PI3Kγ (IC50 = 16 nM); PI3Kα (IC50 = 3.2 μM); PI3Kβ (IC50 = 3.5 μM)

Eganelisib (IPI549) inhibits PI3Kγ with IC50 of 16 nM, with >100-fold selectivity over other lipid and protein kinases (PI3Kα IC50=3.2 μM, PI3Kβ IC50=3.5 μM, PI3Kδ IC50>8.4 μM). Eganelisib is evaluated for activity across all Class I PI3K isoforms. The activity of eganelisib is assessed across all Class I PI3K isoforms. The individual rates constants and for PI3K-α, β and δ are measured using equilibrium fluorescent titration to ascertain the binding affinity of Eganelisib for PI3K-γ. Eganelisib is a remarkably tight binder to PI3Kγ with a Kd of 290 pM and >58-fold weaker affinity for other Class I PI3K isoforms (PI3Kα Kd=17 nM, PI3Kβ Kd=82 nM, PI3Kδ Kd=23 M). Eganelisib exhibits excellent PI3K-γ potency (IC50=1.2 nM) and selectivity against other Class I PI3K isoforms (>146-fold) in PI3K-α, -β, -γ, and -δ dependent cellular phospho-AKT assays. Cellular IC50s for Class I PI3Kα (250 nM), PI3Kβ (240 nM), PI3Kγ (1.2 nM), PI3Kδ (180 nM) are determined in SKOV-3, 786-O, RAW 264.7, and RAJI cells, respectively, by monitoring inhibition of pAKT S473 by ELISA. Furthermore, Eganelisib dose dependently inhibits PI3Kγ dependent bone marrow-derived macrophage (BMDM) migration. Eganelisib is selective against a panel of 80 GPCRs, ion channels, and transporters at 10 μM[1]

Eganelisib (IPI549) exhibits advantageous pharmacokinetic characteristics and potent PI3K-mediated neutrophil migration inhibition. Eganelisib has excellent oral bioavailability, low clearance, and is distributed into tissues with a mean volume of distribution of 1.2 L/kg in vivo (in mice, rats, dogs, and monkeys). Overall, Eganelisib has a good pharmacokinetic profile that enables potent and precise PI3K- in vivo inhibition. The t1/2 of IPI-549 is 3.2, 4.4, 6.7, and 4.3 hours for mice, rats, dogs, and monkeys, respectively. In this model, oral administration of eganelisib at all of the tested doses significantly reduces neutrophil migration in a dose-dependent manner[1].

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Based on the pharmacokinetic properties in mice and pharmacological in vitro profile, compound 26 was well-suited to investigate the impact of potent and selective PI3K-γ inhibition in vivo. IL-8 stimulated neutrophil migration into air pouches in mice has previously been shown to be dependent on PI3K-γ. Thus, to demonstrate PI3K-γ dependent activity of compound 26 in vivo, we evaluated the effect of orally administered compound 26 on IL-8 stimulated neutrophil influx into the air pouches on mice.7,8 Compound 26 significantly reduced neutrophil migration in a dose-dependent manner in this model when administered orally at all of the tested doses (Figure 2a). The degree of inhibition observed directly correlated with plasma concentrations of compound 26 in these mice (Figure 2b) clearly demonstrated that orally administered compound 26 can inhibit PI3K-γ function in vivo. In addition, compound 26 (Eganelisib (IPI-549)) has been shown to inhibit tumor growth in murine syngeneic models through alteration of immune cells in the tumor microenvironment.[1]

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To test this hypothesis, we used Eganelisib (IPI-549), a selective PI3K-γ inhibitor in multiple tumor models19 (Extended data Fig. 2a). Eganelisib (IPI-549) treatment alone led to tumor growth inhibition in 4T1, B16-GMCSF, MC38, CT26, and LLC tumor models (Fig. 2a and Extended data Fig. 2b). Lack of activity in tumors with low suppressive TAMCs (B16-F10, Fig. 2a) suggests PI3K-γ inhibition affects myeloid cells and has no direct effect on tumor cells or other TILs. We also observe a significant reduction in lung metastasis after IPI-549-treatment (Fig. 2b). TIL quantification shows no consistent differences in the total myeloid (CD11b+) or macrophage (CD11b+/F480+) cell populations in either IPI-549- or vehicle-treated 4T1 or B16-GMCSF tumors (Fig. 2c). The effects of IPI-549 on TAMC subsets were comparable to those seen in p110γ −/− mice17,18. PI3K-γ inhibition switches the activation of macrophages from an immunosuppressive M2-like (CD11b+F4/80+CD206+) phenotype to a more inflammatory M1-like (CD11b+F4/80+MHCII+) state (Fig. 2c, Extended data Fig. 2c). We further tested RNA expression of M1 and M2 markers in 4T1 and B16-GMCSF tumors after IPI-549. The expression of prototypic M2-markers (TGF-β, Arg-1, IDO) are reduced, while M1-markers (IL-12, INOS) are higher in IPI-549-treated tumors (Fig. 2d). Given that CD11b+F4/80+ macrophages constitute only a portion of suppressive TAMCs, we further subdivided myeloid cells into granulocytes (CD11b+Ly6G+), monocytes (Ly6Chigh/MHCIIlow = Mono-Lo), immature macrophages (LyC6high/MHCIIhigh = Mono-Hi), M1 macrophages (Ly6Clow/MHCIIhigh = TAM-M1) or M2 macrophages (Ly6Clow/MHCIIlow = TAM-M2)20,21. When we analyzed the myeloid cells with the above gating strategy in 4T1 TILs, we also observed that Eganelisib (IPI-549) shifts myeloid cells toward the TAM-M1 population (Extended data Fig. 3a). Moreover, relative mRNA expression of M1 and M2 markers correlate with a less suppressive function of these cells (Extended data Fig. 3b). We subsequently tested the suppressive function of myeloid cells derived from IPI-549 treated B16-GMCSF tumor bearing mice on naïve CD8 T cells proliferation (Fig. 2e). We show that suppression of CD8+ T cells is abolished in IPI-549-treated mice or when IPI-549 is added to the media. A similar functional observation was made in human myeloid suppressor cells in PBMCs (Extended data Fig. 4b) confirming its potential clinical use. In addition, pharmacodynamic evaluation of whole blood confirms inhibition of PI3K-γ in monocytes after activation with a PI3K-γ stimulus in human volunteers (Extended data Fig. 4a.). To confirm that inhibition of PI3K-γ in myeloid cells is required to delay tumor progression, we depleted myeloid cells (anti-CD11b) prior to implanting LLC-Brei tumors in mice. Treatment with IPI-549 did not delay tumor growth in the absence of TAMCs (Extended data Fig. 2e). Taken together these findings suggest that PI3K-γ inhibition using IPI-549 is mainly effective in a tumor landscape rich in suppressive myeloid cells and allows for more precise delineation of patients in which it will potentially yield the greatest activity. [2]

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We tested the anti-PD-1 and anti-CTLA4 combination with Eganelisib (IPI-549) in the 4T1 and B16-GMCSF models (Fig. 4a). Double checkpoint blockade therapy alone did not result in any complete tumor regressions in 4T1-bearing mice and only 20% of the mice bearing B16-GMCSF benefited from the therapy, further confirming multiple checkpoint resistance in these models. Notably, the addition of Eganelisib (IPI-549) to the combination of anti-CTLA-4 + anti-PD-1 was associated with complete remissions in 30% of 4T1 and 80% of B16-GMCSF tumor-bearing mice. Importantly, tumor-free survivors were resistant to tumor re-implantation (Extended data Figure 9), indicating long lasting adaptive immunity [2].

Biochemical Assay Materials:[1]
The Class I PI3K isoforms α, β, and δ were supplied with the p85α regulatory subunit and the γ isoform was supplied alone. The catalogue number (cat #) of the isoforms used were as follows PI3K-α: p110α/p85α cat #14-602-K, PI3K-β: p110β/p85α cat #14-603-K, PI3K-γ: p120γ cat #14-558-K, PI3K-δ: p110δ/p85α cat #14-604. The Class II PI3K isoforms were supplied as N-terminal 6xHis-tagged proteins; PI3K-C2α amino acids 299-end: cat # 14-906-K, PI3K-C2β full length: cat # 14-907-K and PI3K-C2γ: cat # 14-910-K. ATP disodium salt was prepared to a stock concentration of 30 mM in water, and neutralized to ~ pH 7.0 with sodium hydroxide (NaOH). Concentrations were determined via ultraviolet (UV) absorbance at 260 nm using an extinction coefficient of 15.4 x 103 M -1 cm -1 . Dioctanoyl-phosphatidylinositol-4,5-bis-phosphate (diC8PIP2) was made up as a stock solution of 5 mM in water, and stored at -20°C. Dioctanoyl-phosphatidylinositol (diC8PIP) was made up as a stock solution of 5 mM in water, and stored at – 20°C.

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General Procedure for Class I and Class II PI3K Biochemical Assays at 3 mM ATP: [1]
Promega ADP-Glo Max assay kit was used to determine IC50 values for Class I α, β, δ, and γ and Class II α, β, and γ isoforms of human PI3 kinases. Samples of kinase (20 nM PI3K-α, PI3K-δ, PI3K-C2α, PI3K-C2β, and PI3K-C2γ or 40 nM PI3K-β and PI3K-γ) were incubated with compound for 15 minutes at room temperature in reaction buffer (15 mM HEPES pH 7.4, 20 mM NaCl, 1 mM EGTA, 0.02% Tween 20, 10 mM MgCl2, 0.2 mg/mL bovine-γ-globulins) followed by addition of ATP/diC8-PP2 mixture to give final concentrations of 3 mM ATP and 500 µM diC8PIP2 substrate (for Class I PI3Ks) or 500 µM diC8PIP substrate (for Class II PI3Ks). Reactions were incubated at room temperature for 2 hours followed by addition of 25 µL of Promega kit stop solution. After a 40-minute incubation at room temperature, 50 µL of Promega detection mix was added followed by incubation for 1 hour at room temperature. Plates were then read on Envision plate reader in luminescence mode. Data is converted to % inhibition and then plotted as % inhibition vs compound concentration and fit to a 4 parameter logistic equation to determine IC50 values.

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Biochemical and cell based assays [2]
Biochemical and Cell based assays for the Class I and Class II PI3K isoforms were run as previously described.

At a density of 200,000 cells/200 L/well of RPMI-1640 with 10% FBS, SKOV-3 cells are seeded into 96-well cell culture-grade plates. Cells are incubated for the entire night at 5% CO2 and 37 °C. The cells are treated with substances, resulting in a final concentration of 0.5% DMSO, and then incubated for 30 minutes at 37 °C and 5% CO2. Following the aspiration of the media, 50 L of ice-cold lysis buffer is added to each well. After five minutes of incubation on ice, plates are centrifuged at 3000 rpm for five minutes at 4 °C.

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T cell Suppression Assay [2]
Spleens from naïve mice were isolated and ground through 40-μm filters to generate a single cell suspension. After RBC lysis, CD8+ cells were purified using anti-CD8 (Ly-2) microbeads according to manufacturer’s protocol and labeled with 1 mM CFSE in pre-warmed PBS for 10 min at 37°C. The CFSE-labeled CD8+ T cells were then plated in complete RPMI media supplemented with 0.05 M β-mercaptoethanol onto round bottom 96-well plates (25 × 10E3 cells per well) coated with 1 μg/ml anti- CD3 (clone 1454-2C11) and 5 μg/ml anti-CD28 (clone 37N) antibodies. Purified myeloid cells were added in indicated ratios and plates were incubated at 37 °C. After 48 h, cells were harvested and CFSE signal in the gated CD8+ T cells was measured by flow cytometry. For the human MDSC suppression assay PBMCs were isolated using Lymphoprep™ from donor blood. T cells were isolated by CD3+ selection and frozen in Sigma freezing media for later use. The remaining PBMCs minus T cells were incubated with 20 ng/ml GMCSF and 20 ng/ml IL6 for 6 days to differentiate the myeloid cells and were incubated with or without added Eganelisib (IPI-549). After 6 days, the MDSC cells were isolated by CD33+ selection. These MDSC cells were then mixed with the autologous T cells (at a 1:4 ratio) that had been pre-stained with cell trace violet and activated with anti-CD3 and anti-CD28 beads (Dynal). The T cell proliferation is determined by Cell Trace Violet dye dilution measured by flow cytometry after 72 hours.

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IFN-γ ELISPOT assay [2]
Blood was collected from Vehicle and Eganelisib (IPI-549) (15mg/kg) treated CT26 tumor-bearing mice after 10 days of treatment. PBMC were isolated using Lymphoprep density gradient media. IFN-γ producing cells were quantified using the CTL Mouse Immunospot IFN-γ Single Color ELISPOT kit according to the manufacturer’s instructions. For in vitro re-stimulation, 1×105 PBMC were co-cultured with 1×105 irradiated (2000 rad) CT26 colon carcinoma target cells in CTL test media (CTL) for 16 hours. Irradiated (2000 rad) 4T1 mammary carcinoma target cells were used as negative control targets to assess specificity. IFN-γ spots were quantified using a CTL Immunospot S6 Micro Analyzer and Immunospot Professional Software (CTL).

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Macrophage polarization assay [2]
Bone marrow derived macrophages were prepared from C57Bl/6 mice femur and tibias. Red blood cells were lysed and then the remaining cells were plated in bone macrophage media (BMM) consisting of DMEM, 20% FBS plus pen/Strep and 50 ng/ml M-CSF and incubated for 6 days. Cells were polarized towards an M2 phenotype with the addition of 20 ng/ml IL4 and 50 ng/ml M-MCSF with or without added Eganelisib (IPI-549). Cells were incubated for 48 hours and then RNA was harvested from the cells. qRT-PCR was performed using primers for mouse ARG1 and mouse B-actin.

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PI3K-gamma-specific whole blood PD assay [2]
Whole blood from 6 healthy donors was pretreated with a dose titration of Eganelisib (IPI-549) and then stimulated for 2 minutes with 2.3 ug/ml CXCL12. Cells were lysed, fixed and stained. Human blood samples have been collected after ICF approval. Response to stimulation was determined by measuring phosphorylation of AKT S473 in monocytes (CD14+) by flow cytometry and comparing the value to that of untreated controls. IC50 values for Eganelisib (IPI-549) were calculated for each donor, measuring compound potency against PI3K-γ and in whole blood.

The mice used in this study are C57BL/6J and Balb/c mice, aged 6 to 8 weeks. In the first day of the experiment, tumor cells are injected intradermally (i.d.) in the right flank. Once per day, 15 mg/kg of eganelisib is given orally through gavage. Beginning on day seven after tumor implantation and lasting until day twenty-one. Transportation (5% NMP, 95% PEG) is given to the control groups. Every second or third day, tumors are calibrated with a ruler to measure their volume (length, width, and height). when the total tumor volume reaches 2500 mm3, or when there are signs of distress in the animal. Tumors are then isolated and preserved in ice until needed[2].

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RNAseq [2]
Mice bearing CT26 tumors were treated with vehicle or Eganelisib (IPI-549) (15 mg/kg/day, PO) for 6 or 9 days. Tumors were isolated, and frozen until needed; tumors then thawed and RNA was extracted from all cells. RNAseq was done at Expression Analysis (Q2 Solutions). Sequence reads were aligned to the mouse B38 reference genome using OmicSoft ArrayStudio and the UCSC gene model. Log2(FPKM) was calculated for each gene, and data were mean centered for display in heat maps. The analysis focused on a compilation of about 4,200 mouse genes related to cancer immunology and PI3K pathway signaling compiled from numerous sources including BioCarta pathways, GO gene ontologies, KEGG pathways, WikiPathways, and literature.5

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Mice [2]
C57BL/6J and Balb/c mice (6–8 weeks old) were purchased from Jackson Laboratory. Pmel-1 TCR transgenic mice have been previously reported. Ten to fifteen mice per treatment strategy were used to allow 90% power, and a 5% significance level, and detect differences in tumor-free survival from 10% to 80%. Typically, tumors grow in 100% of control animals. An additional 5 mice per group were for tissue harvest at day 7 and day 14 on treatment. Mice cage, treatment allowance were at random at day 7 after tumor implants.

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Tumor Challenge and Treatment Experiments [2]
On day 0 of the experiments, tumor cells were injected intradermally (i.d.) in the right flank. For the B16 model, 2.5×105 B16-WT or B16-GMCSF cells were injected into C57BL/6J mice. For 4T1 model and for the CT26 model, 5×105 cells were used subcutaneously in Balb/c mice. For studies in immune compromised mice, the CT26 study was done in the Balb/c nu/nu strain and the B16-GMCSF in C57Bl.6 rag1−/− mice. Mice were obtained from Jackson Labs and Charles River Labs. Treatments were given as single agents or in combinations with the following regimen for each drug. The PI3K-γ inhibitor drug Eganelisib (IPI-549) was dissolved at 5% 1-Methyl-2-pyrrolidinone in Polyethylene Glycol 400 and administered by oral gavage once a day at 15 mg/kg . Treatment was initiated on day 7 ending on day 21 post tumor implant. Control groups received vehicle (5% NMP, 95% PEG) without the active product. Anti-CTLA-4 antibody (100 μg/mouse, clone 9H10, Bio X cell) and anti-PD-1 antibody (250 μg/mouse, clone RPM1- 14, Bio X cell) were injected intraperitoneally (i.p.) on days 7, 10, 13 and 16 for the B16, B16-GMCSF and 4T1 models and on days 10, 13 and 16 for the CT26 model. Tumors were measured every second or third day with a caliper, and the volume (length × width × height) was calculated. Mice that had no visible and palpable tumors that could be measured on consecutive measurement days were considered complete regressions. Animals were euthanized for signs of distress or when the total tumor volume reached 2500 mm3. For the re-challenge study: mice with complete responses in the anti-PD-1 treatment group and the anti-PD-1 and Eganelisib (IPI-549) combination group were re-challenged with 2.5 × 105 CT26.WT tumor cells (on day 106 of the study since original tumor implant). Additional mice with complete responses from an additional Eganelisib (IPI-549) and anti-PD-1 group were implanted with 1×105 4T1 tumor cells.

In vitro absorption, distribution, metabolism, and excretion (ADME) properties and pharmacokinetic parameters of compound 26/Eganelisib (IPI-549) were also determined (summarized in Table 5). In vitro, 26/Eganelisib (IPI-549) showed moderate to high cell permeability across Caco-2 cell monolayers, was slowly metabolized in cultured hepatocytes (t1/2 > 360 min), and demonstrated IC50s greater than 20 μM for the CYP isoforms tested (1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4). In vivo (mice, rats, dog, and monkeys), compound 26 had excellent oral bioavailability, low clearance, and distributed into tissues with a mean volume of distribution of 1.2 L/kg (Table 5). Overall, compound 26 had a favorable pharmacokinetic profile to allow potent and selective inhibition of PI3K-γ in vivo.[1]

[1]. Discovery of a Selective Phosphoinositide-3-Kinase (PI3K)-γ Inhibitor (IPI-549) as an Immuno-Oncology Clinical Candidate. ACS Med Chem Lett. 2016 Jul 22;7(9):862-7.

[2]. Overcoming resistance to checkpoint blockade therapy by targeting PI3Kγ in myeloid cells. Nature. 2016 Nov 17;539(7629):443-447.

IPI-549 is under investigation in clinical trial NCT03795610 (Window of Opportunity Study of IPI-549 in Patients With Locally Advanced HPV+ and HPV- Head and Neck Squamous Cell Carcinoma).
Eganelisib is an orally bioavailable, highly selective small molecule inhibitor of the gamma isoform of phosphoinositide-3 kinase (PI3K-gamma) with potential immunomodulating and antineoplastic activities. Upon administration, eganelisib prevents the activation of the PI3K-gamma-mediated signaling pathways, which may lead to a reduction in cellular proliferation in PI3K-gamma-expressing tumor cells. In addition, this agent is able to modulate anti-tumor immune responses and inhibit tumor-mediated immunosuppression. Unlike other isoforms of PI3K, the gamma isoform is overexpressed in certain tumor cell types and immune cells; its expression increases tumor cell proliferation and survival. By selectively targeting the gamma isoform, PI3K signaling in normal, non-neoplastic cells is minimally or not affected, which results in a reduced side effect profile.

C30H24N8O2

528.58

528.202

C, 68.17; H, 4.58; N, 21.20; O, 6.05

1693758-51-8

91933883

Yellow solid powder

3.2

2

6

6

40

1060

1

O=C1C2C(C#CC3C=NN(C)C=3)=CC=CC=2C=C(C(C)NC(C2C(N)=NN3C=CC=NC3=2)=O)N1C1C=CC=CC=1

XUMALORDVCFWKV-IBGZPJMESA-N

InChI=1S/C30H24N8O2/c1-19(34-29(39)26-27(31)35-37-15-7-14-32-28(26)37)24-16-22-9-6-8-21(13-12-20-17-33-36(2)18-20)25(22)30(40)38(24)23-10-4-3-5-11-23/h3-11,14-19H,1-2H3,(H2,31,35)(H,34,39)/t19-/m0/s1

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.73 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 (4.73 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.)