Pictilisib (GDC-0941) (GDC-0941) is a potent pan inhibitor of class I catalytic subunits of PI3K (IC50s: 3/33/3/75 nM for p110α/β/δ/γ).

p110α:3 nM (cell free), p110δ:3 nM (cell free), p110β:33 nM (cell free)

Pictilisib is a potent inhibitor of cell proliferation in these cell lines with submicromolar IC50s. Potent inhibition of Akt (Ser473) phosphorylation was observed in U87MG, PC3, and MDA-MB-361 cells with IC50s of 46, 37, and 28 nM, respectively [1]. In comparison to single-agent treatments, the combination of Pictilisib and docetaxel reduced tumor cell viability by 80% or greater in the breast cancer cell lines tested in vitro. A Bliss sum of 0 was determined in the MDA-MB-453 cell line indicating an additive combination effect whereas Bliss sums > 0 were calculated in the other tumor cell lines indicating a synergistic effect [2]. Treatment with 250 nM Pictilisib for 2 hr resulted in 40%–85% inhibition of pAKT in all cell lines tested. Inhibition of the PI3K/AKT pathway by Pictilisib was reflected as a dose-dependent reduction in cell proliferation/viability. Pictilisib inhibited the growth of both trastuzumab-sensitive and -insensitive cells. The IC50 values for Pictilisib ranged between 150 and 950 nM and did not correlate with trastuzumab sensitivity [3].

Treatment of animals bearing MCF7-neo/HER2 breast cancer xenografts with 7.5 mg/kg docetaxel or 150 mg/kg Pictilisib led to tumor growth delay and tumor stasis, respectively. The combination of 100 mg/kg Pictilisib and docetaxel resulted in tumor stasis during the treatment period that was sustained after dosing ended [2]. AZD8055 (20mg/kg) or Pictilisib (75mg/kg) administration induced a transient increase in blood glucose levels. Treatment with either AZD8055 or Pictilisib led to a marked inhibition of Akt activity as well as phosphorylation of Thr308 and Ser473. Phosphorylation of the Akt substrates PRAS40 and Foxo-1/3a were also inhibited by AZD8055 or GDC-941 [4].

Recombinant human PI3Kα, PI3Kβ, and PI3Kδ are coexpressed in a Sf9 baculovirus system with the p85α regulatory subunit and purified as GST-fusion proteins using affinity chromatography on glutathione-sepharose. Recombinant human PI3Kγ is expressed as monomeric GST-fusions and purified similarly. GDC-0941 is dissolved in DMSO and added to 20 mM Tris-HCl (pH 7.5) containing 200 μg yttrium silicate (Ysi) polylysine SPA beads, 4 mM MgCl2, 1 mM dithiothreitol (DTT), 1 μM ATP, 0.125 μCi [γ-33P]-ATP, and 4% (v/v) DMSO in a total volume of 50 μL. The recombinant GST-fusion of PI3Kα (5 ng), PI3Kβ (5 ng), PI3Kδ (5 ng), or PI3Kγ (5 ng) is added to the assay mixture to initiate the kinase reaction. After incubation for 1 hour at room temperature, the kinase reaction is terminated with 150 μL PBS. The mixture is then centrifuged for 2 minutes at 2000 rpm and read using a Wallac Microbeta counter. The reported IC50 values are calculated using a sigmoidal, dose-response curve fit in MDL Assay Explorer [1].

All drug treatments were tested in quadruplicate during a 4-day incubation period, and the relative number of viable cells was estimated using CellTiter-Glo. Total luminescence was measured on a Wallac Multilabel Reader. Cells were treated simultaneously with docetaxel (dose range = 0.0003–0.020 μmol/L) or GDC-0941 (dose range = 0.083–5 μmol/L) in an 8 × 10 matrix of concentrations chosen to encompass clinically relevant doses (24). The concentration of drug resulting in EC50 was determined using Prism software. Combination synergy of GDC-0941 and docetaxel was determined by Bliss independence analyses. A Bliss expectation for a combined response (C) was calculated by the equation: C = (A + B) ? (A × B) where A and B are the fractional growth inhibitions of drug A and B at a given dose. The difference between the Bliss expectation and the observed growth inhibition of the combination of drugs A and B at the same dose is the 'Delta.Bliss.' Delta.Bliss scores were summed across the dose matrix to generate a Bliss sum. Bliss sum = 0 indicates that the combination treatment is additive (as expected for independent pathway effects); Bliss sum > 0 indicates activity greater than additive (synergy); and Bliss sum < 0 indicates the combination is less than additive (antagonism). Statistical analysis comparing the Bliss sums for each cell line was conducted by the Student t-test [2].

Female nu/nu mice were inoculated subcutaneously with MCF7-neo/HER2 or MX-1 breast cancer cells. When tumors reached a mean volume of 200 to 250 mm3, animals were size-matched and distributed into groups consisting of 10 animals per group. Docetaxel formulated in 3% EtOH, 97% saline was administered intravenously once weekly. GDC-0941, formulated in MCT (0.5% methylcellulose, 0.2% Tween-80) was dosed orally and daily. MAXF1162 is a HER2+/ER+/PR+ patient-derived breast cancer tumor xenograft model established by directly implanting tumors subcutaneously from patient to NMRI nu/nu mice. Tumor volume was calculated as follows: tumor size (mm3) = (longer measurement × shorter measurement2) × 0.5. Tumor sizes were recorded twice weekly over the course of a study. Following data analysis, P values were determined using the Dunnett t test. For pharmacodynamic studies, tumor samples (n = 4) were immediately frozen or fixed in 10% neutral-buffered formalin. Tumors were dissociated in cell extraction buffer, and lysates were analyzed by Western blotting as described above. Immunohistochemistry was conducted using 5-μm paraffin sections of formalin-fixed tissue on a Ventana Benchmark XT instrument by deparaffinization, treatment with antigen retrieval buffer, and incubation with anti-cleaved caspase-3 primary antibody at 37°C. Bound antibody was detected using DABMap technology, and sections were counterstained with hematoxylin [2].