p110α (IC50 = 52 nM); p110β (IC50 = 166 nM); p110δ (IC50 = 116 nM); p110γ (IC50 = 262 nM); Vps34 (IC50 = 2.4 μM); p110α-H1047R (IC50 = 52 nM); p110α-E545K (IC50 = 99 nM); mTOR (IC50 = 4.6 μM)

Buparlisib (NVP-BKM120) exhibits 50-300 nM activity for class I PI3K’s, including the most common p110α mutants. Additionally, NVP-BKM120 is less effective against class III and class IV PI3Ks, with biochemical activity being detected at 2, 5, >5, and >25 μM for the inhibition of VPS34, mTOR, DNAPK, and PI4K, respectively[1]. Buparlisib (NVP-BKM120) induces multiple myeloma (MM) cell apoptosis in a manner that depends on both the dose and the passage of time. At concentrations ≥10 μM, buparlisib (NVP-BKM120) significantly induces apoptosis in all tested MM cell lines at 24 h (P<0.05, compared to control). If not specified otherwise, the following experiments will use ≥10 μM buparlisib (NVP-BKM120) and a 24-hour treatment period. All of the tested MM cell lines exhibit a dose-dependent growth inhibition in response to buparlisib (NVP-BKM120) treatment. Buparlisib (NVP-BKM120) IC50 varies among tested MM cells. ARP-1, ARK, and MM.1R have an IC50 of between 1 and <10 μM, at 24 h of treatment, whereas MM.1S has an IC50 of less than <1 μM, and U266 has an IC50 of between 10 and <100 μM,. In conclusion, NVP-BKM120 treatment inhibits the growth of MM cells and causes them to die off in ways that depend on the dose and the length of time[2].

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The biochemical activity of Buparlisib/15 was assessed across class I PI3K’s, related lipid kinases, and against more than 200 protein kinases. Some of the data are shown in Table 3. Compound 15 exhibited 50–300 nM activity for class I PI3K’s, including the most common p110α mutants. Additionally, 15 exhibited lower potency against class III and class IV PI3K's, where 2, 5, >5, and >25 μM biochemical activity was observed for inhibition of VPS34, mTOR, DNAPK, and PI4K, respectively. No significant activity was observed against the protein kinases tested. [1]

In vitro evaluation of Buparlisib/15 across a range of PI3K deregulated cell lines from a variety of tumor types, including ovarian, glioblastoma, breast, and prostate, was conducted (Table 4). Across all cell lines, pathway modulation and antiproliferative activity was consistent with cellular PI3K inhibition [1].

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BKM120/Buparlisib inhibits the growth of MM cell lines and induces cell apoptosis [2]
To evaluate the effect of BKM120 on myeloma cells, we treated MM cell lines with different doses of BKM120 for 24 or 72 h. BKM120-induced MM cell apoptosis was measured by annexin V binding assay. As shown in Fig. 1a, BKM120 induced MM cell apoptosis in both dose- and time-dependent manners. BKM120 at concentrations ≥10 μM induced significant apoptosis in all tested MM cell lines at 24 h (P <0.05, compared with control). Therefore, we chose 10 μM BKM120 and 24-h treatment in the following experiments if not stated otherwise.

The effect of BKM120/Buparlisib on MM cell growth was tested by MTS assay. As shown in Fig. 1b, BKM120 treatment resulted in a dose-dependent growth inhibition in all tested MM cell lines. BKM120 IC50 (concentration at 50% inhibition) varied among tested MM cells. At 24 h treatment, IC50 for ARP-1, ARK, and MM.1R was between 1 and 10 μM, while IC50 for MM.1S was <1 μM, and IC50 for U266 was between 10 and 100 μM. In summary, our findings indicate that BKM120 treatment resulted in MM cell growth inhibition and apoptosis in dose- and time-dependent manners.

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BKM120/Buparlisib induces primary MM cell apoptosis ex vivo [2]
To evaluate BKM120 activity in primary MM cells, we extended our study to CD138+ primary MM cells freshly isolated from myeloma patients. According to our previous finding, primary MM cells undergo apoptosis ex vivo unless the cells are cocultured with BMSCs. Therefore, CD138+ primary MM cells were cocultured at 1:1 ratio with CD138− BMSCs generated from MM bone marrow aspirates. The cells were treated with different doses of BKM120 from 0 to 1 mM for 24 h. Primary MM cells and BMSCs were identified by APC-CD138 staining. As shown by the representative data obtained from myeloma cells and BMSCs from one out of three patients examined (Fig. 1c), BKM120 induced CD138+ primary MM cell apoptosis in a dose-dependent manner. The primary MM apoptosis rate is slightly elevated even in our control group. This is probably because primary MM cells go into spontaneous apoptosis ex vivo after isolation from the tumor-promoting bone marrow microenvironment. Of interest, BKM120 had significant lower cytotoxicity toward CD138− stromal cells. Figure 1d shows BKM120-induced apoptosis of primary MM cells from three different MM patients. Taken together, these data suggest that BKM120 induces primary MM cell apoptosis and has low toxicity toward non-tumoric BMSCs.

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BKM120/Buparlisib has low toxicity toward normal blood cells of healthy volunteers [2]
To further examine whether Buparlisib/BKM120 induces normal cell apoptosis, PBMCs from different healthy volunteers were incubated with 0–1 mM BKM120 for 24 h. Cells apoptosis rate was measured as described above. As shown in Fig. 2a, BKM120 had comparably low toxicity toward normal PBMCs as to BMSCs. BKM120 at 10 or 100 μM, which were highly apoptotic to MM cells, only resulted in <40% of PBMC apoptosis. Thus, our findings suggest that BKM120 has low cytotoxicity toward normal PBMCs.

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IL-6, IGF, or BMSCs do not protect MM cells from Buparlisib/BKM120-induced apoptosis [2]
IL-6 is an important survival cytokine for MM. Previous work has shown that IL-6 promotes MM cell survival under chemotherapy agent dexamethasone treatment. Therefore, we examined whether IL-6 could attenuate BKM120-induced MM cell apoptosis. For this purpose, different MM cell lines were cultured with or without recombinant human IL-6 at a final concentration of 5 ng/ml in the presence or absence of 10 μM BKM120 for 24 h. As a positive control, MM.1S cells were treated with 40 μg/ml of dexamethasone with or without IL-6 for the same period of time. As shown in Fig. 2b, IL-6 did not affect BKM120-induced MM cell apoptosis, but promoted MM.1S cell survival under dexamethasone treatment.

Previous researches have also shown that IGF is another MM survival cytokine that activates PI3K-Akt pathway. Therefore, we also tested whether presence of IGF affects Buparlisib/BKM120-induced MM cell apoptosis. Different MM cell lines were cultured with or without recombinant human IGF at a final concentration of 10 ng/ml. As shown in Fig. 2c, IGF had no protection on BKM120-induced apoptosis in ARP-1 or U266 cells. Increasing evidence has shown that BMSCs in the myeloma tumor bed provide a tumor promotion microenvironment and protect MM cells from chemotherapy drug-induced apoptosis. Therefore, we also tested whether BMSCs from MM patient bone marrow were able to protect MM cells from BKM120-induced apoptosis. For this purpose, BMSCs generated from MM patients were cocultured with MM cell lines. The cells were treated with or without 10 μM of BKM120 for 24 h. As a positive control, MM.1S cells were cocultured with or without BMSCs and treated with or without 40 μg/ml of dexamethasone for 24 h. After treatment, MM cells were identified as CD138+ cells by APC-CD138 staining. As shown in Fig. 2d, BMSCs were not able to protect MM cells from BKM120-induced apoptosis, but protected MM.1S cells from dexamethasone-induced apoptosis.

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BKM120/Buparlisib causes cell cycle arrest in G1 phase [2]
To study the mechanism of BKM120-induced MM cell growth inhibition and apoptosis, we examined whether BKM120 treatment affects MM cell cycle. As shown in Fig. 3a, ARP-1 cells were cultured with or without 1 μM BKM120 for 24 h. BKM120 treatment resulted in increased G1-phase cells and decreased S-phase cells. Similar findings were observed in other MM cell lines MM.1S and MM.1R (Fig. 3b).

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BKM120/Buparlisib triggers MM cell apoptosis by activating caspases [2]
To elucidate BKM120-induced MM cell apoptosis, MM cell lines, treated with or without BKM120 for 24 h, were assessed for caspase activation by Western blotting analysis. The results showed the cleavage of caspase 3, caspase 7, and caspase 9 (Fig. 4a). PARP cleavage was also detected after BKM120 treatment in all tested cell lines, indicating activation of the caspase cascade. To examine whether BKM120-triggered cell death depends on caspase activation, ARP-1 cells were treated with BKM120 and caspase-3 inhibitor (Fig. 4b). Our result showed that caspase-3 inhibitor repressed BKM120-induced cell apoptosis. Overall, these findings suggest that BKM120 treatment induces MM cell apoptosis through caspase activation.

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BKM120/Buparlisib exposure causes upregulation of BimS and downregulation of XIAP [2]
To analyze the signaling pathways that are modulated by Buparlisib/BKM120 exposure in MM cells, we extended our immunoblotting analysis to cell signaling molecules. First, we examined the inhibitory effect of BKM120 on PI3KAkt-mTOR pathway in MM cells. As shown in Fig. 4c and d, both Akt phosphorylated at Thr473 and Akt phosphorylated at Ser308 were downregulated after BKM120 treatment. Downregulation of total Akt was observed in ARP-1 and MM.1R cells, but not in MM.1S cells (Fig. 4d). This is probably caused by the increase in apoptotic myeloma cells after BKM120 treatment, and/or some cell type-specific regulation of Akt expression/degradation after the treatment. The pP70S6K levels were also decreased after BKM120 treatment in tested MM cells, while total P70S6K expression remained unchanged. Such findings suggest that BKM120 inhibits PI3K-Akt-mTOR pathways in MM cells.

Second, since BKM120 treatment caused cell cycle arrest in G1 phase, we examined the expression of cell cycle regulators. As shown in Fig. 4d, cell cycle repressor p27(Kip1) protein expression was upregulated after BKM120 treatment, while cyclin D1 expression was downregulated.

Next, we examined the expression of apoptosis regulatory factors. Our data showed that the expression of cytotoxic small isoform of Bim, BimS, was upregulated after Buparlisib/BKM120 treatment. Bim is a pro-apoptotic factor belonging to the Bcl-2 family. Bim has three major isoforms, BimEL, BimL, and BimS, generated by alternative splicing. The shortest form BimS is the most cytotoxic isoform. Previous work has shown that the transcription of Bim is regulated by the forkhead transcription factor FKHR-L1, a downstream effector of PI3K. In addition to Bim, the expression of anti-apoptotic XIAP and Bcl-XL, was downregulated after BKM120 treatment (Fig. 4d). Thus, BKM120-induced MM cell apoptosis may be caused by upregulation of cytotoxic BimS and down-regulation of anti-apoptotic XIAP and Bcl-XL.

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Synergistic cytotoxicity of Buparlisib/BKM120 and dexamethasone on MM cells [2]
To test whether BKM120 has a synergistic or addictive effect with other anti-MM chemotherapy agents, ARP-1 cells were treated with Buparlisib/BKM120 (1 μM) in combination with low doses of melphalan, dexamethasone, lenalidomide, or bortezomib. As shown in Fig. 5a, combinational treatment of BKM120 with dexamethasone or bortezomib, but not with other drugs, had synergistic or addictive cytotoxicity in ARP-1 cells. In particular, BKM120 and dexamethasone showed synergistic anti-ARP-1 activity. Next, we extended the experiment to other MM cell lines. As shown in Fig. 5b, although BKM120 or dexamethasone alone at the low doses had only a limited cytotoxicity, combination of both induced significant cell apoptosis in dexamethasone-sensitive cell lines ARP-1 and MM.1S, but not in dexamethasone-resistant cells MM.1R. Cell growth tests also showed that BKM120 and dexamethasone synergistically inhibited MM.1S cell growth (Fig. 5c). In addition, the same combination of drugs only had limited cytotoxicity toward PBMCs (Supplemental Figure 1). [2]
To examine the minimum doses of each drug for a synergistic effect, we treated MM.1S cells with different doses of Buparlisib/BKM120 and dexamethasone for 24 h. The drug synergistic effect was confirmed by Isobologram analysis (Table 1 and Supplemental Figure 2). These results indicated that BKM120 and dexamethasone had synergistic effects when BKM120 was at doses of 0.5 and 1 μM because of the low values of the interaction index.

To elucidate the role of BKM120/Buparlisib and dexamethasone in the synergistic effect on myeloma cells, we treated MM.1S cells with the drugs in a sequential order. MM.1S cells were treated with dexamethasone for the first day, washed, and switched to BKM120 for the second day, or vice versa. Treatments with medium or the single drugs in sequence served as controls. As shown in Fig. 5e, treatment with dexamethasone first followed by BKM120 resulted in higher apoptosis rate than treatments of BKM120 followed by dexamethasone or single drugs alone.

Finally, immunoblotting to analyze caspase-dependent apoptosis was used to elucidate the molecular mechanisms underlying the synergistic effect of the two drugs. As shown in Fig. 5f, Buparlisib/BKM120 and dexamethasone combinational treatment resulted in increased PARP and Bcl-2 cleavage and caspase-3 activation. The total Bcl-2 level remained unchanged. This was probably because that cleaved Bcl-2 was only a small part of the total Bcl-2. These findings indicate an enhanced caspase-dependent apoptosis after dual drug treatment. BimS expression was further upregulated in the combinational treatment, which may be the cause of the synergistic effect. In summary, our findings suggest that BKM120 and dexamethasone have synergistic cytotoxicity in dexamethasone-sensitive MM cells.

In A2780 xenograft tumors, oral dosing of Buparlisib (NVP-BKM120) at 3, 10, 30, 60, and 100 mg/kg results in a dose dependent modulation of pAKTSer473. At doses of 3 and 10 mg/kg, partial inhibition of pAKTSer473 is seen, and at doses of 30, 60, or 100 mg/kg, nearly complete inhibition is seen. Both plasma and tumor drug exposure were well correlated with the inhibition of pAKT (normalized to total AKT)[1]. Buparlisib (NVP-BKM120) (5 M per kg per day for 15 days)-treated mice had significantly lower tumor burdens than control mice, as indicated by tumor volume (P<0.05) and circulating human kappa chain level (P<0.05). Furthermore, NVP-BKM120 therapy significantly increases the survival of tumor-bearing mice (P<0.05)[2].

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The behavior consistent with selective in vitro inhibition of class I PI3K’s translated to in vivo settings in two models of PI3K-AKT pathway driven cancers: the A2780 ovarian carcinoma and the U87MG glioma model, which carry a PTEN deletion. In A2780 xenograft tumors (Figure 4), oral dosing of Buparlisib/15 at 3, 10, 30, 60, and 100 mg/kg resulted in a dose dependent modulation of pAKTSer473. Partial inhibition of pAKTser473 was observed at 3 and 10 mg/kg, and near complete inhibition was observed at doses of 30, 60, or 100 mg/kg, respectively. Inhibition of pAKT (normalized to total AKT) tracked well with both plasma and tumor drug exposure. pAKT modulation was also time dependent, with >90% target modulation achieved with the 60 and 100 mg/kg dose at the 10 h time point when the plasma and tumor exposure was ca. 2 μM [1].

As was the case in vitro, 15/Buparlisib displays in vivo activity across a range of PI3K pathway deregulated tumor xenograft models. In the established U87MG glioma model, significant single agent activity was obtained with 15 at daily oral doses of 30 and 60 mg/kg (Figure 6) in a well tolerated manner. This activity in the U87MG model, coupled with the high permeability and lack of efflux exhibited by 15,suggests that 15 may have utility in PI3K-driven gliomas [1].

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In vivo effects of Buparlisib/BKM120 on established MM [2]
To examine BKM120 in vivo antimyeloma effects, the human MM-SCID mouse model using cell line ARP-1 was established as described previously. When palpable tumors developed (≥5 mm in diameter), mice (ten per group) received daily intraperitoneal injections of Buparlisib/BKM120 (5 μM kg−1 day−1) or vehicle control (DMSO/PBS). As shown in Fig. 6a and b, mice receiving BKM120 treatment had significantly smaller tumor burdens as compared with control mice, which were measured as tumor volume (Fig. 6a, P <0.05) and level of circulating human kappa chain (Fig. 6b, P <0.05). In addition, BKM120 treatment significantly prolonged the survival of tumor-bearing mice (Fig. 6c, P <0.05). [2]
Next, we examined whether Buparlisib/BKM120 and dexamethasone display in vivo synergistic antimyeloma effects. In particular, we wanted to know whether these two drugs could display effective antimyeloma effects in vivo at low doses. SCID mice bearing MM.1S tumor were developed and after palpable tumor developed (tumor diameter ≥5 mm), mice (five per group) were treated with intraperitoneal injections of DMSO/PBS, BKM120 (1 μM kg−1), dexamethasone (50 μg kg−1), or combination of BKM120 and dexamethasone at the same doses seven times, every other day for 15 days. Although BKM120 or dexamethasone alone at the low doses had no therapeutic effects against established myeloma, combinational therapy using both the drugs at the low doses significantly retarded the growth of myeloma, measured as tumor volume (Fig. 6d, P <0.05) and level of circulating human lambda chain (Fig. 6e, P <0.05), in treated mice as compared with control mice or mice treated with BKM120 or dexamethasone alone. In addition, combined treatment significantly prolonged the survival of tumor-bearing mice (Fig. 6f, P <0.05).

BKM120/Buparlisib is dissolved in DMSO and directly distributed into a black 384-well plate at 1.25 µL per well. To begin the reaction, add 25 µL of 10 nM PI3 kinase and 5 µg/mL 1-phosphatidylinositol (PI) in assay buffer (10 mM Tris pH 7.5, 5 mM MgCl2, 20 mM NaCl, 1 mM DTT, and 0.05% CHAPS) into each well. Next, add 25 L of 2 M ATP in assay buffer. The addition of 25 L of KinaseGlo solution stops the reaction after it has run for approximately 50% of the time required to deplete the ATP. After 5 minutes of incubation, the stopped reaction is examined to determine whether any ATP is still present.

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PI3K biochemical assay (ATP depletion assay) [1]
Compounds to be tested were dissolved in DMSO and directly distributed into a black 384-well plate at 1.25 µL per well. To start the reaction, 25 µL of 10 nM PI3 kinase and 5 µg/mL 1-alpha-phosphatidylinositol (PI) in assay buffer (10 mM Tris pH 7.5, 5 mM MgCl2, 20 mM NaCl, 1 mM DTT and 0.05% CHAPS) were added into each well followed by 25 µL of 2 µM ATP in assay buffer. The reaction was performed until approx 50% of the ATP was depleted, and then stopped by the addition of 25 µL of KinaseGlo solution. The stopped reaction was incubated for 5 minutes and the remaining ATP was then detected via luminescence

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PI3K biochemical assay (filter binding assay) [1]
50 µl/well of a 1:1 mixture of 100 µg/ml L-α-phosphatidylinositol and L-α-phosphatidylserine dissolved in chloroform:ethanol (2.2:7.8) was pipetted into 96-well MaxiSorp™ plates. The solvents were evaporated at room temperature and plates were washed with Tris-buffered saline (TBS, pH7.4). PI3Kα was incubated for 60 minat room temperature in coated plates in 50 µl medium containing [γ33P]-ATP (~6 kBq/well), 0.5 µM ATP (or higher as indicated in Fig. 1-2), 5 mM MgCl2, 150 mM NaCl, 25 mM Tris-HCl pH7.4, and 1% DMSO. The reaction was started by adding PI3Kα (0.4 µg/ml, <2 nM) and stopped by adding 50 µl of 50 mM EDTA. Plates were washed twice with TBS and dried; 100 µl/well MicroScint™ PS was added, and bound radioactivity was determined using a TopCount™ counter.

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mTOR TR-FRET assay: [1]
50 nL of compound dilutions were dispensed onto black 384-well low volume non-binding polystyrene plates. Then 5 µL of ATP and GFP4EBP1 with 5 µL mTOR proteins (final assay volume 10 µL) were added and the reaction was incubated at RT in 50 mM HEPES pH 7.5, 10 mM MnCl2, 50 mM NaCl, 1 mM EGTA, 1 mM DTT. Reactions were stopped with 10 µL of a proprietary mixture (IVG), containing the Tb3+ -α-p4EBP1-[pT46] detection antibody, EDTA, in TR-FRET dilution buffer . Plates were then read 15 min later in a Synergy2 reader using an integration time of 0.2 seconds and a delay of 0.1 seconds. The control for 100% inhibition of the kinase reaction was created by replacing the mTOR kinase with an equal volume of reaction buffer. The control for 0% inhibition was created by substituting solvent vehicle (90% DMSO in H2O) without added test compounds.

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DNAPK assay [1]
An in vitro assay kit was used in combination with the purified DNA-PK enzyme. The in vitro kinase assay reactions were performed according to the manufacturer`s protocol but modified as follows: 27 U of purified DNA-PK protein / reaction, 1 µM ATP / reaction, 1 % DMSO or indicated compound / reaction at 37 °C for 30 min.

A2780 cells are cultured in DMEM supplemented with 10% FBS. L-glutamine, sodium pyruvate, and antibiotics. In black-walled, clear-bottom plates, 1000 cells are plated in the same medium at a density of 100 uL per well, and the cells are then incubated for three to five hours. The Buparlisib (NVP-BKM120) supplied in DMSO (20 mM) is further diluted in DMSO (7.5 uL of 20 mM Buparlisib in 22.5 uL DMSO). In order to make nine concentrations, repeat the process of mixing well and adding 10 uL to 20 uL DMSO. It is then followed by the addition of the diluted Buparlisib (NVP-BKM120) solution (2 uL) to the cell medium (500 uL). Equal amounts of this solution (100 uL) are poured on top of the cells in 96-well plates, where they are then incubated at 37°C for three days before being developed with Cell Titer Glo. Luminescence reading with Trilux is used to ascertain whether cell proliferation is being inhibited[1].

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pSer473 Akt Assay [1]
Cells were plated in the same medium at a density of 15,000 cells per well into 96 well tissue culture plates, with outside wells vacant, and allowed to adhere overnight. Test compounds supplied in DMSO were diluted further into DMSO at 500 times the desired final concentrations before dilution into culture media to 2 times the final concentrations. Equal volumes of 2x compounds were added to the cells in 96 well plates and incubated at 37 ºC for one hour. The media and compounds were then removed, the plates chilled and cells lysed in a lysis buffer (150 mM NaCl, 20 mM Tris pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100) supplemented with phosphatase and protease inhibitors. After thorough mixing, lysates were transferred to both pSer473Akt and total Akt assay plates, and incubated overnight with shaking at 4 ºC. The plates were washed with 1 x MSD wash buffer and the captured analytes detected with secondary antibodies. After incubation with the secondary antibody at room temperature for 1-2 hours, the plates were washed again and 1.5x concentration of Read Buffer T (MSD) was added to the wells. The assays were read on a SECTOR Imager 6000 instrument. Ratios of the signal from pSer473Akt and total Akt assays were used to correct for any variability and the percent inhibition of pSer473Akt from the total signal seen in cells treated with compound versus DMSO alone was calculated and used to determine EC50 values for each compound.

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A2780 Cell Proliferation Assay [1]
A2780 cells were cultured in DMEM supplemented with 10% FBS. L-glutamine, sodium pyruvate, and antibiotics. Cells were plated in the same medium at a density of 1000 cells per well, 100 ul per well into black-walled-clear-bottom plates and incubated for 3-5 hours. Test compounds supplied in DMSO (20 mM) were diluted further into DMSO (7.5 ul of 20 mM test compound in 22.5 ul DMSO. Mix well, transfer 10 ul to 20 ul DMSO, repeat until 9 concentrations have been made). The diluted test compound solution (2uL), was then added to cell medium (500 ul) cell medium. Equal volumes of this solution (100 uL) were added to the cells in 96 well plates and incubated at 37 ºC for 3 days and developed using Cell Titer Glo. Inhibition of cell proliferation was determined by luminescence read using Trilux. For the other cell lines, all the methodologies and reagents were described previously (Maira, M. et Al. Cancer Research (2008), 68(19), 8022-8030, 2008).

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Cell growth assay [2]
The growth inhibitory effects of Buparlisib/BKM120 on MM cells or normal PBMCs were assessed by MTS assay following the manufacturer's protocol.

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Apoptosis assays [2]
BKM120/Buparlisib-induced cell apoptosis was detected by annexin V binding assay as previously described.

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Cell cycle analysis [2]
MM cell lines ARP-1, MM.1S, and MM.1R were cultured with or without 1 μM Buparlisib/BKM120 for 24 h. Cells were harvested and permeabilized in 70% ethanol at 4°C overnight, followed by incubation with 50 μg/ml PI and 20 μg/ml RNase-A for 15 min. DNA content was analyzed by flow cytometry and FlowJo software.

Mice: The SCID (severe combined immunodeficiency) mouse model is a female, six to eight week old mouse. One million ARP-1 or MM.1S cells suspended in 50 μL phosphate-buffered saline (PBS) are subcutaneously injected into SCID mice in the right flank. DMSO/PBS or Buparlisib (NVP-BKM120) (5 μM per kg per day) are administered intraperitoneally to mice 15 days after the development of a palpable tumor (tumor diameter ≥5 mm). Each time a blood sample is taken, tumor sizes are also measured every five days. The size of the tumor and the presence of human kappa chain or lambda chain in the bloodstream are used to assess the burden of the tumor.\n

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\n\nIn vivo effects of Buparlisib/BKM120 on established MM [2]
\nSix- to eight-week-old female severe combined immunodeficiency (SCID) mice were subcutaneously inoculated in the right flank with 1 million ARP-1 or MM.1S cells suspended in 50 μl phosphate-buffered saline (PBS). After palpable tumor developed (tumor diameter ≥5 mm), mice were treated with intraperitoneal injection of DMSO/PBS or Buparlisib/BKM120 (5 μM per kg per day) for 15 days. Tumor sizes were measured every 5 days, and blood samples were collected at the same period. Tumor burdens were evaluated by measuring tumor size and detecting circulating human kappa chain or lambda chain.\n

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\nFemale nu/nu mice (6-8 weeks of age, 20-25 g) were used for all in vivo pharmacology studies. Mice were housed in accordance with state and federal guidelines for the humane treatment and care of laboratory animals and received food and water ad libitum. Human ovarian A2780 or glioma U87MG cells were harvested from mid-log phase cultures using trypsin-EDTA. Mice were injected subcutaneously in the right flank with 5x106 A2780 tumor cells suspended in HBSS in a total volume of 100 uL. Compound treatment was initiated when tumor volumes reached to 200–400 mm3 for PK/PD studies and 130-250 mm3 for efficacy studies. All compound treatment was administrated orally. Tumor volumes were determined using StudyDirector software. [1]

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\n\nFor PK/PD dose-dependent studies, A2780-tumor-bearing mice were given a single oral dose of compound at different concentrations (3, 10, 30, 60 and 100 mg/kg or vehicle) and tumors were resected at 10 hr after dosing. Blood samples were taken by cardiac puncture using a syringe primed with heparin sulfate. Resected tumors were snap frozen on dry ice and pulverized using a liquid nitrogen-cooled cryomortar and pestle, and lysed in cold cell extraction buffer containing protease inhibitor tablet (Complete; EDTA-free). Supernatants were taken after centrifugation of tumor lysates at 300xg for 10 min at 4 ºC and the protein concentration in each supernatant was determined by BCA. An equal amount of protein from each tumor lysate was loaded onto 10% Tris-glycine gels, for sodiumdoceylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after which proteins were transferred from the gel onto PVDF membrane. Membranes were probed with antibodies that recognize phosphoAktSer473 or phosphoAktThr308 followed by secondary goat anti-rabbit IgG conjugated to HR. Positive bands were visualized by enhanced chemiluminescence with X-ray film. Similar procedures were used to determine total AKT in the same tumor lysates to serve as normalization for total protein in each determination. The density of the positive band on the X-ray film was scanned and the target modulation for each compound was expressed as percentage inhibition by each compound compared to vehicle treatment. [1]

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\n\nFor efficacy studies in the U87MG or A2780 xenograft models, mice were randomized at mean tumor volumes of approximately 250mm3. Animals were dosed orally daily (q.d.) at 30 or 60 mg/kg. Dose volumes were adjusted based on body weight and were 4-8 mL/kg (0.1 - 0.2 mL). Tumor growth and animal body weight was measured twice weekly with daily clinical observation to monitor potential toxicities related to the drug treatment. Typically, studies were terminated when tumors in vehicletreated group reached 2000 mm3 or adverse compound-related clinical symptoms were observed. Body weights for the A2780 and U87MG efficacy studies with compound 15, Buparlisib/BKM120, were as follows. [1]

Buparlisib is particularly noteworthy because of its high solubility (170 μM on crystalline materials) and its status as one of the most potent bismorpholine compounds in cell-based assays (50 nM for target regulation; 500 nM for cell proliferation). [1]

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The pharmacokinetic properties of Buparlisib/15 were evaluated in a variety of animals (Table 5). Compound 15 exhibited low to moderate clearance (CL) in various animals, with CL values of 11, 3, 13, and 7 mL/(min/kg) in mice, rats, dogs, and monkeys, respectively. Furthermore, compound 15 exhibited moderate to high oral bioavailability in various animals, with oral bioavailability of 80%, 50%, 44%, and 100% in mice, rats, dogs, and monkeys, respectively.

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Pharmacokinetic and Pharmacodynamic Assessment[5]
Overall, the pharmacokinetics of bupalixib showed significant inter-patient variability, but no drug interaction was found between bupalixib and everolimus (Figure 3A; Table 3). Paired skin biopsy samples collected at baseline and at the end of cycle 1 showed that the drug bound to the target with the expected modulation of mTOR/PI3K signaling biomarkers. pS6 and p4EBP1 protein expression was significantly reduced in skin biopsy samples at baseline and after treatment (Figure 3B).

Based on these encouraging rodent pharmacological activities, compound 15/Buparlisib was further investigated. Analysis showed that compound 15 did not exhibit reversible or time-dependent inhibition of CYP450 (3A4, 2C9, 2D6) at concentrations up to 50 μM, nor did it exhibit induction of CYP3A4 at concentrations up to 25 μM. It is highly permeable and has no tendency to efflux, showed no cardiotoxicity, and demonstrated good sensitivity (>10 μM) to both internal safety and external MDS Pharma Services assays for the enzymes, receptors, and transporters included in the assay. Compound 15 has a melting point of 153 °C, a log D (pH 7.4) of 2.9, and a pKa of 5.1. The synthetic route for compound 15 is simple, requiring only four steps (Scheme 1). [1]

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Safety, toxicity, and dose-limiting toxicities (DLT) [5]
The most common adverse events with an incidence exceeding 20% included thrombocytopenia, anorexia, mucositis, nausea/vomiting, fatigue, elevated transaminases (ALT/AST), hyperglycemia, hypokalemia, and musculoskeletal pain (Table 2). No treatment-related deaths occurred, but as of this analysis, 35 patients (81.4%) of all enrolled patients had died. Of the 28 evaluable patients treated during the dose escalation phase, 7 experienced DLT. DLT was defined as grade 3 urinary tract infection, grade 3 fatigue, grade 3 creatinine elevation, grade 3 hyperglycemia, and grade 3 parainfluenza respiratory infection. Details of DLT and the dose levels at which they occurred are provided in Supplementary Table S1. The maximum doses of everolimus 10 mg + 60 mg and everolimus 5 mg + 80 mg in combination with bupalixib were not tolerated. Based on tolerability within the dose-limiting toxicity (DLT) window and long-term tolerability after the 4-week window, the combination of everolimus (5 mg daily) and bupalixib (60 mg daily) was the highest tolerable dose and was identified as the recommended Phase II dose (RP2D).

[1]. Identification of NVP-BKM120 as a Potent, Selective, Orally Bioavailable Class I PI3 Kinase Inhibitor for Treating Cancer. ACS Med Chem Lett. 2011 Aug 26;2(10):774-9.

[2]. Novel phosphatidylinositol 3-kinase inhibitor NVP-BKM120 induces apoptosis in myeloma cells and shows synergistic anti-myeloma activity. J Mol Med (Berl). 2012 Jun;90(6):695-706.

[3]. Combination inhibition of PI3K and mTORC1 yields durable remissions in mice bearing orthotopic patient-derived xenografts of HER2-positive breast cancer brain metastases. Nat Med. 2016 Jul;22(7):723-6.

[4]. Identifying and Targeting Sporadic Oncogenic Genetic Aberrations in Mouse Models of Triple Negative Breast Cancer. Cancer Discov. 2018 Mar;8(3):354-369.

[5]. A Phase I Study of Safety, Pharmacokinetics, and Pharmacodynamics of Concurrent Everolimus and Buparlisib Treatment in Advanced Solid Tumors. Clin Cancer Res. 2020 Jun 1;26(11):2497-2505.

BKM120/Buparlisib is an aminopyridine compound with the structure 4-(trifluoromethyl)pyridine-2-amine, substituted at the 5-position with a 2,6-di(morpholino-4-yl)pyrimidin-4-yl group. It is a selective PI3K inhibitor with antitumor activity. It functions as an EC 2.7.1.137 (phosphatidylinositol 3-kinase) inhibitor and an antitumor drug. It belongs to the morpholinosides, aminopyrimidines, aminopyridines, and organofluorine compounds. Buparlisib has been used in the treatment and basic research clinical trials of various cancers, including lymphoma, metastatic tumors, lung cancer, solid tumors, and breast cancer. Buparlisib is a highly bioavailable, orally bioavailable, specific class I phosphatidylinositol 3-kinase (PI3K) lipid kinase inhibitor with potential antitumor activity. Bupaliximab specifically inhibits class I PI3K in the PI3K/AKT kinase (or protein kinase B) signaling pathway via an ATP-competitive mechanism, thereby inhibiting the production of the second messenger phosphatidylinositol-3,4,5-triphosphate and the activation of the PI3K signaling pathway. This may lead to suppression of the growth and survival of susceptible tumor cell populations. Activation of the PI3K signaling pathway is generally closely associated with tumorigenesis. Dysregulation of the PI3K signaling pathway may lead to resistance to multiple antitumor drugs in tumors. Phosphatidylinositol-3-kinase (PI3K) is an important target for tumor therapy because this signaling pathway is dysregulated in various human cancers. This article describes a series of structure-guided optimizations of 2-morpholino, 4-substituted, 6-heterocyclic pyrimidine compounds, in which the pharmacokinetic properties were improved by modulating the electronic properties of the 6-position heterocycle, and the overall drug-like properties were further fine-tuned by modifying the 4-position substituent. The resulting 2,4-bismorpholino-6-heterocyclic pyrimidine compounds are potent class I PI3K inhibitors, exhibiting mechanistic regulatory effects in PI3K-dependent cell lines and in vivo efficacy in PI3K pathway-dysregulated tumor xenograft models (A2780 ovarian cancer and U87MG glioma). These efforts ultimately led to the discovery of compound 15 (Buparlisib or NVP-BKM120), which is currently in a phase II clinical trial for cancer treatment. [1] In summary, we describe a series of structure-guided optimizations of 6-aminoheterocyclic, 4-substituted, 2-morpholinopyrimidine compounds with high chemiluminescence intensity and low water solubility, ultimately yielding compounds suitable for clinical development. Modifying the amino heterocycle with small groups that regulate the ring electronic properties can increase activity or decrease in vivo chemiluminescence intensity. Introducing a morpholino group at the C4 center of the pyrimidine ring can improve water solubility while maintaining sufficient potency, selectivity and good in vivo properties. The combination of these modifications led to the discovery of a series of substituted 6-aminoheterocyclic 2,4-bismorpholinopyrimidines. Of the compounds in this series, compound 15 (bupaliximab, NVP-BKM120) has entered the human trial stage and is currently undergoing a phase II clinical trial. [1] NVP-BKM120/bupaliximab is a novel phosphatidylinositol 3-kinase (PI3K) inhibitor and is currently undergoing a phase I clinical trial in solid tumors. This study aimed to evaluate the therapeutic effect of BKM120 in multiple myeloma (MM). BKM120 inhibited the growth of MM cell lines and freshly isolated primary MM cells and induced their apoptosis. However, BKM120 had limited cytotoxicity to normal lymphocytes. The presence of bone marrow mesenchymal cells, insulin-like growth factor or interleukin-6 did not affect the apoptosis of tumor cells induced by BKM120. More importantly, BKM120 treatment significantly inhibited tumor growth in vivo and prolonged the survival of tumor-bearing mice. In addition, BKM120 and dexamethasone showed synergistic cytotoxicity in dexamethasone-sensitive myeloma cells. Low doses of BKM120 and dexamethasone (both with limited cytotoxicity when used alone) significantly induced apoptosis in MM.1S and ARP-1 cells. Mechanistic studies showed that BKM120 caused cell cycle arrest by upregulating p27 (Kip1) and downregulating cyclin D1, and induced caspase-dependent apoptosis by downregulating the anti-apoptotic protein XIAP and upregulating the expression of the cytotoxic small subtype Bim (BimS). In summary, our results confirm that BKM120 has anti-multiple myeloma (MM) activity in vitro and in vivo, and suggest that BKM120, used alone or in combination with other MM chemotherapeutic drugs (especially dexamethasone), may be a promising treatment for MM. [2] Buparlisib/BKM120, used alone or in combination with other anti-myeloma chemotherapeutic drugs (especially dexamethasone), may be an effective treatment for multiple myeloma (MM). [2]

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Objective: Synchronous inhibition of mTOR and PI3K in preclinical models has been shown to improve efficacy, providing a theoretical basis for this Phase I study of everolimus and bupalixib (BKM120) in patients with advanced solid tumors. Patients and Methods: We used a Bayesian dose-escalation overdose control design to test escalating doses of everolimus (5 or 10 mg) and bupalixib (20, 40, 60, 80, and 100 mg) in eligible patients. Blood samples were collected on days 8 and 15 of cycle 1 for pharmacokinetic assessment. Paired skin biopsy samples were collected at baseline and at the end of cycle 1 to assess the pharmacodynamic effects of the drugs on the regulation of the mTOR/PI3K pathway. Results: We included 43 patients with a median age of 63 years (range 39–78 years); 25 (58.1%) were female, 35 (81.4%) were Caucasian, and 8 (18.6%) were Black. The most common toxicities were hyperglycemia, diarrhea, nausea, fatigue, and elevated aspartate aminotransferase. Dose-limiting toxicities were observed in 7 patients, including fatigue (3), hyperglycemia (2), mucositis (1), acute kidney injury (1), and urinary tract infection (1). The Phase II recommended dose (RP2D) for this combination therapy was determined to be everolimus (5 mg) and bupalixib (60 mg). Among the 27 evaluable patients, the best response was disease progression in 3 patients (11%) and stable disease in 24 patients (89%). The median progression-free survival and overall survival were 2.7 months (1.8–4.2 months) and 9 months (6.4–13.2 months), respectively. Steady-state pharmacokinetic analysis showed that the dose-normalized maximum concentration and AUC of everolimus and bupalixib in combination were comparable to those of the single drug. [5]

C18H21F3N6O2

410.3936

410.167

C, 52.68; H, 5.16; F, 13.89; N, 20.48; O, 7.80

944396-07-0

Buparlisib Hydrochloride;1312445-63-8

16654980

white solid powder

1.4±0.1 g/cm3

645.7±65.0 °C at 760 mmHg

344.3±34.3 °C

0.0±1.9 mmHg at 25°C

1.574

2.08

1

11

3

29

530

0

FC(C1C(C2C=C(N3CCOCC3)N=C(N3CCOCC3)N=2)=CN=C(N)C=1)(F)F

CWHUFRVAEUJCEF-UHFFFAOYSA-N

InChI=1S/C18H21F3N6O2/c19-18(20,21)13-9-15(22)23-11-12(13)14-10-16(26-1-5-28-6-2-26)25-17(24-14)27-3-7-29-8-4-27/h9-11H,1-8H2,(H2,22,23)

5-[2,6-Di(4-morpholinyl)-4-pyrimidinyl]-4-(trifluoromethyl)-2-pyridinamine.

Buparlisib; BKM120; BKM-120; Buparlisib; 944396-07-0; NVP BKM120; BKM-120; NVPBKM-120; 1202777-78-3; NVPBKM120; NVP BKM120; NVP-BKM120; 5-(2,6-dimorpholinopyrimidin-4-yl)-4-(trifluoromethyl)pyridin-2-amine; BKM-120; NVP-BKM-120; 1202777-78-3; NV-BKM120

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: ~82 mg/mL (199.8 mM)
Water: <1 mg/mL (slightly soluble or insoluble)
Ethanol: 2 mg/mL (4.87 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.09 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 (6.09 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 (6.09 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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Solubility in Formulation 4: ≥ 2.5 mg/mL (6.09 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 5: ≥ 2.5 mg/mL (6.09 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 6: 0.5%CMC Na:6mg/mL

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Solubility in Formulation 7: 2.08 mg/mL (5.07 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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