p110α (IC50 = 5 nM); p110γ (IC50 = 250 nM); p110δ (IC50 = 290 nM); p110β (IC50 = 1200 nM); p110α-H1047R (IC50 = 4 nM); p110α-E545K (IC50 = 4 nM)

We investigated the therapeutic value of BYL719, a new specific PI3Kα inhibitor that blocks the ATP site, on osteosarcoma and bone cells. The in vitro effects of BYL719 on proliferation, apoptosis, and cell cycle were assessed in human and murine osteosarcoma cell. Its impact on bone cells was determined using human mesenchymal stem cells (hMSC) and human CD14+ osteoclast precursors. Two different murine preclinical models of osteosarcoma were used to analyze the in vivo biological activities of BYL719. BYL719 decreased cell proliferation by blocking cell cycle in G0/G1 phase with no outstanding effects on apoptosis cell death in HOS and MOS-J tumor cells. BYL719 inhibited cell migration and can thus be considered as a cytostatic drug for osteosarcoma.[4]

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BYL719/Alpelisib inhibits the proliferation of breast cancer cell lines harboring PIK3CA mutations, correlating with inhibition of various downstream signaling components of the PI3K/Akt pathway.

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NVP-BYL719 potently and selectively inhibits PI3Kα in vitro [3]
PI3Kα, β, δ, and γ enzymes share significant amino acid residue homology with particularly high conservation in the catalytic kinase domain. The 2-aminothiazole scaffold was selected as a starting point for the development of potent and selective PI3K inhibitors based on its binding mode, indicating the potential to use substituents at the amino group to develop interactions with nonconserved amino acids at the ATP pocket entrance (21). Consequently, systematic modification of key moieties and optimization of the drug-like properties led to the identification of NVP-BYL719.

As previously described in ref. 18, in biochemical assays NVP-BYL719/Alpelisib inhibits wild-type PI3Kα (IC50 = 4.6 nmol/L) more potently than the PI3Kδ (IC50 = 290 nmol/L) and PI3Kγ (IC50 = 250 nmol/L) isoforms and shows significantly reduced activity against PI3Kβ (IC50 = 1,156 nmol/L). Here, in addition, we show that NVP-BYL719 potently inhibits the 2 most common PIK3CA somatic mutations (H1047R, E545K; IC50∼4 nmol/L). The compound also lacked activity against the class III family member Vps34 and the related class IV PIKK protein kinases mTOR, DNA-PK, and ATR and was significantly less potent against the distinct lipid kinase PIK4β (Table 1). The kinase selectivity profile of NVP-BYL719 was further examined in in vitro kinase assay panels. Among all the kinases tested (excluding class I PI3K and PI4Kβ) their respective IC50 or Kd values were at least 50-fold higher when compared with PI3Kα (Supplementary Fig. 1, Supplementary Tables 1–4).

To determine the potency and selectivity of NVP-BYL719 in cellular assay systems, Rat1 cells transformed using the activated forms of PI3Kα, PI3Kβ, or PI3Kδ were tested and RPPAs were used to quantify the phosphorylation of Akt (S473) as a marker of PI3K pathway activity (17). As described in ref. 18, NVP-BYL719 potently inhibited Akt phosphorylation in cells transformed with PI3Kα (IC50 = 74 ± 15 nmol/L) and showed significant reduced inhibitory activity in PI3Kβ or PI3Kδ isoforms transformed cells (≥15-fold compared with PI3Kα). Here, we report NVP-BYL719 full dose–response curves as well as its IC80 values on S473P-Akt in Rat1 cells (Supplementary Fig. S2). In addition, treatment of TSC1-null MEF cells with NVP-BYL719 was not associated with a reduction in phosphorylation of RPS6 (S235/236) when compared with the positive control RAD001 (IC50 value < 0.5 nmol/L), suggesting that NVP-BYL719 does not inhibit mTORC1 (Supplementary Fig. S3A and S3B). Similarly, NVP-BYL719 does not seem to interfere with the PIKKs involved in DNA-damage repair (ATM and ATR) processes as determined in ATM- and ATR-dependent assay systems (Supplementary Fig. S3C and S3D). Together, these data strongly support the notion that NVP-BYL719 has the relevant in vitro properties of a selective PI3Kα inhibitor.

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PIK3CA mutant cell lines are selectively sensitive to NVP-BYL719/Alpelisib [3]
The above-mentioned approach was useful in defining what tumors were responsive to PI3Kα inhibition. An independent question is asked when one considers, which therapeutic modality is most selective and hence likely to have the best therapeutic index in a specific cancer genotype. Here, using a novel analytical approach to define the selectivity index of small molecule inhibitors across the CCLE, we compared the selectivity profiles across different compound treatments (∼1,000) encompassing more than 200 mechanisms of actions in PIK3CA mutant versus wild-type cell lines and ranked the compounds based on the magnitude of their effects in these 2 groups (Fig. 6). NVP-BYL719, together with 3 close analogs, showed markedly selective efficacy in PIK3CA mutants when compared with wild-type cell line populations and when compared with pan-PI3K inhibitors. Conversely, MEK inhibitors were differentially more selectively effective in PIK3CA wild-type cell lines compared with mutants.

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BYL719/Alpelisib inhibits osteosarcoma cell proliferation [4]
As expected, Figure 1b shows that BYL719 (chemical structure in Fig. 1a) rapidly inhibited the levels of P-AKT and P-mTOR in all cell lines assessed, confirming the functional activity of BYL719 on osteosarcoma cells (Fig. 1b). XTT assays were then performed to analyze the effects of BYL719 on osteosarcoma cell growth (human: MG63, HOS MNNG; mouse: POS-1, MOS-J and rat: OSRGA) (Fig. 1c). After 72 hr of treatment, BYL719 significantly inhibited the cell growth of all osteosarcoma cell lines tested in a dose-dependent manner (Figs. 1c and 1d) with an IC50 ranging from 6 to 15 µM and with the IC90 from 24 to 42 µM (Fig. 1e) at 72 hr. To determine whether or not the decreased cell viability induced by BYL719 was associated with a cell cycle alteration, flow cytometry of cell DNA content was performed after addition of BYL719 (Supporting Information data 3). The results showed that BYL719 significantly altered the distribution of cell cycle phases and, more specifically, increased cell numbers in the G0/G1 phase from 57 to 70% in MG-63, 44 to 73% in HOS (Supporting Information Data 3A), 45–70% in MOS-J and from 58 to 70% in POS-1 cells (Supporting Information Data 3B). These observations were concomitant with a decrease in cells in the S-G2/M phases. Similar results were obtained on a rat osteosarcoma cell line, OSRGA (Supporting Information Data 4).

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BYL719/Alpelisib acts as a cytostatic drug for osteosarcoma cells [4]
To determine whether these effects were due to inhibition of cell proliferation and/or induction of cell death, the effects of BYL719 were assessed by manual counting of viable cells after trypan blue exclusion staining. BYL719 significantly decreases the number of alive HOS and MOS-J cells in a dose- and a time-dependent manner without affecting the number of dead cells, supporting the idea that BYL719 exerts a cytostatic activity in osteosarcoma cells (Fig. 1d and Supporting Information Data 5A, respectively). In addition, BYL719 failed to induce apoptosis as shown by caspase-3/7 activity assessed in HOS and MOS-J cells (Fig. 1e, left panel and Supporting Information 5B, respectively) and confirming by the absence of cleaved-PARP expression after BYL719 treatment in HOS osteosarcoma cells (Fig. 1e, right panel). Similar data were obtained with MG-63, POS-1 (data not shown) and OSRGA cell lines (Supporting Information Data 4D). Migration assays were also performed on HOS and MOS-J cells to determine the effect of BYL719 on cell motility and demonstrated that BYL719 decreased cell motility (Supporting Information Data 6A and B). We then performed a recovery assay. Surprisingly, the treated cells are able to recovery in the same manner than in the control condition, suggesting that BYL719 as a cytostatic effect only when the drug is present (Fig. 4c). All these data suggest that BYL719 has cytostatic activity in osteosarcoma cells.

In PIK3CA mutant xenograft models in rodents, BYL719 (>270 mg/d) demonstrates statistically significant dose-dependent anti-tumor efficacy. BYL719 has a low half-life of 8.5 hours, a low inter-individual variability in Cmax, and an exposure that increases dose proportionally between 30 mg/d and 450 mg/d in humans. First indications of clinical efficacy for BYL719 (270 mg/d) include 1 confirmed partial response in a patient with ER+ breast cancer and significant PET responses (PMR) and/or tumor shrinkage in 8 of the 17 patients evaluated.

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NVP-BYL719/Alpelisib shows robust PK/PD/Efficacy relationship in PI3Kα-driven tumors [3]
To examine NVP-BYL719 ability to inhibit the PI3K/Akt pathway in a PI3Kα-dependent in vivo model, its pharmacokinetic/pharmacodynamic (PK/PD) relationship was assessed in a Rat1-myr-p110α mechanistic tumor-bearing mouse model. Each female athymic mouse received single or repeated doses of NVP-BYL719 (12.5, 25, or 50 mg/kg, p.o.) and plasma and tumors samples were collected for PK and PD analysis at different time points. Here NVP-BYL719 treatment was associated with dose and time-dependent inhibition of the PI3K/Akt pathway, which notably paralleled time-dependent drug exposure in tumor and plasma (Fig. 1A).

To determine whether dose- and time-dependent pathway inhibition was linked to antitumor activity, Rat1-myr-p110α tumor-bearing nude mice were treated orally every day with the compound for up to 8 consecutive days (Fig. 1B). Treatments of 12.5, 25, and 50 mg/kg were well tolerated and resulted in a dose-dependent and statistically significant antitumor effect with a T/C of 14.1% and regressions of 9.6% and 65.2%, respectively. To assess the relative PI3K selectivity in vivo, we further tested NVP-BYL719 in a corresponding Rat1-myr-p110δ model. NVP-BYL719, when tested at the optimal dose of 50 mg/kg p.o., every day, showed only a modest antitumor effect (T/C of 30%; Fig. 1C).

We next sought to better understand the degree of PI3Kα inhibition that is required for antitumor efficacy. To this end, we first determined the tumor concentrations giving 50% (in vivo IC50) and 80% (in vivo IC80) S473P-Akt inhibition (0.4 and 4 μmol/L, respectively) by measuring the extent of Akt phosphorylation using RPPA and the specific tumor drug concentration in matched samples from multiple animals and at multiple time points (Fig. 1D). Interestingly, when corrected for plasma protein binding of NVP-BYL719 in mouse (PPB = 91.2%), the in vivo IC50 (35 nmol/L) and IC80 (352 nmol/L) values roughly approximate the in vitro cellular IC50 and IC80 of 74 and 301 nmol/L, respectively. We next sought to determine the relationship between exposure, as measured by time over the in vivo IC80, and antitumor efficacy. Here, we found a nearly linear relationship between the antitumor efficacy magnitude and duration of drug exposure over the IC80 (R2 = 0.80, P < 0.001, n = 11; Fig. 1E). From this relationship it seems that 80% inhibition of Akt phosphorylation for at least 29% of the dosing interval is required for NVP-BYL719 to induce tumor stasis, and that this level of pathway inhibition must be sustained for at least 45% of the dosing interval to produce 30% tumor regression in the Rat1-myr-p110α tumor-bearing nude mice. In contrast, in the Rat1-myr-p110δ tumor-bearing nude mice NVP-BYL719 exposure levels did not achieve 80% inhibition of Akt phosphorylation (in vivo IC80 = 29 μmol/L; corrected for NVP-BYL719 plasma protein binding in mouse IC80 = 2,552 μmol/L) most likely explaining the modest antitumor effect observed and in line with the modest activity of the compound on p110δ. To exclude the possibility that our finding could be Rat1 mouse tumor models specific, NVP-BYL719 was administered in vivo at different doses to nude mice and nude rats bearing a diverse range of cancer cell lines–derived tumor xenografts. Here as well, we found a nearly linear relationship between the antitumor efficacy magnitude and duration of drug exposure over the IC80 (R2 = 0.77, P < 0.001, n = 27, Supplementary Fig. S4 and Table S5). These data suggest that sustained inhibition of the PI3K/Akt pathway for a fraction of the dosing interval is required for NVP-BYL719 to produce a robust antitumor effect.

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NVP-BYL719/Alpelisib shows an improved safety profile compared with pan-class I inhibition [3]
The expected on target side effects of anti-PI3K therapy are insulin resistance and hyperglycemia. To assess whether NVP-BYL719 perturbs glucose homeostasis, plasma insulin and glucose blood levels were measured and compared with plasma drug concentrations in matched samples from multiple animals and at multiple time points. The data here revealed that insulin plasma levels increased proportionally with NVP-BYL719 plasma concentrations, whereas blood glucose levels were maintained close to normal up to 20 μmol/L of NVP-BYL719 (Fig. 2A and B). However, above 20 μmol/L, we observed a compound concentration-dependent glucose increase which led to hyperglycemia despite insulin plasma level elevation. Thus, we defined 20 μmol/L as NVP-BYL719-related hyperglycemic threshold in mice.

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Genetic alterations in PIK3CA predict NVP-BYL719/Alpelisib in vivo efficacy [3]
Next, NVP-BYL719 was administered in vivo at the dose of 50 mg/kg (every day, p.o.) to mice bearing a diverse range of cancer cell lines–derived tumor xenografts (Fig. 5A and Supplementary Table S5) with different genetic backgrounds, including the predictive features of the decision tree described previously. Most of the tumor models that carried a PIK3CA mutation or amplification responded to NVP-BYL719 (response defined as T/C < 20%). In contrast, in most of the tumor models that carried a PTEN mutation or were PIK3CA wild type, we observed progressive disease. In vivo, the predictor also significantly enriched for responders (positive predictive value = 89%). These data demonstrate that the NVP-BYL719 predictive features derived from the in vitro profiling and analysis of the CCLE seem relevant for predicting response in vivo (P = 0.01, Fisher test).

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BYL719/Alpelisib simultaneously reduces tumor growth and tumor ectopic bone formations in two murine models of osteosarcoma and slightly modulates systemic bone parameters [4]
We next tested the effects of BYL719 in the murine MOS-J syngenic model of osteosarcoma. BYL719 significantly reduced tumor volumes in a dose-dependent manner compared to a vehicle group (Fig. 2a; p < 0.01 and p < 0.001, respectively for 12.5 and 50 mg/kg BYL719). Indeed, the mean tumor volume decreased from 1747 mm3 in the control group to 938 mm3 for the group treated with 50 mg/kg BYL719 (Fig. 2a, p < 0.001). In addition, µCT analyses were performed on the tibias with the tumors and the contralateral tibias (normal bone) of each mouse. MicroCT analyses of the tibias bearing tumor showed the ectopic bone formation deposited by the tumor cells and clearly demonstrated that BYL719 significantly reduced this tumor ectopic bone (Fig. 2b, left panel). The benefit of BYL719 was confirmed with the calcified tissue parameters measured (Fig. 2b, right panel). The bone volume (BV) was significantly decreased with 50 mg/kg BYL719, 7.08 ± 0.6 to 4.37 ± 0.21 mm3 (p < 0.001) as was the bone surface (BS) from 99.65 ± 5.74 mm2 to 63.91 ± 2.3 mm2 (p < 0.001). Histomorphometric parameters of contralateral tibias were studied to determine the systemic effect of BYL719 on normal bone remodeling without any tumor (Supporting Information Data 7). BYL719 at 50 mg/(kg day−1) did not affect any of the studied trabecular bone parameters (Supporting Information Data 7). However, it significantly reduced several cortical bone parameters including TV (tissue volume), BV, BV/TV, BS/TV, and CTh (p < 0.01) (Supporting Information Data 7). Histological investigations revealed that BYL719 decreased the surface of TRAP+ osteoclasts without affecting the number of osterix+ cells (Figs. 3a and 3b). In addition, the therapeutic benefit of BYL719 was strengthened by the decrease of KI67+ cell number (Fig. 3c) and by a reduction of the tumor vascularization (Fig. 3d).

Based on these results, the therapeutic potential of BYL719 was analyzed in a xenogenic model of osteosarcoma. Nude mice with human HOS tumors were treated with 50 mg/(kg day−1) of BYL719. As with the MOS-J model, BYL719 significantly reduced tumor volumes, from 1445 mm3 for the control group to 650 mm3 for the treated group at the end of the treatment period (Fig. 2c; p < 0.01). These results confirmed the inhibitory effect of BYL719 in a second preclinical osteosarcoma model. MicroCT analysis of the tibias bearing tumor revealed that BYL719 reduced deposition of ectopic bone matrix as shown by the bone parameters values from 64.91 ± 5.2 to 36.4 ± 0.70 mm2 (p < 0.001) and from 6.2 ± 0.33 to 4.0 ± 0.08 mm3 (p < 0.001), respectively, for BS and BV (Fig. 2d, right panel). The effect of BYL719 on normal bone was evaluated by µCT of the contralateral tibia without any tumor confirming the results obtained in the syngenic MOS-J model (Supporting Information Data 8). MicroCT confirmed the effect of BYL719 on cortical bone observed in C57Bl5J mice.

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Therapeutic benefit of combining BYL719/Alpelisib with conventional chemotherapeutic agents [4]
As BYL719 shows a cytostatic effect in osteosarcoma cells, we then assessed the therapeutic benefit to combine BYL719 with ifosfamide (mafosfamide for in vitro experiment) a conventional chemotherapy in the treatment of osteosarcoma. To determine whether this effect was additive or synergistic, the dose-dependent effects with constant ratio design and the combination index (CI) values were performed and calculated according to the Chou and Talalay median effect principal. Figures 4a and 4b show the dose–response curve (combination treatment, BYL719 or mafosfamide monotherapy) and the combination index plots, indicating that BYL719 synergistically enhances the effect of mafosfamide on tumor cell growth (Figs. 4a and 4b). Then we performed a colony formation assay to evaluate capabilities to recover after 2 days of BYL719 ± mafosfamide treatment. While BYL719 did not change the number of colonies, mafosfamide decreased the colony formation compared with control (Fig. 4c). However, the combination of BYL719 with mafosfamide significantly induced the highest decrease of colony formation compared with each single drug alone (Fig. 4c). Moreover, BYL719 potentiates the effect of mafosfamide to induce apoptosis as shown by the increase of cleaved-PARP expression (Fig. 4d).

We then studied the effect of BYL719 (50 mg/(kg day−1)) combined with a suboptimal dose of ifosfamide (IFOS, 30 mg/(kg day−1) for 3 days) the syngenic murine model previously described (Fig. 4e). As expected, 50 mg/kg BYL719 had a significantly inhibitory effect on tumor development as compared to the control group. In contrast, combined treatment of a suboptimal dose of IFOS with 50 mg/(kg day−1) BYL719 significantly decreased tumor growth (1011 mm3) compared to using IFOS alone (1746 mm3) or BYL719 alone (1421 mm3) (Fig. 4e). MicroCT analysis of tumor bones revealed that the combined treatment did not have an impact on the therapeutic response of BYL719, not even synergistic or additive effects were observed on the ectopic bone formed (Figs. 4f and 4g). These results strongly demonstrate the therapeutic interest to combine BYL719 with a conventional chemotherapeutic drug, in order to markedly delay tumor growth and tumor ectopic bone formation.

To evaluate the isoform-specific potency of Alpelisib (NVP-BYL719) in a cell-based system, an N-terminally myristoylated form of each PI3K class IA isoform was expressed in Rat1 fibroblasts as described in ref. 17. The retroviral expression plasmid pBabePuro containing human p110α, p110β, and p110δ with an N-terminal myristoylation (myr) signal followed by an HA-tag were generated. Successfully infected Rat1 cells were selected in medium containing 4 μg/mL of puromycin, expanded and characterized for expression of the p110 isoforms (in 2006). Transgenic expression of the myristoylated protein was confirmed by increased levels of phosphorylated Akt. The TSC1−/−-null MEFs mechanistic model for mTORC1 constitutive activation has been obtained from Dr. D. Kwiatkowski in 2007.[3]

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Alpelisib (NVP-BYL719) potently inhibits the 2 most common PIK3CA somatic mutations (H1047R, E545K; IC50~4 nM). Alpelisib (NVP-BYL719) potently inhibits Akt phosphorylation in cells transformed with PI3Kα (IC50=74±15 nM) and shows significant reduced inhibitory activity in PI3Kβ or PI3Kδ isoforms transformed cells (≥15-fold compared with PI3Kα).

Cell growth and viability, clonogenic assay[4]
Two thousand tumor cells were seeded into 96-well plates and, the day after, the cells were treated with Alpelisib (BLY-719; Piqray; NVP-BYL719) (1–50 µmol/L) for 72 hr. Cell growth/viability was determined using a colorimetric assay using sodium 3′[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro-)benzene sulfonic acid hydrate. Absorbance was read at 490 nm. Cell viability was also determined by trypan blue exclusion assay; viable and nonviable cells were counted manually after 24 and 48 hr of treatment. For clonogenic assay, tumor cells were pretreated with or without 25 µM BYL for 6 hr following by treatment with/without 5 µg/mL mafosfamide for 48 hr in 96-well plate. Then, the tumor cells were splitted in 6-well plate, with 500 cells/well without treatment for 7 days. The number of colonies was counted after crystal violet staining.

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Caspase activity[4]
Two hundred thousand cells were seeded in 6-well plates and cultured with or without Alpelisib (BLY-719; Piqray; NVP-BYL719) for 3–48 hr (25 µM). Caspase activity was assessed using the CaspACE Assay System kit, according to the manufacturer's recommendations. The results were expressed in arbitrary units, corrected for protein concentration quantified by BCA. Cell lysate of cells treated with 1 µg/mL of staurosporine (Invitrogen) overnight was used as the positive control.

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Cell cycle analysis[4]
Subconfluent cultures were incubated with or without 25 µM of Alpelisib (BLY-719; Piqray; NVP-BYL719) for 18 hr, trypsinized, washed, and incubated in PBS containing 0.12% Triton X-100, 0.12 mM EDTA, and 100 µg/mL DNase-free ribonuclease A. Then, 50 µg/mL of propidium iodide were added for 20 min. Cell cycle distribution was determined by flow cytometry and analyzed by DNA cell Cycle Analysis Software.

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Western blots[4]
Two hundred thousand cells were treated with 25 µM of Alpelisib (BLY-719; Piqray; NVP-BYL719) for 3–24 hr and then lysed in RIPA buffer (150 mmol/L NaCl, 5% Tris, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 1 mmol/L Na3VO4, 0.5 mmol/L PMSF, 10 mg/mL leupeptin, 10 mg/mL aprotinin). Total cell lysate (40 μg), determined using the BCA kit, was run on 10% SDS-PAGE and electrophoretically transferred to Immobilon-P membranes. The membrane was blotted with antibodies (Supporting Information Data 1) in PBS, 0.05% Tween 20, and 3% BSA. Antibody binding was visualized using the enhanced chemiluminescence system.

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Alpelisib (0–1000 nM) was applied to cells in escalating concentrations for 72 hours. Using the CyQuant assay, cell viability was measured.

Female athymic nu/nu mice
40 mg/kg
o.g.

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Xenograft Studies[3]
CW2 cells were re-suspended in serum-free RPMI and Growth Factor-Reduced Matrigel (1:1 ratio) and injected subcutaneously into the right flank of 4–6 week old female athymic nu/nu mic. When the average tumor volume reached ~200 mm3, mice received daily doses of vehicle (0.5% Methylcellulose + 0.4% Tween 80, orogastric gavage), neratinib (40 mg/kg; orogastric gavage), alpelisib (30 mg/kg; orogastric gavage), or neratinib + alpelisib . In our previous studies, we have found neratinib to cause anorexia and moderate body weight loss. To avoid these toxicities, all mice were prophylactically supplemented with DietGel 76A (Clear H2O) in addition to regular chow. Tumor diameters were measured twice weekly using calipers and tumor volumes were calculated using the formula: volume = width2 x length/2.

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Cell lines–derived tumor models.[3]
All in life experimentation and efficacy studies were conducted as described previously. Tumor xenografts were grown subcutaneously or orthotopically in nude mice or nude Rowett rats by injection of 3 × 106 to 1 × 107 cells or implantation of tumor fragments of approximately 50 mg. Tumor-bearing animals mice were treated with either vehicle control, alpelisib/NVP-BYL719 , or NVP-BKM120 (p.o., every day) at the doses indicated.

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Patient-derived tumor models.[3]
Patient-derived xenograft (PDX) models were established by implanting surgical tumor tissues from treatment-naïve cancer patients into nude mice. All samples were anonymized and obtained with informed consent and under the approval of the institutional review boards of the tissue providers and Novartis. All PDX models were histologically characterized and external diagnosis was independently confirmed by in-house pathologists and were genetically profiled using various technology platforms after serial passages in mice. PIK3CA mutation was determined by both RNA and DNA deep sequencing technologies and PIK3CA amplification was determined by SNP array 6.0. For efficacy studies, tumor-bearing animals were enrolled when subcutaneously implanted tumors reached about 200 mm3 and treated with alpelisib/NVP-BYL719 at 50 mg/kg daily. The response is reported as percentage change in tumor volume at last day of treatment relative to day 0 (start of treatment).

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Formulation used for in vivo experiments: alpelisib/NVP-BYL719 was formulated for oral administration in solution by solving the compound in N-methyl pyrrolidone, polyethylene glycol 300, solutol HS15, and water (10%:30%:20%:40%, v/v) or in suspension in 1% (w/v) carboxymethylcellulose (CMC) + 0.5% (w/v) Tween 80 similar to NVP-BKM120.[3]

Absorption, Distribution and Excretion
Alpelisib reached a peak concentration in plasma of 1320±912ng/mL after 2 hours. Alpelisib has an AUClast of 11,100±3760h ng/mL and an AUCINF of 11,100±3770h ng/mL. A large, high fat meal increases the AUC by 73% and Cmax by 84% while a small, low fat meal increases the AUC by 77% and Cmax by 145%.
36% of an oral dose is eliminated as unchanged drug in the feces and 32% as the primary metabolite BZG791 in the feces. About 2% of an oral dose is eliminated in the urine as unchanged drug and 7.1% as the primary metabolite BZG791. In total 81% of an oral dose is eliminated in the feces and 14% is eliminated in the urine.
The apparent volume of distribution at steady state is 114L.
The mean apparent oral clearance was 39.0L/h. The predicted clearance is 9.2L/hr under fed conditions.

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Metabolism / Metabolites
Alpelisib is metabolized by hydrolysis reactions to form the primary metabolite. It is also metabolized by CYP3A4. The full metabolism of Alpelisib has yet to be determined but a series of reactions have been proposed. The main metabolic reaction is the substitution of an amine group on alpelisib for a hydroxyl group to form a metabolite known as M4 or BZG791. Alpelisib can also be glucuronidated to form the M1 and M12 metabolites.

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Biological Half-Life
The mean half life of alprelisib is 8 to 9 hours.

Hepatotoxicity
In the prelicensure clinical trials of alpelisib in patients with cancer, liver test abnormalities were frequent although usually transient, asymptomatic, and mild-to-moderate in severity. Some degree of ALT elevation arose in up to 44% of alpelisib- and fulvestrant-treated patients, but were above 5 times the upper limit of normal (ULN) in only 3% to 4%. The aminotransferase elevations rarely necessitated dose modifications or interruptions, and only slightly lower rates of enzyme elevations occurred in patients taking fulvestrant without alpelisib. In these trials that enrolled less than 1000 patients, there were several reports of marked serum aminotransferase elevations that led to early discontinuation. However, the nature and clinical features of the liver injury were not provided and there were no cases of clinically apparent liver injury. Skin rashes were also common with alpelisib therapy, and patients are often given prophylactic antihistamines which appear to result in fewer and milder rashes. However, moderate-to-severe rash can occur and some are accompanied by drug reaction with eosinophilia and systemic signs (DRESS) syndrome, some degree of liver injury (usually anicteric and asymptomatic) being a part of the manifestations.
Likelihood score: E* (unproven but suspected rare cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on alpelisib during breastfeeding. The manufacturer recommends that breastfeeding be discontinued during alpelisib therapy and for 1 week after the final dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Alpelisib is 89% protein bound.

[1]. Co-occurring gain-of-function mutations in HER2 and HER3 modulate HER2/HER3 activation, oncogenesis, and HER2 inhibitor sensitivity. Cancer Cell. 2021 Aug 9;39(8):1099-1114.e8.

[2]. Discovery of NVP-BYL719 a potent and selective phosphatidylinositol-3 kinase alpha inhibitor selected for clinical evaluation. Bioorg Med Chem Lett. 2013 Jul 1;23(13):3741-8.

[3]. Characterization of the novel and specific PI3Kα inhibitor NVP-BYL719 and development of the patient stratification strategy for clinical trials. Mol Cancer Ther. 2014 May;13(5):1117-29.

[4]. BYL719, a new α-specific PI3K inhibitor: single administration and in combination with conventional chemotherapy for the treatment of osteosarcoma. Int J Cancer. 2015 Feb 15;136(4):784-96.

[5]. Targeted therapy in patients with PIK3CA-related overgrowth syndrome. Nature. 2018 Jun;558(7711):540-546.

[6]. Inhibition of BTF3 sensitizes luminal breast cancer cells to PI3Kα inhibition through the transcriptional regulation of ERα. Cancer Lett. 2018 Oct 10;440-441:54-63.

Pharmacodynamics
Alpelisib does not prolong the QTcF interval. Patients taking alpelisib experience a dose dependent benefit from treatment with a 51% advantage of a 200mg daily dose over a 100mg dose and a 22% advantage of 300mg once daily over 150mg twice daily. This suggests patients requiring a lower dose may benefit from twice daily dosing.

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(2S)-N1-[4-methyl-5-[2-(1,1,1-trifluoro-2-methylpropan-2-yl)-4-pyridinyl]-2-thiazolyl]pyrrolidine-1,2-dicarboxamide is a proline derivative.
Alpelisib is a phosphatidylinositol 3-kinase (PI3K) inhibitor with potent antitumor activity. It works by selectively inhibiting class I PI3K p110α, which is the catalytic subunit of PI3K, a lipid kinase that plays a role in various biological processes, including proliferation, survival, differentiation, and metabolism. Alpelisib was designed to target this enzyme that appears to be mutated at a rate of nearly 30% in human cancers, leading to hyperactivation. There are several isoform-specific PI3K inhibitors that are under clinical development or currently approved, such as [idelalisib] used for chronic lymphocytic leukemia (CLL). Approved by the FDA in May 2019, alpelisib is the first approved PI3K inhibitor indicated for the treatment of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative, PIK3CA-mutated, advanced or metastatic breast cancer in combination with [fulvestrant] for postmenopausal women and male patients. To initiate alpelisib therapy, it is required that the presence of a PIK3CA mutation in the tissue and/or liquid biopsy sample collection should be confirmed via FDA-approved diagnostic tests. Alpelisib is marketed under the trade name Piqray and is available as oral tablets. Studies evaluating the therapeutic effectiveness of alpelisib in other cancers, such as ovarian cancer and colorectal cancer, are under ongoing investigations. Alpelisib was granted FDA approval on 24 May 2019. In April 2022, the FDA granted the use of alpelisib in the treatment of PIK3CA-Related Overgrowth Spectrum (PROS) in adults and children who require systemic therapy.

Alpelisib is an oral selective inhibitor of the phosphoinositol-3-kinase (PIK3) which is mutated in several forms of solid tumors and is approved for use in specific forms of advanced or metastatic breast cancer. Serum aminotransferase elevations are common during alpelisib therapy but clinically apparent liver injury with jaundice has not been reported with its use and must be rare, if it occurs at all.
Alpelisib is an orally bioavailable phosphatidylinositol 3-kinase (PI3K) inhibitor with potential antineoplastic activity. Alpelisib specifically inhibits PI3K in the PI3K/AKT kinase (or protein kinase B) signaling pathway, thereby inhibiting the activation of the PI3K signaling pathway. This may result in inhibition of tumor cell growth and survival in susceptible tumor cell populations. Activation of the PI3K signaling pathway is frequently associated with tumorigenesis. Dysregulated PI3K signaling may contribute to tumor resistance to a variety of antineoplastic agents.
ALPELISIB is a small molecule drug with a maximum clinical trial phase of IV (across all indications) that was first approved in 2019 and has 5 approved and 17 investigational indications. Open Targets

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Activating mutations in HER2 (ERBB2) drive the growth of a subset of breast and other cancers and tend to co-occur with HER3 (ERBB3) missense mutations. The HER2 tyrosine kinase inhibitor neratinib has shown clinical activity against HER2-mutant tumors. To characterize the role of HER3 mutations in HER2-mutant tumors, we integrate computational structural modeling with biochemical and cell biological analyses. Computational modeling predicts that the frequent HER3E928G kinase domain mutation enhances the affinity of HER2/HER3 and reduces binding of HER2 to its inhibitor neratinib. Co-expression of mutant HER2/HER3 enhances HER2/HER3 co-immunoprecipitation and ligand-independent activation of HER2/HER3 and PI3K/AKT, resulting in enhanced growth, invasiveness, and resistance to HER2-targeted therapies, which can be reversed by combined treatment with PI3Kα inhibitors. Our results provide a mechanistic rationale for the evolutionary selection of co-occurring HER2/HER3 mutations and the recent clinical observations that HER3 mutations are associated with a poor response to neratinib in HER2-mutant cancers.[1]

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Phosphatidylinositol-3-kinase α (PI3Kα) is a therapeutic target of high interest in anticancer drug research. On the basis of a binding model rationalizing the high selectivity and potency of a particular series of 2-aminothiazole compounds in inhibiting PI3Kα, a medicinal chemistry program has led to the discovery of the clinical candidate NVP-BYL719. [2]

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Somatic PIK3CA mutations are frequently found in solid tumors, raising the hypothesis that selective inhibition of PI3Kα may have robust efficacy in PIK3CA-mutant cancers while sparing patients the side-effects associated with broader inhibition of the class I phosphoinositide 3-kinase (PI3K) family. Here, we report the biologic properties of the 2-aminothiazole derivative NVP-BYL719, a selective inhibitor of PI3Kα and its most common oncogenic mutant forms. The compound selectivity combined with excellent drug-like properties translates to dose- and time-dependent inhibition of PI3Kα signaling in vivo, resulting in robust therapeutic efficacy and tolerability in PIK3CA-dependent tumors. Novel targeted therapeutics such as NVP-BYL719, designed to modulate aberrant functions elicited by cancer-specific genetic alterations upon which the disease depends, require well-defined patient stratification strategies in order to maximize their therapeutic impact and benefit for the patients. Here, we also describe the application of the Cancer Cell Line Encyclopedia as a preclinical platform to refine the patient stratification strategy for NVP-BYL719 and found that PIK3CA mutation was the foremost positive predictor of sensitivity while revealing additional positive and negative associations such as PIK3CA amplification and PTEN mutation, respectively. These patient selection determinants are being assayed in the ongoing NVP-BYL719 clinical trials.[3]

C19H22F3N5O2S

441.47

441.144

C, 51.69; H, 5.02; F, 12.91; N, 15.86; O, 7.25; S, 7.26

1217486-61-7

Alpelisib hydrochloride;1584128-91-5

56649450

white solid powder

1.4±0.1 g/cm3

1.587

-0.02

2

8

4

30

663

1

S1C(=C(C([H])([H])[H])N=C1N([H])C(N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C(N([H])[H])=O)=O)C1C([H])=C([H])N=C(C=1[H])C(C([H])([H])[H])(C([H])([H])[H])C(F)(F)F

STUWGJZDJHPWGZ-LBPRGKRZSA-N

InChI=1S/C19H22F3N5O2S/c1-10-14(11-6-7-24-13(9-11)18(2,3)19(20,21)22)30-16(25-10)26-17(29)27-8-4-5-12(27)15(23)28/h6-7,9,12H,4-5,8H2,1-3H3,(H2,23,28)(H,25,26,29)/t12-/m0/s1

(2S)-1-N-[4-methyl-5-[2-(1,1,1-trifluoro-2-methylpropan-2-yl)pyridin-4-yl]-1,3-thiazol-2-yl]pyrrolidine-1,2-dicarboxamide

Alpelisib; NVP-BYL-719; 1217486-61-7; BYL-719; BYL719; Piqray; Vijoice; NVP-BYL719; Alpelisib (BYL719); NVP-BYL719; NVP-BYL 719; BYL-719; BYL719; BYL 719

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)

Solubility in Formulation 1: ≥ 5 mg/mL (11.33 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 2: 2.08 mg/mL (4.71 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 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 3: ≥ 2.08 mg/mL (4.71 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 4: ≥ 2.08 mg/mL (4.71 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 corn oil and mix evenly.

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Solubility in Formulation 5: 30% PEG400+0.5% Tween80+5% Propylene glycol : 30mg/mL

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Solubility in Formulation 6: 10 mg/mL (22.65 mM) in 0.5% MC 0.5% Tween-80 (add these co-solvents sequentially from left to right, and one by one), Suspension solution; with ultrasonication.

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Solubility in Formulation 7: 10 mg/mL (22.65 mM) in 1% CMC 0.5% Tween-80 (add these co-solvents sequentially from left to right, and one by one), Suspension solution; with ultrasonication.

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