PI3Kβ (IC50 = 4 nM); PI3Kδ (IC50 = 12 nM); PI3Kα (IC50 = 35 nM); PI3Kγ (IC50 = 675 nM)
PI3Kβ (IC50 4 nmol/L)
PI3Kδ (IC50 12 nmol/L)
PI3Kα (IC50 35 nmol/L)
PI3Kγ (IC50 675 nmol/L)[1]

AZD8186 is a potent inhibitor of PI3Kβ with additional activity versus the PI3Kδ isoform. The absolute selectivity profile for PI3Ks is understated in biochemical assays due to the tight-binding kinetics of AZD8186. Selectivity for PI3Kβ and δ is >100-fold higher in 74 protein and lipid kinases than it is in 74 different protein and lipid kinase assays. In a KinomeScan screen, AZD8186 at 10 M displayed no detectable binding to 442 additional kinases. In contrast to other off-target activity, AZD8186 exhibits selectivity for PI3K family kinases. MDA-MB-468 AZD8186 has an IC50 value of 3 nM in the PTEN-null line, where it prevents PI3Kβ-dependent activation of pAKT (Ser473). The PIK3CA-mutant line BT474c has a potency of 752 nM, showing preference for PI3Kβ over PI3Kα. When B cells are stimulated by IgM, PI3Kδ is activated, which phosphorylates AKT. With an IC50 value of 17 nM, AZD8186 prevents pAKT (Ser473) activation induced by IgM in JEKO cells. In tests for cell proliferation, IgM stimulated JEKO cell growth with an IC50 value of 228 nM, while AZD8186 inhibited MDA-MB-468 cell proliferation with a GI50 value of 65 nM. Its IC50 value of 1.981 M, which is consistent with its preference for PI3Kβ over PI3Kα, only inhibited the growth of BT474c cell lines[1].

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In biochemical assays, AZD8186 potently inhibits PI3Kβ (IC50 4 nmol/L) and PI3Kδ (IC50 12 nmol/L) with selectivity over PI3Kα (IC50 35 nmol/L) and PI3Kγ (IC50 675 nmol/L). In a broad panel of protein and lipid kinase assays, selectivity for PI3Kβ and δ was >100-fold versus 74 protein and lipid kinases. At 10 μmol/L, AZD8186 had no significant binding to 442 other kinases in a KinomeScan screen. [1]
In the PTEN-null line MDA-MB-468, AZD8186 inhibited PI3Kβ-dependent activation of pAKT (Ser473) with an IC50 value of 3 nmol/L. Potency in the PI3KCA-mutant line BT474c was 752 nmol/L, demonstrating selectivity for PI3Kβ over PI3Kα. AZD8186 inhibited IgM-stimulated phosphorylation of pAKT (Ser473) activation in JEKO cells with an IC50 value of 17 nmol/L. [1]
In cell proliferation assays, AZD8186 inhibited proliferation of MDA-MB-468 cells with a GI50 value of 65 nmol/L. It inhibited IgM stimulated JEKO cell growth with an IC50 value of 228 nmol/L. It only inhibited BT474c cell growth with an IC50 value of 1.981 μmol/L, consistent with its selectivity for PI3Kβ over PI3Kα. [1]
AZD8186 inhibits ADP-induced human platelet aggregation with a mean IC50 value of 186 nmol/L. [1]
In PTEN-null MDA-MB-468 and PC3 cells, AZD8186 (250 nmol/L) inhibited LPA-mediated activation of AKT. In PTEN-null MEF cell lines, AZD8186 inhibited EGF-mediated activation of pAKT following serum starvation in MDA-MB-468 cell lines. [1]
AZD8186 inhibited pathway activation in PTEN-null prostate lines LNCAP and PC3, and breast lines MDA-MB-468 and HCC70. It suppressed phosphorylation of AKT, PRAS40, S6, and FOXO with IC50 in the range of <10 to 300 nmol/L and full pathway inhibition at 300 nmol/L to 3 μmol/L. AZD8186 inhibited the growth of PTEN-deficient cells lines with GI50 less than 1 μmol/L. [1]
AZD8186 promoted translocation of FOXO to the nucleus in PTEN-deficient HCC70 and LNCAP cells. In LNCAP cells, treatment with 500 nmol/L AZD8186 induced association of FOXO3a with the chromatin fraction. [1]
In a broad cross-disease in vitro cell panel, a cutoff of GI50 < 1 μmol/L highlighted a subset of lines sensitive to AZD8186. The AZD8186-sensitive subset was enriched for PTEN-null cells. PTEN-deficient cells represent 52% (13/25) of the sensitive lines, but only 8% (9/106) of the insensitive lines. A number of lines with genetically WT PTEN were sensitive to AZD8186 (48%; 12/25). [1]

Antitumor activity is evaluated in the PTEN-null TNBC models HCC70 and MDA-MB-468, as well as the prostate models PC3 and HID28, in order to evaluate the single-agent efficacy of AZD8186 in vivo. All four models are slowed down in growth by AZD8186 at doses of 50 and 25 mg/kg twice daily. At 25 and 50 mg/kg, HCC70 is inhibited at 62% (P 0.001) and 85% (P<0.001), respectively, and MDA-MB-468 is inhibited at 47% (P 0.001) and 76% (P<0.001), respectively, at the end of the study, with regression early in the study. PC3's effectiveness in the PTEN-null prostate model is less pronounced, with doses of 25 and 50 mg/kg inhibiting maximum growth by 59% (P 0.001) and 64% (P<0.001), respectively. In contrast, AZD8186 inhibits growth in the PTEN-null prostate explant model HID28 by 79% (P<0.001). When administered to mice, AZD8186 has a brief half-life and a PK profile that produces patchy coverage over a 24-hour period. Animals with PC3 tumors are co-dosed with AZD8186 in the presence of the Cyt P450 inhibitor ABT, which significantly lengthens the time of exposure. Additionally, a tumor growth reduction of 86% (P<0.005) was achieved in the PC3 model using 30 mg/kg of AZD8186+ABT[1].

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In vivo, AZD8186 inhibits PI3K pathway biomarkers in prostate and TNBC tumors. [1]
At 25 and 50 mg/kg twice a day (b.i.d.), AZD8186 inhibited the growth of PTEN-null TNBC models HCC70 and MDA-MB-468, and prostate models PC3 and HID28. In HCC70, inhibition was 62% (P<0.001) and 85% (P<0.001) respectively. In MDA-MB-468, inhibition was 47% (P<0.001) and 76% (P<0.001) respectively. In PC3, maximal growth inhibition was 59% (P<0.001) and 64% (P<0.001) respectively. In HID28, AZD8186 gave 79% (P<0.001) growth inhibition. [1]
Co-dosing AZD8186 with the Cyt P450 inhibitor ABT resulted in significantly increased exposure and increased efficacy in the PC3 model, with 86% (P<0.005) reduction in tumor growth achieved with 30 mg/kg AZD8186 + ABT. [1]
AZD8186 inhibits pAKT (Ser473 and Thr308) and pPRAS40 following acute or chronic dosing in HCC70 and MDA-MD-468 in a dose- and time-dependent manner. Pathway modulation in HID28 and PC3 was similar. Co-dosing with ABT increased the time of pathway suppression in PC3. AZD8186 gives dynamic biomarker modulation consistent with the PK profile. IHC analysis shows that FOXO translocation correlates with the inhibition of pAKT. [1]
To determine if chronic pathway inhibition was required, HCC70 tumors were treated with AZD8186 for 4, 5, and 7 days out of a 7-day cycle. Dosing for 4 days at 50 mg/kg b.i.d. reduced tumor growth by 75% (P<0.05) versus 88% (P<0.05) with constant dosing. Dosed for 5 days at 25 mg/kg b.i.d., tumor growth was reduced by 87% (P<0.05) compared with 93% (P<0.05) with constant dosing. A 2 days on, 5 days off schedule with AZD8186 was inactive. [1]
In HCC70 tumors, combining 25 and 50 mg/kg AZD8186 b.i.d. with 15 mg/kg docetaxel gave greater tumor control compared with each monotherapy. In PC3 tumors, 10 and 30 mg/kg AZD8186 with 15 mg/kg docetaxel showed regressions. Both continuous and intermittent dosing (5 days on 2 days off) of AZD8186 combination with docetaxel in HCC70 tumors gave equal benefit. Benefit was achieved with 2 days exposure of AZD8186 in combination with docetaxel, but this was less effective than 5 days or continuous dosing. [1]

The inhibition of PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ human recombinant PI3K isoforms is evaluated using a Kinase-Glo Plus Assay Kit. 12 point half-log concentration-response curves with a top concentration of 100 μM are constructed by dispensing DMSO solubilised compounds into white 384-well medium-binding microplates using an Echo 555. 3 μL of the appropriate PI3K in Tris buffer (50 mM Tris pH7.4, 0.05% CHAPS, 2.1 mM DTT, and 10 mM MgCl2) is added.

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Inhibition of recombinant PI3Kβ, PI3Kα, PI3Kγ, and PI3Kδ was evaluated in a Kinase Glo-based enzyme activity assay. [1]
AZD8186 was tested across the Dundee Kinase Panel and across the panel of enzymes in the KinomeScan to evaluate a broader selectivity profile. [1]

AZD8186 is applied to cells for 2 hours at concentrations ranging from 3 to 0.01 μM . Following that, cells are lysed on ice using a buffer containing 0.5% Triton X-100, 3 mM EDTA, 3 mM EGTA, 50 mM NaF, 2 mM sodium orthovanadate, 270 mM sucrose, 10 mM β-glycerophosphate, and 5 mM sodium pyrophosphate. Sample loading buffer is used to dilute the lysates before they are separated on 4% to 12% Bis-Tris Novex gels, transferred to nitrocellulose membranes, and overnight probed with primary antibodies. Membranes are washed, then incubated with secondary antibodies that have been HRP-tagged before being observed[1].

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For assaying PI3K pathway suppression in cell lines, BT474c cells were plated and incubated with compound for 2 hours. Cells were lysed, and lysates were transferred to ELISA plates precoated with an anti-Total AKT antibody. The ELISA plates were incubated overnight, washed, and incubated with a phospho-AKT Thr308-specific antibody. Plates were washed again before addition of an anti-mouse-HRP-conjugated secondary antibody. Following incubation, plates were washed, and substrate working solution was added. Development was stopped, and plates were read. [1]
MDA-MB-468 cells were incubated with compound for 2 hours, fixed, blocked, and permeabilized, then stained using a phospho-specific AKT Ser473 antibody and a fluorescently conjugated secondary antibody. Inhibition was measured by a decrease in the number of fluorescently labeled cells containing phosphorylated AKT Ser473. [1]
JEKO cells were preincubated with compound for 1 hour before stimulating with anti-IgM. Plates were placed on ice and centrifuged. Media were removed, and lysis buffer was added. Plates were incubated on ice. AKT was assayed using a Phospho (Ser473)/Total AKT Whole-Cell Lysate Kit. [1]
For Western blot analysis, cells were exposed to AZD8186 for 2 hours, then lysed. Lysates were diluted with loading buffer, separated on gels, transferred onto nitrocellulose membranes, and probed with primary antibodies overnight. Membranes were incubated with HRP-tagged secondary antibodies and visualized. [1]
For LPA stimulation of serum-starved cells, MDA-MB-468 and PC3 cells were cultured, serum-starved overnight, preincubated with vehicle or AZD8186 for 1 hour, stimulated with LPA diluted in serum-free medium containing delipidated BSA. Cell lysates were prepared in RIPA cell lysis buffer containing protease and phosphatase inhibitors, and analyzed by Western blotting. [1]
For FOXO3a translocation assay, cells were seeded into a plate, incubated overnight, exposed to AZD8186 for 2 hours, fixed, permeabilized and blocked, and probed with an antibody against FOXO3a overnight. Following washing, cells were incubated with a secondary antibody conjugated to a fluorescent dye and nuclear stain before washing and imaging using a Cellomics Ar-rayscan. An algorithm measuring the ratio of nuclear to cytoplasmic fluorescence intensity was used to analyze the cells. [1]
Cell panel proliferation assays were performed as outlined in supplementary methods. [1]

Mice：The female CB17 SCID mice ages 6 to 8 weeks are used. HID28 tumor fragments (about 40 mm3) from donor animals are aseptically implanted subcutaneously, at the level of the interscapular region, for the purpose of performing HID28 in vivo experiments. Male outbred athymic (nu/nu) mice, 18 to 25 g (HSD: Athymic Nude-Foxn1nu). For all animal studies, groups must contain a minimum of 8 animals each. AZD8186 is typically prepared once weekly as a suspension in HPMC/Tween and dosed once or twice daily (0 and 6-8 hours). Once a week, AZD8186 is prepared either without ABT at a concentration of 10 mg/mL in 10% DMSO, 60% tri-ethylene glycol (TEG), and 30% water for injection (WFI). AZD8186 is co-dosed with ABT at 0 hours for twice-daily dosing and given alone as the single formulation at 6 to 8 hours. On day 0, 24 hours before the administration of AZD8186, RP-56976 is administered as a single i.v. bolus dose at a rate of 0.1 mL/10 g. It is freshly formulated in physiologic saline at 1.5 mg/mL.

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PC3 cells or HCC70 cells were implanted in the flank of female nude mice. MDA-MB-468 cells were implanted into mammary fat pad. HID28 in vivo experiments were performed under contract by Xentech, where HID28 tumor fragments from donor animals were aseptically implanted subcutaneously in the interscapular region of outbred athymic male mice. [1]
AZD8186 was generally formulated once weekly as a suspension in HPMC/Tween and dosed once or twice daily (0 and 6-8 hours). For groups in which ABT was administered, AZD8186 was formulated once weekly either alone in 10% DMSO/60% triethylene glycol (TEG)/30% water for injection (WFI) or in the presence of ABT. For twice daily dosing (0 and 6-8 hours), AZD8186 was co-dosed with ABT at 0 hours and administered alone at 6 to 8 hours. Docetaxel was formulated fresh in physiologic saline and dosed as a single i.v. bolus dose on day 0, 24 hours before the administration of AZD8186. [1]
Tumor volume was calculated twice weekly from bilateral caliper measurements using the formula: [length × width × width) × π/6]. Growth inhibition from the start of treatment was assessed by comparison of the geometric mean change in tumor volume for the control and treated groups. Statistical significance was evaluated using a one-tailed, two-sample t test. [1]
For pharmacodynamic studies, when mean tumor size reached a certain size, half the tumor was snap-frozen in liquid nitrogen and stored at -80°C, the other half was fixed in formalin buffer and then embedded in paraffin for IHC staining. Total blood was collected by intracardiac puncture and plasma prepared and immediately frozen for PK analysis. For each time point, a minimum of 4 or 5 tumors were used for the analysis. [1]
Frozen tumors were homogenized using Fastprep methodology, and lysates were generated using adjusted lysis buffer. Equivalent amounts of protein were analyzed. The AKT(Ser473)/Total AKT Whole-cell Lysate Kit was used to detect phosphorylated and total AKT. Two ELISAs were used to detect total and phospho-PRAS40. The effect of AZD8186 was calculated by comparing treated groups to control. Mouse tissue was analyzed using the same AKT plates and separate plates for the PRAS ELISA. Phosphorylated (Ser235/236) and total S6 were analyzed with kits. [1]

In mouse, AZD8186 has a short half-life delivering a PK profile that results in intermittent cover over a 24 hours dosing interval. [1]
Co-dosing with the Cyt P450 inhibitor ABT resulted in significantly increased exposure. [1]

[1]. Inhibition of PI3Kβ signaling with AZD8186 inhibits growth of PTEN-deficient breast and prostate tumors alone and in combination with RP-56976. Mol Cancer Ther. 2015 Jan;14(1):48-58.

AZD-8186 is being investigated in the clinical trial NCT03218826 (the PI3Kβ inhibitor AZD8186 in combination with docetaxel for the treatment of patients with PTEN or PIK3CB mutations who have metastasized or are unresectable advanced solid tumors). AZD8186 is a phosphatidylinositol-3 kinase (PI3K) β subtype inhibitor with potential antitumor activity. After administration, AZD8186 selectively inhibits the activity of PI3Kβ in the PI3K/Akt/mTOR signaling pathway, potentially leading to reduced tumor cell proliferation and inducing death in PI3K-expressing cancer cells. Because this drug specifically targets class I PI3Kβ, it may be more effective and less toxic than pan-PI3K inhibitors. PI3K-mediated signaling is frequently dysregulated in cancer cells, leading to increased tumor cell growth and survival, as well as tumor resistance to multiple antitumor drugs.

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AZD8186 is a potent inhibitor of PI3Kβ with additional activity versus the PI3Kδ isoform. The selectivity for PI3Kβ over PI3Kα is mirrored in vivo with AZD8186 modulating AKT and associated pathway biomarkers in a range of PTEN-null tumor models, but not in PTEN WT tumor models in which pan PI3K family inhibitors are active. AZD8186 showed limited effects on normal tissues compared with the pan-PI3K inhibitors. [1]
Loss of PTEN expression is common across many tumor types (TNBC, prostate, head and neck, colorectal cancer, and squamous lung), suggesting that inhibitors of PI3Kβ are relevant in many diseases. [1]
A number of PTEN-null lines were insensitive to AZD8186, consistent with the hypothesis that multiple factors can influence the dependency on PI3Kβ. Candidate pathways include IGFR, EGFR, alternate PI3K pathway drives, and the presence of RAS and RAF mutations. [1]
Achieving antitumor effect with intermittent pathway suppression is advantageous. Given treatment with AZD8186 has the potential to induce feedback through FOXO-regulated genes activating other pro-proliferative signaling pathways, intermittent pathway suppression could reduce the feedback loop-induced resistance. [1]
In TNBC and prostate cancer, docetaxel is a standard-of-care chemotherapy. It is likely that agents targeting PI3Kβ will give greatest benefit when used in combination; therefore, combination with androgen receptor therapy and other molecular-targeted therapies will be explored. [1]
AZD8186 also inhibits PI3Kδ. PI3Kδ signals downstream of the BCRs, and PI3Kδ inhibitors show more profound activity in some hematologic disease (including chronic lymphocytic leukemia and mantle cell lymphoma). The PI3Kβ/δ profile of AZD8186 may provide benefit in hematologic disease. [1]
AZD8186 is currently being tested in a phase I clinical trial. [1]

C24H25F2N3O4

457.4698

457.181

C, 63.01; H, 5.51; F, 8.31; N, 9.19; O, 13.99

1627494-13-6

1627494-13-6

52913813

White to off-white solid powder

1.4±0.1 g/cm3

645.2±55.0 °C at 760 mmHg

344.0±31.5 °C

0.0±1.9 mmHg at 25°C

1.618

2.79

1

8

5

33

748

1

FC1C([H])=C(C([H])=C(C=1[H])N([H])[C@]([H])(C([H])([H])[H])C1=C([H])C(C(N(C([H])([H])[H])C([H])([H])[H])=O)=C([H])C2C(C([H])=C(N3C([H])([H])C([H])([H])OC([H])([H])C3([H])[H])OC1=2)=O)F

LMJFJIDLEAWOQJ-CQSZACIVSA-N

InChI=1S/C24H25F2N3O4/c1-14(27-18-11-16(25)10-17(26)12-18)19-8-15(24(31)28(2)3)9-20-21(30)13-22(33-23(19)20)29-4-6-32-7-5-29/h8-14,27H,4-7H2,1-3H3/t14-/m1/s1

(R)-8-(1-((3,5-difluorophenyl)amino)ethyl)-N,N-dimethyl-2-morpholino-4-oxo-4H-chromene-6-carboxamide

AZD-8186; AZD8186; AZD 8186

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 warming (198.9 mM)
Water: <1 mg/mL
Ethanol: 35 mg/mL warming (76.5 mM)

Solubility in Formulation 1: 15 mg/mL (32.79 mM) in 10% DMSO/60% tri-ethylene glycol (TEG)/30% water (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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Solubility in Formulation 2: ≥ 2.75 mg/mL (6.01 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 3: ≥ 2.75 mg/mL (6.01 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 4: ≥ 2.08 mg/mL (4.55 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 20.8 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.

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Solubility in Formulation 5: ≥ 2.08 mg/mL (4.55 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 20.8 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 6: ≥ 2.08 mg/mL (4.55 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.

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Solubility in Formulation 7: 10%DMSO+ 60% TEG+ 30% WFI: 16mg/mL

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