p110δ (IC50 = 2.5 nM); p110α (IC50 = 1170 nM); p110β (IC50 = 1508 nM); p110γ (IC50 = 2187 nM)
The target of IPI-3063 is the phosphoinoside-3-kinase (PI3K) p110δ isoform. In biochemical assays, its IC₅₀ value for p110δ is 2.5 ± 1.2 nM (n = 5), while for other PI3K isoforms, the IC₅₀ values are significantly higher: 1,171 ± 533 nM (n = 6) for p110α, 1,508 ± 624 nM (n = 5) for p110β, and 2,187 ± 1,529 nM (n = 4) for p110γ [1]
In cell-based assays, the cellular IC₅₀ of IPI-3063 for p110δ is 0.1 ± 0.01 nM (n = 6), and for other class I PI3K isoforms, the cellular IC₅₀ values are at least 1,000-fold higher: 1,901 ± 1,318 nM (n = 4) for p110α, 102.8 ± 35.7 nM (n = 4) for p110β, and 418.8 ± 117.2 nM (n = 2) for p110γ [1]

IPI-3063 is a p110δ selective compound with an IC50 = 0.1 nM in p110δ-specific cell-based assays and cellular IC50 values for the other class I PI3K isoforms are at least 1,000-fold higher. IPI-3063 significantly lowers mouse B cell survival, proliferation, and plasmablast differentiation[1].

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1. Effect on mouse B cell signaling: When mouse primary B cells were stimulated with αIgM + IL-4, IPI-3063 potently reduced the phosphorylation of AKT at serine 473 (p-AKT S473) with a significant effect at 1 nM, and also reduced the phosphorylation of ERK1/2 at Thr202/Tyr204 (p-ERK1/2) with a significant effect at 10 nM. Similar results were observed in B cells stimulated with LPS (for p-AKT), but LPS did not induce ERK1/2 phosphorylation [1]
2. Effect on mouse B cell survival: In assays assessing mouse B cell survival, IPI-3063 reduced BAFF-dependent survival in a dose-dependent manner, achieving a significant decrease when present at 10 nM, and the effect approached that of the pan-PI3K inhibitor GDC-0941. A similar trend was observed in cells incubated with IL-4, while the p110γ inhibitor AS-252424 had no significant effect on survival [1]
3. Effect on mouse B cell proliferation: For mouse B cell proliferation, IPI-3063 blocked proliferation in αIgM + IL-4 stimulated B cells at the lowest concentration tested (1 nM). It significantly reduced cell accumulation at all concentrations tested in αIgM + IL-4 stimulated cells, and had a similar dose-dependent inhibitory effect in LPS-stimulated B cells. However, it did not affect B cell proliferation following stimulation with α-CD40 + IL-4. In LPS + IL-4 stimulated B cells, the inhibitor showed a similar trend but with greater variability [1]
4. Effect on mouse B cell differentiation and antibody class switching: IPI-3063 potently decreased plasmablast differentiation in LPS-stimulated mouse B cells starting at 1 nM (measured by the percentage of the CD138⁺ B220ˡᵒ population), and the highest concentrations inhibited plasmablast differentiation to the same degree as GDC-0941. In B cells stimulated with αCD40 + IL-4, it increased the percentage of B220⁺ cells switching to IgG1 starting at 1 nM, approaching the effect of GDC-0941, but had no significant effect on IgG1 switching in LPS + IL-4 activated cells. Additionally, measuring IgM secretion by ELISA showed a similar trend to plasmablast differentiation inhibition, and the p110γ inhibitor AS-252424 had no significant effect in these assays [1]
5. Effect on human B cell proliferation: In human B cell proliferation assays, IPI-3063 blocked proliferation of human B cells stimulated with human CD40L + anti-human IgM/IgG + hIL-2 + hIL-21 at 1 nM, and significantly reduced proliferation starting at 1 nM when measuring the total number of divided cells and the percent divided. The p110γ inhibitor AS-252424 had no effect on human B cell proliferation [1]

IPI-3063 has good pharmacokinetics in mice[1].

Human recombinant PI3K-α, PI3K-β, PI3K-δ, and PI3K-γ are used. Phosphatidylinositol 4,5 bis phosphate (diC8-PtdIns(4,5)P2) is used. PI3K-α, β, and δ and are heterodimers made up of the p85α regulatory subunit and the full-length p110α, p110β, or p110δ catalytic subunit. The catalytic subunit p110γ has a monomer called PI3K-γ. Samples of kinase (10 nM-α, β, and δ; 20 nM-γ) are incubated with IPI-3063 for 30 min 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-PtdIns(4,5)P2 mixture to give final concentrations of 3 mM ATP and 500 µM diC8-PtdIns(4,5)P2. Reactions are incubated for 2 hours at room temperature, and PI3K activity is measured. Plate readers are used to read plates in luminescence mode.

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1. Preparation of reagents and samples: Human recombinant PI3K-α, -β, -δ, and -γ were used. PI3K-α, β, and δ are heterodimers consisting of full-length p110α, p110β, or p110δ catalytic subunit and the p85α regulatory subunit, while PI3K-γ is a monomer of the p110γ catalytic subunit. Phosphatidylinositol 4,5 bis phosphate (diC8-PtdIns(4,5)P2) was used as a substrate [1]
2. Incubation process: Kinase samples (10 nM for α, β, and δ; 20 nM for γ) were incubated with IPI-3063 for 30 minutes at room temperature in reaction buffer (composed of 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). After that, an ATP/diC8-PtdIns(4,5)P2 mixture was added to achieve final concentrations of 3 mM ATP and 500 µM diC8-PtdIns(4,5)P2 [1]
3. Reaction and detection: The reactions were incubated at room temperature for 2 hours. PI3K activity was assessed using an ADP-Glo Max assay kit, and plates were read on an Envision plate reader in luminescence mode [1]

Purified mouse B cells are incubated for 48 hours in either interleukin-4 (IL-4) or B-cell activating factor (BAFF), along with varying concentrations of IPI-3063 and IPI-443.

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1. Mouse B cell purification and culture: Mouse splenic B cells were purified by negative selection, with purity >95% verified by FACS analysis using anti-B220 antibody. Purified B cells were seeded at a final concentration of 0.5 or 0.25 × 10⁶ cells/mL in RPMI 1640 medium supplemented with 10% (vol/vol) heat-inactivated FCS, 5 mM Hepes, 2 mM l-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 50 µM 2-mercaptoethanol [1]
2. Western blot analysis for signaling molecules: Purified mouse B cells were pretreated with IPI-3063 at various concentrations for 30 minutes, then activated with 5 µg/mL αIgM + 10 ng/mL IL-4 or 5 µg/mL LPS for 1 hour before harvest. Cell lysates were prepared, and western blot was performed to detect p-AKT S473 and p-ERK1/2. ImageJ was used to measure mean fluorescence intensities of each band, and phospho-signal was normalized with actin measurements, with fold change calculated using the stimulated/no drug control [1]
3. B cell survival assay: Total splenocytes were pretreated with IPI-3063 at different concentrations, then cultured with 10 ng/mL IL-4 or 60 ng/mL BAFF for 48 hours. The percentage of viable B cells was calculated by measuring the percentage of B220⁺ 7AAD⁻ cells using FACS [1]
4. B cell proliferation assay (CFSE labeling): Mouse splenocytes or purified B cells were pretreated with IPI-3063 for 30 minutes, then stimulated with different stimuli (αIgM + IL-4 for 72 h, LPS for 72 h, αCD40 + IL-4 for 96 h, LPS + IL-4 for 96 h). B cells were labeled with 2.5 µM CFSE before stimulation. FACS was used to analyze CFSE fluorescence, and the total number of divided cells was determined by the number of B220⁺ 7AAD⁻ CFSEˡᵒ cells [1]
5. B cell differentiation and antibody class switching assay: Purified mouse B cells were pretreated with IPI-3063, then stimulated with 5 µg/mL LPS for 72 h (for plasmablast differentiation) or 5 µg/mL anti-CD40 + 5 ng/mL mIL-4 or 5 µg/mL LPS + 5 ng/mL mIL-4 for 96 h (for IgG1 class switching). FACS was used to detect plasmablasts (7AAD⁻ CFSEˡᵒ CD138⁺ B220ˡᵒ population) and IgG1-switched B cells (7AAD⁻ CFSEˡᵒ B220⁺ IgG1⁺ cells). For IgM secretion, supernatants from LPS-stimulated B cells were collected after 3 days, diluted 1:1,000, and detected by ELISA using anti-mouse IgM coating antibody and HRP-conjugated rabbit anti-mouse IgM secondary antibody [1]
6. Human B cell purification and proliferation assay: Peripheral blood mononuclear cells (PBMCs) were purified from human peripheral blood by density gradient centrifugation using Ficoll-Paque. Human B cells were then purified from PBMCs by negative selection, with purity increased from 4% to >70% verified by FACS using anti-CD19 PE conjugated antibody. Purified human B cells were seeded at 0.1 × 10⁶ cells/mL and cultured with 2 µg/mL human CD40L + 5 µg/mL anti-human IgM/IgG + 100 µg/mL hIL-2 + 100 µg/mL hIL-21 in RPMI 1640 medium with the same supplements as mouse B cell culture. B cells were labeled with CFSE, and after 120 hours of stimulation, FACS was used to analyze the total number of divided cells (CD19⁺ 7AAD⁻ CFSEˡᵒ cells) and the percent divided [1]
7. pAKT S473 ELISA assay: SKOV3 and 786.0 cells were seeded into 96-well plates at 2 million per 200 µl culture media per well; Raji and Raw264.7 cells were seeded at the same density in FBS-free media. After overnight incubation at 5% CO₂ and 37°C, cells were treated with IPI-3063 for 30 minutes. Raji cells were stimulated with 10 µg/mL anti-human IgM for 30 minutes, Raw264.7 cells with 25 nM C5a for 3 minutes (both in the presence of the inhibitor), while SKOV3 and 786.0 cells were not stimulated. Medium was aspirated, 50 µL/well of ice-cold lysis buffer was added, and pAKT level was determined using a phospho-Akt1 (S473) sandwich ELISA antibody kit [1]

Brown Norway rats
50 mg/kg
oral administration

Some studies have mentioned that IPI-3063 has good pharmacokinetic properties in mice, but no specific parameters have been provided, such as absorption, distribution, metabolism, excretion, half-life or oral bioavailability[1].

[1]. Front Immunol. 2017, 8: 747.

[2]. Chem Biol . 2013 Nov 21;20(11):1364-74.

1. IPI-3063 is a potent and selective inhibitor of the PI3K p110δ isoform. It is an effective tool for studying the function of p110δ in immune cells (especially B cells) because it can effectively regulate B cell responses (survival, proliferation, differentiation, antibody class switching) at low nanomolar concentrations [1]. 2. Class I PI3K is an important enzyme that transmits signals from cell surface receptors to downstream mediators, thereby driving cell function. PI3K signaling pathway activation has been found in lymphocytes of patients with B cell malignancies and autoimmune diseases. The p110δ catalytic isoform is crucial for the development, survival, activation, and differentiation of B lymphocytes, and is therefore a reasonable target. The development of IPI-3063 aims to target this subtype. Its potent and selective inhibition of p110δ provides a pathway for studying the role of p110δ in B cell biology and its potential therapeutic applications in B cell-related diseases [1]. Currently, idelalisib is the only selective p110δ inhibitor approved by the FDA for the treatment of certain B cell malignancies. As a novel selective p110δ inhibitor, IPI-3063 has similar effects on B cell function in vitro as the pan-PI3K inhibitor GDC-0941, indicating its potential as a research tool and a potential lead compound for the treatment of B cell-related diseases such as B cell malignancies and B cell-mediated autoimmune diseases [1].

C25H25N7O2

455.52

455.206

C, 65.92; H, 5.53; N, 21.52; O, 7.02

1425043-73-7

IPI-3063; IPI 3063; IPI3063.;

71276090

White to off-white solid powder

1.4±0.1 g/cm3

817.8±65.0 °C at 760 mmHg

448.4±34.3 °C

0.0±2.9 mmHg at 25°C

1.688

3.1

2

7

5

34

962

1

C1(=O)C=CC(C2=C3C(=O)N(C(C)C)C([C@@H](NC4=NC=NC(N)=C4C#N)C)=CC3=CC=C2)=CN1C

OBHAYOJCPNWKBL-HNNXBMFYSA-N

InChI=1S/C25H25N7O2/c1-14(2)32-20(15(3)30-24-19(11-26)23(27)28-13-29-24)10-16-6-5-7-18(22(16)25(32)34)17-8-9-21(33)31(4)12-17/h5-10,12-15H,1-4H3,(H3,27,28,29,30)/t15-/m0/s1

4-amino-6-[[(1S)-1-[8-(1-methyl-6-oxopyridin-3-yl)-1-oxo-2-propan-2-ylisoquinolin-3-yl]ethyl]amino]pyrimidine-5-carbonitrile

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: ~91 mg/mL (~117.1 mM)
Ethanol: ~12 mg/mL (~26.3 mM)