PI3Kδ (Ki = 0.12 nM); PI3Kα (Ki = 0.29 nM); PI3Kγ (Ki = 0.97 nM); PI3Kβ (Ki = 9.1 nM)

Taselisib (GDC0032) is an orally bioavailable, powerful, and selective inhibitor of Class I PI3Kα, δ, and γ isoforms. It inhibits the PI3K β isoform by a factor of 30 less than the PI3Kα isoform. PI3Kα isoform (PIK3CA) mutant and HER2-amplified cancer cell lines are more active against GDC-0032, according to preclinical data. GDC-0032's IC50 value of 2.5 nM limits the growth of MCF7-neo/HER2 cells.[1]

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Selectivity against the broader kinome was also determined by screening against Invitrogen’s SelectScreen panel of kinases. Out of 235 kinases that were screened with 11l/Taselisib (GDC0032) at 1 μM, we found that only five kinases were inhibited greater than 50%: PI3K-C2 beta, PI3Kα, PI3Kδ, PI3Kγ, and hVPS34 at 80.4%, 98.6%, 97.9%, 90.6%, and 69.9%, respectively. Notably, PI3Kβ was not included in the panel of kinases screened. Measurement of IC50 values for potently inhibited kinases showed that we indeed maintained biochemical selectivity over the non-class I PI3Ks, with IC50 values for PI3K-C2 beta and hVPS34 measuring 292 and 374 nM, respectively. Compared to the 290 pM activity against PI3Kα, we achieve a 1000-fold selectivity against these potential off-targets. [1]

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Taselisib (GDC0032) is active in HNSCC cell lines harboring activating PIK3CA aberrations and wild-type PTEN [2]
We tested the antiproliferative activity of GDC-0032 across a panel of 26 HNSCC cell lines, including the majority of commercially available HPV-positive HNSCC cell lines. As expected, there was a gradient of sensitivity in HNSCC cell lines. Cell lines harboring either mutations or amplification of PIK3CA tended to be sensitive to GDC-0032, with IC50 values in the nanomolar range (Fig. 1A). In contrast, 4 of the 6 most resistant cell lines to GDC-0032 had mutation or loss of PTEN, consistent with previous reports of PTEN aberrations leading to resistance to PI3Kα inhibitors through upregulation of PI3Kβ signaling (33–35). The observation of a preferential antiproliferative effect of GDC-0032 in cells with activated PI3KCA has also been observed with other isotype-specific PI3K inhibitors in other tumor types, suggesting that this selectivity could be important in the clinic (36, 37). In contrast, neither PIK3CA nor PTEN status correlated with sensitivity to GDC-0941, a pan-PI3K inhibitor with similar potency against all class IA PI3K isoforms (Fig. 1B). Thus, although PIK3CA and PTEN status may help identify tumors sensitive to GDC-0032, this is not a property shared amongst all classes of PI3K inhibitors.

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Taselisib (GDC0032) induces apoptosis in cell lines with PIK3CA alterations [2]
Next, we investigated the effects of GDC-0032 on downstream PI3K signaling in several genetic contexts. Treatment with GDC-0032 in Cal-33 cells (harboring a PIK3CAH1047R mutation) prevented phosphorylation of AKT and inhibited downstream mTOR targets, such as ribosomal protein S6 kinase (S6K), eukaryotic translation initiation factor 4E-binding protein 1 (4EBP-1), and S6 (Fig. 2A). This translated into an induction of apoptosis, as assessed by cleavage of poly-ADP ribose polymerase (PARP). Similar results were obtained in LB-771, a cell line containing amplification of PIK3CA (Supplementary Fig. S1A). In cell lines containing either PTEN homozygous deletion (UD-SCC-2) or mutation (UPCI-SCC-90), GDC-0032 was appreciably less effective at downregulating AKT/mTOR signaling and inducing cell death (Fig. 2A and Supplementary Fig. S1B). This supports the notion that downregulation of PI3K signaling is necessary for the proapoptotic effects of GDC-0032 in HNSCC.

The dose of 100 nmol/L Taselisib (GDC0032), which inhibits AKT/mTOR signaling in PIK3CA mutant cell lines but not in cells with loss or mutation of PTEN, was chosen for subsequent time-course experiments. This dose of GDC-0032 inhibited phosphorylation of AKT and downstream signaling in Cal-33 (PIK3CA mutant) and LB-771 (PIK3CA amplified) cells, but had little effect on PI3K signaling in UD-SCC-2 (PTEN homozygous deletion) or UPCI-SCC-90 cells (PTEN mutant; Fig. 2B), confirming that this is a concentration of GDC-0032 that inhibits PI3Kα- but not PI3Kβ-dependent signaling.

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Taselisib (GDC0032) radiosensitizes cells with PIK3CA mutation/amplification [2]
Given that inhibition of PI3K signaling has been purported to affect expression of DNA damage repair (DDR) proteins (19, 20, 38) and alter DDR signaling in response to radiation (15, 17), we next sought to study the effect of GDC-0032 on HNSCC cell lines treated with radiation. In Cal-33 cells (PIK3CAH1047R), the combination of GDC-0032 and radiation resulted in both more apoptotic (Annexin V–positive, PI-negative) and nonapoptotic (Annexin V–positive, PI-negative) cell death than either treatment alone (Fig. 3A). GDC-0032 and radiation also slowed cell growth rates more than either treatment alone in PIK3CA-mutant cell lines Cal-33 and HSC-2, but had little effects in PTEN-altered cell lines UPCI-SCC-90 and UD-SCC-2 (Fig. 3B). The increased antitumor effects of combined radiation and GDC-0032 compared with either treatment alone were confirmed using Annexin staining in three additional cell lines bearing activating PIK3CA alterations (LB-771, SNU-1076, and HSC-2), whereas no significant additional activity was observed in cell lines with wild-type PIK3CA and/or inactivating PTEN alterations (HSC-3, UD-SCC-2, HSC-4, and FaDu; Fig. 3C). Similar results were seen with the structurally unrelated p110α inhibitors BYL719 (Supplementary Fig. S2) and A66 (Supplementary Fig. S3), suggesting that GDC-0032 induces cell death following radiation primarily through p110α inhibition, rather than inhibition of other PI3K isoforms or off target enzymes.

Inhibition of other downstream PI3K components using an allosteric AKT inhibitor MK-2206 or the allosteric mTORC1 inhibitor RAD001 also increased radiation-induced apoptosis, albeit to a smaller degree than Taselisib (GDC0032) (Fig. 3D). Similar results were observed with the PIK3CA-mutated cell line HSC-2, although RAD001 did not enhance radiation-induced apoptosis in this cell line (Supplementary Fig. S4).

The gold standard for assessing radiosensitization is clonogenic survival. Using this assay, Cal-33 cells pretreated with Taselisib (GDC0032) had significantly decreased cell survival following radiation (Fig. 3E). Similar radiosensitization was seen when LB-771 cells were treated with GDC-0032 prior to radiation (Supplementary Fig. S5). However, GDC-0032 had no effect on the radiation response of HSC-3, a PIK3CA wild-type cell line resistant to single-agent GDC-0032 (Fig. 3D).

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Taselisib (GDC0032) delays the resolution of DNA double-strand breaks following radiation [2]
PI3K signaling is a key regulator of the DNA damage response (15, 17–21, 39). Therefore, we decided to study whether the GDC-0032–dependent radiosensitization was at least in part attributable to impaired DDR under a state of PI3Kα inhibition. We quantified the amount of DNA double-strand breaks (DSB), as assessed by γH2AX foci, with and without GDC-0032 pretreatment in Cal-33 cells. Cells pretreated with GDC-0032 had significantly more γH2AX foci at 24 and 48 hours after irradiation than control-treated cells (Fig. 4A). This increase in DNA damage upon combination of radiation and GDC-0032 was accompanied by increased formation of p53-binding protein 1 (53BP1) foci, a mediator of the DSB repair downstream of γH2AX (Fig. 4B; ref. 40), and induction of PARP cleavage (Supplementary Fig. S6A). Consistent results were also observed in LB-771 cells (Supplementary Fig. S6B), supporting the notion that GDC-0032 impairs DSB repair in these cells following radiation.

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Taselisib (GDC0032) enhances G2–M arrest following radiation [2]
Ionizing radiation induces two molecularly distinct G2–M checkpoints (41). The first, known as the “early” G2–M checkpoint, consists of a transient, ATM-dependent mitotic block affecting cells in late G2 occurring within minutes of irradiation and can be assessed by the proportion of cells with phosphorylation of histone H3 (HH3) after irradiation (41). The second, more prolonged and known as the “late” G2–M checkpoint, is independent of ATM and results in an accumulation of cells with 4N DNA content (41). It is well accepted that the duration of the G2–M checkpoint reflects the number of unrepaired DSBs (42). Therefore, based on our results we hypothesized that GDC-0032 may alter the DNA damage–induced cell-cycle arrest that occurs following irradiation.
As single agent, treatment with Taselisib (GDC0032) resulted in a mild increase in the proportion of cells in G1 over 72 hours, with a concomitant decrease in the G2 phase of the cell cycle (Fig. 5A).

As expected, radiation induced both the early and late G2–M checkpoints, with a marked decrease in pHH3-positive cells (Supplementary Fig. S7) and an accumulation of cells with 4N DNA content (Fig. 5B). However, by 48 hours after irradiation, the cell-cycle profile was nearly back to baseline. When we examined the impact of PI3Kα inhibition in this setting, we observed that, while treatment with Taselisib (GDC0032) for 24 hours prior to irradiation did not significantly alter the cell-cycle profile of Cal-33 cells (Fig. 5B) or affect the early G2–M arrest as assessed by pHH3-positive cells (Supplementary Fig. S7), the proportion of cells with 4N DNA content at 24 and 48 hours after irradiation was considerably higher with than in control-treated cells. This suggests that the late G2–M arrest induced by radiation is enhanced by pharmacologic inhibition of PI3Kα (Fig. 5B).

Because the late G2–M checkpoint is known to be ATM independent, we investigated whether the enhanced late G2–M checkpoint induced by Taselisib (GDC0032) could be abrogated by either inhibition of other known regulators of the G2–M cell-cycle progression, such as Wee1 and ATR. We found that although both Wee1 inhibition with AZD-1775 and ATR inhibition with VE-821 abrogated the early G2–M checkpoint following irradiation as assessed by pHH3 (Supplementary Figs. S8 and S9), only ATR inhibition with VE-821 reversed the late G2–M arrest induced by the combination of GDC-0032 and radiation (Fig. 5C and Supplementary Fig. S10). Taken together, these results indicate that GDC-0032 enhances the late G2–M checkpoint induced by irradiation in an ATR-dependent fashion, resulting in increased DNA damage overtime.

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Taselisib (GDC0032) activity in vitro [3]
Next, we evaluated the antitumor activity of taselisib in all nine primary cell lines established as long term in vitro cultures (i.e., 4 cell lines harboring HER2/neu gene amplification versus 5 HER2/neu negative cell lines). As shown in Figure 1, using scalar concentrations of taselisib, we found strong growth inhibition in all PIK3CA mutated cell line and/or HER2/neu amplified cell lines when compared to HER2/neu non-amplified cell lines (taselisib IC50 mean±SEM= 0.042 ± 0.006 µM in PIK3CA mutated cell line/FISH+ versus 0.38 ± 0.06 µM in PIK3CA wild type/FISH- tumors, P <0.0001). The USPC-ARK-1 cell line was the most sensitive to taselisib, with a mean inhibitory concentration (IC50) ± standard error (SEM) of 0.014 ± 0.002 µM. The USPC-ARK-4 cell line was found to be the least sensitive, with a mean IC50 of 0.66 ± 0.1 µM. Representative dose response curves are shown in Figure 2A. Dose curves response of the remaining seven cell lines are shown in Figure S1. We further compared the IC50 values of PIK3CA mutated/FISH+ cell lines and PIK3CA wild type/FISH+ cell lines. HER2/neu amplified USC harboring PIK3CA mutations (i.e., USPC-ARK-1, USPC-ARK-20) were significantly more sensitive to taselisib (i.e., mean inhibitory concentration (IC50) ± standard error (SEM) =0.029 ± 0.006 µM) when compared to the PIK3CA wild type HER2/neu amplified cell lines USPC-ARK-2 and USPC-ARK-21 (i.e., mean IC50 values of 0.058 ± 0.007 µM, P =0.01) (Figure 2B).

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Effect of Taselisib (GDC0032) on the cell cycle [3]
To evaluate the potential antiproliferative effects of taselisib in PIK3CA mutated/FISH+ cell line (USPC-ARK-1), the percentage of cell in the G0/G1 phase of the cell cycle was evaluated by flow-cytometry after the exposure to the drug. As shown in Figure 3, treatment with 50 nM, 100 nM and 500 nM of taselisib for 24 hrs was able to significantly increase the fraction of cells in the G0/G1 cell cycle phase in the USC cell lines when compared with the untreated controls (P = 0.0002, P=0.0005, P<0.00001, respectively).

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Taselisib (GDC0032) affects pS6 expression levels in USC cell lines [3]
We next evaluated pS6 expression levels in a representative HER2/neu amplified USC cell line harboring PIK3CA mutation (USPC-ARK-1) before and after exposure to taselisib. Flow-cytometry revealed that taselisib was able to significantly reduce S6 phosphorylation after 4 hrs of treatment at 25 nM (Figure 4). In the FISH+/PIK3CA mutated cell lines, MFI for pS6 before treatment ranged from 99.6 ± 4.1 (mean ± SEM) while after taselisib treatment ranged from 32.3 ± 4.6 (P=0.0004).

GDC-0032 pharmacokinetics is approximately dose proportional and time independent with a mean t1/2 of 40 hours. The addition of GDC-0032 increases the activity of fulvestrant, causing tumor regressions and a 91% delay in tumor growth. The effectiveness of tamoxifen in vivo is also increased when GDC-0032 and tamoxifen are combined (102%TGI for GDC-0032).[1]

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In Vivo Efficacy and Pharmacodynamics [1]
The in vivo efficacy of compound 11l/Taselisib (GDC0032) was compared to compound 1 in the MCF7-neo/Her2 xenograft model grown in nude mice. Daily, oral administration of compound 1 at 45 mg/kg resulted in 69% TGI when compared to vehicle-treated mice (purple curve, Figure 3). Daily administration of compound 11l orally at 1.4, 2.8, 5.8, 11.25, or 22.5 mg/kg resulted in dose-dependent increase in TGI (19%, 76%, 95%, 103%, and 123%, respectively) and tumor regressions when compared to vehicle-treated mice. The increased potency and unbound concentration of compound 11l resulted in comparable TGI (76%) to compound 1 (69%) at a dose that is approximately 16-fold lower (2.8 mg/kg versus 45 mg/kg). Treatment of both compounds 1 and 11l were well tolerated with less than 10% body weight loss observed compared to vehicle controls (data not shown). [1]

The relationship between pharmacokinetics (PK) and pharmacodynamics (PD) of compound 11l/Taselisib (GDC0032) relative to compound 1 was also investigated in the MCF7-neo/Her2 xenograft model (Figure 4). The phosphorylation levels of Akt in MCF7-neo/HER2 tumors, as well as plasma drug concentrations of both compounds, were evaluated 1 h after a single dose of vehicle (MCT), compound 1 at 45 mg/kg, or compound 11l at 1.4, 2.8, 5.8, 11.25, or 22.5 mg/kg. Compound 1 resulted in 58% decrease of phosphorylation of Akt (pAktSer473) levels while a 16-fold lower dose of compound 11l at 2.8 mg/kg resulted in a similar decrease in Akt phosphorylation (59%). The comparable decreases in pAkt between these doses of compounds 11l and 1 are likely due to similar unbound plasma drug concentrations that were achieved in mice after 1 h of dosing (Figure 4).

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Taselisib (GDC0032) enhances the antitumor effects of radiotherapy in vivo [2]
In order to test the ability of GDC-0032 to inhibit PI3K signaling in vivo, we treated nude mice implanted with subcutaneous Cal-33 xenografts with 5 mg/kg of GDC-0032 and harvested the tumors after 2, 6, and 24 hours of treatment. As expected, treatment with GDC-0032 resulted in nearly complete abrogation of AKT and PRAS40 phosphorylation, as well as decreased phosphorylation of 4EBP-1 and S6, at 2 hours after drug administration (Fig. 6A). However, by 6 hours after oral gavage, a rebound in phosphorylation of all of these PI3K targets was detected, probably a reflection of the short half-life of the compound.

We next assessed the efficacy of combined PI3K inhibition and radiation. Mice received daily Taselisib (GDC0032) throughout the experiment and 20 Gy in 5 daily fractions on days 2 to 6. Although treatment with either radiation or GDC-0032 alone resulted in tumor growth delay compared with vehicle-treated mice, only the combination of GDC-0032 and radiation resulted in durable tumor regressions (Fig. 6B). In fact, at the experiment endpoint (90 days of treatment), none of the tumors in the combined radiation and GDC-0032 arm had progressed in comparison to the start of the treatment. We also saw superior activity with transient administration of GDC-0032 during radiation in a HPV+ PIK3CAE545K patient-derived xenograft model (Supplementary Fig. S11). These data also suggest that both mutations (E542K in the helical domain and H1047R in the kinase domain) are functionally active in HNSCC.
Moreover, our in vivo findings suggest that even a transient inhibition of the PI3K/AKT/mTOR pathway (Fig. 6A) is sufficient to sensitize tumors to radiation, an observation with potential clinical implications.

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Taselisib activity in vivo [3]
We further evaluated the effect of Taselisib (GDC0032) in vivo using a xenograft USC model. Ten mice were randomized in two groups, control and taselisib. No signs of general toxicity were seen in any of the 2 treatment groups harboring the USC-xenograft and no animal died during the experiments or had to be prematurely sacrificed due to signs of systemic drug toxicity. One mouse in the control group had to be sacrificed after 7 days because it reached 1 cm3 in tumor volume while the remaining control animals had to be sacrificed within 2 weeks secondary to their tumor volume. Taselisib group showed a significant tumor growth inhibition after 14 days of treatment (P=0.007; Figure 5 panel A) and significantly improved OS when compared to the control group (P<0.0001; Figure 5 panel B). The mean survival of the taselisib-treated mice was 45 days.

Enzymatic activity of the class I PI3K isoforms is measured using a fluorescence polarization assay that monitors formation of the product 3,4,5-inositoltriphosphate molecule as it competes with fluorescently labeled PIP3 for binding to the GRP-1 pleckstrin homology domain protein. As the labeled fluorophore is displaced from the GRP-1 protein binding site as the amount of phosphatidyl inositide-3-phosphate product increases, the fluorescence polarization signal decreases. As heterodimeric recombinant proteins, class I PI3K isoforms are expressed and purified. PIP3 detection reagents, di-C8-PIP2, and tetramethylrhodamine-labeled PIP3 (TAMRA-PIP3) are available from Echelon Biosciences. In the presence of 10 mM Tris (pH 7.5), 25 μM ATP, 9.75 mM PIP2, 5% glycerol, 4 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 2% (v/v) DMSO at 60 ng/mL, PI3K is measured under initial rate conditions. Before reading fluorescence polarization on an Envision plate reader, reactions are stopped with a final concentration of 9 mM EDTA, 4.5 nM TAMRA-PIP3, and 4.2 g/mL GRP-1 detector protein after the assay has been running for 30 min at 25 °C. The fit of the dose-response curves to a 4-parameter equation yields the IC50 values. Each value is the average of three experiments, and all of them have standard deviations that are below the geometric mean.

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Characterization of Biochemical and Cellular Activity in Vitro [1]
Enzymatic activity of the class I PI3K isoforms was measured using a fluorescence polarization assay that monitors formation of the product 3,4,5-inositoltriphosphate molecule as it competes with fluorescently labeled PIP3 for binding to the GRP-1 pleckstrin homology domain protein. An increase in phosphatidyl inositide-3-phosphate product results in a decrease in fluorescence polarization signal as the labeled fluorophore is displaced from the GRP-1 protein binding site. Class I PI3K isoforms were purchased from Perkin–Elmer or were expressed and purified as heterodimeric recombinant proteins. Tetramethylrhodamine-labeled PIP3 (TAMRA-PIP3), di-C8-PIP2, and PIP3 detection reagents were purchased from Echelon Biosciences. PI3Kα was assayed under initial rate conditions in the presence of 10 mM Tris (pH 7.5), 25 μM ATP, 9.75 μM PIP2, 5% glycerol, 4 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 2% (v/v) DMSO at 60 ng/mL. After assay for 30 min at 25 °C, reactions were terminated with a final concentration of 9 mM EDTA, 4.5 nM TAMRA-PIP3, and 4.2 μg/mL GRP-1 detector protein before reading fluorescence polarization on an Envision plate reader. IC50 values were calculated from the fit of the dose–response curves to a 4-parameter equation. Each reported value is an average of three experiments, and all had a standard deviation less than one geometric mean.

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Analysis of Total and Phosphorylated Akt [1]
Tris lysis buffer containing 150 mM NaCl, 20 mM Tris pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, phosphatase inhibitor 1, phosphatase inhibitor II, protease inhibitor, 1 mM NaF, and 1 mM PMSF was added to frozen tumor biopsies. Tumors were dissociated with a small pestle, sonicated briefly on ice, and centrifuged at maximum rpm for 20 min at 4 °C. A 5–20 μg amount of protein from tumor lysates was used to determine phosphorylation status. Samples were assayed in duplicates per manufacturer’s protocol for pAkt/tAkt.

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Liver Microsomal Incubation [1]
LMs (0.5 mL at 20 mg/mL) were premixed with alamethicin dissolved in methanol (100 μg/mg of LMs), G6P dehydrogenase (2.88 units/mg of LMs), and KPi buffer to a final volume of 5 mL. The cofactor mixture was prepared by adding NADP (3.33 mg/mL, 4 mM), UDPGA (12.92 mg/mL, 20 mM), G6P (6.8 mg/mL), and GSH (6.15 mg/mL, 20 mM) to 1 mL of 100 mM KPi incubation buffer. Equal volumes of cofactor solution and LMs solution (2 mg/mL) were added and preincubated for 5 min at 37 °C. The reaction was initiated by adding an equal volume of 3 (5 μM final concentration) in buffer for a final concentration of 0.5 mg/mL of LMs. Final concentrations were 5 mM for GSH and 1 mM for NADP. Following 1 h of incubation at 37 °C, the reaction was quenched by the addition of acetonitrile, followed by centrifugation at 3400g for 10 min. The supernatant was transferred and evaporated. The samples were reconstituted in 10% acetonitrile in water.

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In Vitro Hepatocyte Metabolite Identification Assays [1]
Cryopreserved hepatocytes from male Sprague–Dawley rat (RH; lot MTN) and male beagle dog (DH; lot OES) were used. Cryopreserved human hepatocytes were used. Mobile phase solvents 0.1% formic acid in water and 0.1% formic acid in acetonitrile were used. he tubes were centrifuged for 5 min at 80g, and the supernatants were discarded. Cells were resuspended with 50 mL of prewarmed DMEM medium by gently inverting the tube several times. The tubes were centrifuged for 5 min at 80g, and the supernatants were discarded. Cells were brought up in 2.5 mL of prewarmed DMEM incubation medium. The total cell count and the number of viable cells were determined by the trypan blue exclusion method. Incubations were carried out in scintillation vials containing 0.5 mL of hepatocyte suspension (2.2–2.6 × 106 cells/mL) and 0.5 mL of DMEM media containing test article (20 μM). The scintillation vials were placed on an orbital shaker rotating at 16 rpm in a humidified incubator at 37 °C, 5% CO2. The reaction was quenched with 4 mL of acetonitrile at 0 and 3 h. Diclofenac (10 μM) was the positive control and was incubated with cryopreserved hepatocytes under the same conditions used for Taselisib (GDC0032). The disappearance of diclofenac over 3 h was monitored, and the half-life of the compound was within expected values for each species.

Taselisib (GDC0032) is administered after cells are seeded in 96-well plates in replicates of six with 500–5,000 cells per well overnight. 4% glutaraldehyde is used to fix the cells for 30 minutes after the media have been removed after 4 days. After washing and dissolving in 10% acetic acid, fixed cells are stained with 0.1% crystal violet for 2 minutes.

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Proliferation Aassay with MCF7-neo/HER2 Cells [1]
Cell lines were obtained from the American Type Culture Collection. MCF7-neo/HER2 ectopically expresses HER2 in the MCF7 parental cell line. Cell lines were cultured in RPMI supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, 10 mM HEPES, and 2 mM glutamine at 37 °C under 5% CO2. MCF7-neo/HER2 cells or PC3 cells were seeded in 384-well plates in media at 1000 cells/well or 3000 cells/well, respectively, and incubated overnight prior to the addition of compounds to a final DMSO concentration of 0.5% v/v. MCF7-neo/HER2 cells were incubated for 3 and 4 days, respectively, prior to the addition of CellTiter-Glo reagent and reading of luminescence using an Analyst plate reader. For antiproliferative assays, a cytostatic agent such as aphidicolin and a cytotoxic agent such as staurosporine were included as controls. Dose–response curves were fit to a 4-parameter equation, and relative IC50 values were calculated using Assay Explorer software.

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Proliferation assays [2]
Cells were seeded in replicates of 6 in 96-well plates with 500 to 5,000 cells/well overnight and then treated with Taselisib (GDC0032). After 4 days, the media were removed and the cells were fixed with 4% glutaraldehyde for 30 minutes. Fixed cells were stained with 0.1% crystal violet for 2 minutes, then washed, and dissolved in 10% acetic acid. Absorption of light was quantified using a Biotek Synergy H1 plate reader.

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Annexin V staining [2]
Cells were treated with either Taselisib (GDC0032) or DMSO for 24 hours, irradiated or mock-irradiated, then trypsinized and harvested along with cells in the media after an additional 72 hours. Cells were resuspended in Annexin V buffer, stained with Annexin V–FITC and propidium iodide (PI) according to the manufacturer's instructions, and analyzed for fluorescence with a Fortessa flow cytometer. Resulting data were analyzed using FlowJo software.

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Drug-response assay [3]
The effect of Taselisib (GDC0032) on the viability and IC50 of cells was determined by flow-cytometry assays. Tumor cells derived from 9 primary USC cell lines, established as long term cultures in vitro, were plated in six-well tissue culture plates and treated with taselisib at concentrations of 0.05, 0.1, 0.5, 1.0, 2.0 µM at least 24 hrs after plating. After 72 hours of additional incubation, well contents were harvested in their entirety, centrifuged and then stained with propidium iodide (2 µL of a 500 µg/mL stock solution in PBS) for flow cytometric counts. Viable cells were then quantified using flow-cytometry as percentage of viable cells (mean ± SEM) after exposure to different concentrations of taselisib relative to vehicle-treated cells (i.e., 100% viable). A minimum of 3 independent experiments per USC cell line were performed.

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Flow-cytometry analysis of cell cycle in primary USC cell lines [3]
USC cells were seeded in six-well tissue culture plates and 24 hrs later were treated with Taselisib (GDC0032). After 24 hrs exposure to 50 nM, 100 nM and 500 nM of drug, treated cells and control cells were permeabilized with ice-cold 70% ethanol and fixed for 30 min at 4° C. After spinning at 2000 rpm for 5 min and discarding supernatant, cells were resuspended in 1 ml of PBS. After additional spinning at 2000 rpm for 5 min, 100 µl ribonuclease (100 µg/ml) was added for 5 min incubation at room temperature, before exposure to 400 µl of propidium iodide (50 µg/ml in PBS). Taselisib treated and untreated control cells were acquired with FACSCalibur, using Cell Quest software.

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Flow-cytometry analysis of phosphorylated S6 intracellular levels in primary USC cell lines [3]
Next we evaluated pS6 expression levels in representative HER2/neu amplified USC cell lines harboring PIK3CA mutation (i.e., USPC-ARK-1) before and after exposure to Taselisib (GDC0032) by flowcytometry. After 4 hrs exposure to 25 nM of taselisib, cells were fixed in 4% formaldehyde and permeabilized with ice-cold 90% methanol. Taselisib treated and untreated control cells were incubated with primary rabbit monoclonal antibody against pS6 following the protocol provided by the manufacturers and stained with a fluorescein isothiocyanate-conjugated goat anti-rabbit F(ab’)2 immunoglobulin as a secondary reagent. Cells (i.e., 10,000 events per sample) were analyzed on FACSCalibur, using Cell Quest software.

Six-week-old Nu/Nu mice receive bilateral injections of 5×105 cells resuspended in 200 μL of culture media and Matrigel combined in a 1:1 ratio. Mice are randomly assigned to treatment arms with 8–10 tumors each after the tumors have grown to a size of about 100–200 cm3. Daily oral gavage administration of Taselisib (GDC0032)(5 mg/kg) dissolved in a vehicle containing 0.5% methylcellulose and 0.2% TWEEN-80. [2]

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For in vivo experiments, Taselisib (GDC0032) was dissolved in sterile water, 0.5% methyl-cellulose, and 0.2% Tween-80. [2]
In vivo xenograft studies [2]
Six-week-old Nu/Nu mice were order from Harlan Laboratories. For cell line-derived xenograft studies, mice were injected bilaterally with 5 × 105 cells resuspended in 200 μL of culture media and Matrigel mixed in a 1:1 ratio. After tumors reached approximately 100 to 200 cm3, mice were randomized into treatment arms with 8 to 10 tumors per group. Taselisib (GDC0032) (5 mg/kg) was dissolved in a vehicle containing 0.5% methylcellulose with 0.2% TWEEN-80 and was administered via daily oral gavage. Tumors were irradiated using an X-RAD 320 X-ray system with appropriately sized lead shields.
For patient tumor–derived xenograft studies, mice were implanted with a tumor obtained from a patient with oropharynx squamous cell carcinoma. The tumor DNA was sequenced using CancerSelect-203 R platform and found to have a PIK3CAE542K mutation. After tumors reached approximately 100 to 250 cm3, mice were randomized into treatment arms with 5 to 6 tumors per group. Taselisib (GDC0032) (5 mg/kg) was administered via daily oral gavage for 14 days. Tumors were irradiated 2 Gy daily on days 2 to 4 using a Small Animal Radiation Research Platform at the Johns Hopkins University. Tumor volumes were calculated as (π/6) × length × width2.

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In vivo assay of drug effect [3]
To determine the in vivo activity of Taselisib (GDC0032), a representative PIK3CA-mutated/FISH+ cell line (USPC-ARK-1) was injected into the subcutaneous region of ten 5–8 week old SCID mice. After implantation of cells, tumors were monitored until they reached a tumor volume of 0.1 cm3 prior to initiating the treatment. Mice were then randomized into 2 treatment groups namely, control and Taselisib (GDC0032), keeping average tumor volume similar between groups. Each group consisted of 5 mice. The control group was treated with vehicle (0.5% methylcellulose-0.2% Tween 80) while the Taselisib (GDC0032) experimental group was treated with 11.25 mg/kg of taselisib. Treatments were given orally once a day, 5 days a week. Tumor volume was calculated by the formula V= length × (width)2 × 0.5. Tumor sizes and body weights were recorded three times per week. The mice in both treatment groups were treated for 1 month with taselisib or placebo after which they were observed for overall survival (OS). When tumor reached 1 cm3 or became necrotic the animals were removed from the study and euthanized according to the rules and regulations set forth by the Institutional Animal Care and Use Committee (IACUC).

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In Vivo Xenograft Studies [1]
In vivo efficacy was evaluated in the MCF7-neo/Her2 xenograft model. Five million cells per mouse were resuspended in a 1:1 mixture of Hank’s buffered salt solution and matrigel basement membrane matrix and implanted into number 2/3 mammary fat pad of female athymic (nu/nu) nude mice. Prior to cell inoculation, 17-β-estradiol (0.36 mg/pellet, 60 day release) was implanted into the dorsal shoulder blade area of each nude mouse. After implantation of cells, tumors were monitored until they reached a mean tumor volume of 250–350 mm3 prior to initiating dosing. Test articles such as Taselisib (GDC0032) were dissolved in 0.5% methylcellulose with 0.2% Tween-80 (MCT) and administered daily via oral (PO) gavage.
Tumor volumes were determined using digital calipers using the formula (L × W × W)/2. TGI was calculated as the percentage of the area under the fitted curve (AUC) for the respective dose group per day in relation to the vehicle, such that %TGI = 100(1 – (AUCtreatment/day)/(AUCvehicle/day)). Curve fitting was applied to Log2 transformed individual tumor volume data using a linear mixed-effects (LME) model. Tumor sizes and body weights were recorded twice weekly over the course of the study. Mice with tumor volumes greater than or equal to 2000 mm3 or with losses in body weight greater than or equal to 20% from their weight at the start of treatment were euthanized per IACUC guidelines. For pharmacodynamic marker analysis, MCF7-neo/HER2 xenograft tumors were excised from animals and immediately snap frozen in dry ice.

Pharmacokinetic Properties [1] From the data gathered thus far, 11c, 11k, and 11l/Taselisib (GDC0032) were selected for progression into in vivo pharmacokinetic (PK) profiling. A single dose of each compound was administered to adult female nude mice either intravenously at 1 mg/kg or orally at 25 mg/kg. The data for these three compounds are summarized in Table 3. When compared to the original thiazolobenzoxepin inhibitor 2, the imidazobenzoxazepin inhibitors (11c, 11k, and 11l) all showed improved antiproliferative cellular activity in the MCF7-neo/HER2 line (138, 81, and 25 nM, respectively). Additionally, in line with their low in vitro clearance, the mouse plasma clearance (CLp) values were also low for these optimized compounds. More importantly, in each case, both the unbound clearance (CLu) and correspondingly the unbound oral exposure (AUCu) were significantly (>10×) improved relative to thiazolobenzoxepin 2. Compound 11l/Taselisib (GDC0032) provided the highest unbound exposure of the compounds tested (AUCu = 42 μM·h). On the basis of these results, we selected 11l to evaluate in an in vivo tumor xenograft study in a head-to-head comparison with compound 1.

[1].Discovery of 2-{3-[2-(1-isopropyl-3-methyl-1H-1,2-4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl]-1H-pyrazol-1-yl}-2-methylpropanamide (GDC-0032): a β-sparing phosphoinositide 3-kinase inhibitor with high unbound exposure and robust in vivo antitumor activity. J Med Chem. 2013 Jun 13;56(11):4597-610.

[2]. Taselisib (GDC-0032), a Potent β-Sparing Small Molecule Inhibitor of PI3K, Radiosensitizes Head and Neck Squamous Carcinomas Containing Activating PIK3CA Alterations. Clin Cancer Res. 2016 Apr 15; 22(8): 2009–2019.

[3]. Taselisib, a selective inhibitor of PIK3CA, is highly effective on PIK3CA-mutated and HER2/neu amplified uterine serous carcinoma in vitro and in vivo. Gynecol Oncol. 2014 Aug 27;135(2):312–317.

Taselisib (GDC0032) has been used in trials studying the treatment and basic science of LYMPHOMA, Breast Cancer, Ovarian Cancer, Solid Neoplasm, and HER2/Neu Negative, among others.
Taselisib is an orally bioavailable inhibitor of the class I phosphatidylinositol 3-kinase (PI3K) alpha isoform (PIK3CA), with potential antineoplastic activity. Taselisib selectively inhibits PIK3CA and its mutant forms in the PI3K/Akt/mTOR pathway, which may result in tumor cell apoptosis and growth inhibition in PIK3CA-expressing tumor cells. By specifically targeting class I PI3K alpha, this agent may be more efficacious and less toxic than pan PI3K inhibitors. Dysregulation of the PI3K/Akt/mTOR pathway is frequently found in solid tumors and causes increased tumor cell growth, survival, and resistance to both chemotherapy and radiotherapy. PIK3CA, which encodes the p110-alpha catalytic subunit of the class I PI3K, is mutated in a variety of cancer cell types and plays a key role in cancer cell growth and invasion.

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Dysfunctional signaling through the phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway leads to uncontrolled tumor proliferation. In the course of the discovery of novel benzoxepin PI3K inhibitors, we observed a strong dependency of in vivo antitumor activity on the free-drug exposure. By lowering the intrinsic clearance, we derived a set of imidazobenzoxazepin compounds that showed improved unbound drug exposure and effectively suppressed growth of tumors in a mouse xenograft model at low drug dose levels. One of these compounds, Taselisib (GDC0032) (11l), was progressed to clinical trials and is currently under phase I evaluation as a potential treatment for human malignancies. [1]

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In summary, we discovered a novel class of compounds for the inhibition of PI3K-driven tumors. The key feature for optimization was improving unbound exposure (AUCu) by reducing cLogD as a design parameter. This improvement had profound effects on tumor growth inhibition in vivo. Relative to a closely related benzoxepin scaffold evaluated, imidazobenzoxazepin had better tumor growth inhibition at even lower drug doses. In line with the free drug hypothesis, reducing the intrinsic plasma clearance (in this case, a result of removing metabolic soft spots) and increasing free exposure resulted in more efficacious drugs. This approach was essential because we had access to a substantial set of data for comparing direct analogues in several closely related scaffolds. As a result, we quickly settled on a suitable scaffold to optimize in order to achieve the desired profile. Our optimization efforts led to the identification of 11l (or Taselisib (GDC0032)), currently undergoing clinical development for use in PI3K-related cancers. [1]

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Purpose: ActivatingPIK3CAgenomic alterations are frequent in head and neck squamous cell carcinoma (HNSCC), and there is an association between phosphoinositide 3-kinase (PI3K) signaling and radioresistance. Hence, we investigated the therapeutic efficacy of inhibiting PI3K with Taselisib (GDC0032), a PI3K inhibitor with potent activity against p110α, in combination with radiation in HNSCC.

Experimental design: The efficacy of Taselisib (GDC0032) was assessedin vitroin 26 HNSCC cell lines with crystal violet proliferation assays, and changes in PI3K signaling were measured by Western blot analysis. Cytotoxicity and radiosensitization were assessed with Annexin V staining via flow cytometry and clonogenic survival assays, respectively. DNA damage repair was assessed with immunofluorescence for γH2AX foci, and cell cycle analysis was performed with flow cytometry.In vivoefficacy of GDC-0032 and radiation was assessed in xenografts implanted into nude mice.

Results: Taselisib (GDC0032) inhibited potently PI3K signaling and displayed greater antiproliferative activity in HNSCC cell lines withPIK3CAmutations or amplification, whereas cell lines withPTENalterations were relatively resistant to its effects. Pretreatment with GDC-0032 radiosensitizedPIK3CA-mutant HNSCC cells, enhanced radiation-induced apoptosis, impaired DNA damage repair, and prolonged G2-M arrest following irradiation. Furthermore, combined GDC-0032 and radiation was more effective than either treatment alonein vivoin subcutaneous xenograft models.

Conclusions: Taselisib (GDC0032) has increased potency in HNSCC cell lines harboringPIK3CA-activating aberrations. Further, combined GDC-0032 and radiotherapy was more efficacious than either treatment alone inPIK3CA-altered HNSCCin vitroandin vivo This strategy warrants further clinical investigation. [2]

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Objective
To evaluate the efficacy of Taselisib (GDC0032), a selective inhibitor of PIK3CA, against primary uterine serous carcinomas (USC) harboring PIK3CA mutations and HER2/neu gene amplification.

Methods
Sensitivity to Taselisib (GDC0032) was evaluated by flow-cytometry viability assays in vitro against nine primary USC cell lines. Cell cycle distribution and downstream signaling were assessed by measuring the DNA content of cells and by phosphorylation of the S6 protein by flow-cytometry. Preclinical efficacy of taselisib was also evaluated in vivo in a mouse model.

Results
Four USC cell lines harbored HER2/neu gene amplification by FISH and two of them harbored oncogenic PIK3CA mutations. Taselisib (GDC0032) caused a strong differential growth inhibition in both HER2/neu FISH positive and HER2/neu FISH positive/PIK3CA mutated USC cell lines when compared to lines that were FISH negative and PIK3CA wild type (taselisib IC50 mean±SEM= 0.042 ± 0.006 µM in FISH+ versus 0.38 ± 0.06 µM in FISH- tumors, P <0.0001). Taselisib growth-inhibition was associated with a significant and dose-dependent increase in the percentage of cells in the G0/G1 phase of the cell cycle and dose-dependent decline in the phosphorylation of S6. Taselisib was highly active at reducing tumor growth in vivo in USC mouse xenografts harboring PIK3CA mutation and overexpressing HER2/neu (P=0.007). Mice treated with taselisib had significantly longer survival when compared to control mice (P<0.0001).
Conclusions
Taselisib (GDC0032) represents a novel therapeutic option in patients harboring PIK3CA mutations and/or HER2/neu gene amplification. [3]

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In conclusion, our study shows the first preclinical demonstration that Taselisib (GDC0032), a new PIK3CA inhibitor, may represent a novel, potentially effective therapeutic strategy against PIK3CA mutated and HER2/neu gene amplified USC. Clinical trials of taselisib in USC are warranted.[3]

C24H28N8O2

460.5315

460.233

C, 62.59; H, 6.13; N, 24.33; O, 6.95

1282512-48-4

1282512-48-4

51001932

White to off-white solid powder

1.4±0.1 g/cm3

783.3±70.0 °C at 760 mmHg

427.5±35.7 °C

0.0±2.7 mmHg at 25°C

1.709

0.69

1

6

5

34

751

0

O1C([H])([H])C([H])([H])N2C([H])=C(C3=NC(C([H])([H])[H])=NN3C([H])(C([H])([H])[H])C([H])([H])[H])N=C2C2C([H])=C([H])C(=C([H])C1=2)C1C([H])=NN(C=1[H])C(C(N([H])[H])=O)(C([H])([H])[H])C([H])([H])[H]

BEUQXVWXFDOSAQ-UHFFFAOYSA-N

InChI=1S/C24H28N8O2/c1-14(2)32-22(27-15(3)29-32)19-13-30-8-9-34-20-10-16(6-7-18(20)21(30)28-19)17-11-26-31(12-17)24(4,5)23(25)33/h6-7,10-14H,8-9H2,1-5H3,(H2,25,33)

2-methyl-2-[4-[2-(5-methyl-2-propan-2-yl-1,2,4-triazol-3-yl)-5,6-dihydroimidazo[1,2-d][1,4]benzoxazepin-9-yl]pyrazol-1-yl]propanamide

RG7604; RG7604; Taselisib; 1282512-48-4; 2-(4-(2-(1-isopropyl-3-methyl-1H-1,2,4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)-1H-pyrazol-1-yl)-2-methylpropanamide; Taselisib [INN]; GDC 0032; Taselisib [USAN:INN]; RG 7604; GDC0032; GDC0032; GDC 0032

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: 25~70 mg/mL (54.3~152 mM)
Ethanol: ~7 mg/mL (~15.2 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.43 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 (5.43 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 25.0 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 3: ≥ 2.5 mg/mL (5.43 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.)