p110α (IC50 = 52 nM); p110β (IC50 = 166 nM); p110δ (IC50 = 116 nM); p110γ (IC50 = 262 nM); Vps34 (IC50 = 2.4 μM); p110α-H1047R (IC50 = 58 nM); p110α-E545K (IC50 = 99 nM); mTOR (IC50 = 4.6 μM)

Buparlisib (BKM120) exhibits 50-300 nM activity for class I PI3K’s, including the most common p110α mutants. Additionally, NVP-BKM120 has less potency against class III and class IV PI3K's; inhibition of VPS34, mTOR, DNAPK, and PI4K, respectively, is seen to be inhibited by 2, 5, >5, and >25 μM biochemical activity, respectively[1]. Both dose and time dependencies are present in how buparlisib (BKM120) causes apoptosis in multiple myeloma (MM) cells. Buparlisib (BKM120) significantly induces apoptosis in all tested MM cell lines after 24 hours at concentrations 10 μM (P<0.05, compares with control). If not specified otherwise, the following experiments will use 10 μM buparlisib (BKM120) and a 24-h treatment. All of the tested MM cell lines exhibit a dose-dependent growth inhibition in response to buparlisib (BKM120) treatment. Each tested MM cell has a different buparlisib (BKM120) IC50 value. 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 M. In conclusion, buparlisib (BKM120) treatment causes MM cell growth inhibition and apoptosis in ways that depend on the dose and 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 (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, pAKTSer473 is partially inhibited, and at doses of 30, 60, or 100 mg/kg, it is nearly completely inhibited. Both plasma and tumor drug exposure were well correlated with inhibition of pAKT (normalized to total AKT)[1]. Buparlisib (BKM120) (5 μM per kg per day for 15 days)-treated mice had significantly lower tumor burdens than control mice, as determined by tumor volume (P<0.05) and level of circulating human kappa chain (P<0.05). Additionally, buparlisib (BKM120) treatment 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 is dissolved in DMSO and then immediately distributed at a rate of 1.25 µL per well into a black 384-well plate for the PI3K biochemical assay (ATP depletion assay). 25 µL of 10 nM PI3 kinase, 5 µg/mL 1-phosphatidylinositol (PI), and 25 µL of 2 µM ATP in assay buffer are added to each well to initiate the reaction. Assay buffer is composed of 10 mM Tris pH 7.5, 5 mM MgCl2, 20 mM NaCl, 1 mM DTT, and 0.05% CHAPS. The addition of 25 µL of KinaseGlo solution stops the reaction after it has run for approximately 50% of the ATP. The stopped reaction is allowed to continue for five minutes, after which the remaining ATP is found by luminescence.

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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. Buparlisib (BKM120) is diluted further into DMSO after being supplied in the 20 mM solution (7.5 uL of 20 mM NVP-BKM120 in 22.5 uL DMSO). To make nine concentrations, repeat the process (mix well, transfer 10 uL to 20 uL DMSO, etc.). Then, the cell medium (500 uL) is added, followed by the diluted Buparlisib (BKM120) solution (2 uL). Equal volumes 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: SCID(Six- to eight-week-old female severe combined immunodeficiency) mice are used. One million ARP-1 or MM.1S cells suspended in 50 mL of phosphate-buffered saline (PBS) are subcutaneously injected into SCID mice's right flank. Mice are administered DMSO/PBS or buparlisib (BKM120) (5 μL per kg per day) intraperitoneally for 15 days after the development of a palpable tumor (tumor diameter 5 mm). Blood samples are taken at regular intervals and tumor sizes are measured every five days. The size of the tumor and the presence of circulating human kappa chain or lambda chain are used to assess the burden of the tumor.

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In vivo effects of Buparlisib/BKM120 on established MM [2]
Six- 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.

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Female 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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For 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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For 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 was of particular interest, as the solubility was the highest (170 μM on crystalline material) and it was among the most potent bismorpholine compounds in cell based assays (50 nM target modulation; 500 nM cell proliferation). [1]

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The pharmacokinetic properties of Buparlisib/15 were evaluated across multiple species (Table 5). Low to moderate CL was observed for 15 across species, as CL values of 11, 3, 13, and 7 mL/(min kg) were observed in mouse, rat, dog, and monkey, respectively. Additionally, 15 exhibited medium to high oral bioavailability across species as 80%, 50%, 44%, and 100% was observed in mouse, rat, dog, and monkey, respectively.

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Pharmacokinetic and pharmacodynamic assessments [5]
Overall, there was greater interpatient variability in Buparlisib pharmacokinetic but there was no evidence of DDI between buparlisib and everolimus (Fig. 3A; Table 3). Paired skin biopsy collected at baseline and end of cycle 1 showed evidence of target engagement with expected modulation of biomarkers of the mTOR/PI3K signaling molecules. There was a marked reduction in pS6 and p4EBP1 protein expression between baseline and posttreatment skin biopsy samples (Fig. 3B).

With these encouraging rodent pharmacology activities, compound 15/Buparlisib was studied further. Profiling indicated that 15 exhibited no reversible or time dependent CYP450 (3A4, 2C9, 2D6) inhibition up to 50 μM or CYP3A4 induction up to 25 μM, exhibited high permeability with no propensity for efflux, demonstrated no cardiotoxicity potential, and showed a clean profile (>10 μM) against enzymes, receptors, and transporters included in internal safety and the external MDS Pharma Services panels. The melting point of 15 is 153 °C, its log D (pH 7.4) is 2.9, and its pKa is 5.1. The synthesis of 15 is straightforward, proceeding in four steps for the research route (Scheme 1). [1]

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Safety, toxicity, and DLTs [5]
The most commonly observed adverse event occurring in >20% of subjects included thrombocytopenia, anorexia, mucositis, nausea/vomiting, fatigue, elevated transaminases (ALT/AST), hyperglycemia, hypokalemia, and musculoskeletal pain (Table 2). There were no treatment-related deaths, but 35 (81.4%) of all enrolled patients had died at the time of this analysis. Seven of the 28 evaluable patients treated in the dose-escalation phase experienced a DLT. DLT defining toxicities included grade 3 urinary tract infection, grade 3 fatigue, grade 3 elevated creatinine, grade 3 hyperglycemia, and grade 3 parainfluenza respiratory infection. Details of DLT and the dose level where they were encountered are presented in Supplementary Table S1. The maximum administered combination doses of 10 mg plus 60 mg and 5 mg plus 80 mg everolimus plus Buparlisib, respectively, were not tolerable. On the basis of tolerability within the DLT window and long-term tolerability beyond the 4-week window, the combination of everolimus (5 mg daily) along with buparlisib (60 mg daily) was the highest tolerated dose and was declared the 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.

Phosphoinositide-3-kinases (PI3Ks) are important oncology targets due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein we describe the structure guided optimization of a series of 2-morpholino, 4-substituted, 6-heterocyclic pyrimidines where the pharmacokinetic properties were improved by modulating the electronics of the 6-position heterocycle, and the overall druglike properties were fine-tuned further by modification of the 4-position substituent. The resulting 2,4-bismorpholino 6-heterocyclic pyrimidines are potent class I PI3K inhibitors showing mechanism modulation in PI3K dependent cell lines and in vivo efficacy in tumor xenograft models with PI3K pathway deregulation (A2780 ovarian and U87MG glioma). These efforts culminated in the discovery of 15 (NVP-BKM120), currently in Phase II clinical trials for the treatment of cancer.[1]

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NVP-BKM120 is a novel phosphatidylinositol 3-kinase (PI3K) inhibitor and is currently being investigated in phase I clinical trials in solid tumors. This study aimed to evaluate the therapeutic efficacy of BKM120 in multiple myeloma (MM). BKM120 induces cell growth inhibition and apoptosis in both MM cell lines and freshly isolated primary MM cells. However, BKM120 only shows limited cytotoxicity toward normal lymphocytes. The presence of MM bone marrow stromal cells, insulin-like growth factor, or interleukin-6 does not affect BKM120-induced tumor cell apoptosis. More importantly, BKM120 treatment significantly inhibits tumor growth in vivo and prolongs the survival of myeloma-bearing mice. In addition, BKM120 shows synergistic cytotoxicity with dexamethasone in dexamethasone-sensitive MM cells. Low doses of BKM120 and dexamethasone, each of which alone has limited cytotoxicity, induce significant cell apoptosis in MM.1S and ARP-1. Mechanistic study shows that BKM120 exposure causes cell cycle arrest by upregulating p27 (Kip1) and downregulating cyclin D1 and induces caspase-dependent apoptosis by downregulating antiapoptotic XIAP and upregulating expression of cytotoxic small isoform of Bim, BimS. In summary, our findings demonstrate the in vitro and in vivo anti-MM activity of BKM120 and suggest that BKM120 alone or together with other MM chemotherapeutics, particularly dexamethasone, may be a promising treatment for MM.[2]

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BKM120/Buparlisib is an aminopyridine that is 4-(trifluoromethyl)pyridin-2-amine substituted at position 5 by a 2,6-di(morpholin-4-yl)pyrimidin-4-y group. A selective PI3K inhibitor with anti-tumour properties. It has a role as an EC 2.7.1.137 (phosphatidylinositol 3-kinase) inhibitor and an antineoplastic agent. It is a member of morpholines, an aminopyrimidine, an aminopyridine and an organofluorine compound.
Buparlisib has been used in trials studying the treatment and basic science of Lymphoma, Metastases, Lung Cancer, Solid Tumors, and Breast Cancer, among others.
Buparlisib is an orally bioavailable specific oral inhibitor of the pan-class I phosphatidylinositol 3-kinase (PI3K) family of lipid kinases with potential antineoplastic activity. Buparlisib specifically inhibits class I PI3K in the PI3K/AKT kinase (or protein kinase B) signaling pathway in an ATP-competitive manner, thereby inhibiting the production of the secondary messenger phosphatidylinositol-3,4,5-trisphosphate and 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.

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In summary, we have described the structure guided optimization of a series of 6-aminoheterocyclic, 4-substituted, 2-morpholino pyrimidines which exhibited high CL and low aqueous solubility into a compound suitable for clinical development. Modification of the aminoheterocycle with small groups that modulated the ring electronics either improved potency or reduced the in vivo CL values. Incorporation of a morpholine group at the C4 central pyrimidine position increased the aqueous solubility while retaining sufficient potency, selectivity, and favorable in vivo properties. The combination of these modifications led to the discovery of a series of substituted 6-aminoheterocyclic, 2,4-bismorpholino pyrimidines. From this series, compound 15 (Buparlisib, NVP-BKM120) has advanced into humans and is currently being assessed in phase II trials. [1]

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In conclusion, our study demonstrates the anti-MM activity of BKM120 in vitro and in vivo. Buparlisib/BKM120 alone or together with other antimyeloma chemotherapeutics, particularly dexamethasone, may be promising therapeutic agents for MM.[2]

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Purpose: Concurrent inhibition of mTOR and PI3K led to improved efficacy in preclinical models and provided the rationale for this phase I study of everolimus and buparlisib (BKM120) in patients with advanced solid tumor. Patients and methods: We used the Bayesian Escalation with Overdose Control design to test escalating doses of everolimus (5 or 10 mg) and buparlisib (20, 40, 60, 80, and 100 mg) in eligible patients. Pharmacokinetic assessment was conducted using blood samples collected on cycle 1, days 8 and 15. Pharmacodynamic impact on mTOR/PI3K pathway modulation evaluated in paired skin biopsies collected at baseline and end of cycle 1. Results: We enrolled 43 patients, median age of 63 (range, 39-78) years; 25 (58.1%) females, 35 (81.4%) Caucasians, and 8 (18.6%) Blacks. The most frequent toxicities were hyperglycemia, diarrhea, nausea, fatigue, and aspartate aminotransferase elevation. Dose-limiting toxicities observed in 7 patients were fatigue (3), hyperglycemia (2), mucositis (1), acute kidney injury (1), and urinary tract infection (1). The recommended phase II dose (RP2D) for the combination was established as everolimus (5 mg) and buparlisib (60 mg). The best response in 27 evaluable patients was progressive disease and stable disease in 3 (11%) and 24 (89%), respectively. The median progression-free survival and overall survival were 2.7 (1.8-4.2) and 9 (6.4-13.2) months. Steady-state pharmacokinetic analysis showed dose-normalized maximum concentrations and AUC values for everolimus and buparlisib in combination to be comparable with single-agent pharmacokinetic.[5]

C₁₈H₂₂CLF₃N₆O₂

446.85

446.144

C, 48.38; H, 4.96; Cl, 7.93; F, 12.75; N, 18.81; O, 7.16

1312445-63-8

Buparlisib;944396-07-0

66577015

White to yellow solid powder

2.674

2

11

3

30

530

0

NC(N=C1)=CC(C(F)(F)F)=C1C2=NC(N3CCOCC3)=NC(N4CCOCC4)=C2.[H]Cl

DGPLYAXBXJXEID-UHFFFAOYSA-N

InChI=1S/C18H21F3N6O2.ClH/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);1H

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.59 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 (5.59 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 (5.59 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: 0.5% CMC Na : 6mg/mL

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