PI3Kα (IC50 = 0.5 nM); PI3Kδ (IC50 = 0.7 nM); PI3Kβ (IC50 = 3.7 nM); PI3Kγ (IC50 = 6.4 nM); mTOP (IC50 = 45 nM)
COPANLISIB HCl targets class I PI3K isoforms (p110α, p110β, p110γ, p110δ). Biochemical IC50 values: PI3Kα = 0.5 nmol/L, PI3Kβ = 3.7 nmol/L, PI3Kγ = 6.4 nmol/L, PI3Kδ = 0.7 nmol/L. IC50 against mTOR = 45 nmol/L. In cellular assays, it inhibited PI3Kα‑driven pAKT (S473) in KPL4 cells with IC50 = 0.6 nmol/L and PI3Kβ‑driven pAKT in LPA‑stimulated PC3 cells with IC50 = 7.8 nmol/L (pAKT T308) [1].

In both KPL4 cells and LPA-stimulated PC3 cells, BAY 80-6946 reduces pAKT levels. BAY 80-6946 exhibits antiproliferative activity and induces apoptosis in a subset of human cancer cell lines that have PIK3CA mutations and/or overexpression of HER2. Combining HER2-targeted therapies with BAY 80-6946 inhibits growth more effectively than either therapy when used separately and can improve cells' sensitivity to trastuzumab and lapatinib.

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COPANLISIB HCl potently inhibited the catalytic activity of class I PI3K isoforms with IC50 values of 0.5 nmol/L (p110α), 3.7 nmol/L (p110β), 6.4 nmol/L (p110γ), and 0.7 nmol/L (p110δ). It showed weak activity against mTOR (IC50 45 nmol/L) and no significant inhibition (≤30% at 1 μmol/L) of PI4K‑II, PIP4‑5K, PIP5‑4K, or 220 other kinases in a Millipore panel [1].
In rat ELT3 cells (TSC2‑deficient, PI3K‑independent mTORC1 activation), COPANLISIB HCl at 5 nmol/L completely inhibited AKT phosphorylation, but only modestly reduced p‑p70S6K at 500 nmol/L and had no effect on p‑4E‑BP1, whereas the dual PI3K/mTOR inhibitor BEZ‑235 completely blocked p70S6K and 4E‑BP1 at 500 nmol/L [1].
In cellular PI3Kα and PI3Kβ assays: In KPL4 cells (PIK3CA‑mutant/HER2+), COPANLISIB HCl reduced basal pAKT (T308 and S473) with IC50 values of 0.4 and 0.6 nmol/L, respectively, and inhibited p‑4E‑BP1 (T70) with IC50 4.6 nmol/L. In LPA‑stimulated PC3 cells (PTEN‑null, PI3Kβ‑driven), it inhibited pAKT (T308 and S473) with IC50 values of 7.8 and 10 nmol/L, respectively, and p‑4E‑BP1 with IC50 34 nmol/L [1].
Antiproliferative activity: In a panel of human tumor cell lines, COPANLISIB HCl showed IC50 values <10 nmol/L in several breast, endometrial, and hematologic cancer lines. In breast cancer cells, it had mean IC50 = 19 nmol/L in PIK3CA‑mutant lines (n=9) and 17 nmol/L in HER2+ lines (n=7), compared to 774 nmol/L in PIK3CA wild‑type/HER2‑negative lines (n=11) [1].
Apoptosis induction: In BT‑20 (lapatinib‑resistant, PIK3CA‑mutant) breast cancer cells, COPANLISIB HCl at 20 and 62 nmol/L induced 2‑ and 3‑fold increases in caspase‑9 activity (24 h), increased phospho‑p53 (Ser15) and cleaved PARP at 200 nmol/L (48 h). In BT474 cells (HER2+), it induced caspase‑9 activation with EC50 340 nmol/L; combination with lapatinib showed synergy (combination index 0.20‑0.29) [1].
COPANLISIB HCl also potently induced nuclear localization of FOXO1A with EC50 <1 nmol/L (Supplementary Fig. S2) [1].

Copanlisib (BAY 80-6946; 0.5-6 mg/kg; intravenous injection; every second day, every third day; for 60 days; athymic nude rats) treatment exhibits significant antitumor activity in the rat KPL4 tumor xenograft model[1].
In nude rats bearing H460 NSCLC xenografts, a single intravenous dose of COPANLISIB HCl (6 mg/kg) resulted in tumor concentrations ~100‑fold higher than plasma at 48 h, with slow tumor clearance. pAKT (S473) in tumors was inhibited >90% at 24 h and remained suppressed for 48‑72 h. Immunohistochemistry showed 65% reduction in Ki67 and 75% reduction in phospho‑histone H3 at 24 h. FDG‑PET revealed dose‑dependent inhibition of tumor uptake [1].
In KPL4 breast cancer xenografts (HER2+/PIK3CA‑mutant) in nude rats, COPANLISIB HCl administered i.v. every second day (Q2D) for 5 doses produced TGI of 77%, 84%, 99%, and 100% at 0.5, 1, 3, and 6 mg/kg, respectively. Complete tumor regression was observed in 10/10 rats at 3 and 6 mg/kg, with all rats tumor‑free on day 73 [1].
In HCT116 colon cancer xenografts (PIK3CA/KRAS mutant), COPANLISIB HCl at 3 and 6 mg/kg Q2D for 5 doses gave TGI of 75% and 88%, with tumor growth delays of 10 and 11 days [1].
In patient‑derived xenograft models (Lu7860 erlotinib‑resistant NSCLC, and MAXF1398 luminal breast cancer), COPANLISIB HCl at 14 mg/kg Q2D for 10 days produced TGI of 88% and 71%, respectively [1].
Weekly dosing schedule (two i.v. doses on one day per week) of COPANLISIB HCl at 9 mg/kg BID once weekly in HCT116 model gave equivalent TGI to 6 mg/kg Q2D. When combined with paclitaxel in Lu7343 NSCLC (PIK3CA E545K), COPANLISIB HCl (10 mg/kg BID once weekly for 3 weeks) plus paclitaxel (25 mg/kg weekly) produced 100% response rate (5 CR, 5 PR) at end of treatment, and long‑lasting regression (60% CR, 40% PR at day 55) [1].

The effect of BAY 80-6946 on PI3Kα, PI3Kβ, and PI3Kγ activity is measured by the inhibition of 33P incorporation into phosphatidylinositol (PI) in 384-well MaxiSorp® plates coated with 2 µg/well of PI and phosphatidylserine (PS) (1:1 molar ratio). In each PI3K isoform assay, 9 µL of reaction buffer (50 mM MOPSO, pH 7.0, 100 mM NaCl, 4 mM MgCl2, 0.1% BSA) is used, containing either 25 ng of purified human p110γ protein or 7.5 ng of His-tagged N-terminally truncated p110α or p110β protein. Adding 5 µL liters of a 40-µM ATP solution with 20 µCi/mL [33>/sup>P]-ATP initiates the reaction. By adding 5 µL of a 25-mM EDTA solution, the reaction is stopped after 2 hours of incubation at room temperature. After washing the plates, Ultima GoldTM scintillation cocktail (25 µL) is added. With the help of a BetaPlate Liquid Scintillation Counter, the radioactivity incorporated into the immobilized PI substrate is identified.

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PI3K biochemical lipid kinase assays: Inhibition of ³³P incorporation into phosphatidylinositol was measured. For PI3Kδ, a homogeneous time‑resolved fluorescence (HTRF) assay was used (Upstate/Chemicon PIProfiler). For PI4K‑II, PIP4‑5K, and PIP5‑4K, similar conditions were used with recombinant enzymes and appropriate lipid substrates (PIP4 or PIP5) [1].
Selectivity profiling: COPANLISIB HCl was tested at 1 μmol/L against a panel of 220 kinases (Millipore) and showed <30% inhibition for all except PI3K and mTOR. No inhibition of PI4K‑II, PIP4‑5K, or PIP5‑4K was observed [1].

The CellTiter-Glo® luminescent cell viability kit is used to calculate the rate of cell proliferation over a 72-hour period. In separate microtiter plates, cells are briefly plated. After an overnight incubation at 37 °C, the luminescence values in the t=0 hour plates are calculated. The cells are incubated in the t=72 hour plates for 72 hours at 37 °C after the addition of test substances that have been diluted in growth medium. After a 10-minute reaction with CellTiter-Glo® solution, Luminescence values are calculated using a Wallac 1420 Victor2TM 1420 multilabel HTS counter. Subtracting the luminescence values from the corresponding values in the t=72 hour plates, one can calculate the percentage inhibition of cell growth. Calculating the percentage of cell growth inhibition is done by comparing values between drug-treated cells and controls.

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Cellular pAKT and p‑4E‑BP1 quantification: AlphaScreen SureFire assays (PerkinElmer) were used to measure phospho‑AKT (T308, S473) and phospho‑4E‑BP1 (T70) in KPL4 cells (basal) and in LPA‑stimulated PC3 cells (after overnight serum starvation, 1 h inhibitor pre‑incubation, then 50 μmol/L LPA for 10 min). IC50 values were calculated [1].
Western blotting: Cells were treated with compound for 2 h, lysed, and probed with antibodies against p‑AKT(S473), total AKT, p‑p70S6K(T389), total p70S6K, p‑4E‑BP1(Thr37/46), p‑4E‑BP1(Ser65), total 4E‑BP1 [1].
Cell proliferation assay: Cells were seeded in 96‑well plates, treated with compound for 72 h, and viability measured using CellTiter‑Glo Luminescent Cell Viability Kit (Promega). IC50 values were determined [1].
Apoptosis assays: Caspase‑9 activity was measured using Caspase‑Glo9 Assay Kit (Promega) after 24 h treatment. Phospho‑p53 (Ser15) and cleaved PARP were measured using MSD Apoptosis Kit (K15102D‑2) after 48 h treatment [1].
FOXO1A nuclear localization assay: Cells were treated with compound and nuclear translocation was assessed (details in Supplementary, EC50 <1 nmol/L) [1].

Athymic nude rats injected with KPL4 tumor cells[1]
0.5 mg/kg, 1 mg/kg, 3 mg/kg or 6 mg/kg
Intravenous injection; every second day, every third day; for 60 days

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Animals: Athymic nude rats and nude mice (details not specified). Tumor xenografts were established by subcutaneous injection of tumor cells (e.g., KPL4, HCT116, Lu7860, MAXF1398). Dosing started when tumors were established (size not specified). COPANLISIB HCl was dissolved in PEG400/acidified water (0.1 N HCl, pH 3.5; 20/80, v/v) or 5% mannitol vehicle. Route: intravenous bolus injection. Dosing schedules: Q2D (every second day) for 5‑10 doses, or weekly (two doses on one day). Doses ranged from 0.5 to 14 mg/kg. Body weight and tumor volumes measured twice weekly. Tumor volume calculated as (l × w²)/2. Tumor growth inhibition (TGI) and response rates (RECIST criteria) calculated [1].
Pharmacokinetic studies: In rats and mice, blood collected at 5,15,30 min and 1,4,7,24,30 h after single i.v. dose (6 mg/kg in rats, 14 mg/kg in mice). Concentration determined by LC‑MS/MS. Tumor and plasma concentrations measured at various time points for PK/PD analysis [1].

Pharmacokinetic parameters in rats (nude rats, i.v. 6 mg/kg): Vss = 32 L/kg, plasma clearance (CL) = 3.95 L/kg·h, terminal t½ = 6.0 h. After multiple dosing (Q2D×5), similar exposure, no accumulation. In mice (nude mice, i.v. 14 mg/kg): CL = 16 L/kg·h, t½ = 0.7 h, Vss = 12.9 L/kg (Supplementary Table S2). High plasma‑free fraction: rat 35%, mouse 14%, dog 33%, human 16%. Tumor exposure was ~100‑fold higher than plasma at 48 h after a single i.v. 6 mg/kg dose in H460 xenografts, and drug cleared more slowly from tumor than plasma [1].

Maximum tolerated dose (MTD) in nude rats was 6 mg/kg (Q2D). At MTD, maximum mean body weight loss of 6‑10% occurred during first few dosing days, then returned to normal by end of dosing. No lethality observed. In mice, MTD was >14 mg/kg with Q2D schedule [1]. No other toxicity data (e.g., organ toxicity) reported.

[1]. BAY 80-6946 is a highly selective intravenous PI3K inhibitor with potent p110α and p110δ activities in tumor cell lines and xenograft models. Mol Cancer Ther. 2013 Nov;12(11):2319-30.

Copanlisib dihydrochloride is the dihydrochloride of Copanlisib. It is an antitumor drug, an apoptosis inducer, and an EC 2.7.1.137 (phosphatidylinositol 3-kinase) inhibitor. It contains Copanlisib.
See also: Copanlisib dihydrochloride (note moved to).

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COPANLISIB HCl (BAY 80‑6946) is a novel imidazolinonquinazoline class PI3K inhibitor identified via high‑throughput screening and optimized for drug‑like properties. It exhibits >2,000‑fold selectivity against other lipid and protein kinases except mTOR (45 nM). In cellular assays, it is >100‑fold selective for PI3K over mTOR. The compound is highly efficacious in xenografts with PIK3CA mutations or HER2 amplification, including complete tumor regression in KPL4 breast cancer model. Its favorable preclinical profile led to clinical development (phase I trials with weekly i.v. dosing). The drug shows synergy with paclitaxel and MEK inhibitors. A first‑in‑human phase I study was ongoing at the time of publication (NCT00962611, NCT01411410, NCT01392521) [1].

C23H30CL2N8O4

553.45

552.176

C, 49.92; H, 5.46; Cl, 12.81; N, 20.25; O, 11.56

1402152-13-9

Copanlisib;1032568-63-0

135565785

Off-white to light yellow solid powder

4

9

7

37

974

0

Cl.Cl.O1CCN(CC1)CCCOC1C=CC2C(C=1OC)=N/C(=N\C(C1=CN=C(N)N=C1)=O)/N1CCNC1=2

STGQPVQAAFJJFX-UHFFFAOYSA-N

InChI=1S/C23H28N8O4.2ClH/c1-33-19-17(35-10-2-6-30-8-11-34-12-9-30)4-3-16-18(19)28-23(31-7-5-25-20(16)31)29-21(32)15-13-26-22(24)27-14-15;;/h3-4,13-14H,2,5-12H2,1H3,(H2,24,26,27)(H,28,29,32);2*1H

BAY 80-6946; BAY80-6946; BAY-80-6946; BAY806946; BAY-806946; BAY 806946; Copanlisib HCl; Copanlisib dihydrochloride; Copanlisib; trade name Aliqopa

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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: 100 mg/mL (180.69 mM) in PBS (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: 10% Trifluoroacetic acid water solution: 1mg/mL

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