders of magnitude higher than for soluble arginases in cell lysates; this may protect hepatic urea cycle Arg1 from inhibition [1]
- Hepatic Arg1 is tightly associated in a multi-enzyme complex at the mitochondria with substrate channeling, potentially making it less accessible to CB-1158 compared to cytoplasmic or extracellular arginase [1]
- CB-1158 is currently in clinical trials for patients with solid tumor malignancies (NCT02903914) as a monotherapy and in combination with anti-PD-1 [1]
- Arg1-expressing myeloid cells are abundant in human tumors across multiple histologies (NSCLC, breast, gastric, colorectal, prostate, pancreatic, ovarian, bladder, renal cell carcinoma, etc.) [1]
- Cancer patients have significantly elevated plasma Arg1 and reduced plasma L-arginine compared to healthy volunteers, suggesting immune suppression associated with higher circulating Arg1 [1]

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Numidargistat is an aminopyrrolidine compound in which the pyrrolidine ring is substituted at positions 1, 3, and 4 with (2S)-2-aminopropionyl, amino, carboxyl, and 3-boronpropyl groups, respectively (3R,4S-stereoisomers). It is a selective arginase inhibitor developed by Calithera Biosciences for the treatment of solid tumors. It possesses various pharmacological activities, including as an EC 3.5.3.1 (arginase) inhibitor, an antitumor drug, and an immunomodulator. It belongs to the borate, pyrrolidine carboxylic acid, N-acylpyrrolidine, aminopyrrolidine, amino monocarboxylic acid, and primary amino compounds classes. CB-1158 is currently being investigated in the clinical trial NCT03910530 (an INCMGA00012, INCB001158, and combination therapy study in Japanese patients with advanced solid tumors). Numidargistat is an oral arginase inhibitor. Arginase, a manganese-dependent enzyme, hydrolyzes the amino acid arginine into ornithine and urea, possessing potential immunomodulatory and antitumor activities. After administration, numidagistat inhibits the breakdown of arginine by arginase produced by myeloid cells, thereby restoring arginine levels. This allows arginine to stimulate nitric oxide synthesis and the secretion of pro-inflammatory cytokines and chemokines, thereby inducing T cell proliferation and activation. Therefore, this drug may inhibit the immunosuppressive effects of tumor-infiltrating myeloid cells and promote lymphocyte-mediated antitumor immune responses. Arginase is produced by neutrophils, macrophages, and myeloid-derived suppressor cells (MDSCs) and plays a role in inflammation-related immunosuppression.

C11H22BN3O5

287.1204829216

287.165

C, 46.02; H, 7.72; B, 3.76; N, 14.64; O, 27.86

2095732-06-0

Numidargistat dihydrochloride;Numidargistat 2HCl (CB1158, INCB01158)

131801114

Off-white to light yellow solid powder

5

7

6

20

381

3

OC([C@@]1(CN(C([C@H](C)N)=O)C[C@@H]1CCCB(O)O)N)=O

ZZJLMZYUGLJBSO-LAEOZQHASA-N

InChI=1S/C11H22BN3O5/c1-7(13)9(16)15-5-8(3-2-4-12(19)20)11(14,6-15)10(17)18/h7-8,19-20H,2-6,13-14H2,1H3,(H,17,18)/t7-,8-,11-/m0/s1

(3R,4S)-1-(L-alanyl)-3-amino-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid

CB-1158; INCB01158; CB1158; INCB 01158; CB 1158; INCB-01158; Numidargistat; 2095732-06-0; CB-1,158; INCB001,158; INCB-001,158;

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)

DMSO : ~100 mg/mL (~348.29 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.71 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 25.0 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: ≥ 2.5 mg/mL (8.71 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 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: ≥ 2.5 mg/mL (8.71 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 25.0 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.)